*6.1.1. Mediators eliciting and perpetuating interstitial pneumonia in systemic sclerosis*

The key mechanisms in CTD-IP involve an interplay between various cell types and humoral factors; the pathogenesis is initiated by microvascular injury, leading to endothelial cell damage and alveolar epithelial injury [12, 132, 133]. This is followed by activation of the coagulation cascade, release of various cytokines, e.g., IL-1, IL-4, IL-6, IL-13, chemokines, and lysophosphatidic acid (LPA), and growth factors including transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), and insulin-like growth factor (IGF-1), which leads to activation of fibroblasts, resulting in the development of fibrosis [12, 132, 134]. Many epithelial-derived factors influence the behavior of fibroblasts, with soluble mediators known to exhibit profibrotic activities [134]. The pivotal mediator of fibrosis is the multifunctional cytokine, TGF-β, which, along with platelet-derived growth factor, endothelin-1 (ET-1), plays a major role in the pathogenesis of SSc. There is evidence that epithelial-tomesenchymal transdifferentiation (EMT) occurs in lung fibrosis, and this process is mediated by TGF-β and potentially ET-1 [135, 136]. TGF-β responses are mediated by canonical Smad signaling [137]. Binding of TGF-β to its receptor elicits signaling through phosphorylation and nuclear translocation of cytoplasmic Smad protein, triggering transcription of genes such as type I collagen, fibronectin, α-smooth muscle actin, and CTGF, which promote fibrogenesis [138]. ET-1 is a potent vasoconstrictor produced by endothelial cells, epithelial cells, and mesenchymal cells. ET-1 binds to ET-1A and ET-1B receptors, recruits fibroblasts, stimulates extracellular matrix production, and also stimulates TGF-β production in lung fibroblasts [139]. Elevated levels of ET-1 have been found in the blood vessels, lung, kidneys, and skin of SSc patients [140]. LPA is produced by activated platelets and fibroblasts. The LPA receptor is expressed in fibroblasts, endothelial cells, and epithelial cells. Both are involved in the development of lung fibrosis in mouse model of IPF, suggesting LPA mediates fibroblast recruitment [141]. IGFs have been implicated in pulmonary fibrosis because increased levels of IGF-1 are detected in the serum as well as in the BAL of SSc patients [142]. CTGF, also known as CCN2, which plays a pivotal role in the stimulation of extracellular matrix production and myofibroblast differentiation, is involved in angiogenesis and forming the connective tissue [143]. The levels are elevated in the skin and lungs from SSc patients as well as in the sera [144].

**6. Pathogenesis of interstitial pneumonia associated with connective** 

**6.1. Classic mechanisms in the pathogenesis of interstitial pneumonia in connective tissue** 

Numbers of studies on the pathogenesis of IP have been performed in SSc as well as in mouse models of IP, providing evidence for plausible mechanisms that may lead to pulmonary fibrosis in CTD. This is natural because, among the whole CTDs, SSc has the highest prevalence of IP, and currently, the lung disease consists the major cause of death in patients with SSc, being shifted away from mortality due to renal crisis which was more common in the past. The high morbidity and mortality due to IP in SSc have been eliciting not only multidisciplinary clinical studies but also basic researches. Many lines of evidence acquired from the studies on SSc, and relevant researches on fibrosis have been implying the following potential scenario of

The key mechanisms in CTD-IP involve an interplay between various cell types and humoral factors; the pathogenesis is initiated by microvascular injury, leading to endothelial cell damage and alveolar epithelial injury [12, 132, 133]. This is followed by activation of the coagulation cascade, release of various cytokines, e.g., IL-1, IL-4, IL-6, IL-13, chemokines, and lysophosphatidic acid (LPA), and growth factors including transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), and insulin-like growth factor (IGF-1), which leads to activation of fibroblasts, resulting in the development of fibrosis [12, 132, 134]. Many epithelial-derived factors influence the behavior of fibroblasts, with soluble mediators known to exhibit profibrotic activities [134]. The pivotal mediator of fibrosis is the multifunctional cytokine, TGF-β, which, along with platelet-derived growth factor, endothelin-1 (ET-1), plays a major role in the pathogenesis of SSc. There is evidence that epithelial-tomesenchymal transdifferentiation (EMT) occurs in lung fibrosis, and this process is mediated by TGF-β and potentially ET-1 [135, 136]. TGF-β responses are mediated by canonical Smad signaling [137]. Binding of TGF-β to its receptor elicits signaling through phosphorylation and nuclear translocation of cytoplasmic Smad protein, triggering transcription of genes such as type I collagen, fibronectin, α-smooth muscle actin, and CTGF, which promote fibrogenesis [138]. ET-1 is a potent vasoconstrictor produced by endothelial cells, epithelial cells, and mesenchymal cells. ET-1 binds to ET-1A and ET-1B receptors, recruits fibroblasts, stimulates extracellular matrix production, and also stimulates TGF-β production in lung fibroblasts [139]. Elevated levels of ET-1 have been found in the blood vessels, lung, kidneys, and skin of SSc patients [140]. LPA is produced by activated platelets and fibroblasts. The LPA receptor is expressed in fibroblasts, endothelial cells, and epithelial cells. Both are involved in the development of lung fibrosis in mouse model of IPF, suggesting LPA mediates fibroblast recruitment [141]. IGFs have been implicated in pulmonary fibrosis because increased levels of IGF-1 are detected in the serum as well as in the BAL of SSc patients [142]. CTGF, also

classic mechanisms in the pathogenesis of CTD-IP [12, 132, 133].

