In the current study we have found that serum levels of CXCL-8, TGF-β, TNF-α and IFN-γ were higher and IP-10 expression was lower in IPF patients. Moreover, the intracellular expression of IL-17 in CD3 + CD4 + ROR-γt + cells was higher in IPF patients. This altered expression of these T cell-derived mediators did not correlate with altered expression of the T cell subset studied transcription factors GATA3, T-bet, ROR-γt and FOXP3 in PBMCs since their levels were similar in IPF and in HC subjects.
Pulmonary fibrosis is important in lung-associated diseases which are characterized by the scarring and thickening of lung tissue. Diverse etiologies may account for the pathogenesis of the disease such as environmental exposures, autoimmune disorders, infections, or idiopathic factors all of which involve immune dysregulation (27).
Th2 cells expressing the GATA3 transcription factor are important in the generation of fibrosis. In contrast, Th1 and its mediators play a dual role in the pathogenesis of lungs fibrosis. Lung fibroblasts tend to induce a Th1-type response under normal conditions reflecting their role in normal scar healing (13).However, downregulation of the anti-fibrotic mediators such as IFN-γ in the Broncho alveolar lavage (BAL) fluid or circulation of IPF patients suggests a complex role of Th1 cells in the development of fibrosis (28). Overall, although T cells represent a small population in healthy lungs, the numbers of T cell subsets are higher in the lungs and Broncho alveolar lavage (BAL) of IPF patients (29–31). In this study we found a positive correlation between T-bet and GATA3 expression.
Further evidence is provided in animal models of lung fibrosis. Survival is significantly decreased in GATA-3 transgenic mice compared with wild-type mice following bleomycin (BLM)-induced lung fibrosis (32). This was linked to a greater level of pulmonary fibrosis in GATA-3 transgenic mice than in wild-type mice 28 days after bleomycin treatment (32). Thus, GATA3 expression regulates lung fibrosis. In contrast, lung concentrations of anti-fibrotic IFN-γ was significantly decreased in these GATA-3 transgenic mice compared with wild-type by 7 days after either saline or bleomycin treatment (32). These results demonstrate that Th2/GATA-3 transcription factor has a central role in the development of fibrosis in bleomycin models (26). Mice lacking T and B cells (RAG2−/− mice) are more susceptible to bleomycin-induced scleroderma and RAG2/T-bet double-deficient mice show an even higher sensitivity to bleomycin than observed in T-bet−/− mice (33). This data demonstrated that T-bet plays an important role in preventing bleomycin-induced scleroderma. In current study, we did not find any changes in the expression of T-bet in PBMCs of IPF patients compared with healthy control subjects.
Regarding our findings about not finding a significant difference between healthy subjects and fibrosis patients in terms of T-bet and GATA3 expression, it's important to note that fibrosis is a complex and heterogeneous condition with various underlying causes and tissue-specific characteristics. The expression levels and functional roles of T-bet and GATA3 may vary depending on the specific fibrotic disease, tissue site, and stage of fibrosis. Additionally, other factors, such as the composition of the immune cell population and the presence of other transcription factors or signaling molecules, can influence the overall outcome (27, 34) .
IFN-gamma is a pro-inflammatory cytokine produced primarily by T lymphocytes and natural killer (NK) cells. In pulmonary fibrosis, IFN-gamma is thought to promote fibrosis by stimulating the production of extracellular matrix components and inducing fibroblast activation (35). It can also enhance the production of other pro-fibrotic factors such as TGF-beta. Additionally, IFN-gamma contributes to the recruitment and activation of immune cells, perpetuating the inflammatory response in the lung tissue. In controversy; some studies have shown an anti-fibrotic role for IFN-γ. IFN-γ is a key anti-fibrotic agent in the lungs, liver and kidneys (19) and particularly altered IFN-γ1b expression is central to the pathogenesis of IPF and is associated with changes in mortality (36). IFN-γ1b has a pleiotropic action with antimicrobial, antiangiogenic, antifibrotic, and/or immunomodulatory effects. This data suggests a possible therapeutic role of IFN-γ in IPF patients (36).
