Inflammation-Associated Wound Healing through a Monocytic Lens

*Avi Petroff*

#### **Abstract**

The development of macrophages from monocytes during wound healing is a complicated and convoluted process. Classically or alternatively activated macrophages result from a complex network of cytokine signaling between circulating monocytes entering tissue, resident macrophages, and stromal fibroblasts. This network of signaling constitutes a continuous communication between these cell types, influencing factors such as inflammatory duration, healthy or fibrotic tissue repair, and downstream macrophage functionality. "Forward talk" from monocytes to fibroblasts, as well as "back talk" from fibroblasts to monocytes, can greatly influence the behavior of each cell type. This cell-cell communication, though difficult to fully encapsulate *in vitro*, can be facilitated through implementation of specific cell culture techniques. 3D cell culture systems enable a more representative assessment of myofibroblast phenotypes that would likely be seen during wound repair. Co-culture systems further enable cell-cell interactions in the inflammatory and wound repair cascades to be assessed in coordination with each other. Looking ahead, these cell culture techniques, alongside novel concepts such as organ-on-a-chip models, can provide deeper insight into the myriad molecular mechanisms we claim to understand currently. Our improved understanding of these cellular interactions can lead to improved clinical outcomes for pathologies associated with these complex cell types.

**Keywords:** monocyte, macrophage, fibroblast, inflammation, communication, cell culture, wound repair, fibrosis, cytokine, cellular development

#### **1. Introduction**

To truly appreciate the history of our understanding of macrophages, we must first understand how macrophages manifest from earlier cellular lineages. Just as humans have common ancestors and lineages with other primates, so too do macrophages with other cells derived from myeloid progenitor cells. All this is to say, just as humans are complex organisms, macrophages have fantastic intricacies and potential fates. The whole world is its oyster, or petri dish, if you will.

So, how do macrophages come to be? We'll go over some key points here, and some will be expanded upon in subsequent sections within this chapter. Discussion in this chapter will pertain to macrophages, monocytes, and fibroblasts in a broad cellular sense but will also have a focus on human physiology. Briefly, macrophages comprise

a heterogenous and highly variable group of myeloid cells that function within the innate immune system [1]. The functions of macrophages are largely attributed to inflammation, which will be discussed further below.

#### **2. Mention of macrophage manifestation**

What makes macrophages particularly fascinating is the plasticity of their manifestation. In other words, macrophages have the innate ability to alter their phenotype during development in response to, and in conjunction with, myriad environmental signals [1]. This plasticity in "activation" of macrophages is a growing area of research in fields such as immunology, disease progression, tissue and extracellular matrix homeostasis, and resolution of inflammation [2].

More specifically, the activation of macrophages can come about by stimuli such as cytokines—special cellular signaling proteins—that influence levels of gene and protein expression [2]. The unique composition of cell surface receptors, intracellular enzymes, and cytokines allows us to create distinctions that organize our understanding of macrophage activation.

The exact categories of macrophage activation go beyond the scope of this chapter, and such distinctions are still a point of ongoing scientific discussion. However, for the purpose of our understanding, there are two primary routes of macrophage activation. The first is commonly known as "classically activated" or "type 1" macrophages (M1), and the second is known as "alternatively activated" or "type 2" (M2) [2]. These states of activation have been described as polarized extremes within a continuum of macrophage functionality [3]. Indeed, various subsets of nomenclature have been established, such as M2a and M2b, that expand upon the basic M1 and M2 classifications, accounting for the possibility of activation within a spectrum of phenotypes [2]. For the purpose of this chapter, we'll delve deeper into M1 and M2, bearing in mind the continuum within which we are exploring.

Classically activated macrophages are purported to be involved in the canonical response to tissue injury and/or infection. As expected, this pathway of activation is characterized by macrophage expression of many pro-inflammatory cytokines, such as TNF-α, and interleukins such as IL-1β, IL-6, and IL-12 [1]. These M1 macrophages are heavily involved in the inflammatory cascade discussed in subsequent sections within this chapter, primarily through the production of reactive oxygen and nitrogen species [1].

