Unique and Shared Fibroblast Mechanisms across Human Tissues and Pathologies

**Chapter 3**

## The Role of Fibroblasts in Atherosclerosis Progression

*Tadeja Kuret and Snežna Sodin-Šemrl*

#### **Abstract**

The following chapter addresses vascular fibroblasts in a healthy, quiescent state, as well during vascular inflammation, focusing on atherosclerosis. The development of atherosclerosis, an inflammatory disease of medium- and large-sized arteries, has traditionally been viewed as an "inside-out" mechanism, with prominent roles of the innermost layer of the artery, consisting of endothelial cells. However, emerging evidence suggests a new paradigm of "outside-in" mechanism, including an earlier role for fibroblasts, constituents of the outermost adventitial layer of the artery. Phenotypic and functional changes of fibroblasts in adventitia may even occur prior to, or alongside endothelial activation. Activated adventitial fibroblasts, implicated in atherosclerosis progression, begin to transform into myofibroblasts, upregulate production of different proinflammatory cytokines, chemokines, growth factors, proteolytic enzymes, extracellular matrix proteins and reactive oxygen species, leading to extensive matrix remodeling, chemotaxis and recruitment of immune cells. Due to their suitable location for drug delivery systems, preventing fibroblast activation, modulating their activity or inducing myofibroblast dedifferentiation could represent a promising therapeutic approach for atherosclerosis regression.

**Keywords:** atherosclerosis, fibroblasts, inflammation, disease progression

#### **1. Introduction**

Fibroblasts are mesenchymal cells that are morphologically characterized as adherent, flat, elongated (spindle-shaped) cells with leveled, oval nuclei. One of their major roles is to produce and integrate structural proteins, such as collagen, elastin, and proteoglycans into the extracellular matrix (ECM) of most mesenchymal tissues and thus maintain their structural integrity [1].

In healthy arteries, fibroblasts can be found in the adventitia, the outermost layer of the vessel wall. Adventitial fibroblasts display numerous subtypes, even in a quiescent state, however, very little is known about their exact involvement in atherosclerosis development and progression [2]. Most of the attributed functions of adventitial fibroblasts have been largely extrapolated from findings describing fibroblasts in different tissues and organs, such as the skin. However, fibroblasts from different anatomic sites and tissues are functionally and phenotypically distinct. For example, cultured fetal and adult human skin fibroblasts derived from different anatomical

sites expressed distinct transcriptional patterns of genes involved in extracellular matrix synthesis, lipid metabolism, and cell signaling pathways regulating proliferation, cell migration and fate determination [3]. The discovered topographic differences of fibroblasts might be connected to the positional memory since adult fibroblast maintain key features of the HOX gene pattern expression, established during embryogenesis. Indeed, many HOX genes that encode a family of evolutionarily conserved transcription factors, are differentially expressed in fibroblasts derived from different anatomical sites, indicating that fibroblasts from each topographic site express a unique HOX gene expression pattern [3, 4].

Fibroblasts are metabolically active cells that play a central role in, not only matrix maintenance and remodeling and regulating ECM, but also in managing interstitial fluid volume and pressure, new tissue formation and wound healing. They have been found to be associated with many connective tissue pathologies, either due to their direct implication in the disease mechanism or due to the resulting fibrosis associated with damage in other cell types [5]. Recently, novel mechanisms proposed a prominent role of fibroblasts also in the development and progression of atherosclerosis. Atherosclerosis is a chronic, fibro-proliferative disease of the arterial vessel walls that underlies the development of many cardiovascular diseases (CVDs) and affects the structure and function of the involved arteries [6].

Vascular inflammation, leading to atherosclerosis, has been traditionally viewed as an "inside-out" response, beginning with the activation of endothelium and an inflammatory response that spreads outwards, from the intima towards media and adventitia, ultimately forming fibrous plaque and damaging all three vessel wall layers [7, 8]. The classical mechanism of atherogenesis has been challenged recently with emerging evidence supporting a new hypothesis of an "outside-in" mechanism, in which vascular inflammation actually begins in the adventitia and progresses inward towards the media and intima [8, 9], suggesting a more prominent role of fibroblasts than previously thought.

So, in order to pinpoint, the potential role of fibroblasts during atherosclerosis progression, we need to first look at the arterial wall composition and function.

#### **2. Vessel wall structure and fibroblasts**

#### **2.1 Arterial vessel wall structure**

Characterization of the resident, stromal cell populations and subpopulations in a blood vessel is an important step in understanding cellular contribution to vascular development and disease. Since atherosclerosis is prevalently a disease of large- and medium-sized arteries [10], we will focus here on the description of the structure of these vessels and corresponding vascular stromal cell populations.

The walls of large- and medium-sized arteries are a heterogeneous three-layered structure consisting of the tunica intima, media and adventitia. Each layer is unique in its histologic, biochemical and functional properties and is differentially involved in maintaining vascular homeostasis and regulating the vascular response to stress or injury [8]. The tunica intima or innermost layer represents a monolayer of endothelial cells, which are in direct contact with the blood flow. The intima is separated from the tunica media by a basement membrane and an internal elastic lamina. The tunica media consists of multiple layers of vascular smooth muscle cells (VSMC). The tunica adventitia or the outermost layer is separated from the media by an external elastic

#### *The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

lamina and represents the most complex layer of the blood vessel [11]. The adventitia is composed primarily of fibroblasts in a loose connective tissue matrix, and it also contains resident immune cells (e.g. dendritic cells, macrophages, mast cells), pericytes, small blood vessels with endothelial cells (*vasa vasorum*), several progenitor cells and adrenergic nerve cells (**Figure 1**) [13].

For many years, adventitial cells were thought to have a limited physiological "static" function serving as structural support to the blood vessel, supplying oxygen and nutrients to the media of large vessels, and sustaining sympathetic innervation of the vessel wall [14]. However, latest *in vivo* and *in vitro* studies have shown that the adventitia represents a dynamic microenvironment that regulates both structural and functional properties of all three arterial layers [15]. The adventitia is an important source of cells that migrate towards the intima and media. For example, stem and progenitor cells that function and reside in the adventitia can transform into medial and intimal cells, such as VSMCs, and endothelial cells [16–18]. The adventitia of coronary arteries also contains cholinergic nerve terminals that release acetylcholine, diffusing to the intima layer of endothelium, where it induces the release of nitric oxide, causing VSMC relaxation and vasodilatation [19]. After stimulation with angiotensin II, adventitial fibroblasts can synthesize and release endothelin-1 (ET-1) that is important in mediating VSMC contraction [20]. The important role of adventitia is further supported by the role of *vasa vasorum* network, serving as a pipeline for inflammatory cell infiltration during vascular inflammation. In atherosclerosis, inflammation contributes to increased neovascularization and enhanced permeability of the adventitial *vasa vasorum*, allowing more inflammatory cells to enter the atherosclerotic plaque. Indeed, by suppressing the neovascularization of

#### **Figure 1.**

*Heterogeneous cellular composition of the vascular adventitia. Compared to arterial intima and media, which are composed of endothelial and smooth muscle cells, respectively, the arterial adventitia is composed of a variety of heterogeneous cell populations, including fibroblasts, immunomodulatory cells (e.g. dendritic cells, macrophages),*  vasa vasorum*, pericytes and adrenergic nerves. The figure was adapted from [12] and created using Biorender.*

*vasa vasorum*, Moulton et al. [21] observed reduction in numbers of macrophages in atherosclerotic plaques and inhibition of atherosclerosis progression in experimental mice models [21]. Additionally, adventitial resident cells participate in initiation and regulation of vascular development, response to injury and tissue repair and thus, importantly contribute to disease development, especially intimal hyperplasia. This is mediated by their ability of responding to external physiological stress with intensive tissue repair or arterial remodeling [9, 16, 22]. Importantly, resident adventitial cells (e.g. fibroblasts) are often the first cells in the vascular wall to become activated in response to hormonal and inflammatory stimuli, as well as environmental stress, such as hypoxia/ischemia and hypertension [8, 15].

#### **2.2 Fibroblasts in healthy arterial vessel walls**

Understanding the role of fibroblasts in normal and pathologic conditions is often obstructed by the lack of reliable and specific markers. The fibroblast is therefore still one of the most difficult cell types to define *in vivo*, likely due to their heterogeneity (multiple subtypes) and plasticity [23].

In a healthy artery, adventitial fibroblasts are found in a non-active, quiescent state, and are usually defined by their location in the vessel wall since they can be separated from the more generally recognized smooth muscle cell layer by an external elastic lamina [24, 25]. All currently used markers to identify fibroblasts, including vimentin, platelet derived growth factor receptor α (PDGFRα), fibroblast specific protein 1 (FSP1), discoidin-domain receptor, and prolyl-4-hydroxylase, are potentially problematic, as they are also expressed in other cell types and are not present in all fibroblasts [3, 26, 27]. Therefore, to identify fibroblasts, investigators have to rely on the lack of markers for other cell lineages (e.g. non-lymphoid, non-endothelium, and non-epithelium), along with morphologic, functional, and biochemical characteristics [3].

Adventitial fibroblasts show differences in morphology, size, function and activity in the healthy, as well as stressed conditions or disease states [15]. For example, An et al. [28] found two major fibroblast subpopulations in the adventitia of rat thoracic aorta. The two populations were described as epithelioid-like cells and spindle-like cells, however only epithelioid-like fibroblasts were sensitive to stimulation with angiotensin II, a hormone involved in the development of hypertension and atherosclerosis [28]. Studying fibroblasts from the adventitia of bovine pulmonary artery, Das et al. [29] concluded that numerous phenotypically and biochemically distinct fibroblast subpopulations can be found and only a selective increase in the number of resident fibroblast subpopulations with enhanced growth capability was observed under hypoxic conditions [29].

With the development and increasing popularity of single cell RNA sequencing technology, it might now be possible to find markers, specific to adventitial fibroblasts, as well as to characterize in depth, their subpopulations in normal and diseased states [30]. Kalluri et al. [31] explored the cellular atlas of healthy mice aortas using single cell RNA sequencing. They showed that fibroblasts represent approximately 33% of all aortic cells and were defined by higher expression of PDGFRα and collagens/collagen-binding proteins (e.g. Col1a1, Col1a2, Dcn, Lum) whereas the expression of VSMC-associated contractile proteins (e.g. Myh11, Cnn1) was reduced. These fibroblasts clustered into two subpopulations and are probably derived from the adventitia, however their exact location needs to be confirmed by immunohistochemistry or in-situ hybridization [31]. Another study using the high resolution single

#### *The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

cell analysis approach, was performed by Gu et al. [32] in aortic adventitial cells from wild type and apolipoprotein E-deficient (*ApoE−/−)* mice. They determined four heterogeneous mesenchymal populations with differential gene expression suggesting potential functions in ECM organization, immune regulation and bone formation. Furthermore, interaction of resident, mesenchymal cells with immune cells was enhanced in the adventitia of *ApoE−/*− mice. These data revealed a heterogeneous cellular landscape of the adventitia and confirmed fibroblast variability present already in the healthy, quiescent state [32].

#### **3. Adventitial fibroblasts in vascular pathology**

#### **3.1 Contribution of adventitial fibroblasts to vascular pathology**

In response to injury or environmental stress, adventitial fibroblasts can become activated, displaying altered phenotypic and functional properties. Activated fibroblasts intensely proliferate and increase production and deposition of ECM proteins, as well as proinflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), and growth factors, such as vascular endothelial growth factor (VEGF) [12, 33]. These molecules directly affect the phenotype of other resident cells in the vessel wall, such as VSMC and endothelial cells, promote neointima formation, regulate *vasa vasorum* expansion and affect the recruitment of infiltrating immune cells [8, 24]. Despite an excessive proliferation of activated fibroblasts, no hyperplasia of adventitia is usually observed in vascular pathologies. It is likely that many different mediators can also activate replication repressor signals in fibroblasts to limit or control replication. For instance, protein tyrosine phosphatases that regulate growth factor signaling in vascular remodeling, are upregulated in adventitial fibroblasts in response to vascular injury presumably to mitigate proliferative responses [34]. It was also demonstrated that adventitial fibroblasts under hypoxic conditions activate protein kinase C-zeta and MAPK phosphatase-1 that repress proliferative signals and limit the proliferation of fibroblasts [35, 36].

Vascular fibroblasts may also produce large amounts of reactive oxygen species (ROS) that seem to regulate their proliferation and ECM deposition [37, 38]. Activated fibroblasts increasingly interact with other cell types in the arterial wall, such as endothelial cells and VSMC and regulate their functions, as well as recruit immune cells into the vessel wall [39]. After their activation, some fibroblasts differentiate into myofibroblasts that contain bundles of stress fibers and can be identified by expression of contractile proteins, such as α-smooth muscle actin (αSMA). Multiple stimuli in the microenvironment, such as mechanical stress, growth factors (e.g. TGF-β), proinflammatory cytokines (e,g, IL-1β, IL-6, TNF-α), adhesion molecules, and ECM molecules can cause differentiation of a fibroblast towards the myofibroblast phenotype. Myofibroblasts contribute to extensive remodeling, intimal hyperplasia, and luminal stenosis due to their invasion and migration into the intima and increased production and secretion of ECM proteins [40–43].

The recognition that fibroblasts are not only able to generate, but also to sustain inflammatory responses, provides insight into why vascular inflammatory responses, in certain situations, fail to resolve. It is suggested that chronic inflammation occurs due to dysregulated fibroblast activity in which they fail to switch off their inflammatory programme, leading to the inappropriate survival and retention of leukocytes within inflamed tissue [44]. It is also clear that the activated adventitial fibroblasts

play an important role in regulating *vasa vasorum* growth, which can contribute to ongoing vascular remodeling by acting as a conduit for delivery of inflammatory and progenitor cells [45].

Recent studies shed light on the implication of adventitial fibroblasts in different vascular pathologies, characterized by arterial remodeling and neointimal formation [43]. One of the most persistent findings in experimental *in vitro* and *in vivo* models is intensive adventitial remodeling, found very early in response to vascular injury or stress [46–48]. Adventitial remodeling in the vasculature has been characterized by increased proliferation of fibroblasts, which appear to be the first cells in the vessel wall that respond to different stimuli by their activation [49]. Direct evidence of fibroblast migration into the intima was provided in a study, performed by Li et al. [50], in which primary syngeneic adventitial fibroblasts were transduced with β-galactosidase (*LacZ*) and introduced into the adventitia of rat carotid arteries immediately after baloon injury. MRNA expression of *LacZ* and in situ enzymatic activity of β-galactosidase were detected in the media and the neointima, 7 days after injury. On the contrary, in the arteries that were not injured, the expression of *LacZ* and enzymatic activity of β-galactosidase were restricted to the adventitia [50]. Similar findings were later reported by Han et al. [51] showing that adventitial fibroblasts migrated to the media and intima on seventh day after balloon injury in the rat carotid artery. The results were obtained by direct labeling of adventitial fibroblasts using *in vivo* gene transfer technique, as well as transmission electron microscopy [51]. Furthermore, Dutzmann et al. [52] discovered that early activation of adventitial fibroblasts after wire-induced injury in C57BL/6 mice stimulated their proliferation and release of proinflammatory cytokines and growth factors, and the subsequent proliferation of VSMC, resulting in neointima formation [52].

