RGC32 Promotes Bleomycin-Induced Systemic Sclerosis in a Murine Disease Model by Modulating Classically Activated Macrophage Function

Abstract

Systemic sclerosis (SSc) is a multisystem autoimmune disorder that is characterized by inflammation and fibrosis in the skin and internal organs. Previous studies indicate that inflammatory cells and cytokines play essential roles in the pathogenesis of SSc; however, the mechanisms that underlie the inflammation-driven development of SSc are not fully understood. In this study, we show that response gene to complement 32 (RGC32) is abundantly expressed in mouse macrophages in the early stage of bleomycin-induced SSc. Importantly, RGC32 is required to induce the inflammatory response during the onset of SSc, because RGC32 deficiency in mice significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammatory mediators inducible NO synthase (iNOS) and IL-1β in macrophages. RGC32 appears to be a novel regulator for the differentiation of classically activated macrophages (M1 macrophages). IFN-γ and LPS stimulation induces RGC32 expression in primary peritoneal macrophages and bone marrow-derived macrophages. RGC32 deficiency impairs the polarization of M1 macrophages and attenuates iNOS and IL-1β production. Mechanistically, RGC32 interacts with NF-κB proteins and promotes iNOS and IL-1β expression by binding to their promoters. Collectively, our data reveal that RGC32 promotes the onset of SSc by regulating the inflammatory response of M1 macrophages, and it may serve as a promising therapeutic target for treating SSc.

Figure 1. RGC32 deficiency ameliorated bleomycin-induced skin and lung fibrosis

(A–B) Mice were injected with bleomycin (Bleo, 0.02U) subcutaneously for the times indicated. RGC32 mRNA (A) and protein (B) expression during the early stage of skin fibrosis were detected by qPCR (A) and Western blotting (B), respectively. RGC32 protein levels in B were quantified by normalizing to GAPDH. *P<0.05, **P<0.01 compared to the control (PBS-treated or day 0, n=6). (C) RGC32 deletion (RGC32−/−) blocked bleomycin-induced skin fibrosis as compared to wild type mice (WT). Skin sections were collected 28 days after bleomycin injection. The skin thickness was measured by ultrasonic inspection. (D) Quantification of the skin thickness in bleomycin-treated WT and RGC32−/− mice compared to PBS control. **P < 0.01 (n=6). (E) RGC32−/− inhibited bleomycin-induced collagen deposition in skin as shown by Masson's trichrome staining. H&E staining showed skin structure. (F) RGC32−/− blocked bleomycin-induced Collagen I (COL1A1) expression in skin tissues as determined by Western blotting. COL1A1 protein levels were quantified by normalizing to GAPDH. *P < 0.05, n=6. (G–H) RGC32−/− blocked bleomycin-induced lung fibrosis. Lung fibrosis was induced in WT and RGC32−/− mice by bleomycin injection (0.2U) for 24 days. RGC32−/− inhibited bleomycin-induced collagen deposition in lung as shown by Masson's trichrome staining. H&E staining showed the lung structure (G). (H) RGC32−/− blocked bleomycin-induced COL1A1 expression in lung tissues as determined by Western blotting. COL1A1 protein levels were quantified by normalizing to GAPDH. **P < 0.01, n=6.

Figure 2. Macrophage RGC32 modulated the inflammation in bleomycin-induced skin sclerosis

Mice were injected with bleomycin (Bleo, 0.02U) subcutaneously for 0–14 days (A) or one day (B–G). (A) F4/80 mRNA expression was induced during the initial stage of skin fibrosis as detected by qPCR. *P<0.05, ***P<0.001 compared to PBS-treated skin tissues at the corresponding time points (n=6). (B) RGC32 was induced in F4/80-positive macrophages in the bleomycin-treated skin. Frozen sections of skin tissues were co-immunostained with F4/80 and RGC32 antibodies. (C–D) RGC32 deficiency (RGC32−/−) inhibited bleomycin-induced accumulation of F4/80+ macrophages in skin tissues. The percentage of F4/80+ macrophages was measured by flow cytometry. Shown are representative FACS analyses (C) and the percentage of F4/80+ macrophages (D) in PBS- or bleomycin-treated skins of wild type (WT) and RGC32−/− mice, *P<0.05, n=6. (E) RGC32−/− attenuated bleomycin-induced iNOS and IL-1β protein expression in mouse skins, as detected by Western blot. The protein levels of iNOS and proIL-1β were quantified by normalizing to GAPDH. *P<0.05, **P<0.01, n=6. (F–G) RGC32−/− attenuated bleomycin-induced iNOS and IL-1β expression in macrophages as co-immunostained with F4/80 and iNOS antibodies (F), or F4/80 and IL-1β antibodies in skin frozen sections (G). (H) RGC32−/− attenuated bleomycin-induced arginase expression in macrophages. Frozen skin sections were co-immunostained with F4/80 and arginase antibodies. (I) RGC32−/− attenuated bleomycin-induced arginase protein expression in mouse skins, as detected by Western blot. The protein levels of arginase were quantified by normalizing to GAPDH. *P < 0.05, n=6.