Alternatively activated macrophages, in contrast to M1 macrophages, secrete minimal pro-inflammatory cytokines [1]. Instead, M2 macrophages secrete numerous anti-inflammatory cytokines such as IL-10, CCL18, and CCL22 [1]. Further, alternatively activated macrophages play a crucial role in counteracting pro-inflammatory and cellular immune mechanisms, with inhibitory and regulatory functions in such pathways [4]. In this respect, M1 macrophages are broadly categorized as the proinflammatory side of the spectrum, whereas M2 macrophages pertain mostly to the anti-inflammatory side of the spectrum [1].

Though more complicated than necessary for the purpose of discussion in this chapter, there are various *in vitro* protocols employed to induce M1 and M2 phenotypes [5]. Because M1 and M2 phenotypes represent such polarized and opposite activation states within the macrophage continuum, these basic classifications provide an opportunity to study and assess macrophages in a wound healing context. However, we must remember that *in vivo* macrophage phenotypes in tissue wounds would likely demonstrate a much more complex and variable phenotype.

#### *Inflammation-Associated Wound Healing through a Monocytic Lens DOI: http://dx.doi.org/10.5772/intechopen.102645*

To conclude our introduction to the manifestation of macrophages, it should additionally be noted that IL-10 is a much more complex cytokine than previously thought. Difficult to describe and fully encapsulate in experimental data or scientific publications, IL-10 can behave as both a pro-inflammatory and anti-inflammatory cytokine in various environmental conditions [6, 7]. With the presence of IL-10 during the developmental process of macrophages, it is exciting to consider that there is potentially still much to learn about macrophages, and that our history of understanding these cells continues into our future.

#### **3. Inflammation during wound healing**

Now that we understand a bit more about the manifestation of macrophages, let us now turn our attention to the myeloid cells from which macrophages are themselves derived—monocytes. Monocytes are another complex and intriguing cell type and will be one of our focuses for the bulk of this chapter. However, it would be inappropriate to discuss the functions and fates of monocytes without also discussing the role of another cell type involved in would healing—fibroblasts. Fibroblasts are largely involved in the structuring and maintenance of the extracellular matrix (ECM) of tissue and play a vital role in the wound healing process.

In most human tissues, healthy wound healing is predicted to occur following hemostasis (blood clotting) via orderly and efficient progression through various stages of a signaling cascade (**Figure 1**). These stages include local inflammation, inflammatory resolution, tissue cell proliferation, and tissue remodeling [8]. Fibrosis—the formation of dysfunctional and often distorted scar tissue—occurs when these sequential events are dysregulated by dynamic signaling pathways [9, 10].

The inflammatory phase of wound healing is when circulating monocytes, as well as neutrophils, infiltrate tissue at the site of injury via cytokine recruitment [8]. Upon arrival, these circulating monocytes, in addition to local tissue monocytes, may differentiate into tissue macrophages (**Figure 2**) [8]. Classically or alternatively activated macrophages then play a role in tissue debridement, phagocytosis of foreign particles, and interaction with other cell types at the wound site such as fibroblasts and lymphocytes. This communication is in the form of secreted cytokines and growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor

#### **Figure 1.**

*Signaling cascade following hemostasis during wound healing. Progression through these stages, either subsequently or in an overlapping fashion, leads to the healthy remodeling of tissue. Dysregulation at various points within this cascade can result in aberrant wound repair, as is seen in fibrosis.*

(FGF), and transforming growth factor β (TGFβ) [8]. Macrophages also play a role in antigen presentation for T-lymphocytes for development of downstream memory immune responses [8]. Without the vital role of macrophages, there would be slowed growth of damaged tissue, persistence of cellular and particulate debris, and slowed progression through the subsequent stages of wound healing (**Figure 1**).