Pulmonary artery hypertension (PAH) is one of the vascular pathologies, characterized by extensive arterial remodeling and neointima formation, in which fibroblasts were shown to play an important role [53]. For example, in the neonatal bovine hypoxic PAH model, adventitial fibroblasts were found to undergo the earliest and most significant increases in proliferation, among all the vascular wall cell types [54]. Fibroblasts derived from experimental hypoxia-induced PAH and patients with PAH, display a hyperproliferative, apoptosis-resistant, and proinflammatory phenotype, defined by increased production of IL-6, IL-1β, CCL2/MCP1, CCL12/SDF1, VCAM1 and osteopontin [12, 55]. Moreover, when naïve bone marrow derived macrophages were exposed *in vitro* to conditioned medium generated by adventitial fibroblasts from human PAH patients and hypoxia induced PAH animals, they increased the transcription of several markers of activation (e.g. Cd163, Cd206, Il4ra and Socs3) [56]. These findings suggest that activated adventitial fibroblasts in PAH secrete various soluble factors required for macrophage activation and polarization leading to the propagation of inflammation from adventitia towards media and intima, supporting the "outside-in" hypothesis [53].

#### **3.2 The role of fibroblasts in atherosclerosis and potential fibroblast-targeted therapy**

Several findings suggest a role of fibroblasts in all stages of atherosclerosis, from initial phase to fibrous cap and plaque formation. It is becoming evident that adventitial cells, including adventitial fibroblasts are one of the first cells to respond to injury and become activated in the initial stage of atherosclerosis, even before the formation of atherosclerotic lesions, supporting the new "outside-in" hypothesis [46, 48].

#### *The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

Studies have shown that in various presentations of CVDs, the adventitia becomes heavily populated with multiple immune cell types, including monocytes, macrophages and T-cells, while adventitial fibroblasts proliferate increasingly and differentiate into myofibroblasts [33, 57–59]. Furthermore, several studies reported on increased *vasa vasorum* neovascularization in early atherosclerosis prior to the development of endothelial dysfunction [60, 61]. Neovascularization may act as a pipeline, allowing the entry of immune cells into the site of injury, as the density of *vasa vasorum* is highly correlated with the extent of inflammatory infiltrates in *ApoE−/−* mice [21]. These studies indicate that increased neovascularization of *vasa vasorum* in adventitia, that promotes inflammatory response and plaque angiogenesis, can occur before the endothelial activation and dysfunction in the intima [8]. Adventitial fibroblasts can regulate the growth and neovascularization of *vasa vasorum* through the release of soluble angiogenic growth factors, such as VEGF, TGF-β and plateletderived growth factor (PDGF). Furthermore, with the release of chemokines, such as monocyte chemoattractant protein (MCP1), fibroblasts facilitate infiltration of circulating leukocytes, further increasing the growth of the *vasa vasorum* and perpetuating the inflammatory response [37].

Xu et al. [62] investigated the role of adventitial fibroblasts in atherosclerotic lesion formation by comparing the characteristics of adventitial fibroblasts from *ApoE−/−* and wild type mice. They found αSMA expressing adventitial fibroblast in *ApoE−/−* mice, but not in wild type mice. The gene expression of collagen I and collagen III was upregulated in adventitial fibroblasts from *ApoE−/−* mice, compared to the wild type mice. Furthermore, adventitial fibroblasts from *ApoE−/−* mice synthesized more TGF-β, MCP1, and PDGFβ and exhibited proliferatory and migratory properties [62]. MCP1 is important in regulation of migration and infiltration of monocytes into the vessel wall, which differentiate into macrophages, form foam cells and importantly contribute to fatty streak formation [63]. The effects of adventitial fibroblasts are also ascribed to ROS produced by adventitial fibroblast NADPH oxidases that play important roles in neointimal formation and growth in vascular pathologies, including atherosclerosis [37]. Xu et al. [64] studied ROS production and expression of NADPH oxidase subunit p47phox in the hyperlipid diet-induced atherosclerosis in the *ApoE−/−* mouse model. The activated fibroblasts from aortas of *ApoE−/−* mice displayed upregulated NADPH oxidase activity, augmented ROS production, and increased p47phox levels, compared with wild-type mice. ROS production was also associated with the increased proliferation and migration of adventitial fibroblasts. In addition, silencing of p47phox decreased the proliferation and migration of fibroblasts from *ApoE−/−* mice [64]. Fibroblasts, in response to ROS proliferate and release a number of growth factors and other mediators that influence vascular function, including ET1, PDGF, endothelial growth factor (EGF), fibroblast growth factor2 (FGF2), prostaglandin H2 (PGH2), and cyclophilins [65, 66]. In addition, ROS can also stimulate phenotypic switch of VSMC from the contractile to the proliferative and migratory one, suggesting that fibroblasts can indirectly influence other cell types inside the vessel wall [38].

In the initial phase of fibrosis, injured arteries start with tissue remodeling, and the formation of initial fibrous plaque actually represents a protective process; however, as in all chronic inflammatory conditions, fibrotic components in the plaque produce surplus levels of cytokines and proteolytic enzymes, causing excessive remodeling and tissue damage [59]. In advanced stages of atherosclerosis, fibrosis plays a central role and fibroblasts are the major cell population involved in remodeling of ECM in the fibrous plaque [59]. The most important functions of fibroblasts

in progressed atherosclerosis include regulation of the inflammatory response, ECM protein production, and maintenance of the structural integrity of the plaque as well as regulated balance of MMP production, to enable beneficial tissue remodeling, alongside preventing plaque rupture, for instance [59, 67]. The potential role of fibroblasts in the development of atherosclerosis is shown in **Figure 2**.

Regulation of fibroblasts activities might be beneficial in controlling or reversing the progression of atherosclerosis, hence, the adventitial fibroblast may be an attractive target for therapeutic intervention. Furthermore, the location of the adventitia as the outermost arterial layer makes it suitable for drug delivery and gene therapy [24]. It has already been shown that local adventitial drug delivery into coronary arteries results in better efficiency compared to luminal or intimal delivery methods [71]. Low efficiency of gene transfer to cells in adventitia by intraluminal administration has been reported and efficient transfection of these cells is achieved only when endothelium is denuded or damaged. In atherosclerotic arteries, intimal hyperplasia might present an additional barrier for intraluminal delivery. To overcome these problems, delivery from the adventitial side might be considered. On the other hand, numerous attempts

#### **Figure 2.**

*The potential role of fibroblasts during atherosclerosis progression. Emerging evidence suggests that adventitial fibroblasts are activated in the initial stage of atherosclerosis, supporting the new "outside-in" hypothesis, which proposes that vascular inflammation begins in the adventitia and progresses inward towards the media and intima. In contrast, the original "inside-out" hypothesis of atherosclerosis proposes that the inflammatory response spreads from the intima outward towards adventitia with a more prominent role of endothelial and smooth muscle cells. The figure was adapted from [68–70] and created using Biorender. Legend: ECM, extracellular matrix; LDL, low density lipoprotein; ROS, reactive oxygen species; VSMC, vascular smooth muscle cells.*

#### *The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

to transfect or deliver the therapeutic agents to the media from the adventitial side of large blood vessels have failed because of the impenetrable nature of the external elastic lamina, separating adventitia from the media layer. This barrier hence allows for selective adventitial delivery and specific targeting of cells residing in adventitia [72].

Perivascular delivery of an adenoviral vector expressing a NADPH oxidase inhibitor in the rat carotid artery adventitia significantly reduced neointimal formation after balloon angioplasty. This specific vector targeted adventitial fibroblasts, and it did not affect VSMC in the media [73]. Targeting proteins, expressed by activated fibroblasts could attenuate vascular inflammatory responses and ameliorate vascular disease, including atherosclerosis. Recently, it was discovered that inhibition of expression or activity of fibroblast activation protein (FAP), that is expressed in activated but not in quiescent fibroblasts and was found to be associated with atherosclerotic plaques, can attenuate progression of atherosclerosis by increasing plaque stability in experimental mice models of atherosclerosis [74]. Reports on animal models of cardiomyopathy have indicated that reversibility of fibrosis was possible, with losartan (a selective angiotensin II type 1 receptor antagonist), which suppressed TGF-β expression [75, 76]. However, angiotensin II type 1 receptor is expressed also on VSMC and TGF-β can be produced by multiple cell types, in addition to fibroblasts.

A substantial number of adventitial fibroblasts can differentiate into myofibroblasts during initial stages of atherosclerosis, upon the influence of proinflammatory cytokines, chemokines adhesion molecules, growth factors and ECM proteins [33, 77]. For example, TGF-β induces the transition of a fibroblast into the myofibroblast by stimulating αSMA expression and collagen production [41, 42]. These highly proliferative αSMA-positive cells were found to be widely distributed in atherosclerotic plaques [24]. However, myofibroblasts in the atherosclerotic plaques can derive from multiple other sources, including VSMC [39], the endothelial to mesenchymal transition [78], as well as resident macrophages [79] (**Figure 3**). Myofibroblasts can contribute to changes in the function and structure of the vessel wall that occur during atherosclerosis (i.e arterial remodeling) due to their contractile properties and enhanced ECM protein production [80]. Myofibroblasts migrate from the adventitia to the media and intima and contribute to intimal hyperplasia [81, 82].

Although myofibroblasts were previously considered to be terminally differentiated cells, their capacity for dedifferentiation, defined as the loss of αSMA, is now well recognized and necessary to resolve idiopathic pulmonary fibrosis [83]. Several factors, such as prostaglandin E2 (PGE2), nuclear factor erythroid 2-related factor2 (Nrf2) and FGFs have shown the ability to dedifferentiate established lung and corneal myofibroblasts and might be promising therapeutic targets also for adventitial myofibroblasts in atherosclerosis [84]. For example, treatment with PGE2 was shown to inhibit proliferation and collagen I expression in fibroblasts extracted from histologically normal lung tissue [85]. In TGF-β or ET-1-activated lung myofibroblasts, treatment with PGE2 induced a dose-dependent decrease in αSMA and collagen I expression that was associated with inhibition of focal adhesion kinase signaling [86]. Sulforaphane, a Nrf2 activator, induced myofibroblast dedifferentiation in cultured lung fibroblasts from patients with idiopathic pulmonary fibrosis, as well as inhibited TGF-β-mediated profibrotic effects [87]. Corneal myofibroblasts that were grown in the presence of FGF1 or FGF2 and heparin reduced expression of αSMA, TGF-β receptors, and cadherins, thus promoting the quiescent fibroblast phenotype [88]. FGF21 has been shown to induce angiotensin-converting enzyme 2 (ACE2), and thus inhibit vascular remodeling, hypertension and fibrosis, and when stimulating with adiponectin, FGF21 may inhibit aortic inflammation in atherosclerosis, as well as

#### *Fibroblasts - Advances in Inflammation, Autoimmunity and Cancer*

#### **Figure 3.**

*Various cells from the arterial wall can differentiate into myofibroblasts in atherosclerosis. Macrophages, resident fibroblasts, smooth muscle cells and endothelial cells can differentiate into myofibroblasts, depending on tissue microenvironment and inflammatory mediators. Myofibroblasts are characterized by increased expression of* α*-smooth muscle actin and synthesize and release large amounts of ECM proteins, growth factors, proinflammatory cytokines, proteolytic enzymes and their inhibitors, as well as reactive oxygen species. They are responsible for extensive ECM remodeling, increased chemotaxis and recruitment of immune cells and provide signals for further myofibroblast differentiation. The figure was adapted from [41] and created using Biorender. Legend:* α*SMA:* α*-smooth muscle actin; ECM: extracellular matrix; EMT, endothelial mesenchymal transition FGF: fibroblast growth factor; IL: interleukin; NOS: nitric oxide synthase; MMPs: matrix metalloproteases; ROS: reactive oxygen species; TGF-*β*: transforming growth factor-*β*; TIMPs: tissue inhibitors of MMPs; VEGF: vascular endothelial growth factor.*

decrease cardiac dysfunction in myocardial infarction, attenuate smooth muscle cell proliferation and migration and lower macrophage oxidizes low-density lipoprotein uptake [89]. In addition, Fgf21 knock-out mice have been reported to show impaired lipid metabolism [90]. So, FGF21 has shown promise, as a potential therapeutic for atherosclerosis, but would need further investigation in regard to its effects on leukocytes and activities of its receptors.

#### **3.3 The origin of fibroblasts in atherosclerosis**

Fibroblast heterogeneity in quiescent and diseased state might be a result of their various origins, as well as plasticity, since they can transform into different cell types, subsequently to their adaptation to stress or injury. Evidence suggests that fibroblasts involved in atherosclerosis may originate from different adventitial mesenchymal stem/progenitor cells, however, recent studies revealed they can also originate from VSMC or endothelial cells [30].

Several distinct progenitor/stem cell populations with the capacity to differentiate into endothelial cells, VSMC, fibroblasts, and macrophages reside in a specialized

#### *The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

niche in the adventitia at the media-adventitia border [91]. A population of vascular progenitor cells in the aortic adventitia of *ApoE−/−* mice expressing the stem cell markers Sca1 and CD34 was described that might differentiate to vascular fibroblasts [92]. However, the exact identity of adventitial progenitor/stem cells is still controversial, since fibroblast also have the ability to acquire stem cell properties by upregulating Sca1 [93–95]. Moreover, mesenchymal stem cells and fibroblasts are similar in terms of morphology and share the expression of a number of surface markers, such as CD90, CD73, CD105, vimentin and FSP1. Some researchers therefore suggest that these adventitial stem cells positive for Sca1 are actually fibroblasts [30, 93]. For example, Tang et al. [96] showed that 40% of Sca1-positive adventitial stem cells also express PDGFRα, found to be expressed on the surface of fibroblasts [96]. Using single cell RNA sequencing, Gu et al. [32] identified four mesenchymal clusters in the aortas of *ApoE−/−,* as well as wild type mice, but did not annotate them as stem or progenitor cells. However, one of the clusters displayed high expression of Sca1 indicating stem cell properties [32]. The separation between adventitial fibroblasts and progenitor/stem cells seem to be much smaller than previously thought and cell transition of stem cells into fibroblasts or vice versa appears to be common in atherosclerosis [30]. It would be interesting to further investigate how this transdifferentiation would affect the pathological process of atherosclerosis and whether it could be targeted to reverse atherosclerosis progression.

