Regulatory Effects of IL-17A Monoclonal Antibody and Fibronectin on Chronic Graft-Versus-Host Disease

Objectives: To investigate the role of IL-17A monoclonal antibody and fibronectin (FN) in chronic graft-versus-host disease (cGVHD), the therapeutic effect of exogenous FN peptides(FN29andFN36, block IL17A/IL17RA interaction) in cGVHD and the mechanism how FN peptides alleviate cGVHD, so as to provide new ideas and theoretical basis for the treatment of cGVHD.

Methods: FN KO mice were identified via extracting DNA, PCR and gel electrophoresis. Mice plasma FN level was detected by ELISA to verify the knockout (KO) efficiency induced by tamoxifen. After TBI conditioning, recipients were injected with a certain dose of bone marrow cells and spleen cells from donors, then acute GVHD (aGVHD) and cGVHD murine models were established, including BALB/c to C57BL/6 and DBA/2 to BALB/c cGVHD murine models. The clinical manifestations of GVHD were observed and scored. HE staining and masson's trichrome staining were used to score pathological GVHD. GVHD severity between FN KO mice and FN loxP+/+, Cre- mice were compared to investigate the effect of FN on GVHD.

Different cGVHD models, including BALB/c to C57BL/6, and DBA/2 to BALB/c GVHD models were established. Then exogenous FN peptides were injected into recipients via tail vein injection and intraperitoneal injection to rise FN level in vivo. The GVHD severity of mice receiving FN peptides was compared with that receiving PBS, so as to investigate the effect of FN on GVHD and the therapeutic effect of FN peptides on GVHD.In a mouse model of NCG infused with human peripheral blood mononuclear cells, the degree of GVHD in the group receiving IL-17A monoclonal antibody was compared with the control group receiving NS by intraperitoneal injection of IL-17A monoclonal antibody.

Finally, flow cytometry and immunofluorescence were used to investigate the mechanism how FN peptides and IL-17A monoclonal antibody regulate cGVHD. Using flow cytometry analysis, the proportion of B cells, F4/80+ macrophages in spleen were analyzed. Multiplex cytometric bead array was used to detect IL-6, IL-10, MCP-1, TNF, IFN levels. By immunofluorescence staining technique, infiltration of F4/80+ macrophages, infiltration of F4/80+CD206+ M2 macrophages and antibody deposition in recipients' target tissues, including the skin, liver, lung, kidney were detected.

Results: Recipients with cGVHD showed clinical manifestations, including weight loss, hair loss, hunch back and skin scleroderma. Inflammatory cells infiltration, tissue damage and fibrosis could be detected pathologically.

Compared with the control group, down-regulation of FN level with FN KO mice had no significant effect on the survival of recipients with aGVHD, but the clinical and pathological manifestations of cGVHD in FN KO mice were more severe significantly.

The up-regulation of FN levels using exogenous FN peptides had no significant effect on the survival of aGVHD. While FN peptides which was given via tail vein injection in vivo prolonged survival of mice with cGVHD. Besides, in cGVHD models, including both BALB/c to C57BL/6 mice model and DBA/2 to BALB/c mice model, recipients receiving exogenous FN peptides via intraperitoneal injection showed milder cGVHD severity than that receiving PBS. Intraperitoneal injection of IL-17A monoclonal antibody had an effect on the survival curve of cGVHD, and the intraperitoneal injection of IL-17A monoclonal antibody group had a longer survival and milder cGVHD.

The level of MCP-1 in receipents receiving exogenous FN peptides decreased significantly. Infiltration of M2 macrophages in target tissues decreased, and the deposition of antibodies in target organs also decreased.

Conclusion:IL-17A monoclonal antibody attenuates cGVHD in mice. FN had no significant effect on aGVHD, and exogenous FN peptides had also no significant effect on aGVHD. However, FN showed a protective effect on cGVHD. FN KO aggravated cGVHD. While up-regulation of FN level by exogenous FN peptides could weaken the severity of cGVHD, and FN peptides showed a therapeutic effect on cGHVD which is not model-dependent. FN peptides may inhibit the migration and infiltration of M2 macrophages in target tissues by reducing MCP-1, which may contribute to reduction of TGF-β secreted by M2 macrophages in target tissues, and antibodies' deposition in target organs also decreased. As a result, cGVHD could be attenuated.

No relevant conflicts of interest to declare.

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