The plasticity of activated macrophages is quite impressive. The elegant balance between classically and alternatively activated macrophages is crucial for preventing pathologies or acute reactions within wounded tissue [11]. This balance is made possible by the macrophage phenotypic plasticity; classically activated macrophages within the wound site can kill pathogens, neutralize toxins, and debride the wound [11]. In turn, alternatively activated macrophages are recruited via cytokines to repair tissue and heal the wound [11]. To prevent recruitment of circulating monocytes beyond this point, classically activated macrophages can switch their apparent phenotype to alternatively activated macrophages, based on signals from the local stromal tissue (**Figure 2**) [11]. This interesting signaling between stromal tissue, macrophages, and circulating monocytes will be discussed further later in this chapter.

In many human tissues, wound healing initiates the TGFβ signaling pathway, distinct from macrophage-secreted TGFβ [10]. Exposure of tissue fibroblasts to TGFβ induces another type of cellular activation. In this situation, tissue fibroblasts can become activated into myofibroblasts, characterized by expression of α-smooth muscle actin (α-SMA) [8]. Myofibroblasts play a prominent role in wound healing by synthesizing and secreting large quantities of ECM material, and additionally obtain a phenotype of increased contractility. The ECM secretions and increased contractility of these cell types facilitate wound healing, though excessive levels are characteristic of tissue fibrosis [8].

#### **Figure 2.**

*Schematic of the central role of macrophages in wound healing. Circulating monocytes that differentiate into macrophages, in addition to tissue resident macrophages, can be classically or alternatively activated. Activated macrophages then contribute to various processes associated with wound healing. Cellular crosstalk between monocytes, macrophages, and stromal myofibroblasts further contributes to the complexity of the wound repair process.*

#### *Inflammation-Associated Wound Healing through a Monocytic Lens DOI: http://dx.doi.org/10.5772/intechopen.102645*

The magnitude and duration of the proliferative stage of wound healing is correlated to the duration of the inflammatory phase [12]. Indeed, the amount of collagen deposited by myofibroblasts can be reduced by attenuating the inflammatory response [13]. During remodeling, fibrovascular tissue formed during the proliferative stage matures into scar tissue [12]. Normal reduction in myofibroblast numbers through apoptosis is important during this ECM remodeling, and prolongation of myofibroblast survival leads to excessive scar tissue formation [12].

Before moving to the next section of this chapter, let us connect these concepts and ideas surrounding cell types to form a clearer picture for our understanding. It certainly wasn't an exaggeration on my part when I described monocytes, macrophages, and fibroblasts as complex cell types! In a vacuum, each of these cells can behave in unique ways, demonstrating variable phenotypes even when assessed individually as a mono-culture of cells. This is a focal point of many *in vitro* studies that aim to articulate the innate behavior of these cells. However, the world of possibilities becomes even larger when considering these cells in coordination with each other. The myriad interactions between just these three cell types *in vivo* presents a niche area of modern cellular research, quite literally.

#### **4. Stromal influence on monocyte signaling: a game of broken telephone**

The canonical understanding of wound healing involves the "forward talk" from circulating immune cells to stromal cells, such as fibroblasts, to induce paracrine signaling or activation of these fibroblasts into myofibroblasts [8]. Indeed, it has long been accepted that the processes of monocyte recruitment, differentiation into classically or alternatively activated macrophages, and signaling to fibroblasts follow an organized and stringent process [14]. However, our general understanding is somewhat lacking when considering the "back talk" from stromal cells to monocytes, and the effects of this communication on monocyte cytokine production and downstream macrophage differentiation (**Figure 2**).