The evidence that fibroblasts might originate from VSMC came from Wirka et al. in 2019 [97]. They reported that VSMC can transform into fibroblast-like cells (termed "fibromyocytes"), found in the arteries of *ApoE−/−* mice, as well as atherosclerotic human coronary arteries. Fibromyocytes expressed lower levels of VSMC differentiation markers and increased expression of genes, associated with fibroblast cluster, such as lumican, decorin and biglycan. However, fibromyocytes were transcriptionally different from the fibroblasts indicating that either they will further dedifferentiate into fibroblasts or they might represent another distinct population of fibroblasts [97]. Furthermore, it is currently still unclear, whether fibroblasts might also transform into VSMC.

Endothelial cells can serve as a possible source of atherosclerotic fibroblasts since they can undergo endothelial-mesenchymal transition, promoting atherosclerosis progression [98]. This was elegantly shown in 2016 by Evrard et al. [78] using a tamoxifeninducible endothelial lineage tracking system in *ApoE−/−* mice. After 8 weeks of high fat diet, the mouse atherosclerotic plaques consisted of one third endothelial-derived cells positive for FAP and a range of other fibroblast markers. These cells further expanded in number in advanced atherosclerotic plaques. *In vitro* modeling confirmed that endothelial-mesenchymal transition is driven by TGF-β signaling, oxidative stress and hypoxia that are all characteristic for atherosclerosis. Furthermore, the extent of this transition correlated with an unstable plaque phenotype in humans, driven by altered collagen and MMP production that might be associated with clinical events [78].

Whether functional differences between fibroblasts, originating from different sources exist and what is their exact contribution to development and/or progression of atherosclerosis, still remain two important and open questions.

#### **4. Conclusions**

Recently, the well-established "inside-out" hypothesis of atherosclerotic development and progression has been revitalized to involve an "outside-in" component,

including an earlier role for fibroblasts in the tunica adventitia layer. It is now thought that early "outside-in" events, with more prominent roles of fibroblasts in adventitia may even occur prior to, or alongside endothelial activation. Adventitial fibroblasts involved in atherosclerosis comprise a very heterogeneous population, due to their differential origins (mesenchymal stem/progenitor cells, smooth muscle cells, endothelial cells, macrophages) and an extensive repertoire of possible cell transitions into αSMA+ myofibroblasts, or even back to stem cells. It is thought that when resident fibroblasts begin to transform into αSMA+ myofibroblasts, this allows for intense release of VEGF, TGF-β, collagens, IL-1, IL-6, MMPs, ROS and angiotensin II, among other factors, that could lead to perpetual autocrine differentiation and inflammatory, proliferative states, responsible for ECM remodeling, chemotaxis and recruitment of immune cells. Their heterogeneity and consequently, a general lack of specific markers, both contribute to difficulties in studying their exact phenotypes and functions in atherosclerosis. The recent development and accessibility of single cell RNA sequencing technology provides new opportunities to find answers to the remaining questions in an unbiased manner. Furthermore, modulating fibroblast activity, preventing their activation or inducing myofibroblast dedifferentiation in atherosclerotic arteries could represent a promising therapeutic approach for atherosclerosis regression. The plasticity of the atherosclerotic plaque may reform and dynamically remodel many times, before either rupturing or, on the other hand, stabilizing with a plaque cap or even regressing, depending on, among many factors, also molecules in the microenvironment (micro-exoproteome) and presence of certain cellular profiles that may help lean the process either way.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Tadeja Kuret1,2 and Snežna Sodin-Šemrl2,3\*

1 Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Slovenia

2 FAMNIT, University of Primorska, Koper, Slovenia

3 Department of Rheumatology, University Medical Centre Ljubljana, Slovenia

\*Address all correspondence to: ssodin1@yahoo.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of Fibroblasts in Atherosclerosis Progression DOI: http://dx.doi.org/10.5772/intechopen.98546*

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#### **Chapter 4**

## Heterogeneity of Fibroblasts in Healthy and Diseased Kidneys

*Takahisa Yoshikawa, Yuki Sato and Motoko Yanagita*

#### **Abstract**

Chronic kidney disease (CKD) is a worldwide health problem affecting 9.1% of the world's population. The treatments to prevent the progression of CKD remain limited, however. Resident fibroblasts in the kidneys play crucial roles in the pathological conditions commonly recognized in CKD, such as renal fibrosis, renal anemia, and peritubular capillary loss. Fibroblasts in the kidney provide structural backbone by producing extracellular matrix proteins and produce erythropoietin for normal hematopoiesis under physiological conditions. In the diseased condition, however, fibroblasts differentiate into myofibroblasts that produce excessive extracellular matrix proteins at the cost of the inherent erythropoietin-producing abilities, resulting in renal fibrosis and renal anemia. Pericytes, which are mesenchymal cells that enwrap peritubular capillaries and highly overlap with resident fibroblasts, detach from peritubular capillary walls in response to kidney injury, resulting in peritubular capillary loss and tissue hypoxia. Several reports have demonstrated the beneficial roles of fibroblasts in the regeneration of renal tubules Renal fibroblasts also have the potential to differentiate into a proinflammatory state, producing various cytokines and chemokines and prolonging inflammation by forming tertiary lymphoid tissues, functional lymphoid aggregates, in some pathological conditions. In this article, we describe the heterogenous functions of renal fibroblasts under healthy and diseased conditions.

**Keywords:** chronic kidney disease, erythropoietin, renal anemia, fibrosis, myofibroblast, tertiary lymphoid tissue

#### **1. Introduction**

Chronic kidney disease (CKD) is a worldwide public health problem. In 2017, the prevalence of CKD was estimated to be 9.1% in the world's population, and has increased by 29.3% from 1990 to 2017 [1]. The prevalence of CKD in elderly individuals over 65 years old is especially high and is predicted to increase further as a result of the increasingly aged society [2]. CKD is a risk factor for end-stage renal disease (ESRD) and is also recognized as an independent risk factor for cardiovascular diseases and their associated mortality [3]. Patients with ESRD need renal replacement therapies such as dialysis and renal transplantation to survive. The cost of these therapies is enormous and the financial burden is a critical problem for patients

and society [4]. Nevertheless, treatment to prevent the progression of CKD and the occurrence of CKD-associated complications remain limited.

Fibroblasts are distributed in various organs throughout the body and contribute to both homeostasis and disease. In the kidney, resident fibroblasts play crucial roles in both health and disease, and their phenotypes are heterogenous and plastic [5]. Under physiological conditions, renal fibroblasts provide structural support for the entire kidney architecture and produce erythropoietin (EPO). In contrast, in diseased kidneys, fibroblasts lose these physiological functions and transdifferentiate into myofibroblasts. These phenotypic changes result in fibrosis, renal anemia, and peritubular capillary loss, all of which are common pathological conditions of CKD, irrespective of the etiology [6]. Renal fibroblasts also act as inflammatory cells and produce proinflammatory cytokines and chemokines under some pathological conditions [5, 7]. In aged injured kidneys, fibroblasts play a crucial role in prolonging inflammation by inducing tertiary lymphoid tissue (TLT) formation [8]. These features highlight the importance of understanding the behavior of fibroblasts in the kidney in order to identify efficient therapeutic strategies to prevent CKD progression. In this article, we describe the current understanding of the heterogeneous functions of fibroblasts in healthy and diseased kidneys.

#### **2. Fibroblasts in the kidney**

#### **2.1 Characteristics and functions of resident fibroblasts in kidneys**

Renal resident fibroblasts are spindle-shaped cells that exist in the interstitial space, which is defined as the area between nephrons. Nephrons are functional units of the kidney and are composed of glomerular and tubular cells. Fibroblasts provide the structural backbone of the kidney by producing extracellular matrix (ECM) proteins and interact with surrounding cells to maintain the homeostatic state in healthy kidneys. Identification of resident fibroblasts is performed based on their location, shape, and positive expressions of several fibroblast markers such as CD73 and PDGFRβ [9]. As these markers are neither homogeneously positive nor specific for resident fibroblasts, confirming the negative expression of other cell-lineage markers such as CD45, a hematopoietic cell marker, is also necessary to identify renal resident fibroblasts.

In addition to the role of structural cells, fibroblasts have unique organ-specific functions. In the kidney, small subset of the renal resident fibroblasts residing in the corticomedullary area produces Epo, a hormone essential for erythropoiesis in response to hypoxia [10]. Although there are few Epo-producing cells and they exist only in the deep cortex in the physiological state, under severe hypoxic conditions such as severe anemia, the number of Epo-producing cells increases and they can be detected in the cortical area [11]. The increase in the number of Epo-producing cells under hypoxic conditions is likely due to the increase in the number of the cells that have acquired Epo-producing ability but not to cellular proliferation, because Epo production is activated in anemic mice under administration of a cell cycle inhibitor or γ-ray irradiation [10]. Interestingly, while other growth factors for hematopoietic cells (such as granulocyte colony-stimulating factor) are produced in bone marrow, where hematopoietic cells are generated and the growth factors are required, EPO is produced from the kidney. One possible explanation for this is that kidneys are physiologically hypoxic compared with other organs, which allows them to be more

sensitive to small changes in oxygen delivery than other organs and is advantageous to the production of EPO in response to hypoxia [12]. Another explanation is that kidneys function as a "critmeter," with the ability to set hematocrit within the normal range by regulating plasma volume and the red blood cell mass in a common site [13].

#### **2.2 Origin and heterogeneity of fibroblasts in the kidney**

In 1974, Le Douarin et al. reported that, in transplantation experiments of quail neural tubes to chicks, quail neural crest–derived cells were identified in the renal interstitial space [14]. Consistently, we found that fibroblasts in neonate kidneys express p75 neurotrophin receptor (p75NTR), a neural crest marker [15]. Moreover, Epo-producing cells express neuronal markers such as microtubule-associated protein 2 and neurofilament light polypeptide [10]. Based on these previous findings, we conducted a lineage tracing study using *myelin protein zero (P0)-Cre* mice, which label neural crest–derived cells, and found that resident fibroblasts in the cortex and corticomedullary area are lineage-labeled with *P0-Cre* [15]. We also demonstrated that, in embryonic kidneys, *P0-Cre* lineage–labeled cells appeared in the interstitial space along the outer capsule and ureter from E13.5 and more than 99% of the resident fibroblasts in the cortex and outer medulla were labeled with *P0-Cre* in adult kidneys. Consistently, Epo-producing fibroblasts were also labeled with *P0-Cre* in the experiment using *P0-Cre:R26R:EPO-GFP* mice [15]. In contrast, renal fibroblasts in the medulla are not labeled with *P0-Cre*. Wnt4 expression is identified in the medullary stromal mesenchyme in embryotic kidneys and reactivated in medullary fibroblasts after renal injury [16, 17]. These findings suggest the regional heterogeneity of fibroblasts and their origin in the adult kidney. The functional heterogeneity of PDGFRβ<sup>+</sup> Epo-producing cells was also reported. Precise histological analysis showed that different subpopulations of fibroblasts produce Epo responding to different stimuli [18].

#### **2.3 Pericytes in kidneys**

Pericytes are mesenchyme-derived cells that enwrap capillaries with their processes embedded in the vascular basement membrane. Resident fibroblasts and pericytes share several characteristics, including their interstitial location and cell surface markers such as CD73 and PDGFRβ, and, as such, these two types of cells are often confused. Pericytes support the capillary structure and regulate vascular tone with their contraction force [19]. Moreover, they interact with endothelial cells to maintain capillary homeostasis [6]. Humphreys et al. reported that the origins of pericytes in the kidneys were FoxD1-expressing cells in an experiment using *FoxD1- Cre* mice [20]. On the other hand, it was reported that *P0-Cre* lineage–labeled cells in E13.5 embryonic kidneys transiently expressed FoxD1 [15]. Based on these findings, it is assumed that resident fibroblasts and pericytes are highly overlapped populations in the kidneys.

#### **3. Renal fibrosis as a hallmark of CKD**

Renal fibrosis is a common pathological condition of CKD, irrespective of the etiology. It is defined as excessive accumulation of ECM such as collagen and fibronectin in the interstitial space and is recognized as a predictive indicator of renal prognosis [21]. Previous studies have shown that dysfunction of the renal fibroblasts can induce several pathological conditions associated with CKD, such as renal fibrosis, renal anemia, and peritubular capillary loss. Against this background, renal fibroblasts have been focused on as hopeful therapeutic targets for CKD and its complications.

#### **3.1 Myofibroblasts in kidneys and their origin**

Myofibroblasts are recognized as the main contributor to fibrosis in multiple organs. They are characterized by dense endoplasmic reticulum and contractile microfilament bundles [22]. Their most prominent feature is the expression of α-smooth muscle actin (α-SMA) that forms myofilament bundles and promotes their high contractility [23]. Although myofibroblasts are almost undetectable in healthy kidneys, they expand dramatically in diseased kidneys and drive fibrosis by producing a large amount of ECM proteins and through their own proliferation. The origin of myofibroblasts has been discussed for decades, and several genetic lineage tracing studies recently revealed that resident fibroblasts and pericytes are the main sources for myofibroblasts [9]. We reported that *P0-Cre* lineage–labeled cells, which were progenitors of resident fibroblasts (as mentioned above), could differentiate into myofibroblasts in several kidney injury models [15]. Humphreys et al. reported that *FoxD1-Cre* lineage–labeled pericytes are the main sources for myofibroblasts [20]. These studies demonstrated that most myofibroblasts are derived from these renal fibroblasts and pericytes. Although several studies have reported other types of cells as progenitor cells for myofibroblasts, such as epithelial cells, endothelial cells, and hematopoietic cells, recent lineage tracing experiments demonstrated that tubular epithelial cells do not become myofibroblasts *in vivo* [20, 24, 25]. The endothelialto-mesenchymal transition, in which endothelial cells transdifferentiate into myofibroblasts, was also reported to contribute less to myofibroblast pools than resident fibroblasts [24]. Additionally, Kramman et al. used single-cell RNA sequencing (scRNA-seq) and parabiosis techniques to demonstrate the limited contribution of circulating monocytes to myofibroblast pools with very few matrix genes expression in murine fibrotic kidneys [25].

Notably, although genetic lineage tracing is not feasible in humans, a recent study utilizing scRNA-seq of human kidney samples supports the notion that these findings in mice appear to be conserved in humans. Kuppe et al. conducted scRNA-seq on human kidneys in patients with CKD and demonstrated that Notch3<sup>+</sup> pericytes and Meg3<sup>+</sup> fibroblasts were the main sources for highly ECM-producing myofibroblasts using pseudo-time trajectory analysis and diffusion map analysis [26]. These studies support the idea that most renal myofibroblasts derive from renal resident fibroblasts or pericytes.

