(WHS-K1.06) EXTRACELLULAR GRANZYME B CONTRIBUTES TO DELAYED HEALING OF CUTANEOUS LEISHMANIASIS IN A MURINE MODEL
Thursday, May 16, 2024
9:15 AM – 10:15 AM East Coast USA Time
Background and Hypothesis: Cutaneous leishmaniasis (CL) is an infectious disease caused by different species of leishmania parasite, resulting in a variety of chronic skin lesions that leave severe scarring and disfigurement. Granzyme B (GzmB), a serine protease, has been implicated in pathogenesis of CL caused by Leishmania braziliensis. Although the cytotoxic role of intracellular GzmB in parasite killing and targeted apoptosis in CL is well known, the contribution of extracellular GzmB to chronic inflammation, tissue damage, and scarring seen in CL is poorly understood. Here, we hypothesized that GzmB is elevated in CL caused by Leishmania major (L. major) and contributes to impaired healing through the cleavage of cell-cell and cell-basement membrane adhesion proteins.
Methods: Paraffin-embedded human skin biopsy specimens with confirmed diagnosis of L. major induced CL were subjected to the histological analysis using hematoxylin& eosin, immunohistochemistry (IHC), and immunofluorescent staining for GzmB, cellular markers of different immune cells, and GzmB substrates. Cell- free in vitro cleavage assay was used to identify new GzmB substrates. Twelve wild type and 11 GzmB knockout C57BL/6 mice were inoculated with L. major and the clinical course of the disease was documented over 8 weeks. Skin samples were collected for histological analysis at study endpoint.
Results: GzmB was highly expressed in human CL lesions compared to normal skin. The expression of E- cadherin, a cell-cell junction protein was significantly reduced in areas with higher number of GzmB expressing cells ( n=11, p<0.001). Collagen VII and collagen XVII, were also reduced in areas with accumulation of GzmB+ cells. Cell-free in vitro cleavage assay resulted in identification of two novel substrates of GzmB, desmoglein 4 and annexin A2. Further, IHC staining of human samples showed reduction of desmoglein 4 in samples demonstrating psoriasiform dermatitis, and annexin A2 on endothelial cells of thrombotic blood vessels. In vivo study revealed that while 54% of GzmB KO mice showed no visible lesion/scarring, all WT mice still had clinical lesion/scarring at the study end point.
Conclusion: Our results show that extracellular GzmB is associated with epidermal changes and vascular thrombosis in L. major induced CL and deletion of GzmB significantly reduces the healing time of the lesions in a murine model of CL. Collectively, our findings suggest a prominent role for extracellular GzmB in pathogenesis of skin lesions caused by L. major.