As a brief aside, a commonly used cell line for assessing monocyte cellular processes is the THP-1 cell line. THP-1 cells are immortalized human monocytes derived from an acute monocytic leukemia patient [15]. These cells are invaluable because they potentiate a simplistic but widely available *in vitro* cell model of inflammatory infiltrate. These cells allow for meaningful immune modulation analyses because they demonstrate few changes during cell culture periods, maintain minimal genetic variation, and present few obstacles in terms of ethical issues or donor availability [15]. Lastly, these cells are very useful because they can be classically or alternatively activated into macrophages through various experimental techniques. We will not go into these specific laboratory protocols, but it must be understood that THP-1 monocytes provide an informative tool for exploring macrophage differentiation.

However, as is the case with many *in vitro* studies, it is difficult to fully recapitulate cell-cell interactions that would occur *in vivo*, and thus studies using THP-1 cells should be assessed with this in mind. In any experimental model that does not incorporate all physiological components, there are elements missing that can only be compensated to a certain degree. Even the most optimal *in vitro* models fails to include the plethora of microenvironmental and cellular niches that can contribute to cellular processes *in vivo*. Nevertheless, there is much to learn about the characteristics of monocytes using THP-1 cells, and so we'll include these cells in our discussion.

Previous studies have shown that gene expression in THP-1 monocytes encoding macrophage differentiation markers is influenced by co-culture with fibroblasts [16]. However, there are currently few studies of this type that assess the relative effects of co-culture on both cell types, particularly in the context of wound healing. Could it be the case that multiple cell types influence each other's behavior in cell culture? Let us explore this for a moment.

As outlined earlier, fibroblast activation into myofibroblasts is largely induced by mechanical properties of the ECM through various mechanotransduction pathways [14]. Strain caused by fibroblast focal adhesions within the collagen meshwork of the ECM enables efficient mechanical activation of latent TGFβ1 through integrin-mediated cell-pulling [14]. Increased TGFβ1 signaling can then induce further fibroblast activation into myofibroblasts in a positive feedback loop. As well, myofibroblast activation facilitates active ECM remodeling during wound healing, producing mechanical cues for other cell types such as circulating blood monocytes [14]. Such signaling characterizes the "back talk" from ECM myofibroblasts to monocytes. Erroneous and persistent communication from ECM signals to monocytes can promote further myofibroblast activation and fibrosis [14], characterizing a feedback loop of "forward talk". Thus, a 3D cell culture model that closely resembles physiological ECM would likely be most appropriate for the assessment of myofibroblast activation via TGFβ1 signaling pathways.

That there have been relatively few studies assessing the relative effects of co-culture on circulating monocytes and fibroblasts is unfortunate. It is troubling because of a phenomenon previously demonstrated via *in vitro* fibrosis models in which immunologically-activated myofibroblasts promoted monocyte migration into tissue through cellular "back talk" [17]. This "back talk" could have been the result of pro-inflammatory stimuli of myofibroblasts inducing subsequent changes in monocytic gene expression and physiological chemotactic recruitment [18]. In other words, the importance of co-culturing these cell types together is emphasized by the role of myofibroblasts as immunoregulatory "sentinel cells" [18].

All this is to say, there are complex cell-cell interactions between stromal fibroblasts in a 3D ECM and circulating blood monocytes. Indeed, stromal cells, specifically fibroblasts, are becoming increasingly prominent in research regarding pathogenesis of tissue inflammation, immunomodulation of tissue microenvironments, transition from acute to chronic inflammation, and inflammation persistence associated with rheumatoid arthritis [19]. In this context, fibroblasts play a functional role in the progression of chronic inflammation, in addition to their role in tissue fibrosis highlighted earlier.

Beyond involvement in chronic inflammation and tissue fibrosis, fibroblasts and stroma in a broad sense play a functional role in another chronic pathology: malignant disease. In this instance, stromal cells lay down the components of the non-tumor ECM that potentiates growth of solid tumors [19]. However, stromal cells can also be a key driver of tumor progression through the inhibition of apoptosis in malignant cells in breast carcinoma [20]. Further, stromal cells and cancer-associated fibroblasts are purported to facilitate tumorigenesis and eventual metastasis through production of oncogenic signals and promotion of angiogenesis [19].

