Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis

In this study, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. We hypothesized that activin A may be involved in the regulation of inflammatory and fibrotic responses during EAO by regulating the potential pro-fibrotic properties of newly-recruited CCR2+ macrophages. To examine these possibilities, we compared the progression of EAO in WT and Ccr2−/− mice as well as in mice overexpressing follistatin to reduce activin bioactivity. Our results demonstrated that CCR2 and activin A, acting through macrophages, are crucial for the development of testicular inflammation and fibrosis in EAO. In short, testicular inflammation and fibrosis were reduced in the absence of Ccr2, as indicated by lower numbers of immune cells expressing ECM proteins (fibronectin, collagen I) and decreased production of collagen, MMPs, as well as inflammatory mediators (indicated by changes in Il-10, Ccl2, and Inhba expression). Furthermore, activin A acted as an inducer of CCR2 and fibrosis-related mediators (fibronectin, CXCR4, MMP2, and PDGF) in macrophages. Inhibition of activin A in vivo by overexpression of FST during EAO decreased testicular fibrosis and expression of collagen I by TMs.

EAO is a well-established mouse model of autoimmune-based male infertility featuring marked testicular deposition of ECM proteins (including collagens and fibronectin), increased numbers of TMs, and elevated levels of inflammatory mediators such as TNF, CCL2, and activin A. These pathological alterations combine to cause tissue destruction, inhibition of spermatogenesis, and subsequent infertility [8, 13, 21, 24]. Overall, our study revealed CCR2 and activin A to be important players in the progression of fibrosis in testicular inflammation. Furthermore, our findings underlined the pro-fibrotic role of monocyte-derived macrophages in the development of testicular fibrosis.

CCR2 is a receptor for CCL2, an inflammatory chemokine that mediates the chemotaxis of leukocytes to injury sites [54]. In this study, the absence of CCR2 during testicular inflammation not only preserved the tissue from the deposition of ECM and production of activin A, IL-10, and CCL2 but also protected the testis from damage by reducing the accumulation of immune cells. This is in accordance with previous reports demonstrating reduced inflammatory and fibrotic responses in different organs in the absence of CCR2 signaling [14, 28, 30, 55,56,57]. Here, we showed that in contrast to WT animals, Ccr2−/− mice exhibited normal levels of Il-1a, Il-10, Ccl2, and Inhba mRNA in the testes following EAO induction, pointing to the essential contribution of CCR2+ cells to the induction of the inflammatory response.

Since the mechanisms leading to the fibrotic response and the origin of ECM proteins during the progression of testicular inflammation were unknown, we wanted to address these questions. ECM-producing cells originate from a variety of sources, including bone marrow-derived circulating cells known as fibrocytes [15, 58]. Fibrocytes are characterized by the co-expression of hematopoietic markers such as CD45, CD34, or CD11b and mesenchymal markers such as collagen or vimentin [59]. These cells mainly migrate to injury sites by chemotaxis via chemokine ligand/receptor pathways, including CCL2/CCR2 or CXCL12/CXCR4 [59, 60]. Fibrocytes can also originate from a subpopulation of monocytes via monocyte-to-fibroblast transition [61,62,63]. Our results indicate that during the severe stage of EAO, immune cells (particularly macrophages) act as important sources of fibronectin and collagen I production. Since the CD34 marker was not included in our analysis, we cannot definitely exclude the possibility that a proportion of the ECM-expressing CD45+ cells may have been fibrocytes. The significant reduction in the accumulation of CD45+ cells and TMs expressing fibronectin, collagen I, and CXCR4 observed in EAO Ccr2−/− testis indicates the importance of CCR2 and CXCR4 signaling in testicular ECM expression. In addition, the very high levels of Cxcl12 and Cxcr4 mRNA detected in WT EAO testis were significantly reduced in the absence of Ccr2, underlining the importance of newly-recruited CCR2+ cells as a source of these molecules during the testicular fibrotic response. Similarly, the expression of fibronectin in TMs under pathological conditions in the human testis (impaired spermatogenesis and collagen accumulation) points to the possible involvement of macrophages in fibrotic remodeling. These data are in agreement with earlier reports demonstrating CCR2-dependent inhibition of BMDM and fibrocyte infiltration in kidney injury, colon fibrosis, and colitis [28, 30, 64,65,66]. Moreover, CXCR4+ macrophages have also been shown to act as a pro-fibrotic cell subpopulation in the kidney and lung [67,68,69]. Future studies are required to establish whether CCR2+CXCR4+ TMs are the key players involved in the EAO fibrotic response. However, currently, we cannot exclude the possibility that the production of Cxcl12 by CCR2+ cells may account for the reduced infiltration of CXCR4+ cells in Ccr2−/− mouse testes.

