Systemic sclerosis (SSc) is a systemic connective tissue disease characterized by organ fibrosis, vascular abnormalities, and autoimmunity (). Although its etiology remains unclear, recent studies have implicated innate immunity, as well as monocyte and macrophage activation in SSc pathogenesis. Circulating monocytes are multifunctional precursors that play important roles in both immune and inflammatory responses, as well as tissue remodeling. Bone marrow-derived CD14+ monocytes form a population of monocytes with heterogeneous surface markers, phagocytic capacity, and differentiation potential at the migration site. In addition, CD14+ monocytes can differentiate into macrophages, dendritic cells, collagen-producing fibrocytes (
Systems Level Analysis of Systemic Sclerosis Shows a Network of Immune and Profibrotic Pathways Connected with Genetic Polymorphisms.
identified a gene co-expression network involved in macrophage activation, adaptive immunity, interferon response, extracellular matrix (ECM) deposition, TGFβ signaling, and cell proliferation in the skin of SSc patients (
Nintedanib inhibits macrophage activation and ameliorates vascular and fibrotic manifestations in the Fra2 mouse model of systemic sclerosis.
), suggesting that the therapeutic inhibition of macrophage activation is associated with clinical benefits in SSc patients. However, the key regulators of aberrant monocyte and macrophage activation remain unclear.
Interferon regulatory factor 8 (IRF8) is a 50 kDa transcription factor that was originally identified as a negative regulator of type I interferon (
Inhibition of IRF8 Negatively Regulates Macrophage Function and Impairs Cutaneous Wound Healing.
). Since IRF8 may contribute to SSc progression, including fibrosis, vascular abnormalities, and impaired wound healing, we evaluated IRF8 levels in SSc monocytes and investigated the effects of IRF8 regulation on the pathophysiological roles of monocytes and macrophages in SSc fibrosis.
IRF8 is downregulated in peripheral blood mononuclear cells (PBMCs) of patients with diffuse cutaneous SSc (dcSSc)
First, we evaluated IRF8 mRNA levels in the PBMCs of patients with SSc by real-time quantitative PCR (RT-qPCR). No significant differences were observed between IRF8 mRNA expression in PBMCs from patients with SSc and healthy subjects (Figure 1a). However, IRF8 mRNA expression was significantly lower in patients with dcSSc than in those with limited cutaneous SSc (lcSSc) and in healthy subjects (P = 0.0349 and 0.0041, respectively). Moreover, SSc patients with anti-topoisomerase I (Topo1) or anti-RNA polymerase III (RNAPIII) antibodies, which are more likely to be present in dcSSc patients, had lower IRF8 levels than those with anticentromere antibodies (Figure 1b).
IRF8 is downregulated in circulating monocytes and corelates negatively with the modified Rodnan total skin thickness score (mRSS)
To evaluate which type of PBMCs was important for IRF8 downregulation, we purified circulating CD14+ monocytes from PBMCs by depleting magnetically-labeled cells and determined IRF8 mRNA and protein levels by RT-qPCR and immunoblotting, respectively. IRF8 mRNA expression was significantly lower in monocytes of patients with dcSSc than in those of patients with lcSSc and in healthy subjects (Figure 1c; P = 0.0259 and 0.0193, respectively). Similarly, IRF8 protein levels were significantly lower in monocytes of patients with dcSSc than in those of the healthy subjects (Figure 1d; P = 0.0401). Importantly, IRF8 mRNA expression in circulating monocytes was significantly and negatively correlated with mRSS (Figure 1e; r = -0.45, P = 0.020) but not with clinical characteristics such as patient age, disease duration, or lung function test results (% predicted forced vital capacity (%FVC) and lung carbon monoxide diffusing capacity (%DLco)) in patients with interstitial lung disease (ILD; Supplementary Figure S1a). There was a trend toward lower IRF8 expression in SSc patients with ILD compared to patients without, although the reduction was not significant (P = 0.061, Supplementary Figure S1b). These results suggest that IRF8 levels in monocytes may have a strong impact on skin sclerosis.