While reading this, you may notice that we have veered onto tangential thinking. Our main discussion involves monocytes, fibroblasts, and macrophages in the context of wound healing following acute inflammation. How, then, does chronic inflammation and malignant disease progression relate to this? The answer lies within the interactions between the cell types involved in these processes. The "forward talk"

and "back talk" in each of these situations is critical for the progression of cell migration, development, signaling, and proliferation. Just as communication is vital for a healthy relationship between humans, so too is it vital for these cellular processes—even if pathological!

To realign our thinking regarding fibroblasts, it is purported that even in the absence of external stimuli, fibroblasts are capable of promoting monocyte migration through the production of similar protein signals to cytokines, called chemokines [17]. Further, this behavior is not limited to one cell type, but instead is an intrinsic property of fibroblasts [17]. This alludes to the concept of immunologically-activated myofibroblast "back talk" inducing monocytic migration, which then ties into our understanding of downstream fates of monocytes into classically or alternatively activated macrophages, for example.

With this in mind, lets tie a few more strings together. At a site of wound injury, circulating blood monocytes are recruited to the site of injury via the process of inflammation [21]. Through a multitude of cytokine signals between monocytes, tissue macrophages, and stromal cells, monocytes quickly acquire certain macrophage phenotypic characteristics once at the wound site [21]. The coordination between blood-derived and tissue macrophages allows for the synthesis and release of many different types of regulatory cytokines and chemokines that are crucial for the wound healing process [21]. This "forward talk" from monocytes and macrophages allows for stromal fibroblasts to acquire a myofibroblast phenotype in many cases, and facilitates processes such as matrix deposition, tissue contraction, and cellular reorganization. The last point of discussion for the purpose of this chapter will thus be how the "forward talk" and "back talk" demonstrate an elegant interplay, influencing the quality and quantity of wound repair.

#### **5. Implications toward our understanding of macrophages**

Up to this point, we have discussed the key players of the inflammatory cascade, some cell types involved in wound repair, and the significance of the "forward talk" and "back talk" between some of these cellular driving forces. What implications, then, does all of this have with regards to our understanding of macrophages?

The answer to this question, as is the case with many scientific inquiries, lies in how we observe and assess the communicatory phenomena surrounding monocytes and macrophages. We've already alluded to how each cell type can innately behave one way when assessed in mono-culture, and how this behavior can be altered when assessed in co-culture. However, the extent to which we can improve our experimentation on these cell types is dependent on how accurately we can recapitulate some of the physiological processes that occur *in vivo* using mimetic cell culture models.

Two potential ways through which the scientific community can better understand the complex interplay of monocytes, macrophages, and fibroblasts is through the use of co-culture systems, but also through the use of 3D cell culture systems. Such 3D systems could more intimately mimic the biological interactions that occur in living tissues and can provide novel perspectives beyond those attainable through 2D cell cultures [16, 22]. Current cell culture models that lack 3D and co-culture techniques are likely to underestimate potential immunomodulatory effects of stromal fibroblasts on monocytes and macrophage development due to the omission of complex bi-directional signaling that can occur between these cell types in living tissue [22].

For example, a novel macrophage and fibroblast co-culture model was designed and employed to assess the effect of material surface properties on inflammatory response regulation *in vitro* [23]. This co-culture system was used because it more closely mimicked autocrine, paracrine, and juxtacrine signaling between these two cell types [23]. As well, the comparison between macrophage behavior in monoculture vs. co-culture was made possible [23]. Together, this exemplifies the usefulness of co-culture models when understanding the inflammatory processes that occur physiologically, particularly in the context of biomaterial assessment.

A similar model designed by many of the same authors was also used to assess physiological host responses of biomaterials *in vitro* [24]. This model maintained the advantage of spatially-controlled interactions between macrophages and fibroblasts in a co-culture system [24]. Further, data showed that macrophages could induce wound healing and even fibrotic responses via upregulation of fibroblast outgrowth, cytokine production, and myofibroblast activation [24]. Use of this model was also advantageous because additional factors, such as cytokines, could also be assessed *in vitro* for their effects on cell-cell communication in a co-culture system [24].

