The Fibrosis and Immunological Features of Hypochlorous Acid Induced Mouse Model of Systemic Sclerosis

Abstract

Fibrotic animal models are critical for the pathogenesis investigations and drug explorations in systemic sclerosis (SSc). The bleomycin (BLM)-induced mouse model is the classical and most widely used fibrosis model. However, traditional subcutaneous injection of BLM rarely induced diffuse skin and lung lesions. Hypochlorous acid (HOCl)-induced mice are a more representative model that have diffuse cutaneous lesions, lung fibrosis and renal involvement. However, the fibrotic and immunological features of this model are not fully elucidated. Here, we injected BALB/c mice subcutaneously with HOCl used at different concentrations of HOCl (1:55, 1:70, and 1:110 NaClO: KH2PO4, hereafter named HOCl55, HOCl70, and HOCl110, respectively) for 6 weeks to induce fibrosis, and also used HOCl110 at different time course (4, 5, and 6 weeks). Morphological changes were observed via HE and Masson's trichrome staining. Immunohistochemistry or real-time PCR was used to detect inflammatory infiltrates, important fibrosis pathways and pro-inflammatory mediator expression. Flow cytometry was used to detect the alteration of immune cells in mouse spleen. Skin and lung fibrosis were most obvious in the HOCl55 group compared to lower concentration groups. In the HOCl110 group, dominant inflammatory infiltrates were found after 5 weeks, and significant fibrosis was found after 6 weeks. Then we explored the fibrosis and immunological profiles in the HOCl110 (6 weeks) group. Important fibrosis pathway proteins such as TGF-β, NF-κB, Smad3, p-Smad3, STAT3, and p-STAT3 were significantly elevated at week 6 in the HOCl110 group. Increased infiltration of CD4+T cells, CD8+T cells, CD20+B cells, and myofibroblasts was found both in skin and lung tissues. However, decreased CD4+T cells, CD8+T cells, monocytes and macrophages and increased CD19+B cells were found in the spleen tissues. The mRNA expression of fibrosis mediators such as IL-1β, IL-6, IL-17, IL-33, TNF-α, and CTGF was also upregulated in skin and lung tissues. In conclusion, HOCl induced fibrosis mouse model displayed systemic immune cell infiltration, pro-inflammatory mediator release, vasculopathy and fibrosis, which better mimicked human SSc than BLM-induced mice.

Keywords: HOCl-induced mice; SSc; fibrosis; immune cell infiltration; pro-inflammatory mediators.

Figure 1

HOCl-induced dermal inflammation and fibrosis in a dose-dependent manner. Different dilution of HOCl (HOCl 55, HOCl 70, HOCl 110), BLM or PBS was subcutaneously injected in BALB/c mice daily for 6 weeks. (A) Skin swollenness, thickening, subcutaneous fat loss were found in the skin of HOCl and BLM group by HE and Masson staining. Lung structure was disordered, in which abundant inflammatory cells infiltrated, collagen bundles deposited in alveolar or blood vessels, and microvessels reduced in the lung of HOCl group. (B) Significant increase in dermal thickness was observed in HOCl and BLM group compared with those injected with PBS. (C,D) Skin and lung hydroxyproline content was increased in HOCl and BLM group (^*P < 0.05).

Figure 2

HOCl-induced dermal inflammation and fibrosis in a time-dependent manner. Mice were subcutaneously injected HOCl 110 for 4, 5, or 6 weeks. (A) Significant increase of inflammatory cell infiltration was found at 4 weeks and reached the peak at 5 weeks. Collagen deposition was found at 4 weeks and became obvious at 6 weeks. (B) An increasing dermal thickness was observed with time increasing. (C,D) The hydroxyproline content was also increased in the skin and lung tissues after 4 weeks (^*P < 0.05).

Figure 3

The expression of important fibrosis pathways mediators in the skin and lung tissues of HOCl induced mice. (A) TGF-β, NF-κB, Smad3, phospho-Smad3, STAT3, and phospho-STAT3 were strongly increased in the skin of HOCl and BLM groups, as well as the lung tissues of HOCl group. (B,C)The statistical analysis of the protein expression of important fibrosis mediators in skin and lung tissues (^*P < 0.05, MOD: mean optical intensity).

Figure 4

Inflammatory infiltrates in the skin and lung tissues of HOCl-induced mice. T cell (CD4+/CD8+), B cell (CD19+) and myofibroblast (SMA+/Vimentin+) were detected by immunohistochemical analysis. (A) The expression intensity and range of CD4 and CD8 was increased in the skin of the HOCl group and BLM group. High levels of SMA and Vim were also found in the two groups. (B,C) The MOD in HOCl and BLM group was significantly higher than PBS group. (^*P < 0.05, MOD: mean optical intensity).

Figure 5

The alteration of immune cell in the spleen and pro-inflammatory mediators in the skin and lung tissues of HOCl-induced mice. (A) The percentage of CD4+ T cells, CD8+ T cells, CD19+ B cells, macrophages, monocytes and neutrophils in the spleen of HOCl-induced mice. (B) The levels of IL-1β, IL-6, IL-17, IL-33, TNF-α, CTGF, and COL1A2 mRNA in the skin and (C) lung tissues. All experiments were repeated at least three times. Data are presented as 2 ^(−ΔΔCT) relative to the levels of GAPDH. ^* indicates P < 0.05 vs. PBS group.