Graduation Date

Fall 12-15-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

St. Patrick Reid

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes Chikungunya fever (CHIKF), characterized by high fever, headache, rash, and joint pain. With global travel on the rise, infections caused by CHIKV have emerged in new regions, expanding their geographic range and infecting susceptible hosts. Currently, no approved prophylactic or therapeutic measures exist to prevent or treat these infections, with prevention strategies primarily focused on vector control. However, these measures have proven inadequate due to the emergence of mutations that enhance viral transmission or vector range. Therefore, urgent attention is needed to develop therapies to treat the infection. Although CHIKV has been reported to cause bone pathology, the underlying molecular mechanisms are not well understood. This study aims to investigate the effects of CHIKV infection on the function of bone marrow-derived mesenchymal stem cells (BMSCs), which are known to contribute to bone formation and regulate immune responses during infection and inflammation. In the following studies, we hypothesize that CHIKV alters the properties and function of BMSCs, leading to bone pathology and suppression of immune responses during infection. We investigated the effect of infection on matrix mineralization using staining and enzymatic assays during in vitro differentiation of BMSCs into osteogenic cells. In the presence of CHIKV infection, we observed reduced production of alkaline phosphatase enzyme and deposition of calcium crystals, essential for matrix mineralization during bone formation. Thus, osteogenic differentiation of BMSCs is impaired during CHIKV infection, as evidenced by the decrease in matrix mineralization (production of alkaline phosphate and calcium crystals). Using NanoString gene expression, we demonstrate that CHIKV infection alters the expression of several host factors associated with bone homeostasis, including major osteogenic transcription factors. Validation of gene expression using qRT-PCR analysis showed significant alteration in CTGF, ATF4, and c-FOS expression. Notably, the expression of c-FOS was upregulated 20-fold in infected cells compared to mock cells. c-FOS is a transcription factor that is found to be involved in a few viral pathogenesis. So, we treated cells with T-5224, a known inhibitor of c-FOS, and detected the effect of T-5224 on viral nsP1 gene expression during CHIKV infection. We observed a decreased viral nsP1 gene expression in T-5224 treated cells, indicating a role of c-FOS on nsP1 expression. In our second project, flow cytometry analysis revealed that infection induces the immunosuppressive effect of BMSCs on PBLs, leading to a decreased PBL population, and increased expression of markers associated with immunosuppressive function, including IDO1, iNOS, PDL-1, TGFβ1, PDL-1, and GAL9. The expression of immunosuppressive genes i.,e. IDO1, iNOS, and PGE2 were upregulated during CHIKV infection, with a ∼20-fold induction in the expression of IDO1. IDO1 is an enzyme that plays a critical role in the modulation of immune responses and the induction of immunosuppression. We also observed a significantly high expression of surface marker GAL9 (∼40 fold) during infection. GAL9 is involved in immune regulation and inflammation modulation, interacting with immune cells, and regulating cell activation, apoptosis, and cytokine production. Thus, we developed a novel in vitro cell model for studying CHIKV pathogenesis and demonstrated a possible mechanism of bone pathology during CHIKV infection. Furthermore, we identified host factors as potential targets for antiviral/anti-inflammatory treatment modality in CHIKV infection.

Comments

2023 Copyright, the authors

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Available for download on Wednesday, December 03, 2025

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