*6.1.1. Mediators eliciting and perpetuating interstitial pneumonia in systemic sclerosis*

**tissue disease**

164 Contemporary Topics of Pneumonia

**disease**

The earliest events of the parenchymal lung involvement in CTD include inflammation and associated alveolar epithelial injury which occurs due to undetermined causes or can be caused by some environmental pathogens. The alveolar epithelial damage and inflammation let resident fibroblasts of pulmonary interstitium to locate to the alveolar wall, and the fibroblasts become activated through a variety of mediators such as TGF-β [145, 146]. The activation of resident fibroblasts was shown to be induced by the recruitment of active TGF-β from the lung tissue [147]. The resident lung fibroblasts play a pivotal role in lung fibrosis, and they are considered to be a more primitive or less differentiated lineage of fibroblasts that are prepared for repair at injury response [148]. The recruitment of activated fibroblasts and myofibroblasts that produce large amount of extracellular matrix proteins occurs in the process. These population of cells are not only derived from resident interstitial fibroblasts but also come from circulating progenitor cells which include mesenchymal stem cells recruited from the bone marrow and cells of a monocyte lineage that localize to the lung [149]. Myofibroblasts persist as critical profibrotic cells in affected lung tissue. It is conceivable that minor injury and subsequent disease process lead to the development of a lung microenvironment prone to fibrosis. That series of events results in an accumulation of constituents of the extracellular matrix (ECM), which remodels normal tissue architecture, which in turn culminates in pulmonary organ failure. Essentially, the lung is primed to develop fibrosis in response to injury, and it is likely that the intrinsic response is more severe in CTDs than normal individuals. Thus, in SSc, such genetic or intrinsic differences can be reflected to the serological phenotypes such as the expression of autoantibodies. Patients with SSc having anti-topoisomerase antibodies are liable to develop significant lung fibrosis, while those with anti-RNA polymerase III antibodies are less [150].

#### *6.1.2. Involvement of immune mechanisms in interstitial pneumonia in systemic sclerosis*

The immune system is also implicated in the pathology of SSc. Several lines of evidence suggest that a specific population of activated T cells exhibiting type 2 helper T (Th2) phenotype potentially mediates tissue fibrosis, secreting IL-4 and IL-13 both of which activate fibroblasts and collagen production by inducing TGF-β [151]. In SSc, T cells with memory phenotype were found in lung biopsy specimens from patients with lung involvement [152]. In some studies reported, increased numbers of lung memory CD8 T cells are associated with more severe pulmonary fibrosis [153–155]. Luzina et al. have shown an increase in CD8 T cells in the lungs of SSc patients by using T cells isolated from BAL fluid and demonstrated that a subset of patients at higher risk of progressive lung disease had activated, long-lived CD8 T cells which could promote fibrosis through production of profibrotic factors such as IL-4 and oncostatin M, as well as activation of TGF-β [156]. Regulatory T cells (Tregs) which maintain self tolerance can be impaired in their ability to suppress CD 4 effector T cells [157]. Currently, the precise knowledge of the role of effector cells in innate and adaptive immune system in SSc-associated IP is rather insufficient. More fundamental investigations in this aspect are needed to address to many queries as to the whole scenario of the development of IP in CTDs.

Besides, as concerns idiopathic pulmonary fibrosis (IPF), the latest evidence of immune mechanisms in IPF was reviewed in the recent literature, which includes involvement of both innate immunity and adaptive immunity at several levels of the processes toward development of fibrinogenesis in the human lung of IPF or in its model mice, as summarized in **Table 5** [158]. Briefly, in adaptive immune system, the role of T cells seems complex and subset dependent; Th2 and Th17 cells were shown to promote pulmonary fibrosis, although Th1, Th22, and γδ-T cells have been found to attenuate fibrotic disease. Treg and Th9 subsets have been proposed to exert both anti- and profibrotic effects. In innate immunity, M2 macrophages and neutrophils have been suggested to enhance pulmonary fibrosis, whereas M1 macrophages were assigned a protective role, but contradictory findings have also been described [158].

After all, a variety of studies on the pathogenesis of IPF have been conducted, and many experimental models were generated to explore the mechanisms. However, it is yet highly questionable whether the evidence provided from the studies of IPF is applicable to the etiology of CTD-associated IP. Furthermore, it is still unclear whether the animal models such as the mouse bleomycin model can truly replicate the autoimmune progressive forms of the ILDs seen in human CTDs.
