Baricitinib attenuates pulmonary fibrosis in scleroderma mice by regulating macrophage polarization

Abstract

Objective To determine whether baricitinib regulates macrophage polarization through Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway to reduce the severity of pulmonary fibrosis in mice with scleroderma. Methods Thirty 8-week-old female C57BL/6 mice were randomly divided into control group (Ctl, n=6), scleroderma group (SSc, n=12) and scleroderma+baricitinib group (SSc+Ba, n=12). The mice of the SSc and SSc+Ba group were inflicted with a subcutaneous injection of 7.5 mg/kg bleomycin in the back, and an intragastric gavage of 5 mg/kg baricitinib. ELISA was used to detect the plasma levels of inflammatory factors, IL-1β, IL-4, IL-6 and IL-10. HE staining and Masson staining were employed to determine the thickness of skin dermis and pathological changes and collagen content in lung tissue. The mRNA expression of IL-1β, IL-4, IL-6 and IL-10 in lung tissue was measured by RT-PCR, and the protein levels of macrophage markers CD86, CD163 and JAK2/STAT3 in lung tissue were detected by Western blotting. Afterwards, flow cytometry was applied for the expression of F4/80, CD86 and CD163 to determine the polarization of macrophages. Immunofluorescence assay was used to detect CD86 and CD163 to clarify the polarization of macrophages in lung tissue. Results Compared with the Ctl group, the dermis at the injection site in the back was significantly thickened (P < 0.001), the score of pulmonary fibrosis was obviously increased (P < 0.001), and the collagen content in skin and lung tissue was notably elevated in the SSc group (P < 0.001). The above changes were alleviated to varying degrees in the SSc+Ba group (P < 0.01). The SSc group had significantly higher plasma levels of IL-1β (P < 0.05) and IL-6 (P < 0.001) as well as elevated their mRNA levels in lung tissue (P < 0.05) than the Ctl group. Baritinib treatment decreased the plasma concentrations of the above factors (P < 0.01) and their mRNA levels in lung tissue (P < 0.01). JAK2/STAT3 was highly expressed in the SSc group (P < 0.01), and baricitinib reduced the activation of JAK2/STAT3 pathway (P < 0.05). Western blotting and immunofluorescence assay indicated that CD86 protein (marker of M1 macrophages) was highly expressed (P < 0.01) and CD163 protein (marker of M2 macrophages) was in low expression (P < 0.05) in lung tissue in the SSc group than the Ctl group. In the SSc+Ba group, CD86 protein was reduced (P < 0.05) and CD163 protein was increased (P < 0.05) in lung tissue. Flow cytometry showed that the proportion of F4/80+CD86+CD163- M1 macrophages in the spleen was increased (P < 0.001), and the proportion of F4/80+CD86- CD163+ M2 macrophages was decreased in the SSc group when compared with Ctl group (P < 0.01). In the SSc+Ba group, the proportion of M1 was decreased (P < 0.01), and that of M2 was increased (P < 0.001). Conclusion Baricitinib inhibits M1 macrophage polarization through JAK2/STAT3 signaling pathway, thus reducing the fibrosis of lung tissue in SSc mice.

Keywords

• scleroderma
• interstitial lung disease
• baricitinib
• jak2/stat3
• macrophages