Our results also showed that expression of Mmp2, Mmp14, Mmp9, and Timp1 was increased in WT EAO testes, indicating activation of the ECM machinery. The gelatinases MMP2 and MMP9 mainly cleave collagen IV, an important component of the basement membrane, making it easier for leukocytes to transmigrate [70, 71]. Activation of MMP2 requires the involvement of MMP14 [72]. Aggregated ECM proteins require degradation by MMPs to prevent fibrosis; on the other hand, TIMP1 inhibits the degradative function of MMP proteins, thereby exacerbating the fibrotic process. However, elevated levels of MMPs could also contribute to immune cell migration or activate PTCs /fibroblasts to produce more ECM proteins, thereby promoting tissue fibrosis [73, 74]. Thus, the process of testicular fibrosis appears to depend on the dynamic balance between MMP and TIMP activities. In this study, loss of Ccr2 abrogated the elevated expression of Mmp2 and Mmp14 occurring in EAO testes. Interestingly, the expression of these genes positively correlated with levels of Adgre1 mRNA, indicating that macrophages are involved in MMP and TIMP1 production and thereby in the promotion of testicular fibrosis at the inflammation stage.

The changes in integrins and PDGF gene expression observed in EAO may also contribute to the fibrotic phenotype, as these groups of molecules are known as important promoters of fibrosis [52, 53, 75, 76]. The increased levels of Itgav/Itgb3 (encoding integrin αvβ3) and Pdgfb/Pdgfrb mRNAs detected during EAO (dependent on Ccr2 expression) were likely due to the infiltration of macrophages, as Itgav and Pdgfrb correlated positively with Adgre1 expression. Furthermore, PDGF acts as a strong chemoattractant for fibrocytes in pulmonary fibrosis, while pharmacological blockade of the PDGF/PDGFR axis is considered a promising treatment option [77].

Taken together, these in vivo data indicate that CCR2 contributes to the development of testicular fibrosis during inflammation by mediating leukocyte infiltration, cytokine release, and ECM protein production by immune cells, particularly TMs. In addition, our results suggest that CCR2 + TMs may produce multiple factors, including the CXCL12/CXCR4 axis, MMPs, αv integrin, PDGFB, and PDGFRβ, which potentially act as downstream signaling molecules to regulate immune processes during testicular inflammation (summarized in Fig. 8).

Fig. 8figure 8

Model of the role of activin A, CCL2/CCR2, and macrophages in fibrotic remodeling during EAO. Increased expression of TNF during testicular inflammation induces SCs to release higher levels of CCL2 and activin A. In response to elevated levels of CCL2, macrophages expressing CCR2 are attracted to the site of injury and infiltrate the inflamed testes. Activin A stimulates CCR2 expression on macrophages and drives them to a pro-fibrotic phenotype by inducing the expression of CXCR4, collagen I, fibronectin, MMPs, integrins and PDGFs. Together, these processes facilitate the development of fibrosis in EAO testes. In EAO mice, inflammatory response and fibrotic remodeling can be inhibited by the knockout of Ccr2, blocking macrophage infiltration, or by the use of FST, an activin A antagonist. (GC dying germ cells, SC Sertoli cells, PTC peritubular cells, Mφ macrophages)

In addition to CCR2, activin A is another important fibrotic regulator [6, 23]. In the current study, activin A stimulated CCR2 expression (at the mRNA and protein level) in macrophages and reduced Ccl2 levels. Similarly opposing effects of activin A on Ccr2 and Ccl2 expression have been demonstrated in human monocyte-derived macrophages [78]. Our findings indicated that activin A can increase the responsiveness of inflammatory BMDMs to CCL2 by enhancing the expression of CCR2. Furthermore, according to our results, activin A also induced the expression of CXCR4 in macrophages. As CXCR4 is highly expressed by macrophages during testicular inflammation, reduced infiltration by monocytes/macrophages in Ccr2−/− mice during EAO may explain why the level of testicular Cxcr4 mRNA did not increase in CCR2-depleted mice following induction of EAO (see Fig. 8).