#### **3.2 Progenitor of myofibroblasts; Gli1+ fibroblasts in the perivascular niche**

Mesenchymal stem cells (MSCs) are defined as cells with self-renewal and clonogenic capacity. Gli1+ fibroblasts are MSC-like cells that reside in both the pericyte niche and the adventitia of larger vessels across multiple organs, including the kidney, and exhibit trilineage differentiation potential *in vitro* [27, 28]. A fate tracing study utilizing *Gli1-CreERt2:tdTomato* reporter mice revealed that, although Gli1+ fibroblasts represented only 0.2% of the PDGFRβ<sup>+</sup> renal fibroblast population in healthy kidneys, after renal injury, they proliferated dramatically, mainly in the medulla and inner cortex, and differentiated into αSMA<sup>+</sup> myofibroblasts. Additionally, using

*Gli1-CreERt2:iDTR* (inducible diphtheria toxin receptor) mice, the ablation of Gli1+ cells by diphtheria toxin (DT) administration dramatically reduced renal fibrosis by approximately 50% after unilateral ureteral obstruction (UUO), which is an *in vivo* experimental model of renal fibrosis. These data suggested that Gli1<sup>+</sup> fibroblasts predominantly proliferated and contributed to renal fibrosis, suggesting the heterogeneity of the potential to transdifferentiate into myofibroblasts among PDGFRβ<sup>+</sup> fibroblasts in the kidney.

#### **3.3 The roles of proximal tubule injury in CKD progression**

Acute kidney injury (AKI) is a highly prevalent disorder and is one of the risk factors for the progression of CKD [29]. The underlying molecular mechanisms for CKD transition after AKI have been investigated for decades. The proximal tubules are the most vulnerable segment in the nephron, and are assumed to trigger the AKI to CKD progression. To investigate whether injured proximal tubules can trigger renal fibrosis, we selectively damaged proximal tubules by DT administration in *Ndrg1- CreERt2:iDTR* mice, in which DTR is specifically expressed on proximal tubules in the kidneys [30]. Low-dose single DT administration caused mild proximal tubule injury and reversible fibrosis whereas high-dose single DT or repeated low-dose DT administration caused sustained renal fibrosis. This study showed that injury of the proximal tubules is sufficient to cause several features of CKD, and that the frequency and severity of proximal tubule injury are associated with the degree of AKI to CKD progression. As an explanation for the association between proximal tubule injury and CKD, tubulointerstitial interactions in injured kidneys have been reported to contribute to renal fibrosis [31]. Yang et al. demonstrated that, in a multiple profibrotic AKI model, injured proximal tubules underwent cell cycle arrest in G2/M and acquired the profibrotic secretory phenotype by upregulating profibrotic cytokine production, such as transforming growth factor β-1 (TGFβ-1) and connective tissue growth factor [32]. Additionally, the administration of a p53 inhibitor, a therapy employed for bypassing G2/M arrest, attenuated renal fibrosis in a renal ischemic reperfusion injury model. Injured proximal tubules have been reported to secrete several other profibrotic ligands expressed during renal development. The Wnt family plays a crucial role in kidney development, and many Wnt family genes are upregulated in fibrotic kidney models [33]. Zhou et al. demonstrated that the selective ablation of Wntless, a cargo receptor necessary for Wnt secretion, in renal tubular epithelial cells but not in interstitial fibroblasts attenuated renal fibrosis in a UUO model, suggesting that the Wnt family secreted by renal tubules contributed to renal fibrosis. Moreover, Maarouf et al. showed that Wnt1 expression genetically induced in proximal tubules was sufficient for renal fibrogenesis by inducing interstitial myofibroblast activation and proliferation [34]. According to these reports, one of the mechanisms of the AKI to CKD transition is that ligands secreted from injured renal tubules contribute to renal fibrogenesis by activating myofibroblasts.

#### **4. Two common CKD complications, renal anemia and peritubular capillary loss, are also caused by dysfunction of renal fibroblasts/ pericytes**

Renal anemia is a common complication that affects the majority of patients with CKD [35]. The cause of renal anemia is the relative deficiency of EPO. Several recent studies have shown that dysfunction of renal fibroblasts contribute to this complication. EPO production is stimulated by hypoxia and regulated by hypoxia-inducible factors (HIFs). In normoxic conditions, HIFs are hydroxylated by HIF-prolyl hydroxylase domain–containing proteins (PHDs), and hydroxylated HIFs are degraded by the ubiquitin-proteasome system [36, 37]. In hypoxic conditions, the hydroxylation and degradation of HIFs is inhibited, resulting in the transcriptional activation of HIF-inducible genes, including *EPO*. In renal fibrosis models, the Epo-producing fibroblasts transdifferentiate into myofibroblasts in response to kidney injury and decrease the capacity to produce Epo at the same time [11, 15, 38]. Souma et al. demonstrated that activation of HIFs by the genetic inactivation of PHDs in Epo-producing cells restored Epo production in Epo-producing cell–derived myofibroblasts in a renal fibrosis model [39]. Additionally, severe anemia or the administration of selective estrogen receptor modulators, the neuroprotective agents, neurotrophin, and the renoprotective agent, hepatocyte growth factor, restored the ability to produce Epo in myofibroblasts [15]. These results demonstrated that the ability to produce Epo in myofibroblasts has plasticity and can be restored by therapeutic interventions. As for the mechanism of the decrease of Epo production in myofibroblasts, Souma et al. showed that NFκB signaling repressed Epo production in fibroblasts in UUO models. Moreover, *Epo-Cre:R26-IKK2ca/+* mice, in which NFκB signals in Epo-producing cells were selectively activated, showed that 20% of Epoproducing cells were positive for α-SMA, suggesting that NFκB signaling also contributed to the transition of EPO-producing fibroblasts into myofibroblasts [11]. Another possible mechanism for the repression of Epo production in myofibroblasts is that hypermethylation in the *Epo* promotor, which is induced by TGFβ-1 stimulation, inhibits *Epo* expression in myofibroblasts in the fibrotic kidney [38]. Fuchs et al. also showed that Epo production in renal fibroblasts was suppressed by TGFβ signaling in renal fibrosis models, utilizing *PDGFR*β*-Cre:TGF*β*-R2*fl/fl mice, and hypothesized that it occurred before the phenotypic shift of fibroblasts to myofibroblasts because the frequency of α-SMA+ myofibroblasts did not differ between the knockout mice and control mice [40].

Although the administration of erythropoiesis-stimulating agents (ESAs) is a currently well-established and effective clinical treatment, it might be associated with several adverse effects, such as hypertension and thrombotic complications [41]. To avoid safety concerns associated with ESAs, PHD inhibitors, which upregulate EPO production via the stabilization of HIFs, have been developed and used for the treatment of renal anemia [36, 42–45].

Another common pathological feature of CKD is the loss of peritubular capillaries [46]. Renal pericytes enwrap peritubular capillaries and support them structurally. In response to injury, pericytes detach from capillaries and their processes, which form networks surrounding the capillaries, start to direct from their associated capillaries to the adjacent tubules, concomitant with transdifferentiation into myofibroblasts [39]. This pathological change makes peritubular capillaries unstable and causes capillary rarefaction and loss [47]. Reduced peritubular capillary blood supply can cause chronic hypoxia in renal parenchymal cells such as tubules and stromal cells. Hypoxia aggravates renal fibrosis by stimulating fibroblasts and altering their gene expressions associated with ECM metabolism [48]. For example, hypoxia upregulated collagen type 1 and the tissue inhibitor of metalloproteinase-1 expression and also downregulated matrix metalloproteinase-1 *in vitro*, which led to the accumulation of ECM proteins. Excessive fibrosis also reduces the efficiency of oxygen diffusion due to the expanded distance between capillaries and tubules and induces renal damage,

#### *Heterogeneity of Fibroblasts in Healthy and Diseased Kidneys DOI: http://dx.doi.org/10.5772/intechopen.99492*

which can drive further CKD progression. Recently, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, which prevents glucose reabsorption by SGLT2 in proximal tubules, was reported to prevent tissue hypoxia and renal fibrosis in an ischemic reperfusion injury (IRI) model by ameliorating renal capillary rarefaction and the detachment of pericytes from endothelial cells with the promotion of vascular endothelial growth factor-A expression in proximal tubular cells [49]. Additionally, SGLT2 inhibitor treatment was reported to increase hematocrit concomitantly with the elevation of serum EPO concentrations in patients with type 2 diabetes mellitus [50, 51]. On the basis of these reports, SGLT2 inhibitors are expected to play a beneficial role in the restoration of the physiological functions of fibroblasts.

#### **5. Beneficial function of myofibroblasts in kidneys**

Contrary to the long-held assumption that fibrosis is detrimental to the host, recent evidence suggests that fibrosis also has host-protective roles in some cases. To investigate the role of fibroblasts during the early phase of kidney injury, we utilized *P0-Cre:iDTR* mice [52], which allow us to induce resident fibroblast-specific dysfunction at the desired time point by DT administration. Utilizing this system, we found that the dysfunction of resident fibroblasts in the acute phase of injury impaired tubular regeneration. During the transition from fibroblasts to myofibroblasts, fibroblasts upregulate the expression of retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in retinoic acid (RA) synthesis. Given that RAs are essential for kidney development, RAs derived from myofibroblasts might promote tubule regeneration. Retinoic acid receptor (RAR) γ and the downstream molecules such as αB-crystallin were expressed in proximal tubules in injured kidneys, and the administration of an RAR inverse agonist to the proximal tubule cell line attenuated proliferation *in vitro*. Another example of the beneficial role of fibroblasts during injury was shown by the experiment utilizing the intravital imaging method [53]. Schiessl et al. demonstrated that, in response to laser-induced tubular cell injury, PDGFRβ<sup>+</sup> interstitial cells migrated towards tubular injury sites and enclosed the injured tubules. Additionally, PDGFRβ inhibitors compromised the recruitment of the interstitial cells and tubule regeneration. These results suggest that fibroblasts have the potential to promote tubule regeneration, at least in the acute phase of kidney injury, via PDGFRβ signaling.

#### **6. Renal fibroblasts associated with inflammation**

Although myofibroblasts are established as the primary effector cells driving fibrosis, several recent studies have demonstrated that resident fibroblasts in the kidney also have a proinflammatory phenotype. Souma et al. reported that Epo-producing fibroblast–derived myofibroblasts upregulated the expression of the target genes of NFκB such as *Il6* and *Ccl2* in an UUO model [11]. Leaf et al. reported that renal pericytes could work as innate immune cells and respond to damage-associated molecular patterns (DAMPs) sensitively in the acute phase of renal injury [54]. In response to the stimulation with DAMPs, renal pericytes activated NLRP3 inflammasome and secreted IL-1β and IL-18 in a TLR2/TLR4 and Myd88-dependent manner and aggravated renal fibrosis in the IRI model. *In vitro*, Myd88 knockout pericytes and Tlr2/Tlr4 double knockout pericytes also reduced their ability to migrate and the expression of the genes associated with fibrosis in response to TGF-β or DAMPs. These results

suggested that both the inflammatory and fibrogenic properties of pericytes were dependent on Myd88, TLR2, and TLR4. Further studies are necessary to elucidate the significance of proinflammatory fibroblasts in renal injury and CKD progression.

It is of note that an anti-inflammatory role of fibroblasts and pericytes was also reported. Using *FoxD1-Cre:CD73*fl/fl mice, Perry et al. demonstrated that CD73, an enzyme converting AMP to adenosine on fibroblasts and pericytes, was necessary to suppress inflammation and attenuate renal fibrosis after kidney injury [55]. Additionally, the absence of CD73 on the fibroblasts was associated with an increase in α-SMA expression *in vitro*. The authors hypothesized that the adenosine locally generated by CD73 on fibroblasts and pericytes might act on adenosine receptors in an autocrine and paracrine manner and attenuate macrophage infiltration and profibrotic properties. Importantly, renal fibroblasts can act as modulators of inflammation and contribute to normal tissue repair by interacting with the surrounding cells.

### **7. Age-dependent phenotype of fibroblasts: tertiary lymphoid tissue: associated fibroblasts**

#### **7.1 Tertiary lymphoid tissue formation in aged injured kidneys**

An epidemiological study showed that elderly patients with AKI had an increased risk for CKD progression [56]. The mechanism for the maladaptive repair after AKI in the elderly remains unknown. To identify the mechanism, we compared the renal response to injury between young and aged mice. As in humans, while young kidney repaired itself after injury, aged kidney exhibited sustained tubular injury and fibrosis [8]. Unexpectedly, we found multiple TLTs in aged kidneys but not young kidneys in the chronic phase after kidney injury. TLT is an ectopic lymphoid tissue that develops at the site of chronic inflammation. TLTs are mainly composed of lymphocytes that are structurally and functionally supported by unique phenotypic fibroblasts inside TLTs (**Figure 1**). Unlike simple infiltration of the inflammatory cells, TLTs can promote lymphocyte proliferation and differentiation, resulting in the generation of antibodysecreting plasma cells, as recognized in secondary lymphoid organs [57]. Importantly,

#### **Figure 1.**

*Tertiary lymphoid tissues in aged murine kidney 45 days after ischemic reperfusion injury. (A) Immuno fluorescence staining of B220 (green) and CD3*ε *(red). Tertiary lymphoid tissues (TLTs) are mainly composed of B220+ B cells and CD3*ε *+ T cells. (B) Immunofluorescence staining of p75NTR (green) and CD21 (red). Fibroblasts inside TLTs express p75 neurotrophin receptor (p75NTR). In mature TLTs, some of the fibroblasts differentiate into p75NTR− /CD21+ follicular dendritic cells. Scale bar: 50* μ*m.*

although TLTs are identified in various disease conditions, such as autoimmune diseases, infections, and cancers, TLTs can play beneficial or pathological roles in a context-dependent manner [58]. For example, in chronic inflammatory or autoimmune diseases, TLTs contribute to disease persistence and have detrimental effects on the host [59]. In contrast, during infections, TLTs are assumed to play beneficial roles to eliminate pathogens by promoting immune responses [60]. The role of TLTs in aged injured kidneys remains unclear, and will be discussed in the next section.

#### **7.2 Characteristics and origin of fibroblasts inside tertiary lymphoid tissues**

Fibroblasts inside TLTs exhibit unique characteristics that are distinct from those outside TLTs, such as the strong expression of p75NTR (**Figure 1**) [8]. After kidney injury, resident fibroblasts acquire the ability to produce RAs by upregulating RALDH2, which is assumed to promote the transition of the adjacent fibroblasts into p75NTR+ TLT-associated fibroblasts. Some of the p75NTR+ TLT-associated fibroblasts acquire abilities to secrete homeostatic chemokines such as CCL19 and CXCL13, which are the driving force for recruiting lymphocytes and promoting TLT formation. Inside of more mature TLTs, CD21+ /p75NTR− follicular dendritic cells (FDCs) appear as part of stromal cells (**Figure 1**). FDCs are stromal cells residing in the B cell follicles of secondary lymphoid organs; they drive germinal center reactions [61]. These TLTassociated fibroblasts in the kidneys are *P0-Cre* lineage–labeled cells, suggesting that renal resident fibroblasts can differentiate into these various types of fibroblasts [8].