A step in the right direction toward a more comprehensive experimental system for monocytes, macrophages, and fibroblasts is the recent development of organon-a-chip (OOC) models. These models employ microfluidic *in vitro* systems that markedly improve our ability to assess how immune cells interact with parenchymal cells to mediate immune responses to inflammation [25, 26]. These models are powerful and flexible, but they can be difficult to manufacture. In addition, they are not usually designed to accommodate cell-type specific nor sensitive analyses of cellular responses to co-culture conditions, such as changes in gene expression of cytokine markers [22].

#### **6. Conclusion**

Our understanding of macrophages has immensely improved over the past 140 years. So too has our understanding of myriad cellular interactions, such as inflammation, wound healing, and tissue homeostasis. This has, in part, been made possible by our simultaneous understanding of other crucial cell types, such as monocytes and stromal fibroblasts, and the role these cells have in our greater understanding of macrophages.

The importance of experimental models that employ both 3D and co-culture techniques for assessing cellular responses to inflammatory cytokine stimuli thus cannot be understated. To that end, we must appreciate the potential to accumulate incomplete or perhaps even fallacious data from mono-culture or 2D systems that do not incorporate various physiological factors that are at play during the "forward talk" and "back talk" of these cellular processes.

Looking ahead, our understanding of macrophages can and should be expanded upon. By designing cell culture systems that better enable assessment of the many cell-cell communications during inflammation, wound healing, and tissue homeostasis, we can continue to explore these incredible feats of nature. Advancements in these areas could improve clinical outcomes for patients suffering from numerous pathologies discussed in this chapter, such as chronic inflammation, tissue fibrosis, and even cancer formation. I look forward to the "forward talk" that we have yet to discover.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

I would like to personally thank my undergraduate research thesis advisors, Dr. Cindy Hutnik and Dr. David O'Gorman, for their role in shaping my fascination for this area of scientific discovery. You both challenged me to learn and explore topics surrounding wound healing, inflammation, and fibrosis and enabled me to branch into fields of research that I had never thought to consider prior.

I would also like to personally thank Jelena Vrdoljak, the Author Service Manager for IntechOpen, for her role in supporting the production of this chapter. Her patience, guidance, and communication made submission to this book seamless and organized.

## **Author details**

Avi Petroff Sackler School of Medicine, New York State/American Program, Tel Aviv University, Tel Aviv, Israel

\*Address all correspondence to: avipetroff@mail.tau.ac.il

© 2022 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.

## **References**

[1] Genin M, Clement F, Fattaccioli A, Raes M, Michiels C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer. 2015;**15**:577

[2] Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity. 2014;**41**(1):14-20

[3] Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: Tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in Immunology. 2002;**23**(11):549-555

[4] Gordon S, Martinez FO. Alternative activation of macrophages: Mechanism and functions. Immunity. 2010;**32**(5):593-604

[5] Ploeger DT, Hosper NA, Schipper M, Koerts JA, de Rond S, Bank RA. Cell plasticity in wound healing: Paracrine factors of M1/M2 polarized macrophages influence the phenotypical state of dermal fibroblasts. Cell Communication and Signaling: CCS. 2013;**11**(1):29

[6] Cavaillon JM. Pro- versus antiinflammatory cytokines: Myth or reality. Cellular and Molecular Biology (Noisyle-Grand, France). 2001;**47**(4):695-702

[7] Fernandes JC, Martel-Pelletier J, Pelletier J-P. The role of cytokines in osteoarthritis pathophysiology. Biorheology. 2002;**39**(1-2):237-246

[8] Seibold LK, Sherwood MB, Kahook MY. Wound modulation after filtration surgery. Survey of Ophthalmology. 2012;**57**(6):530-550 [9] Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. The Journal of Pathology. 2003;**200**(4):500-503

