Roles of TRIM24 in macrophage activation and experimental visceral leishmaniasis

Muscutt, Edward William

Abstract

Visceral leishmaniasis (VL) causes immune and haematological dysfunction and is fatal in >95% of cases if left untreated. While macrophages are vital for efficient Leishmania parasite clearance, parasite-macrophage interactions are critical for VL progression. The transcription factor tripartite motif protein 24 (TRIM24) was recently predicted to be downregulated in an experimental VL mouse model. However, roles of TRIM24 in macrophage activation remain poorly understood, and have not been explored in VL. Here, we use TRIM24 knockout (KO) C57BL/6 mice to investigate roles of TRIM24 in bone marrow-derived macrophage (BMDM) activation in vitro, and the effects of TRIM24 deletion in an experimental model of VL in vivo using a combination of microscopy, flow cytometry, and single cell RNA sequencing (scRNA-seq).
Firstly, we show that TNF and IL-6 production in KO BMDMs is unaffected after TLR4 stimulation. However, more nitric oxide was released by KO BMDMs, attributed to higher iNOS expression. Accompanying increases in IFNβ release and interferon response-related transcriptional signatures indicated an increased propensity for KO BMDMs to produce and respond to interferons. Uptake of L. donovani amastigotes was unaffected in KO BMDMs. ScRNA-seq of L. donovani-infected BMDMs revealed a metabolic shift in KO BMDMs towards parasite-permissive oxidative phosphorylation, while also potentiating interferon responses.
Flow cytometric analysis of total KO lymphoid organs revealed the dispensability of TRIM24 during homeostatic leukocyte development. However, assessment of relative fitness during 50:50 mixed bone marrow (BM) chimeric reconstitution revealed an advantage of KO leukocytes in lymphoid organs, with 80% of BM CD45+ cells deficient in TRIM24 at 10 weeks post-reconstitution. L. donovani infection reduced this skew in spleen and liver but not in BM, identifying a potential role for TRIM24 in BM retention that persists during infection independently of the CXCR4-CXCL12 axis. Immune responses characteristic of VL were unaffected by TRIM24 deletion (IFNγ, TNF, IL-10, IL-6 production, granuloma size and number, Kupffer cell iNOS expression), and hepatic parasite burden was slightly increased. Elevated mRNA abundance of interferon-stimulated genes was observed in KO spleen 24 hours post-infection.
Finally, we used scRNA-seq to report a transcriptional landscape of L. donovani-infected WT murine BM, providing evidence for an emergency myelopoiesis-promoting environment. Haematopoietic stem and progenitor cells were directly responding to CD4+ T cell-derived IFNγ, and we identify CD4+ T cells as a major source of Csf1 during infection. Ccl5 was expressed by several cell types, and parasite-permissive metabolic shifts were also observed, all contributing factors to myelopoiesis. These metabolic shifts were exacerbated by TRIM24 deletion, probably through modulation of mTOR signalling. This data therefore provides an interesting avenue for future investigation of TRIM24 in immunometabolism.

Citation

Muscutt, E. W. Roles of TRIM24 in macrophage activation and experimental visceral leishmaniasis. (Thesis). University of Hull and University of York. https://hull-repository.worktribe.com/output/4557553

• Thesis Type: Thesis
• Deposit Date: Feb 22, 2024
• Publicly Available Date: Feb 22, 2024
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4557553
• Award Date: Feb 1, 2024