Enhanced Th17 responses are associated with skin fibrosis in patients with systemic sclerosis (SSc), however, it’s relation with lung fibrosis is unclear (37). BLM treatment of mice resulted in higher numbers of Th17 cells in the circulation, skin and lungs together with increased serum levels of IL-17A, TGF-β1, IL-6 and RORγt (37). In the current study we did not find any differences in expression of the Th17-inducing ROR-γt in PBMC from IPF patients although a greater intracellular expression of IL-17 in cells from IPF patients was seen. IL-17A plays a key role in various inflammatory conditions by promoting the expression of inflammatory factors, including cytokines, chemokines, matrix metalloproteinases, and acute phase proteins as well as playing a role in pulmonary fibrosis (38).
Interestingly, the expression of ROR-γt positively correlated with level of intracellular expression of IL-4 which supports earlier data suggesting that IL-4 has a dual role the pathogenesis of lung injury and fibrosis (48). During the later stages of fibrosis, IL-4 plays a profibrotic role since IL-4−/− mice developed significantly less pulmonary fibrosis relative to wild type mice. However, IL-4 does not directly stimulate proliferation, α-smooth muscle actin, and type I collagen expression in lung fibroblasts isolated from the wild-type mice and fibroblasts from IL-4−/− mice had a markedly attenuated response to pro-fibrotic factors (39). Overall, IL-4 may play a role in the pathogenesis of lung fibrosis by limiting early recruitment of T lymphocytes but also by stimulating chronic fibrosis (39). These data indicate that the development of lung fibrosis is under the control of Th1, Th2 and Th17 responses although other cell types such as macrophages may also be important (40).
We show higher serum levels of TGF-β, TNF-α, CXCL-8 and IFN-γ with lower levels of IP-10 in IPF patients. Increased CXCL-8 gene expression by alveolar macrophages is observed in IPF (41) and CXCL-8 levels are elevated in serum, BALF and tissue of IPF patients compared with healthy controls (41). Serum CXCL-8 levels have been reported as indicative of disease activity (42). In the current study the serum levels of CXCL-8 were higher than HC. CXCL-8 can have pro-fibrotic properties during angiogenesis by acting through chemokine (C-X-C motif) receptor 2 (CXCR2) (43, 44). In contrast, to the higher tissue levels of CXCL-8 in IPF tissues, IP-10 levels are reduced (41) although up-regulation of pulmonary chemokine IP-10 was directly associated with lung damage (45).
Specific blockage of the IP-10 receptor CXCR3 significantly decreased the severity of chronic pulmonary inflammation by decreasing the recruitment of inflammatory cells. IP-10 is proposed as a key player responsible for the development of tree shrew-based collagen-induced arthritis (TsCIA)-CPI (45). In the BML model of lung fibrosis, IP-10 lung levels correlated with a higher angiogenic response. Systemic administration of IP-10 significantly reduced BLM-induced pulmonary fibrosis without any changes in lung lymphocytes or NK cell numbers and reduced angiogenesis (46).
No direct effects of IP-10 on pulmonary fibroblasts have been reported although IP-10 inhibits fibroplasia and deposition of extracellular matrix via modulating angiogenesis (46). Interestingly, IP-10 can directly interact with syndecan-4, a Heparan sulfate proteoglycan, on the cell surface in lung interstitial tissues and inhibit fibrosis (40). Jiang et al observed that a marked decrease in neutrophil recruitment and an increase in both myofibroblast and induction of interstitial fibrosis in BLM-treated syndecan-4–null (Sdc4–/–) mice. Furthermore, fibroblast migration in IPF was inhibited in the presence of an IP-10 protein defective for CXCR3 binding. Thus, administration of an IP-10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis (47).
We found that the expression of the Treg cell-associated transcription factor FOXP3 was negatively correlated with age of IPF patients. The effect of aging on Treg cell function in the pathophysiology of IPF not well documented. Higher serum levels of TNF as we observed may account for this correlation (48). In current study the levels of TNF-α was positively correlated with age and in line with other studies. TNF-α is important cytokine in the development of pulmonary fibrosis (49) although exogenous TNF-α attenuated the development of pulmonary fibrosis in mice (49).
There is some limitations in the current study including the restricted number of patients, the mismatch between the age of IPF patients and HCs and the failure to include diverse ethnic groups. However, our study indicates the elevation of proinflammatory and anti-inflammatory cytokines in IPF patients and lower level of IP-10 chemokine in serum of IPF patients.
In conclusion, the level of IP-10 in patients with IPF is low; and may this account for the development and control of lung fibrosis. The interactions of soluble inflammatory or anti-inflammatory mediators in the pathogenesis of lung cancer are not well understood. Thus, extensive future work is required to establish the interactions of the myriad of implicated mediators including in vivo studies with KO animals.