Previously, we showed that activin A-induced the expression of fibrosis-specific genes in mouse PTCs and NIH 3T3 fibroblasts. Furthermore, expression of the activin dimer subunit was increased, not only in inflamed mouse testes but also in human testes with impaired spermatogenesis and focal leukocytic infiltrates [13, 24]. Notably, the severity of the induced inflammation and damage was directly proportional to the level of activin A [13, 24]. These findings, together with the effects of Ccr2 deficiency during EAO (including reduced infiltration of macrophages, lower levels of ECM deposition and Inhba) implicate activin A as a key regulator of testicular inflammation and fibrosis, acting through TMs. In vivo inhibition of activin A by overexpression of follistatin clearly demonstrated the role of activin A in the development of testicular inflammation; for example, suppression of the accumulation of TM-derived collagen I by follistatin indicated that activin A affected the progression of testicular fibrosis by regulating pro-fibrotic properties of macrophages. Moreover, the expression of Inhba correlated positively with testicular Mmp and Timp1 expression during EAO. Therefore, elevated levels of MMPs and TIMP1 in EAO testes were associated with both macrophage infiltration and activin A expression.

Activin A treatment influenced the transcriptome of BMDMs in vitro and increased the expression of Fn1, Ccr2, Cxcr4, Mmp2, Mmp14, Itgav, Pdgfa, Pdgfb, and Pdgfrb genes as well as the levels of fibronectin, CCR2, CXCR4, and MMP2 proteins. Moreover, activin A enhanced the migratory ability of macrophages in response to CCL2 stimulation. Interestingly, in addition to increased expression of fibrosis-related genes, activin A-induced transformation to spindle-shaped morphology and diminished F4/80 fluorescence in BMDMs, indicating possible conversion to a fibroblastic phenotype in vitro. Similarly, activin A reportedly reprogrammed pre-tumorigenic macrophages to a tumor-associated phenotype, promoted conversion of CD4+CD25− naïve T cells into regulatory T cells, and induced a pro-fibrotic transcriptome in fibroblasts, thereby transforming them into myofibroblasts [79,80,81]. Furthermore, macrophages from the synovium of active rheumatoid arthritis patients, which exhibited pro-inflammatory, macrophage-polarizing properties, were inhibited by the addition of an anti-activin A neutralizing antibody [82].

Combining these results, we postulate that activin A promotes the progression of testicular fibrosis by inducing the production of fibrosis-related mediators in macrophages. Furthermore, although activin A treatment reduced the expression of Mmp9, Timp1, and Itgb3 in BMDMs, other factors, such as TNF, may increase the levels of MMP9 and TIMP1 in macrophages or other testicular cells, thereby promoting their expression in EAO testes [83]. In addition, cells such as fibroblasts can produce large amounts of β3 integrin in response to inflammatory stimuli, which could underlie the high levels of Itgb3 mRNA observed in EAO testes [84].

Critically, it should be noted that utilizing BMDMs matured in the presence of M-CSF as a surrogate for TMs may not exactly replicate all functional aspects of the TMs. However, M-CSF is a critical regulator of the maturation and tissue-specific functions of TMs [85]. As TMs make up a very small proportion of the normal mouse testis cell population, in vitro experimentation on isolated TMs would require an unreasonably large number of mice. In accordance with 3R principles, TMs were replaced by BMDMs, drastically reducing the number of animals required.

Taken together, our in vivo and in vitro results illustrate that activin A promotes the differentiation of macrophages to a pro-fibrotic phenotype through the induction of a variety of inflammatory mediators and receptor molecules (summarized in Fig. 8). Activin A-stimulated BMDMs are potential fibroblast precursors, which likely facilitate tissue fibrosis. In addition, by inducing CCR2 expression, activin A potentially affects the recruitment of monocytes/macrophages to injury sites. Although our data indicate that CCR2, activin A, and macrophages are responsible for the testicular fibrotic response, the precise immune mechanisms that regulate this process require further investigation. Future studies are needed to address the direct influence of activin A using targeted depletion of activin A receptors (in monocytes/macrophages) to analyze their role in inflammatory responses and fibrotic remodeling.

In conclusion, our data indicate that CCR2 and activin A regulate the development of fibrosis during testicular inflammation and underline the crucial pro-fibrotic function of macrophages in inflammation-associated fibrotic remodeling in EAO. Future therapeutic targeting of CCR2 and/or activin A may offer a possible strategy to address testicular fibrosis.

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