[10] Schlunck G, Meyer-ter-Vehn T, Klink T, Grehn F. Conjunctival fibrosis following filtering glaucoma surgery. Experimental Eye Research. 2016;**142**:76-82

[11] Chistiakov DA, Myasoedova VA, Revin VV, Orekhov AN, Bobryshev YV. The impact of interferon-regulatory factors to macrophage differentiation and polarization into M1 and M2. Immunobiology. 2018;**223**(1):101-111

[12] Zada M, Pattamatta U, White A. Modulation of fibroblasts in conjunctival wound healing. Ophthalmology. 2018;**125**(2):179-192

[13] Serhan CN, Savill J. Resolution of inflammation: The beginning programs the end. Nature Immunology. 2005;**6**(12):1191-1197

[14] Pakshir P, Hinz B. The big five in fibrosis: Macrophages, myofibroblasts, matrix, mechanics, and miscommunication. Matrix Biology. 2018;**68-69**:81-93

[15] Chanput W, Mes JJ, Wichers HJ. THP-1 cell line: An in vitro cell model for immune modulation approach. International Immunopharmacology. 2014;**23**(1):37-45

[16] Kuen J, Darowski D, Kluge T, Majety M. Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model. PLoS One. 2017;**12**(7):e0182039

[17] Enzerink A, Salmenpera P, Kankuri E, Vaheri A. Clustering of *Inflammation-Associated Wound Healing through a Monocytic Lens DOI: http://dx.doi.org/10.5772/intechopen.102645*

fibroblasts induces proinflammatory chemokine secretion promoting leukocyte migration. Molecular Immunology. 2009;**46**(8-9):1787-1795

[18] Kaufman J, Graf BA, Leung EC, Pollock SJ, Koumas L, Reddy SY, et al. Fibroblasts as sentinel cells: Role of the CDcd40-CDcd40 ligand system in fibroblast activation and lung inflammation and fibrosis. Chest. 2001;**120**(Suppl. 1):53S-55S

[19] Patel R, Filer A, Barone F, Buckley CD. Stroma: Fertile soil for inflammation. Best Practice & Research. Clinical Rheumatology. 2014;**28**(4):565-576

[20] Wiseman BS, Werb Z. Stromal effects on mammary gland development and breast cancer. Science. 2002;**296**(5570):1046-1049

[21] Brancato SK, Albina JE. Wound macrophages as key regulators of repair: Origin, phenotype, and function. The American Journal of Pathology. 2011;**178**(1):19-25

[22] Petroff A, Pena Diaz A, Armstrong JJ, Gonga-Cavé BC, Hutnik C, O'Gorman DB. Understanding inflammation-associated ophthalmic pathologies: A novel 3D co-culture model of monocyte-myofibroblast immunomodulation. Ocular Immunology and Inflammation. 2021:1- 12. DOI: 10.1080/09273948.2021.1980816. PMID: 34648419

[23] Zhou G, Loppnow H, Groth T. A macrophage/fibroblast co-culture system using a cell migration chamber to study inflammatory effects of biomaterials. Acta Biomaterialia. 2015;**26**:54-63

[24] Zhou G, Liedmann A, Chatterjee C, Groth T. In vitro study of the host responses to model biomaterials via a fibroblast/macrophage co-culture

system. Biomaterials Science. 2016;**5**(1):141-152

[25] Polini A, Del Mercato LL, Barra A, Zhang YS, Calabi F, Gigli G. Towards the development of human immune-systemon-a-chip platforms. Drug Discovery Today. 2019;**24**(2):517-525

[26] Sasserath T, Rumsey JW, McAleer CW, Bridges LR, Long CJ, Elbrecht D, et al. Differential monocyte actuation in a three-organ functional innate immune system-on-a-chip. Advancement of Science. 2020; **7**(13):2000323
