Targeting hedgehog signaling/KL4-dependent M2 macrophage polarization for treatment of chronic graft-versus-host diseases and tissue fibrosis

Abstract Background: Chronic graft-versus-host disease (cGVHD) represents the leading cause of non-relapse-related mortality and morbidity following allogeneic hematopoietic stem cell transplantation (HCT) (Flowers M, Bone Marrow Transplant, 2021; Blood, 2015). It is typically manifested as an autoimmune-like syndrome. In addition to effects mediated by T cells, cGVHD involves B-cell stimulation, autoantibody production, and systemic fibrosis. There is currently no satisfactory therapy for fibrosis in cGVHD due to poorly defined molecular mechanisms. Recent reports suggest the enrichment of M2 macrophage (MФ) in cGVHD (Du J, Blood, 2017; Alexander KA, JCI, 2014) and clinical efficacy of depletion of MФ with anti-CSF1R antibody in recurrent/refractory cGVHD patients (Wolff D, NEJM, 2024); however, whether and how M2 MФ polarization drives the development and tissue fibrosis remains to be elucidated. We observed the critical role of Hedgehog (Hh) signaling in M2 MФ polarization (Petty A, JCI, 2019). Here, we examined the effect of Hh-dependent M2 MФ polarization in the pathogenesis of cGVHD and the therapeutic potential of targeting Hh signaling/KLF4-dependent M2 MФ polarization to treat the disease and tissue fibrosis. Methods: BALB/c (H2d) recipient mice were irradiated (750 cGy x-ray), 6 hours later injected with T/B cell-depleted bone marrow cells (TBCD-BM) and whole splenocytes from C57BL/6 (B6, H2b) donor mice, and myeloid-specific knockout (KO) and transgenic B6 strains of LysMcre+Smofl/fl mice [Smoothened (Smo) KO, SmoΔM], LysMcre+Smo-M2C mice (Smo overexpression, SmoCM), LysMcre+Klf4fl/fl mice (KLF4 KO, Klf4ΔM), and LysMcre+SmoCMKlf4fl/fl (KLF4 KO in SmoCMcells, SmoCMKlf4 ΔM) mice. Human skin biopsies from 9 patients (pts) who had undergone HCT and developed cGVHD, and 3 volunteers were obtained at The Ohio State University (OSU) Dermatology Biorepository between July 01, 2023, and March 30, 2024, with approval of the Institutional Review Board of the OSU. In addition, 4 lung tissue paraffin blocks from bronchiolitis obliterans (BO) lungs of cGVHD pts and 4 normal lung controls from cardiac sudden death pts were obtained from the OSU biorepository. Results: Significantly higher MФ (CD11b+F4/80+), M2 MФ (CD11b+F4/80+CD206+/Arg1+), and lower M1 MФ (CD11b+F4/80+iNOS+/MHC II+), along with high expression of Hh signaling genes (Smo, SHH), KLF4, M2 MФ markers (F4/80, CD206, Arg1), and fibrogenic factors (TGFb1, Ym1) were observed in multiple tissues (spleen, lung, skin, etc.) of murine cGVHD vs TBCD-BM controls by flow cytometry and 7-color multiplex immunofluorescence (IF) analysis. SmoCM transgenic mice promoted, while the conditional KO or transgenic mice (SmoΔM, Klf4ΔM, SmoCMKlf4ΔM) blocked M2 MФ polarization, inhibited the expression of fibrogenic factors, suppressed disease development and tissue fibrosis, and extended cGVHD mice survival. Tissue immune microenvironment analysis by multiplex IF revealed the enrichment of M2 MФ and Smo+ MФ and their association with tissue fibrosis of the lung and skin, as well as skin damage severity scores (Lee SJ, Blood, 2017) in cGVHD pts cohorts. Moreover, cGVHD mice were treated with vehicle control, Smo inhibitor (Smoi, Glasdegib), KLF4 inhibitor (KLF4i, Kenpaullone), and their combination for 28 days, starting on day 12 or 28 post-HCT for preventative or curative therapy. Results showed that both Smoi and KLF4i monotherapies significantly improved survival and reduced clinical scores versus vehicle, and their combination was markedly more effective than either alone in both preventative and curative settings. Histological analysis exhibited that both Smoi and KLF4i significantly reduced collagen fibrosis and tissue damage, with their combination substantially outperforming either alone. These findings demonstrated the therapeutic and synergistic efficacy of the Smoi and KLF4i in treating murine cGVHD. Conclusion: Our study using mouse models and patient cohorts underscored the relevance of the Hh/KLF4-dependent M2 polarization in cGVHD progression and tissue fibrosis, demonstrating that genetic and therapeutic targeting of Smo/KLF4-mediated M2 MФ polarization blocked the progression of cGVHD and tissue fibrosis. Our results identified a novel mechanism of cGVHD, providing important insights and clinical relevance for precisely targeting M2 macrophage polarization as a new therapeutic approach for treating cGVHD and preventing tissue fibrosis in the disease.