Figure 3. Macrophage RGC32 is required for skin and lung sclerosis development

(A–B) Full chimeric mice were generated by transplanting either WT or RGC32−/− BMCs to lethally irradiated WT mice. 8 weeks after the transplantation, recipient mice were treated with PBS or 0.02U bleomycin for 28 days to induce skin fibrosis. (A) Recipient mice receiving RGC32−/− BMCs exhibited a significant reduction in bleomycin-induced collagen deposition in skins compared to mice receiving WT BMCs as shown by Masson's trichrome staining. H&E staining indicated the skin structure. (B) Recipient mice received RGC32−/− BMCs showed a significant decrease in bleomycin-induced collagen I (COL1A1) expression in skin tissues as determined by Western blotting. COL1A1 protein levels were quantified by normalizing to GAPDH. **P<0.01, n=3. (C) The expression of iNOS and IL-1β was decreased in bleomycin-treated recipient mice receiving RGC32−/− BMCs compared to those receiving WT BMCs. The skin tissues were collected 1 day after bleomycin injection. The iNOS and proIL-1β protein expression were detected by Western blot and quantified by normalizing to GAPDH. *P < 0.05, n=6. (D–E) Full chimeric mice were generated similarly as described in A–B. 8 weeks after the transplantation, recipient mice were treated with PBS or 0.2U bleomycin for 24 days to induce lung fibrosis. (D) RGC32 deficient BMCs attenuated bleomycin-induced lung fibrosis as shown by the improved structure (H&E staining) and the reduced collagen deposition (Masson's trichrome staining). (E) COL1A11 protein expression in bleomycin-treated lung tissues was determined by Western blot. COL1A1 levels were quantified by normalizing to GAPDH. *P<0.05, n=6.

Figure 4. RGC32 mediated macrophage classical activation

(A–B) RGC32 was induced with IFNγ (100 ng/mL) and LPS (100 ng/mL) (I+L) in cultured PEMs and BMDMs. RGC32 protein levels in I+L-treated PEMs (A) and BMDMs (B) were measured by Western blot and quantified by normalizing to GAPDH, respectively. **P < 0.01, ***P < 0.001 compared with vehicle-treated cells (Ctrl), n=4. (C–F) RGC32 deficiency (RGC32−/−) inhibited I+L-induced iNOS and IL-1β production. PEMs (C–D) or BMDMs (E–F) isolated from WT or RGC32−/− mice were treated with vehicle (Ctrl) or I+L (100 ng/mL each) to induce macrophage classical activation. The expression of iNOS and IL-1β were determined by Western blot and quantified by normalizing to GAPDH, respectively. *P < 0.05, **P < 0.01, n=5.

Figure 5. RGC32 was essential for the activation of NF-κB pathway in classically activated macrophages

(A–B) RGC32 deficiency (RGC32−/−) blocked of IκB (pIκB) and NF-κB (pNF-κB) phosphorylation in skin tissues. WT and RGC32−/− mice were injected with bleomycin (Bleo, 0.02U) subcutaneously. Skin tissues were collected 1 day after the bleomycin treatment. The expression of pIκB, IκB, pNF-κB and NF-κB was measured by Western blotting (A) and quantified by normalizing to GAPDH (B). *P<0.05, **P<0.01, n=6. (C–F) RGC32−/− significantly inhibited IFNγ+LPS (I+L)-induced increase in pIκB and pNF-κB levels in macrophages. PEMs (C, D) or BMDMs (E, F) isolated from WT and RGC32−/− mice were treated with 100 ng/mL IFNγ and 100 ng/mL LPS for 30 min. IκB, NF-κB, pIκB, and pNF-κB levels in PEMs (C) and BMDMs (E) were detected by Western blot and quantified by normalized to GAPDH (D–F), respectively. *P<0.05, **P<0.01, n=6. (G–I) RGC32−/− blocked I+I-induced NF-κB nuclear translocation. BMDMs isolated from WT and RGC32−/− mice were treated with 100 ng/mL IFNγ and 100 ng/mL LPS for 30 min. Cytoplasmic and nuclear protein fractions were prepared to detect the NF-κB distribution by Western blot (G). Cytoplasmic NF-κB level was quantified by normalizing to GAPDH (H), and nuclear NF-κB level was quantified by normalizing to Lamin B (I). *P < 0.05, n=4.

Figure 6. RGC32 interacted with NF-κB and bound to inflammatory gene promoters in classically activated macrophages

(A–D) NF-κB co-immunoprecipitated with RGC32 in classically activated macrophages. BMDMs from WT mice were stimulated with 100 ng/mL IFNγ and 100 ng/mL LPS for 6 h. Normal IgG isotype or antibodies against RGC32 (A) or NF-κB (C) were used for immunoprecipitation (IP). NF-κB (A) or RGC32 antibody (C) was used for immunoblotting (IB). (B, D) Quantification of NF-κB and RGC32 levels shown in (A) and (C) by normalizing to GAPDH, respectively. (E, F) RGC32 and NF-κB bound to the same regions of iNOS or IL-1β promoters. BMDMs were treated with 100 ng/mL IFNγ and 100 ng/mL LPS for 30 min followed by chromatin immunoprecipitation assay (ChIP) using control IgG, NF-κB, or RGC32 antibodies. PCR was performed to detect the RGC32 and NF-κB binding regions in iNOS (E) and IL-1β (F) promoters. (G) Blockade of NF-κB activation diminished RGC32 binding to the iNOS and IL-1β promoters in BMDMs. BMDMs were treated with 200 µM ammonium pyrrolidine dithiocarbamate (PDTC) for 1 hour, followed by treatment with 100 ng/mL IFNγ and 100 ng/mL LPS for 30 min. RGC32 antibodies were used for ChIP. PCR was performed to detect RGC32 binding to NF-κB binding regions in iNOS and IL-1β promoters. (H) Blockade of NF-κB activation inhibited RGC32-enhanced iNOS and IL-1β expression in RAW264.7 cells. RAW264.7 were transfected with control (−) or RGC32 expression plasmid. 24 h later, cells were treated with 200 µM PDTC for 1 hour followed by induction with 100 ng/mL IFNγ and 100 ng/mL LPS for 6 h. The iNOS and IL-1β protein expression were detected by Western blot and quantified by normalizing to GAPDH. *P < 0.05, **P < 0.01, n=4 (for panel B, D and H).