#### **7.3 Clinical significance of tertiary lymphoid tissues in CKD and the elderly**

Several studies have reported that TLTs are induced in various kidney diseases [62–65]. Additionally, we reported that TLTs developed not only in murine kidneys but also in human kidneys in an age-dependent manner [8]. In the analysis on kidneys from nephrectomy cases for renal cell carcinoma and autopsy, excluding pyelonephritis, glomerulonephritis, autoimmune kidney diseases, and hematological malignancies, TLTs were identified only in the elderly over 60 years old. The components of human TLTs are quite similar to those of murine TLTs. To evaluate TLTs objectively, we classified renal TLTs into three stages based on the immunostaining patterns as follows [66]. TLTs not containing CD21+ FDCs or a germinal center response, dense Ki67+ proliferative B cell clusters, were defined as stage 1. TLTs containing CD21+ FDCs but no germinal center response were defined as stage 2. TLTs containing both CD21+ FDCs and a germinal center response were defined as stage 3. In this classification, the severity of the TLT stages and the area of TLTs were related with the severity of ischemic injury in murine renal IRI models. In humans, more and higher-stage TLTs were identified in the kidneys of patients with CKD than without CKD among elderly patients 60 years or older in the analysis using kidneys from nephrectomy cases due to renal cell carcinoma [66]. These data demonstrated that the developmental stage of TLTs was associated with the severity of kidney injury, thereby indicating that TLTs have potential as a marker of severity of renal injury.

#### **7.4 Potential of tertiary lymphoid tissues as therapeutic targets for CKD**

Although TLTs are assumed to be associated with the severity of renal injury, it has been challenging to determine whether renal TLTs are pathogenic and if they directly affect renal function. We reported that, in unilateral renal IRI models of aged mice,

the administration of GK1.5, anti-CD4 monoclonal antibody, diminished TLT formation and inflammatory marker expressions and improved renal fibrosis [8]. This result suggests that renal TLTs could be pathogenic and the therapies targeting renal TLTs thus have the potential to improve renal function in patients with CKD. As this intervention is not specific to TLTs and affects systemic immune systems, however, a more specific therapy for TLTs is necessary to determine whether TLTs in aged injured kidneys are detrimental or not.

#### **8. Conclusion**

Resident fibroblasts in the kidney are essential components to maintain homeostasis under physiological conditions. In CKD, dysfunction of renal fibroblasts causes the main pathological conditions of renal fibrosis, renal anemia, and peritubular capillary loss. Importantly, renal fibroblasts are heterogeneous and have the potential to change their phenotypes depending on the local microenvironment (**Figure 2**) [5]. Although myofibroblasts mainly contribute to renal fibrosis and deteriorate renal function by producing excessive ECM, they can also have host-protective roles in the early phase of kidney injury. Renal fibroblasts can differentiate into proinflammatory fibroblasts that secrete inflammatory cytokines and chemokines, which can promote TLT formation under several diseased conditions. Fibroblasts or pericytes also have an anti-inflammatory function via CD73 expression. A better understanding of the heterogeneity and roles of renal fibroblasts might lead to the development of a new therapeutic approach for kidney diseases. Recent novel technologies such as scRNAseq have revealed the heterogeneity of renal fibroblasts that had not previously been identified by conventional technologies [26]. The application of these technologies to various clinical renal diseases is expected to further clarify the heterogeneity of renal fibroblasts, which will result in an enhanced understanding of the pathophysiology of kidney diseases and the development of novel treatments.

#### **Figure 2.**

*Heterogeneity of fibroblasts in kidneys. Renal fibroblasts derive from P0- or FoxD1-Cre lineage–labeled cells. Renal fibroblasts change their phenotypes depending on the local microenvironment. Myofibroblasts have the potential to produce excessive extracellular matrix proteins at the cost of EPO production. In response to released DAMPs after injury, fibroblasts can differentiate into proinflammatory fibroblasts that produce IL-18 and IL-1*β*. In contrast, in aged kidneys, retinoic acids produced by activated fibroblasts after injury induce the differentiation of adjacent fibroblasts into p75NTR+ fibroblasts, which produce homeostatic chemokines such as CXCL13 and CCL19, resulting in tertiary lymphoid tissue (TLT) formation. These chemokine-producing fibroblasts can further differentiate into CD21<sup>+</sup> /p75NTR− FDCs in mature TLTs.*

#### **Conflict of interest**

YS is employed by the TMK Project, which is a collaboration between Kyoto University and Mitsubishi Tanabe Pharma. MY receives research grants from Mitsubishi Tanabe Pharma and Boehringer Ingelheim. TY reports no conflicts of interest.

#### **Appendices and nomenclature**


### **Author details**

Takahisa Yoshikawa1 , Yuki Sato1,2 and Motoko Yanagita1,3\*

1 Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

2 Medical Innovation Center, TMK Project, Graduate School of Medicine, Kyoto University, Kyoto, Japan

3 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan

\*Address all correspondence to: motoy@kuhp.kyoto-u.ac.jp

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Heterogeneity of Fibroblasts in Healthy and Diseased Kidneys DOI: http://dx.doi.org/10.5772/intechopen.99492*

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#### **Chapter 5**

## Fibroblast-Like Synovial Cell Subsets in Rheumatoid Arthritis

*Søren Lomholt, Morten A. Nielsen, Maithri P. Aspari, Peter B. Jørgensen, Adam P. Croft, Christopher Buckley and Tue W. Kragstrup*

#### **Abstract**

Fibroblasts like synoviocytes (FLS) play several significant roles in rheumatoid arthritis (RA) pathophysiology. This chapter will describe known roles of FLS in disease initiation, joint inflammation, disease persistence and joint destruction. It will describe the newly characterized subsets of FLS based on single cell RNA sequencing studies, and their association to specific aspects of the disease. Finally, we will discuss the future of targeting FLS in the treatment of RA. The FLS in the synovial lining layer are identified by surface complement decay-accelerating factor (CD55) along with lubricin and metallopeptidase expression. Pathological activation of this lining layer subset result in bone and cartilage damage in mice. FLS of the sublining layer are often characterized by THY1 expression, but recent studies have highlighted a heterogeneity where several distinct subsets are identified by additional markers. Sublining FLS expressing human leukocyte antigen-DRA (HLA-DRA) produce C-X-C motif chemokine 12 (CXCL12) and receptor activator of nuclear factor-κB ligand (RANKL) and seems to constitute a pro-inflammatory subset that is associated with inflammation and tertiary lymphoid structures. Another subset of FLS characterized by CD34 expression may discriminate a common progenitor fibroblast subset. Taken together, studies isolating and characterizing gene expression in synovial FLS report both associations of unknown importance and markers that may impose protective or destructive features. This supports evidence of FLS as active players in RA pathology capable of cellular recruitment, local cellular crosstalk and promotion of joint destruction. These discoveries may serve as an atlas for synovial activation in RA and have identified several potential fibroblast markers for the development of targeted treatment.

**Keywords:** Fibroblast like synoviocytes, Rheumatoid arthritis, Inflammation, Autoimmunity, Tertiary lymphoid structures, Fibroblast activation protein, Fibroblast targeted treatment

#### **1. Introduction**

In normal resting conditions the synovial membrane is a thin layer of wellordered cells historically called type A and B synoviocytes. These cells form a barrier between the articular cavity and a sublining layer, the latter being heterogeneous and composed of several cell linages. Fibroblasts, immune cells and mature vasculature (capillaries, arterioles and venules) made up of pericytes and endothelia are some of the various cell types constituting this layer [1–3].

#### **2. Rheumatoid arthritis**

Rheumatoid arthritis (RA) has a multifactorial etiology and is one among the most common systemic autoimmune diseases [4, 5]. The factors that mediate the initiation of RA is yet to be unraveled. However, the pathology of RA involves abnormalities in both the innate and the adaptive immune system, and both of these systems are implicated with the progression and persistence of the disease [6, 7]. The synovial membrane is the primary site of pathology during the synovitis stage of the disease and characterized by proliferation of tissue resident, synovial cells and the infiltration of inflammatory cells from the blood. RA is a chronic, progressive disease leading to degradation of articular cartilage and bone along with several systemic manifestations [8].

In RA, the inflamed synovial membrane undergoes hyperplasia and transforms into less structured lining layer and sublining tissues both rich in fibroblasts like synoviocytes (FLS) [9, 10]. This inflamed synovial membrane eventually begins to invade the cartilage surfaces and the underlying bone, commonly referred to as pannus [11, 12].

Present day treatment strategies for RA primarily focuses on suppression of cytokine signaling and T- and B-cell activity. These therapies have highlighted the importance of immune response in driving the progression of RA. However, they also clearly demonstrate that in a large proportion of patients these treatments are incapable of inducing disease remission [8, 13]. Synovial phenotyping of RA patients based on histology has highlighted a fibroblast dominated synovial pathotype [14]. This pathotype is believed to include a large proportion of the non-responders to conventional and biologic disease modifying anti-rheumatic drugs [15–17]. This is supported *in vitro* where anti-tumor necrosis factor alpha (TNFα) treatments were ineffective in cultures dominated by FLS [18]. Furthermore, a recently published, biopsy driven clinical trial in RA patients with inadequate response to anti-TNFα treatment, showed significantly higher response rates when patients with B-cell poor synovium were treated with IL-6 receptor inhibitor tocilizumab compared to the B-cell depleting agent rituximab [19].

In the following sections, we will first describe RA FLS in general before the era of single cell RNA sequencing (scRNA-seq). We will summarize the known and proposed roles of FLS in RA initiation, joint inflammation, disease persistence and joint destruction. Finally, we will describe the newly characterized subsets of FLS based on scRNAseq studies their connection to specific aspects of clinical disease, future outlooks in the context of RA diagnosis, RA tissue phenotyping and therapy targeting FLS.

#### **3. Fibroblast like synovial cells in rheumatoid arthritis**

#### **3.1 Disease initiation**

The central role of FLS in RA pathology is highlighted in murine studies demonstrating that activation of FLS is sufficient to initiate local joint inflammation leading to persistent arthritis [20, 21]**.**

Furthermore, FLS greatly contribute to the transformation of the thin synovial membrane into a multi-layered invasive hyperplastic pannus [22]. This expansion of FLS in the inflamed synovium is likely a result of at least one of the following processes. First, pathological subsets of FLS seem to proliferate to some extent and develop a local resistance to apoptosis [23–25]**.** Secondly, pluripotent mesenchymal stem cells may migrate into the synovium from the circulation, where they differentiate into mature pathological subsets of FLS [26]**.** Lastly, a local mesenchymal progenitor cell population may undergo activation and differentiation into distinct phenotypes of FLS [27]. Collectively, this leads to a local increase in pathological FLS in the RA synovium.

#### **3.2 Joint inflammation**

Pathogenic FLS constitute the majority of cells found in the inflamed synovial tissues, and play an important role in the inflammatory cascade, linking innate and adaptive immunity [6, 10]. FLS are capable of significantly affecting the local inflamed environment through production of cytokines and chemokines leading to recruitment and activation of immune cells [9, 28]. Specifically, pathogenic FLS are able to provide an adequate survival signal for synovial T-cells [29], a signal that is superior to the one produced by non-inflammatory fibroblasts [30]. This interaction between FLS and lymphocytes can inhibit the resolution of local inflammation [30, 31] through both paracrine and direct cell–cell interactions [32]. This pathogenic role of the FLS is facilitated by the up-regulation of several adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) [6, 33]. In addition to recruitment and co-activation of T-cells in the inflamed joint, FLS have been shown to be able to present antigens on class II major histocompatibility complex (MHC-II) to CD4+ T-cells [34].

Furthermore, FLS are involved in the formation of tertiary lymphoid structures (TLS) in the RA synovium. Stromal cell populations such as the fibroblastic reticular cell support organization of these lymphocyte aggregates similarly to that of secondary lymphoid organs with distinct T- and B-cell niches [35]. Thymocyte differentiation antigen 1 (THY1, also known as CD90) and podoplanin (PDPN) positive fibroblast associated with TLS in RA (**Table 1**) produce several chemokines such as C-X-C motif ligand (CXCL) 13 and C-C motif ligand (CCL)21 implicated with lymphocyte recruitment and organization [47, 48]. Another marker associated with the TLS associated fibroblast is the receptor activator of nuclear factor kappa-β ligand (RANKL), which is important in both bone homeostasis and lymph node development [35, 49].

Collectively, FLS may be involved in both the pro-inflammatory initiation in the synovium, lymphocyte recruitment and the organization of TLS. A fibroblast driven RA phenotype resulting in persistent inflammation and a lymphoid rich synovium similar to what have been shown by histology.

#### **3.3 Disease persistence**

The highly proliferating and pathogenic RA FLS are very different from their quiescent state during non-inflamed conditions where FLS control the structural integrity of the joint lining and sublining layer [22]. The immunological events initiating a pathogenic state of RA FLS is still not fully understood, but proliferation and transformation of the FLS may occur prior to immune infiltration [50].


*The table contains a list of surface and transcriptional profiles of fibroblast subsets, fibroblast like cell subsets and macrophage subsets (pre-scRNA-seq) related to rheumatoid arthritis. For scRNA-seq studies, fibroblast subset names refer to the original articles. "+" and "- "shows whether the cells of interest are positive or negative for the cellular markers. The cellular markers which are discussed in the text are also listed under abbreviations.*

#### **Table 1.**

*Surface and transcriptional profiles of FLS subsets (and related cellular subsets) in rheumatoid arthritis.*

In RA, subsets of FLS can differentiate to become inflammatory, migratory, and invasive, thus collectively fostering disease aggravation in various animal models of RA [45, 51, 52]. Constitutive activation is a hallmark of RA FLS and leads to production of several inflammatory cytokines, such as interleukin (IL)-1β, TNFα and IL-6

#### *Fibroblast-Like Synovial Cell Subsets in Rheumatoid Arthritis DOI: http://dx.doi.org/10.5772/intechopen.99240*

and chemokines such as monocyte chemoattractant protein 1 (MCP-1/CLL2) [9] and CXCL12 [53]. Even though the activation of RA FLS is greatly affected by pro-inflammatory factors in the local environment, epigenetic changes are also important [54]. Epigenetic changes lead to constitutive activation even when the cells are removed from the inflamed environment and remain without addition of proinflammatory stimuli [52]**.** Moreover, a recent study suggests a link between epigenetic-driven positional identity of FLS (e.g. small versus large joints and proximal versus distal joints) and clinical disease patterns [55]. This link is further supported by the finding of oncogenes at sites of tissue destruction [56, 57] together with a highly activated nuclear factor κ beta pathway in RA FLS [58].