IRF8-silenced monocytes exhibit a pro-fibrotic M2 phenotype
Since IRF8 expression was reduced in circulating monocytes of dcSSc patients, we sought to evaluate the phenotype of monocytes due to IRF8 downregulation. To achieve this, IRF8 expression in healthy human circulating monocytes was silenced through RNA interference and the expression levels of pro-fibrotic and inflammatory cytokines were determined by RT-qPCR and immunoblotting. The results confirmed that IRF8 was successfully knocked down at the mRNA (Figure 2a) and protein (Figure 2b) levels, respectively. Compared to control monocytes, IRF8-silenced monocytes displayed significantly higher levels of MCP1 and pro-fibrotic and inflammatory cytokines, such as TNF-α, TGF-β, and IL-6 (Figure 2c). Furthermore, IRF8 downregulation significantly increased the mRNA expression of IRF4, Kruppel-like factor 4 (KLF4), and CCAAT/enhancer binding protein β (C/EBPβ) (Figure 2d), which are important transcriptional factors that regulates M2 macrophage polarization.
IRF8-silenced monocyte-derived macrophages (MDMs) display M2 differentiation and enhanced levels of pro-fibrotic factors
Next, we evaluated whether IRF8-silencing regulates M1/M2 macrophage differentiation in monocytes (siIRF8MDM1 or siIRF8MDM2) in vitro by analyzing M1/M2 cell surface markers by flow cytometry (Figure 3a). Induction of M1/M2 markers and cytokine expression were further confirmed in Supplementary Figure S3. The percentage of CD163+CD68+ cells (M2 macrophage surface markers) was significantly higher in siIRF8MDM2 cells than in M2 macrophages derived from control monocytes (Figure 3b). In addition, the mean fluorescence intensity (MFI) of CD163 was upregulated in siIRF8MDM2 cells (Figure 3c). Conversely, the percentage of CD80+CD68+ cells, (M1 macrophage surface markers) was significantly lower in siIRF8MDM1 cells than in M1 macrophages derived from control monocytes (Figure 3b), consistent with the low MFI of siIRF8MDMI cells (Figure 3c). Together, these findings indicated predominant M2 polarization in IRF8-silenced monocytes.
We also analyzed the gene expression levels of SSc-related cytokines, chemokines, and ECM components by RT-qPCR in these macrophages. TNF-α, TGF-β, IL-6, MCP-1, VEGF, alpha smooth muscle actin (αSMA), and early growth response protein 1 (EGR1) expression levels were significantly higher in siIRF8MDM1 cells than in control M1 macrophages (Figure 3d). In contrast, siIRF8MDM2 cells displayed higher MCP-1, VEGF, αSMA, EGR1, and collagen type I alpha 1 chain (COL1A1) levels (Figure 3e). Bead-based immunoassays revealed that TNF-α, TGF-β, and MCP-1 protein levels were significantly upregulated in siIRF8MDM1 cells compared with control M1 macrophages (Figure 3f). A similar, however, non-significant, trend was observed in siIRF8MDM2 (Figure 3g). Altogether, these results suggested that IRF8-silenced MDMs induce the recruitment of chemokines, inflammatory cytokines, and pro-fibrotic factors, which may contribute to SSc fibrosis.
IRF8-silenced MDMs affect fibrotic function in fibroblasts
To determine whether macrophages affected fibrotic function in fibroblasts, we co-cultured primary human fibroblasts with either IRF8-silenced MDMs (siIRF8MDM1 or siIRF8MDM2) or control macrophages (M1 or M2), without cell-cell contact. Interestingly, RT-qPCR revealed that the mRNA expression of Sp1, an important transcriptional factor in fibrosis, was significantly higher in fibroblasts co-cultured with siIRF8MDM1 and siIRF8MDM2 cells than in the control macrophages. Moreover, EGR1 levels tended to be higher in siIRF8MDM1 and siIRF8MDM2 cells, although this relationship was not statistically significant (Figure 3h).