Altered metabolic activity with increased glycolysis is another hallmark of RA FLS [59]. Metabolic reprogramming of FLS were recently connected to complement C3 and C3a receptor-activation. Here repeated inflammatory challenges resulted in a distinct pro-inflammatory phenotypic priming of FLS in mice models of arthritis [60].

On the opposite side, several factors attempt to facilitate remission of proinflammatory FLS. One such potential immune regulator is the MerTK expressing synovial macrophage which *in vitro* reduce matrix metalloproteinase (MMP) production by lining layer FLS [61].

Thus, even though FLS are responsive to their inflammatory context they may possess a distinct positional identity which enables a cytokine-independent intrinsic activation contributing to disease persistence in RA.

#### **3.4 Joint destruction**

The severe joint destruction of late-stage RA is in part attributed to the pannus tissue which is rich in FLS. RA FLS are identified as invaders of the joint cartilage *in vivo* [62, 63], an invasive behavior that has been confirmed *in vitro* [64] and in mice [52]. FLS mediate cartilage degradation which is attributed to a combination of facilitating adhesion factors and production of proteases, here among several well-known matrix metalloproteinases (MMPs) [9, 52, 64]. Cartilage degradation is ameliorated when fibroblast activation protein (FAP) deficiency is induced in the human TNFα transgenic mice model of arthritis [65]. The invasiveness of pathological RA FLS is further emphasized by human FLS migrating to other joints in mouse models of RA and degrading the implanted human cartilage [51]. Migration that may be facilitated by specific anticitrulinated protein antibodies [66]. Notably, the *ex vivo* invasiveness of FLS correlates with joint erosions [67].

Increased osteoclastic activity leading to bone erosions in RA is another major factor in joint destruction. Here FLS produce CXCL12, RANKL, dickkopf related protein (DKK) 1, etc. which may increase both osteoclast migration, differentiation, proliferation/activation and inhibit osteoblast function [53, 68, 69].

#### **4. Single cell analysis of synovial fibroblast subsets in rheumatoid arthritis**

#### **4.1 Phenotyping of fibroblast like synovial cells**

Increasing spatial and molecular resolution in present day cellular analysis are changing our view of the synovial membrane in RA. Most notable is the identification of different fibroblast subsets within the inflamed synovial membrane. Recent work

and ongoing studies are utilizing scRNA-seq, CyTOF and flow cytometry cell sorting to further investigate and distinguish these subsets and their role in disease pathology.

Recent scRNA-seq studies have identified several distinct disease-associated subsets in the inflamed synovial membrane, often grouped as lining layer or sublining layer FLS [10, 43, 44], **Figure 1**. The present studies utilize flow cytometry assisted cell sorting and transcriptomic clustering strategies based on exclusion of hematopoietic lineage cells (CD45), endothelial cells (CD31), red blood cells (CD235a), and pericytes (CD146) while using PDPN or collagen production as a positive marker (**Table 1**).

#### **4.2 Lining layer fibroblasts**

A common finding in scRNA-seq studies confirms the presence of complement decayaccelerating factor (CD55) and absence of THY1 expression in FLS of the lining layer (**Table 1**). Of note, Mizoguchi et al. [44] did not report histological data of CD55 distribution, but a high level of CD55 gene expression in CD34- THY1- lining layer fibroblasts. All scRNA-seq studies (**Table 1**) of joint tissue reported lubricin (PRG4) expression in the lining layer subset [10, 43–45]. All present studies showed similar patterns of gene expression pertaining to the potential markers of FLS presented in the following section.

Zhang et al. [10] and the reanalysis of the same human data by Croft et al. [45] both reported a distinct lining fibroblast subset, SC-F4 and F4 respectively. This lining fibroblast subset was associated with expression of chloride intracellular ion channel 5 (CLIC5) and heparin binding epidermal growth factor-like growth factor (HBEGF). Mizoguchi et al. [44] and Stephenson et al. [43] also reported increased hyaluronan synthase 1 (HAS1) and metallopeptidase expression.

#### **Figure 1.**

*The figure is a schematic presentation of fibroblast subsets identified by scRNA-seq studies of synovial tissue from patients with rheumatoid arthritis. The subsets have been divided into lining layer FLS and sublining layer FLS. No scRNA-seq studies yet have examined fibroblast subsets from the synovial fluid. Based on grouping markers and transcription profiles listed in Table 1, we propose 4 sublining phenotypes. Cells have been divided by dashed lines when the cellular markers were not listed in all the original studies. THY-1 and PRG4 expression gradients from the lining layer to the sublining layer is shown by the color density of the red and blue bars. The cellular markers are discussed in the text and listed under abbreviations. TLS: Tertiary lymphoid structures.*

HAS1 is important for hyaluronan production and is a response to pro-inflammatory stimuli in RA synoviocytes. This activation results in hyaluronan cell coating, leukocyte/monocyte recruitment and facilitation of fibroblast-monocytes binding [70].

CD55, a C3 convertase inhibitor, has received increasing interest in cancer, where CD55/CD97 binding is associated with several oncogenic properties such as invasion and migration [71]. In RA, CD55 positive FLS are exclusive to the lining layer and in proximity to CD97 positive macrophages, suggesting a possible mechanism of crosstalk [36]. CD55 is not exclusive to RA [72], but it has been suggested as a protective factor in a mice model of immune complex mediated arthritis [73].

In the context of joint tissue, the mucin-like glycoprotein, PRG4, has been proposed as having a dual role comprising of well-known lubricating property and as a moderator of inflammation via NF-κβ pathways through interaction with both CD44 and toll-like receptors [74].

CLIC5 is present in several intracellular organelles, but predominantly located at the mitochondrial inner membrane, where it has been associated with modulation of reactive oxygen species [75]. However, no functional studies have been published regarding CLIC5 in RA.

The epidermal growth factor family member, HBEGF, is present and involved in several physiological processes such as wound healing, tumor formation and angiogenesis. One common topic is its association with cell migration, as seen in keratinocyte/fibroblast models and in enterocytes in necrotizing enterocolitis [76]. In RA, HBEGF positive macrophages have recently been shown to increase synovial fibroblast invasiveness in an *in vitro* model [77].

Several matrix metalloproteinases, MMP1, MMP3 and MMP14 was connected to a specific subset of FLS by Mizoguchi et al. [44]. These destructive enzymes have previously been connected to cartilage degradation in RA, but MMP14 was also noted by Mizoguchi et al. as a migratory factor [44].

Taken together, studies isolating and characterizing gene expression in lining layer fibroblasts report both associations of unknown importance and markers that may impose protective and destructive features. This suggests that the lining layer fibroblast subset is an active subset in RA pathology capable of cellular recruitment and significant local cellular crosstalk.

#### **4.3 Sublining layer fibroblasts**

The scRNA-seq studies have reported several distinct sublining subsets presented in **Table 1**. The initial study by Stephenson et al. [43] identified THY1 as a marker of sublining fibroblasts and the subsequent scRNA-seq studies confirmed THY1 as a specific, albeit not universal marker of sublining fibroblasts [10, 44, 45].

Zhang et al. characterized this heterogeneity of the sublining layer fibroblasts and defined three THY1+ groups with additional subset markers; CD34 defined the SC-F1 cluster, human leukocyte antigen (HLA)-DRAhigh defined the SC-F2 cluster and DKK-3 defined the SC-F3 cluster. The SC-F2 in particular was significantly increased in leukocyte-rich RA ssynovium compared to leukocyte-poor RA synovium and osteoarthritis (OA) synovium [10], suggesting these to encompass TLS-associated fibroblast subsets. Reanalysis of these human data by Croft et al. [45] enabled the distinction of four sublining layer fibroblast groups (F1–3,-5, **Table 1**).

As with the lining layer, large sets of multiomics data are available. Several markers connected to joint inflammation and destruction have been identified in these subsets. However, the markers most consistently reported are THY1, HLA-DRA, CD34, DKK3.

THY1 is a glycoprotein present on the membrane of several different cells including endothelial and mesenchymal cells [78]. Among the functions associated with THY1 expression is cellular contact, CD97 binding, integrin binding, trans-endothelial migration and MMP-9 and CXCL8 secretion after binding to neutrophiles [78].

As with THY1, CD34 is an established marker in different cell types including several stromal cells, epi/endothelial cells and fibrocytes [79]. Its function is largely unknown but has been linked to proliferation, adhesion, differentiation and is proposed as a marker of progenitor subsets in both mesenchymal, epithelial and endothelial cells [79].

MHC molecule (both class I and II) functions are typically attributed to antigen presentation. Several MHC molecules have been associated with autoimmune disease. Examples are the association of the MHC-I molecule HLA-B27 with ankylosing spondylitis, reactive arthritis and juvenile idiopathic arthritis subsets [80], and the association of MHC-II molecules HLA-DR1 and DR4 association with RA [81]. The specific function of HLA-DRA in RA FLS is yet to be investigated.

The DKK family of glycoproteins are well known modulators of WNT pathways connected to embryogenesis, bone formation and eye and skin development [82]. DKK-1 has been extensively described in fibroblasts from RA patients and is a key player in joint remodeling [69]. DKK-3 has been reported as a chondroprotective factor in OA [83] and suggested as a B-cell modulator whose absence aggravates autoimmune symptoms in a murine systemic lupus erythematosus model [84] and a CD8 T-cell modulator involved in antigen tolerance [85].

Enrichment of several genes related to pro-inflammatory cytokines and proteins related to bone metabolism in RA have been reported in sublining fibroblasts including IL-6, MCP-1/CCL2, CXCL12 and RANKL. Two proteins not mentioned above is the RANKL decoy receptor osteoprotegerin (OPG) which inhibit osteoclastogenesis in synovial macrophages [86] and the relatively new osteoglycin (OGN) that may both be part of the vascular system and may affect osteoblast differentiation [87].

The interferon regulatory factor 1 (IRF1) is a significant component of the interferon signature/inflammation pathway, through which TNF induces production of CXCL9–11 and in its absence diminishes B-cell activating factor expression [88].

The heparin-binding growth factor midkine (MDK) is less investigated than the above-mentioned cytokines but has been identified in human synoviocytes and associated with leukocyte migration to the synovium and osteoclastogenesis in mice [89].

C3, a unifying step for all three complement activating pathways has previously been located around microvasculature in the sublining of the RA synovium [90].

The cellular adhesion molecule 1 (CADM1) is a transmembrane member of the immunoglobulin superfamily with no known relation to RA. It has been identified as a tumor suppressor gene in solid cancers such as squamous cell carcinoma a, but may contribute to infiltration in adult T-cell leukemia/lymphoma [91].

To summarize, the sublining layer is a heterogeneous compartment of the inflamed RA synovium, regarding both cell linages and especially fibroblast subsets (**Table 1** and **Figure 1**). Several distinct fibroblast subsets have been identified, but recuring markers such as HLA-DRA, CD34 and DKK-3 are relatively unknown in the RA context. Results from scRNA-seq studies propose that the sublining layer fibroblast subsets are significantly involved in cellular crosstalk, leukocyte recruitment, para- and autocrine pro-inflammatory stimulation, and joint tissue destruction. Notably, some distinguishing factors such as DKK-3 may be enriched to form a regulatory anti-inflammatory and pro self-tolerance subset with similar chondroprotective effects and immune modulation of antigen tolerance mentioned in the previous

section. An HLA-DRAhigh/CXCL12/RANKLhigh associated subset may constitute the pro-inflammatory TLS associated fibroblast subsets and CD34 may discriminate a common progenitor fibroblast subset. Together, the presence of both pro-inflammatory subsets and potential anti-inflammatory and progenitor subsets suggests an ongoing cellular balancing throughout the sublining layer, which may open avenues for new research in treatment strategies targeting FLS.

#### **4.4 Fibroblast like synoviocytes in rheumatoid arthritis compared to other arthritides**

In RA, synovial division into lining/sublining layers suggests differentiated roles of subsets of FLS regarding cytokine production, joint destruction, and possible regulatory mechanisms.

The expansion of these distinct subsets is different in RA compared with OA. Mizoguchi et al. reported a greater fraction of the THY1<sup>+</sup> CD34− (perivascular) subset but less of the THY1− CD34− (lining) subset in RA compared with OA [44]. Notably, here the proportion of THY1+ CD34− (perivascular) FLS correlated with leukocyte infiltration and ultrasonic and histological synovitis [44].

Similarly, Zhang et al. reported an overabundance of the THY1<sup>+</sup> CD34− HLA-DRAhigh (SC-F2) subset with upregulated expression of CXCL12 and IL-6 and a THY1<sup>+</sup> CD34+ (SC-F1) subset in RA. In contrast, lining FLS (SC-F4) were more fabundant in OA [10].

The causal link between distinct subsets and RA pathogenesis was investigated in mice by Croft et al. Here the mouse thy1− subset homologous to human lining FLS (F4) were correlated to joint damage and mouse thy1<sup>+</sup> sublining FLS correlated to inflammation [45]. Notably, the elimination of FAP expressing subsets reduced pannus formation and joint destruction [45]. This suggests that FAP is a marker of pathologically active FLS in RA [45, 92, 93].

Comparison of subsets of FLS in RA and psoriatic arthritis are underway [94] and may potentially assist in discriminating these arthritides.

#### **5. Fibroblasts derived from synovial fluid versus synovial tissue**

Arthrocentesis is a common therapeutic procedure in treatment of RA. Fibroblast cultured from synovial fluid aspirates initially express similar phenotypical traits compared to tissue derived synovial fibroblast cultures [95, 96]. Despite these similarities, synovial fluid derived fibroblasts are likely a proxy regarding changes in the synovium and results must be interpreted as such. In both research and clinical settings synovial biopsies are both economical and well tolerated [97–100]. However, synovial fluid analysis of both cellular and soluble components is very useful in clinical settings where the length of consultations/sterile procedural environments/ analytic facilities may limit the use of synovial biopsies. To the authors knowledge, no studies have yet reported scRNA-seq analysis of synovial fluid fibroblasts.

#### **6. Circulating mesenchymal fibroblast like cells in rheumatoid arthritis**

In excess to tissue resident FLS, Orange et al. recently highlighted the presence of circulating fibroblast-like cells in the blood of RA patients shortly before symptomatic disease flare [46]. Interestingly these pre inflammatory mesenchymal (PRIME) cells show enrichment of previously reported markers of distinct sublining subsets of FLS e.g., DKK-3, CD34 and HLA-DR. This suggests that PRIME cells may constitute a heterogeneous pool of circulating FLS-like cells with distinct functions. Subsets of FLS migrating from the RA affected synovium, or a common homogeneous pool of circulating progenitor FLS awaiting recruitment signals from local sites of inflammation could potentially be the origin of these cells, although this remains to be investigated. Regardless, PRIME cells may not only be a useful marker predicting disease flares in RA, but also potentially explain how synovitis is transmitted from joint to joint [51].

#### **7. Future therapeutic perspectives**

The insights recently generated through high resolution scRNA-seq have revolutionized our understanding of specific subsets of FLS in RA and their involvement in driving different aspects of RA pathobiology. This understanding has also provided the basis for generating specific targetable markers of pathological subsets of FLS in RA. Targeting strategies that could be used as either monotherapy or as an add-on treatment to present day cytokine or lymphocyte inhibitors [101].

FLS could be targeted by drugs used in fibrotic conditions such as nintedanib or pirfenidone [102]. However, these drugs are likely affecting a completely different aspect of fibroblast functions. Therefore, new drugs are needed. An example is the addition of the cyclin-dependent kinase inhibitor, Seliciclib, which is currently being evaluated [103].

The well-known FAP marker of activated stromal cells has a diagnostic and prognostic potential through precise and low background positron emission tomography tracers developed in cancer-immunology [104]**.** The recent development of specific quinoline-based positron emission tomography tracers that act as FAP inhibitors have demonstrated promising results both preclinically and clinically in different cancers but could also be promising as diagnostic and prognostic markers of RA [105]. Further, the clinical potential of targeting FAP expressing FLS would be a targeted treatment eliminating pathologically activated RA FLS, in both the lining and the sublining layer [45, 93].

Among other interesting targets, NOTCH3 is one of the most recently *in vivo* validated pathological targets. NOTCH3 is expressed on the surface of RA FLS and linked with THY1 expression. NOTCH3 may also be a useful target in a therapeutic senescence strategy through selective activation of the g-protein coupled receptor melanocontin type 1 receptor [106]. Furthermore, in an animal model of RA injection of NOTCH3-neutralizing monoclonal antibody attenuated the severity of arthritis. Taken together, the *in vitro* studies on NOTCH3, including its connection to spatial distribution of FLS and the above-mentioned animal study underline NOTCH3 as a promising therapeutical target in RA [106, 107]. Targeting the complement C3 - C3a receptor axis may serve as another preventive or complementary strategy, where metabolic priming of FLS can be avoided or reduced [60]. Another possible strategy of targeting FLS is drug delivery via the extra domain A fibronectin splice variant identified in OA and RA [108, 109] and utilized in cancer [110].

Several other reagents targeting FLS are currently being tested ranging from metabolite modulators to treatments targeting intracellular signal transduction or epigenetic changes [111].

Collectively, these therapies targeting subsets of FLS are emerging as promising diagnostic and therapeutic tools. Tools for optimized and stratified treatments in RA based on which cellular mechanisms and which fibroblast subsets are pathologically activated in the individual patient.

#### **8. Conclusions**

Collectively, pathological FLS presented in this chapter are deeply connected to the RA pathophysiology of disease initiation, joint inflammation, disease persistence and joint tissue destruction.

Recent scRNA-seq studies have identified several distinct subsets of FLS causally linked to major elements of RA pathogenesis e.g., inflammation and joint destruction, while other subsets may present regulatory, pro-inflammatory TLS associated or common progenitor FLS.

These first steps in a scRNA-seq era of RA research warrants both rejoice and due diligence. Due diligence because we henceforth must appreciate the cellular diversity and the complex cellular crosstalk of the RA synovium. Like FLS, monocytes/ macrophages and lymphocytes exhibit distinct subsets in RA, which may be as important in understanding the spectrum of RA disease, e.g., lymphocyte dominated vs. lymphocyte poor synovium and erosive vs. non-erosive disease. Furthermore, we must appreciate the heterogeneity of FLS and cellular organization (here among TLS formation) of the sublining layer.

Rejoice because the recent subset studies have produced a language and knowledge and a novel nomenclature for FLS in future research. A breakthrough that might enable clinicians in the future to modulate specific aspects of RA through fibroblast subset targeted treatment.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Abbreviations**


**97**

#### *Fibroblasts - Advances in Inflammation, Autoimmunity and Cancer*


### **Author details**

Søren Lomholt1 , Morten A. Nielsen1 , Maithri P. Aspari1 , Peter B. Jørgensen2 , Adam P. Croft<sup>3</sup> , Christopher Buckley3 and Tue W. Kragstrup1 \*

1 Department of Biomedicine, Aarhus University, Aarhus, Denmark

2 Section for Immunology, Technical University of Denmark, Denmark

3 Rheumatology Research Group, University of Birmingham, Birmingham, UK

\*Address all correspondence to: kragstrup@biomed.au.dk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 6** Fibroblasts in Sjögren's Syndrome

*Kerstin Klein*

#### **Abstract**

The Sjögren's syndrome is an autoimmune disease characterized by chronic inflammation of the exocrine glands, leading to dryness of mucosal surfaces, and often to severe systemic manifestations. Here, the immunomodulatory function of fibroblasts derived from salivary glands, a primary site affected by the Sjögren's syndrome, is discussed. Specific subsets of these fibroblasts drive the formation of tertiary lymphoid structures, which are associated with severe disease and which constitute a risk factor for the development of lymphoma in Sjögren's syndrome. Single cell RNA-sequencing has provided new insights into subsets of fibroblasts in inflamed salivary glands and has provided evidence for the existence of shared inflammationassociated fibroblasts across chronically inflamed tissues. These findings support the concept of targeting the fibroblast compartment in Sjögren's syndrome and other chronic inflammatory diseases. In addition to the immunomodulatory role of fibroblasts, the interaction of the epithelium with fibroblasts is essential for salivary gland homeostasis. Fibroblasts provide essential signals for the regeneration of salivary gland epithelial cells, which is disturbed in Sjögren's syndrome, and leading to the loss of saliva secreting cells and subsequent hyposalivation.

**Keywords:** Sjögren's syndrome, autoimmunity, inflammation, tertiary lymphoid structure, salivary gland

#### **1. Introduction**

#### **1.1 Sjögren's syndrome**

The Sjögren's Syndrome (SjS) is a systemic autoimmune disease, most commonly presenting between the fourth and six decades of life. It affects predominantly women, with an estimated female to male ratio of 9:1 [1]. With a prevalence of 0.3 to 1 per 1000 people [1], the SjS represents the second most common rheumatic autoimmune condition after rheumatoid arthritis (RA). The SjS can either occur as single disease, often termed as primary SjS, or is associated with other autoimmune diseases, such as RA, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), or dermatomyositis [2]. Up to now, no disease modifying therapies for SjS have been approved and treatment is mainly symptomatic [3].

The hallmark of SjS is a hypofunction of exocrine glands, in particular salivary and lacrimal glands [3]. Dryness of mouth (xerostomia) and eyes (xerophthalmia), alongside fatigue and pain are the major symptoms affecting more than 80% of

patients with SjS [2]. The majority of patients with SjS present with glandular symptoms, which are often present over many years before diagnosis [4]. Whilst often considered as "benign features", these symptoms underpin great patient-reported disability. Other signs of systemic dryness, with scant information regarding the etiology and affecting patients' quality of life, involve the skin, the nose, the throat, the trachea and the vagina [5].

A major classification criterion for SjS is the infiltration of salivary glands with lymphocytes (focus score ≥1, in minor labial salivary gland biopsy), a condition called sialadenitis. The second major classification criterion is the presence of anti-SSA/Ro auto-antibodies, which is mandatory in patients with a lack of sialadenitis [3]. In 30 to 40% of patients, systemic epithelial and extra-epithelial manifestations occur that can affect the joints, skin, lungs, kidneys and nervous system [2].

People with SjS have increased morbidity and mortality compared to the general population [4]. Although being a rare event, the risk of B-cell lymphomas is 15 to 20 times higher in patients with SjS as the general population and accounts for the leading cause with an impact on patients's survival [2, 4]. The most common type of lymphomas in patients with SjS are mucosa-associated lymphoid tissue (MALT) lymphomas. Chronic activation of B cells at the primary sites affected by SjS, such as the salivary glands, was attributed to the development of lymphoma. Several risk factors have been defined for the development of lymphoma in patients with SjS; among them is the presence of ectopic germinal centers in tertiary lymphoid structures (TLS) [2].

#### **1.2 Epithelial cell activation is central in the pathogenesis in SjS**

The SjS develops in genetically predisposed individuals upon exposure to stress factors. Hormones as well as infectious agents, and in particular viruses, are assumed to play key roles in the pathogenesis of the SjS. Activated epithelial cells in salivary glands are the central cell type in the current concept underlying the pathogenesis. They are on the one hand drivers of the ongoing inflammation and on the other hand, due to the excess of apoptosis of epithelial cells in salivary glands, a source of auto-antigens for infiltrating lymphocytes [6]. Epithelial cells respond to and produce pro-inflammatory cytokines and chemokines and thus, promote inflammation. They produce MHC-II and co-stimulatory molecules, enabling them to directly interact with and activate T-cells. Furthermore, they produce B-cell activating factor (BAFF), inducing the activation and survival of B cells [7]. Many of these characteristics have been previously described for fibroblasts, e.g. in the synovium of rheumatoid arthritis patients, and analogies of synovial fibroblasts with salivary gland-derived fibroblasts have been recognized [7, 8]. However, the ability of salivary gland-derived fibroblasts to exert similar functions is only at the beginning to be characterized in detail.

#### **2. Fibroblasts in tertiary lymphoid structures**

#### **2.1 Tertiary lymphoid structures are often present in autoimmune diseases**

TLS, or ectopic lymphoid organs (ELS), often develop at sites of inflammation in target tissues. Their formation has been associated with chronic inflammation, autoimmune disease, cancer, and transplant rejection [9]. TLS are sites of ectopic

#### *Fibroblasts in Sjögren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.98946*

autoantibody production and expansion of potential autoreactive B cell clones [7, 10]. The formation of TLS in salivary glands is an established model for studying TLS formation in autoimmunity in general. The frequency of the presence of TLS varies among different autoimmune diseases, with a high frequency in autoimmune thyroiditis and low presence in systemic lupus erythematosus [10]. Approximately 30–40% of patients with SjS exhibit TLS in their salivary glands, the primary sites of the disease [11, 12]. A similar percentage of TLS is found in patients with rheumatoid arthritis, in which TLS are associated with a lympho-myeloid pathotype that represents a distinct disease entity as the diffuse myeloid and pauci-immune fibroid pathotpyes [13]. Hence, the presence and absence of TLS in salivary glands of patients with SjS might underlie different pathophysiological processes in different, not yet characterized, disease subsets. The formation of TLS is across autoimmune diseases associated with more severe disease and poor prognosis [7].

TLS often share several typical structural characteristics with secondary lymphoid tissues (lymph nodes, tonsils, spleen, Peyer's patches, mucosa-associated lymphoid tissues), including highly organized lymphocytic aggregates, with T and B cell segregation, the development of high endothelial venules, and follicular dendritic cell networks. In contrast to secondary lymphoid tissues, the lymphocytic aggregates found in TLS can range from a simple aggregates to highly ordered structures with bona fide germinal centers that support the production of autoreactive plasma cells [9, 10]. TLS formation and secondary lymphoid tissue development follow numerous overlapping signaling pathways, however, the cellular sources of signaling molecules differ [10]. In contrast to secondary lymphoid structures whose development is initiated at the embryonic stage, TLS develop postnatally in response to inflammatory signals, where they provide a specialized pro-inflammatory environment that plays a key role in perpetuating disease progression in autoimmune conditions [7, 14]. Podoplanin (pdpn)-expressing fibroblastic reticular cells in secondary lymphoid organs and pdpn+ stromal fibroblasts in TLS provide signals and the scaffold structure that foster the interaction of T cells with dendritic cells, and hence drive innate and adaptive immune responses [15, 16].

#### **2.2 Tertiary lymphoid structures are associated with severe disease in Sjögren's syndrome**

The routine histopathological examination of minor salivary gland biopsies carries a substantial prognostic value regarding disease severity and outcome [17]. Higher inflammatory scores, and the presence of germinal center-like structures in particular, in salivary glands of patients with SjS were associated with more severe disease, illustrated by elevated titers of rheumatoid factor, anti-Ro/SSA and anti-Ro/ SSB auto-antibodies, enhanced levels of local and systemic pro-inflammatory mediators and a reduced saliva secretion [11, 17, 18]. Germinal center-like structures have been identified in approximately 25% of patients with SjS [17, 19]. Their presence at time of diagnosis, or sole high lymphocytic scores, were shown to account as independent risk factors for the development of Non-Hodgkin's lymphomas in patients with SjS [17, 19].

Given the pivotal role of TLS in SjS, the identification of factors and pathological mechanisms triggering and regulating their formation and those of germinal center-like structures is of high interest in order to identify potential targets for drug development.

#### **2.3 Adenoviral infection using retrograde excretory duct cannulation is a model of SjS**

Studying the chronology of TLS formation and associated cell types in animal models, together with complementary evaluation of human salivary gland specimens, provided new insights into pathomechanisms associcated with sialadenitis in SjS, and unraveled the analogy of TLS formation to the development of secondary lymphoid organs. A model that proved to be of particular value for studying salivary gland inflammation in SjS, and the formation of TLS in autoimmune processes in general, is the selective submandibular gland administration of a replication-defective adenovirus 5 (AdV5) through retrograde excretory duct cannulation in wild-type C57/Bl76 mice. These mice were shown to resemble several hallmarks of SjS, including lymphocytic infiltration of salivary glands, TLS formation, anti-nuclear autoantibody (ANA) formation, and reduction in salivary flow indicative of excretory gland dysfunction [20]. Cannulated mice developed SjS-like periductal lymphoid aggregates within two weeks after AdV5 delivery. Within three weeks, the inducible TLS acquired progressively hallmarks of functional germinal centers, with segregated B and T cell areas, high endothelial venules in T-cell rich areas, and follicular dendritic cell networks in up to 70% of the lymphocytic aggregates. Local expression of activation-induced cytidine deaminase (AID), the enzyme required for Ig somatic hypermutation and class-switch recombination, pointed to the functional activation of B cells in TLS [20].

#### **2.4 Fibroblasts drive the formation of tertiary lymphoid structures**

During secondary lymphoid organ development in embryogenesis, mesenchymal precursor cell maturate into intercellular adhesion molecule-1 (ICAM-1)high, vascular cell adhesion molecule-1 (VCAM-1)high organizer cells, in a process that is dependent on lymphoid tissue inducer cells and lymphotoxin β receptor (LTβR) signaling. This leads to a sustained stromal cell production of interleukin 7 (IL7), C-X-C motif chemokine ligand 13 (CXCL13) and to a lesser extent C-C motif chemokine ligand 21 (CCL21) [21]. The subsequent migration of lymphocytes into the anlagen is responsible for the full differentiation of fibroblastic reticular cells within distinct areas of the secondary lymphoid organ [22–24].