Our results showed that IRF8 expression was diminished in circulating monocytes of patients with dcSSc, and IRF8-silencing induced a pro-fibrotic phenotype in MDMs. Therefore, we analyzed the effect of IRF8-silencing in monocytes on fibrosis in vivo by generating transgenic mice with diminished IRF8 expression in their myeloid cells (IRF8flox/flox;Lyz2Cre/+ (IRF8cKO mice)). Figure 4 shows the back skin phenotype of the mice at 12 weeks. Although no differences were observed in dermal thickness (Figure 4a and b) or total collagen content (Figure 4c), COL1A1 and COL1A2 mRNA expression levels were significantly higher in IRF8cKO mice than in the wild type mice (Figure 4d). The levels of matrix metalloproteinase (MMP) 9, which cause matrix degradation, were significantly upregulated in the IRF8cKO mice skin (Figure 4d), suggesting that a balance between matrix production and degradation may affect the skin phenotype. These findings indicated that IRF8 downregulation in monocytes may induce pro-fibrotic conditions that are insufficient to induce skin fibrosis in vivo. Therefore, we induced skin fibrosis in the mice via intradermal BLM injection. Histological analysis (Figure 5a and b) and the hydroxyproline assay (Figure 5c) revealed significantly increased dermal thickness and hydroxyproline content in BLM-treated mice compared to PBS-treated controls, and more aggravated fibrosis in BLM-treated IRF8cKO mice. Moreover, the back skin of IRF8cKO mice displayed significantly increased expression of IL-6 and αSMA (Figure 5d). Finally, BLM-treated IRF8cKO mice displayed significantly more infiltrating macrophages than BLM-treated control mice (Figure 5e and f). BLM-treated IRF8cKO mice skin exhibited significantly increased CD163 levels and decreased CD86 levels, confirming M2 dominance in BLM-treated IRF8cKO mice (Figure 5g).
In this study, we demonstrated that IRF8 expression is downregulated in monocytes of dcSSc patients and is significantly negatively correlated with mRSS, indicating that IRF8 plays a key role in the effect of monocytes in skin fibrosis. Moreover, we showed that IRF8-silenced monocytes predominantly differentiated into pro-fibrotic macrophages with increased levels of pro-fibrotic cytokines and chemokines. Furthermore, IRF8cKO mice with BLM-induced skin fibrosis displayed increased dermal thickness and dermal fibrosis with increased hydroxyproline content and pro-fibrotic gene expression. To our knowledge, this is the first study to reveal that aberrant expression of the transcriptional factor, IRF8, in monocytes directly aggravates fibrosis by inducing macrophage abnormalities in SSc skin.
Our results further showed that IRF8 was downregulated in monocytes of dcSSc patients, while IRF8-silencing in monocytes induced their differentiation predominantly into M2 macrophages. These findings were consistent with those of a recent study that revealed that IRF8 knockdown reduced M1-specific gene expression and produced an M2-dominant environment in wound sites (
Inhibition of IRF8 Negatively Regulates Macrophage Function and Impairs Cutaneous Wound Healing.
). Furthermore, IRF8 downregulation due to hypermethylation has been associated with increased IL-6 production in monocyte-derived dendritic cells (DCs), while IRF8 demethylation has been reported to reverse this effect and suppress Th1 responses (
Ocular Behcet’s disease is associated with aberrant methylation of interferon regulatory factor 8 (IRF8) in monocyte-derived dendritic cells.
). These results support our finding that IRF8 downregulation in monocytes may cause a shift to a Th2/M2 phenotype, which is important for establishing SSc symptoms. Our findings also revealed that IRF8-silenced monocytes displayed pro-fibrotic features and increased pro-fibrotic cytokine production in vitro. Indeed, it has been reported that IRF8 is required for M1 polarization induced by Notch-RBP-J signaling (
Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization.
), which leads to an M2 shift by downregulating IRF8. Interestingly, we found that IRF8-silenced monocytes displayed significantly upregulated levels of C/EBPβ, an important transcriptional factor for M2-specific gene expression (
Identification of an atypical monocyte and committed progenitor involved in fibrosis.
). Thus, we postulate that IRF8-silenced monocytes may share pro-fibrotic characteristics with SatMs.
Our analysis of cell surface markers showed that IRF8 downregulation in vitro caused a predominantly M2 differentiation. However, further analysis revealed mixed M1 and M2 phenotypes. In IRF8-silenced M1 MDMs, not only were the levels of M1 cytokines, such as TNF-α and IL-6, elevated but also those of M2-like pro-fibrotic factors, including MCP-1, EGR1, TGF-β, and αSMA. Although other conditions may affect this phenotype in vivo, it remains unclear how these cells are relevant for maintaining fibrosis at wound sites. Our observations of TGF-β-producing M2-like M1 macrophages agree with those of a recent report which revealed an increased number of macrophages with mixed M1/M2 features in patients with SSc (
Augmented production of chemokines (monocyte chemotactic protein-1 (MCP- 1), macrophage inflammatory protein-1α (MIP-1α) and MIP-1β) in patients with systemic sclerosis: MCP-1 and MIP-1α may be involved in the development of pulmonary fibrosis.
). Moreover, MCP-1 not only acts as a chemoattractant for monocytes, but also promotes collagen expression in fibroblasts via the TGF-β signaling pathway (
Control of T(H) 2 polarization by the chemokine monocyte chemoattractant protein-1.