In TLS of salivary glands of patients with SjS, a network of pdpn<sup>+</sup> and fibroblast activating protein (FAP)+ fibroblasts were identified that support the formation of TLS [24]. The same markers have been previously identified on fibroblast reticular cells in lymph nodes [15, 25], which provide, by the secretion of CCL19 and CCL21, the key factors for the migration and retention of T cells in secondary lymphoid organs [16, 25, 26]. Among the pdpn+ fibroblasts in TLS, two functionally distinct populations have been identified in human salivary glands that provide the signals for lymphocyte survival and organization within TLS, respectively (**Figure 1**) [24]. The first cluster of pdpn+ fibroblasts, was characterized by high expression of FAP, ICAM-1, VCAM-1 and CD34 [24]. Pdpn+CD34+ fibroblasts in TLS produced IL7 and BAFF, underlying their function in supporting lymphocyte survival and homeostasis [24]. The second cluster of pdpn+ CD34− fibroblasts was characterized by high expression levels of CXCL13, CCL19 and CCL21. Expansion of a similar network of pdpn+ fibroblasts has been observed upon salivary gland infection of mice with Ad5V. Of note, this expansion occurred before lymphocyte infiltration, suggesting a pivotal, early role for fibroblasts in SjS. Fibroblasts in TLS of cannulated mouse salivary

#### *Fibroblasts in Sjögren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.98946*

**Figure 1.**

*Stromal fibroblast populations contribute to the formation of tertiary lymphoid structures (TLS). Created by BioRender.com. DCs, dendritic cells; ILCs, innate lymphoid cells.*

glands expressed CXCL13, CCL19, BAFF, IL7 and LTβR, with an increased expression of lymphoid chemokines specifically in the ICAM-1+ VCAM-1+ subpopulation of pdpn+ fibroblasts [24].

#### **2.5 IL13 and IL22 induce the formation of tertiary lymphoid structures**

Pdnp+ fibroblasts in human TLS expressed receptors for and responded to stimulation with IL13, IL4, IL22, TNF, and LTα1β2 [24, 27]. Elevated levels of IL13 have been detected in serum of patients with SjS, where they correlated with titers of anti-Ro/SSA auto-antibodies [28], and in Id3 knockout mice, a model for T cell mediated SjS [29, 30]. Also, high levels of IL22 in sera of patients with SjS have been shown to correlate with clinically relevant parameters, such as reduced salivary flow, hypergammaglobulinemia, as well as serum titers of rheumatoid factor, anti-Ro/SSA and anti-Ro/SSB auto-antibodies [31]. Together these data suggested a potential link between increased levels of IL13 and IL22 with the auto-antibody production in SjS.

IL13 stimulation of cultured human salivary gland fibroblasts, in synergy with TNF and LTα1β2, induced the expression of VCAM-1, ICAM-1 and pdpn *in vitro* (**Table 1**). This function was confirmed *in vivo*. Innate lymphoid cells, fibroblasts and epithelial cells have been identified as the source of IL-13 in the developing TLS. IL13 expression was induced rapidly within a few hours upon AdV5 administration in mice, leading to the priming of immunofibroblast progenitors by activation of IL4 receptor signaling, and the subsequent expression of pdpn, ICAM-1 and VCAM-1. AdV5 administration in IL4R−/− mice led to a disturbed TLS assembly, and salivary glands were characterized by reduced expression levels of CXCL13 and


#### **Table 1.**

*Chemokines involved in the formation of tertiary lymphoid structures in salivary glands of patients with Sjögren's syndrome.*

CCL21, smaller inflammatory foci and an abolished autoantibody production. The early priming of immunofibroblasts by IL13 was independent of the presence of lymphocytes [24].

In contrast to IL13, IL-22 stimulation of cultured human salivary gland fibroblasts induced proliferation but did not induce the expression of pdpn, ICAM-1 and VCAM-1 *in vitro* [24]. Studies in cannulated mice revealed that the induction of IL22 was, similarly to the induction of IL13, an early event after AdV5 delivery in TLS formation. The main sources of IL22 in the developing TLS were T cells, along with innate lymphoid cells and natural killer cells. IL22 induced the expression of CXCL13 in stromal fibroblasts and the expression of CXCL12 in epithelial cells, leading to the subsequent B cell aggregation and auto-antibody production [27]. Together these data demonstrated that IL13 mediates the early priming of fibroblasts in the developing TLS, and IL22 is responsible for the expansion of the fibroblastic network. These early steps in TLS development were independent of LTα1β2 and RORγ<sup>+</sup> lymphoid tissue inducer cells [24], which regulate the final maturation of fibroblastic reticular cells in the development of secondary lymphoid organs [21]. However, studies in LTβr −/− and RORγ−/− mice have underlined their critical role in the final differentiation and stabilization of the functional phenotype of immunofibroblasts at later stages of the TLS assembly [24].

#### **3. Overlap of fibroblasts from patients with SjS and other inflammatory diseases**

#### **3.1 Single cell RNA sequencing identifies fibroblast clusters across diseases**

The asset of single cell RNA sequencing (scRNA-seq) technologies has enabled the identification of different fibroblasts populations associated with chronic inflammation across different diseases and anatomical sites [32–39]. Given the

#### *Fibroblasts in Sjögren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.98946*

pro-inflammatory role of fibroblasts and their ability to carry a certain degree of inflammatory memory [40], interfering with fibroblasts has become a new potential therapeutic strategy in chronic inflammatory diseases.

In a recent study, scRNA-seq data sets derived from four different inflammatory diseases, namely rheumatoid arthritis, interstitial lung disease, ulcerative colitis and the SjS were integrated, with the aim to provide a stromal cell atlas to identify pathogenic fibroblast subsets shared across diseases [8]. For each inflamed tissue, non-inflamed control tissues were included in the analysis. With respect to the SjS, biopsies derived from minor salivary gland biopsies of patients with SjS were compared to those from patients with sicca symptoms, characterized as non-autoimmune dryness, and who did not fulfill the classification criteria for SjS. Given the lack of a universal fibroblast marker, fibroblasts in this study were characterized by the expression of collagen (COL) 1A1 and defined as non-epithelial, non-immune, non-endothelial, and non-mural cells based on the respective specific markers for those cell types. By pooling the scRNA-seq data sets from salivary glands, lungs, the synovium and the gut, 14 clusters of fibroblasts have been identified, each of them consisting of genes that were shared across different tissues in addition to tissue-specific genes (**Table 2**). Among these clusters, two of them expanded across tissues in inflamed versus respective non-inflamed controls.



#### **Table 2.**

*Shared fibroblasts clusters between salivary gland (SG), lung, synovium, and gut, as defined by Korsynsky et al. [8].*

#### **3.2 CXCL10+ CCL19+ fibroblasts: Interactors with immune cells**

The first of these shared clusters is characterized by the marker genes CXCL10 and CCL19. Based on a gene set enrichment and pathway analysis, CXCL10+ CCL19+ fibroblasts were identified as a subset that potentially directly interacts with immune cells. Among the enriched pathways were "lymphocyte chemotaxis", "antigen presentation", and "positive regulation of T cell proliferation". Furthermore, scRNA-seq data suggested that CXCL10+ CCL19+ fibroblasts respond to key pro-inflammatory cytokines, including interferon (IFN) γ and IFNα, TNF, IL1, and IL1. The responsiveness to IFNγ and IFNα was specific to CXCL10+ CCL19+ fibroblasts [8]. This might be of high relevance in the context of the SjS, given the pronounced role of type I and II interferon signatures detected in SjS, and their association with more severe disease [41, 42]. CXCL10+ CCL19+ fibroblasts functionally resembled the pdpn+ CD34− CCL19 expressing fibroblasts that have been described to be involved in the formation of TLS in salivary glands of patients with SjS [24].

#### **3.3 SPARC+ COL3A1+ fibroblasts: a vascular-interacting population**

The second shared cluster of fibroblasts that was identified to be expanded across inflamed tissues was characterized by the expression of secreted protein acidic and cysteine rich (SPARC) and COL3A1. SPARC<sup>+</sup> COL3A1+ fibroblasts resembled a potentially endothelium-driven activated fibroblast state, characterized by the enrichment of pathways associated with extracellular matrix binding and remodeling. In addition, key developmental and morphogen signaling pathways were enriched, including hedgehog, transforming growth factor (TGF) β, WNT, bone morphogenic protein (BMP) and Notch signaling. By comparing these shared human fibroblast clusters to the temporal activation of fibroblast clusters in the mouse model of dextran sulfate sodium (DSS)-induced colitis, the expansion of SPARC+ COL3A1+ fibroblasts was identified as an early event in the inflammatory process, in which vascular remodeling preceded leukocyte infiltration [8].

#### **4. Fibroblasts in the regeneration of salivary gland epithelial cells**

A key process in gland development is the epithelial-mesenchymal interaction [43]. The stromal-derived extracellular matrix is essential for the growth, morphogenesis and differentiation of salivary gland tissues [44]. Extracellular matrix remodeling and fibrosis are pathological features found in minor salivary gland biopsies of patients with SjS, that are associated with salivary gland inflammation, reduced stimulated salivary flow but not with age [44–46]. Point mutations in people with hypohidrotic ectodermal dysplasia (HED) lead to a disturbed signaling between the salivary epithelium and mesenchymal fibroblasts, affecting their gland development. Salivary and sweat glands have the same embryonic origin and people with HED present with defects in salivary glands, sweat glands, teeth and hair [47].

Several studies have pointed out that the correlation of salivary flow with the degree of inflammation in salivary glands of patients with SjS is low [48–51], suggesting that other mechanisms than inflammation underlie hyposalivation in SjS. Salivary gland epithelial cells of patients with SjS are more prone to anoikis, a detachmentinduced apoptosis, after activation of Toll-like receptor 3 signaling [52]. In healthy salivary glands, salivary gland stem cells reside in ducts of salivary glands and differentiate into saliva secreting acinar cells to maintain homeostasis. In SjS, salivary gland stem cells are fewer in numbers and exhibit an aged phenotype, with a reduced capacity to self-renew and proliferate [53]. This suggests that saliva production in patients with SjS might not be restored solely be the use of anti-inflammatory drugs. In regenerative medicine approaches, the co-culturing of stem cells together with fibroblasts is essential for engineering secreting salivary epithelial cells [54, 55]. Hence, fibroblasts might also be involved in the disturbed regeneration of salivary gland epithelial cells in SjS and are likely to have functions beyond promoting inflammation.

#### **5. The potential role of fibroblasts in extra-glandular manifestations of the SjS**

Extra-glandular manifestations are found in 30–40% of patients with SjS, and can be divided into epithelial and extra-epithelial manifestations that can affect the central and peripheral nervous system, the lungs, lymph nodes, kidneys, joints, the skin and the muscles [2, 4]. A role of fibroblasts in extra-glandular manifestations has not been studied yet, maybe due to limited assess of available tissue samples from affected sites.

Articular manifestations, such as arthralgias and synovitis, are the most common extra-glandular manifestations and affect 30–60% of patients with SjS [2, 56]. Arthritis in patients with SjS is often classified as non-erosive [57]. However, recent more sensitive methods such as ultrasound and magnetic resonance imaging (MRI) have detected erosions in more than one third of SjS patients with joint pain and no previous diagnosis of arthritis [57, 58]. In patients with SjS, arthritis most frequently occurs in proximal interphalangeal (PIP) and metacarpophalangeal (MCP) joints and wrists [57], a pattern that is overlapping with the one found in hands of RA patients [59]. Synovial fibroblasts are the major stroma cells of the joint and play a pivotal role in the pathogenesis of rheumatoid arthritis by promoting the ongoing inflammation and cartilage degradation [60]. The existence of shared fibroblast clusters in salivary glands of SjS patients and synovial tissues of rheumatoid arthritis patients, suggests a role of fibroblasts also in articular manifestations of the SjS. However, this potential role of fibroblasts remains to be proven.

#### **6. Conclusions**

The Sjögren's syndrome is a chronic inflammatory autoimmune disease with huge unmet needs for patients and clinicians. No therapies for the treatment of the SjS have been approved so far. The pathogenic processes in the exocrine glands have only partially unraveled. Whereas the contribution of salivary gland epithelial cells has been studied in detail, the functional role of fibroblasts in maintaining epithelial cell function, as well as their role in the regulation of the inflammatory process has only recently been recognized. The potential of targeting the fibroblast compartment in salivary glands of patients with SjS has been underscored by studies characterizing their role in the establishment of TLS as well as by scRNA-seq of minor salivary gland tissues. Together these studies pointed to an early, to a large extent lymphocyteindependent, role of fibroblasts in the pathogenesis of the SjS.

Integration of the scRNA-seq data sets across inflamed human tissues, including minor salivary gland tissues from patients with SjS, together with scRNA-seq data sets from mouse models, have suggested a two stage mechanism for fibroblast activation and fibroblast-mediated regulation of inflammation. In this model, the expansion of SPARC+ COL3A1+ vascular-associated fibroblasts initiates vascular remodeling and subsequent leukocyte infiltration and precedes the expansion of CXCL10+ CCL19+ immune-interacting fibroblasts.

CXCL10<sup>+</sup> CCL19+ immune-interacting fibroblasts functionally resemble pdpn+ CD34<sup>−</sup> CCL19 expressing fibroblasts that are critically involved in TLS assembly. Formation of TLS is initiated after experimental salivary gland infection by IL13 and IL22 that prime immunofibroblast progenitors and induce the expansion of the fibroblast network, respectively. This supports the concept of fibroblast-targeting strategies to treat TLS-associated autoimmune diseases such as the SjS.

#### **Conflict of interest**

The author declares no conflict of interest.

*Fibroblasts in Sjögren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.98946*

### **Author details**

Kerstin Klein1,2

1 Department of BioMedical Research, University of Bern, Bern, Switzerland

2 Department of Rheumatology and Immunology, University Hospital Bern, Bern, Switzerland

\*Address all correspondence to: kerstin.klein@dbmr.unibe.ch; kerstin.klein@insel.ch

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Mojca Frank Bertoncelj and Katja Lakota*

*Fibroblasts - Advances in Inflammation, Autoimmunity and Cancer* presents recent advances in understanding the roles of fibroblasts and mesenchymal stem cells in tissue homeostasis and the development of human disease. The book delves into general principles of fibroblast and mesenchymal stem cell biology and their diversity across the human body. It highlights these cells' unique and shared characteristics across organs (e.g., vasculature, kidney, joints and exocrine glands) and specific pathologies (e.g., tissue damage, inflammation, fibrosis and cancer). A particular focus is set on the roles of fibroblasts in disease chronicity, recurrence, progression, therapeutic resistance and utilisation of the advancing knowledge for developing new therapeutic approaches within and beyond disease boundaries.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK © 2021 IntechOpen © monsitj / iStock

Fibroblasts - Advances in Inflammation, Autoimmunity and Cancer

IntechOpen Series

Biochemistry, Volume 25

Fibroblasts

Advances in Inflammation,

Autoimmunity and Cancer

*Edited by Mojca Frank Bertoncelj* 

*and Katja Lakota*