). Thus, monocytes and macrophages with reduced IRF8 expression may be a source of increased MCP-1 levels in SSc and promote monocyte recruitment to the skin and collagen production by fibroblasts. Consistent with this notion, we observed significantly more skin infiltrating macrophages in BLM-induced IRF8cKO mice than in wild type (WT) mice and significantly higher COL1A1 and αSMA expression in IRF8-silenced macrophages. Although the expression of these genes may directly contribute toward skin fibrosis, they can also act as a marker of macrophage to myofibroblast transition (MMT). Several recent studies have reported MMT in renal fibrosis, from bone marrow-derived M2 macrophages (
TGF-β/Smad3 signalling regulates the transition of bone marrow-derived macrophages into myofibroblasts during tissue fibrosis.
), consistent with our M2 IRF8-silenced MDMs results. Thus, IRF8-silenced monocytes may differentiate into myofibroblasts via M2 macrophages.
Our non-contact co-culture experiments revealed Sp1 gene upregulation in human skin fibroblasts co-cultured with IRF8-silenced MDMs, indicating that these macrophages may activate fibroblasts by producing pro-fibrotic factors. However, a recent report indicated that direct contact between migrated macrophages and myofibroblasts via Cadherin-11 is important for fibrosis by establishing a niche with active TGF-β (
Cadherin-11-mediated adhesion of macrophages to myofibroblasts establishes a profibrotic niche of active TGF-β.
). Whether macrophages directly affect fibroblasts was not resolved in this study, and the role of direct contact between IRF8-silenced MDMs and fibroblasts in fibrosis is a subject of future studies.
To evaluate the role of IRF8-silencing in monocytes and macrophages in vivo, we generated myeloid cell-specific IRF8cKO mice (IRF8flox/flox; Lyz2Cre/+). Although COL1A1 and COL1A2 mRNA levels were significantly higher in the untreated skin of these mice at 12 weeks, dermal thickness was unchanged, suggesting that the deletion of IRF8 was insufficient to induce fibrosis in vivo. Nonetheless, the role of IRF8 downregulation as a key driver of fibrosis cannot be ruled out. Accordingly, since genetic abnormalities cannot completely explain SSc susceptibility, epigenetic mechanisms that link genetics and environmental triggers may be involved. BLM-treated IRF8cKO mice exhibited higher CD163, IL-6, and αSMA expression levels, dermal thickness, and hydroxyproline content than WT mice, suggesting that other factors such as environmental triggers may play critical roles in IRF8-associated fibrosis.
One of the limitations of our study is that the in vitro macrophage model might not fully reflect the differentiation phenotype and function of macrophages in vivo. In addition, it is difficult to precisely determine if the infiltrating macrophages in the skin of patients with SSc and IRF8cKO mice are derived from circulating monocytes. However, dermal macrophages have been reported to be primarily generated from bone marrow monocytes (
Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.
); therefore, a large proportion of macrophages infiltrating the skin of SSc patients and IRF8cKO mice likely originate from circulating monocytes. Moreover, IRF8-silenced human MDMs and MDMs derived from IRFcKO mice may differ phenotypically. Since IRF8cKO mice-derived MDMs exhibited similar, not identical, surface markers and cytokine expression patterns as those of IRF8-silenced human MDMs (Supplementary Figure S4d), their roles in vivo might also be dissimilar. Finally, the patients enrolled in this study had diverse backgrounds regarding systemic treatment and disease duration, which may have affected IRF8 expression levels in PBMCs and monocytes. Although IRF8 levels were not significantly different between patients with or without treatment and displayed no significant correlation with disease duration, sequential samples from the same patients should be investigated to confirm these effects.
The findings of this study highlight the important role of IRF8 in monocytes and macrophages during SSc fibrosis and suggest the modulation of IRF8 expression as a novel therapeutic target. A recent report revealed several enhancer regions essential for IRF8 expression, one of which is a ∼50 kb IRF8 enhancer that specifically regulates IRF8 expression in murine monocytes and macrophages, and whose deletion resulted in decreased IRF8 protein expression in these cells (
Cryptic activation of an Irf8 enhancer governs cDC1 fate specification.
). We speculate that there might be abnormalities in this enhancer in monocytes of SSc patients. Moreover, IRF8 gene promoter hypermethylation has been observed in some autoimmune diseases and 5-Aza-2’-deoxycytidine demethylation has demonstrated beneficial effects (