Browsing by Author "Parihar, Suraj"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Open Access
    Protein Kinase C - delta (PKC-δ): a critical hub for immunomodulatory functions in macrophages during Mycobacterium tuberculosis infection
    (2023) Hazra, Rudranil; Parihar, Suraj
    Tuberculosis (TB) has reached epidemic levels and emerged as the second deadliest infectious disease globally after CoVID-19. By evolving the ability to evade host defense via intrinsic mechanisms, Mycobacterium tuberculosis (Mtb), the etiological agent of TB has been deleterious to human health and has necessitated novel therapeutic interventions, the primary notion to combat Mtb infection. Hence, the identification of host-modulating candidate genes involved in immune evasion and putative pathogen-killing pathways during Mtb infection is crucial. Additionally, macrophages are the first line of defense against Mtb infection through activating effector genes, which lead to pathogen killing and acquiring long-lasting immunity. One such candidate gene with potential novel therapeutic intervention, Protein Kinase C – δ (PKCδ) has been recognized as a critical marker with clinical and experimental evidence in recent years. An experimental mouse model of global PKCδ knockout (PKCδ-/- ) revealed mechanistic alterations enhancing the susceptibility to various infectious diseases including Mtb infection, suggesting a protective phenotype of PKCδ against invading pathogens. However, the macrophage-specific role of PKCδ during Mtb infection remains unknown and has not been delineated yet. Because the pulmonary microenvironment during Mtb infection is majorly governed by macrophages, initiating innate and skewing adaptive immune response, we have exploited the role of PKCδ in macrophages using the macrophage-specific PKCδ knockout mice (LysMcrePKCδflox/flox). Our success in characterizing this experimental murine strain has resulted in the establishment of an immunologically comparable PKCδ functional study platform, which has been adopted herein to investigate the immunomodulatory effects of Mtb infection in the ablation of PKCδ in macrophages. An early lymphocytic immune response increased neutrophil turnover, and reduced inflammatory macrophages are all accompanied by PKCδ deficiency in macrophages, which was abolished in the chronic stage of infection. Bonemarrow-derived macrophages from LysMcrePKCδflox/flox murine model further showed that the disease susceptibility is a consequence of an array of cellular intrinsic mechanisms and dysregulated proteome which are modulated by PKCδ. Furthermore, increased expression in bronchoalveolar lavage (BAL) samples from active TB patients and increased bacterial burden in PKCδ silenced human monocyte-derived macrophages with decreased pro-inflammatory cytokine response strongly signify PKCδ as a key hub for immunomodulatory functions during Mtb infection and a potential host-directed therapeutic (HDT) target against TB.
  • No Thumbnail Available
    Item
    Open Access
    The role of Cysteinyl leukotriene type 1 receptor (CysLTR1) during Listeria monocytogenes and mycobacterium tuberculosis infections in mice
    (2025) Poswayo, Sibongiseni Kwakho Luntukazi; Parihar, Suraj; Ozturk, Mumin; Brombacher, Frank
    During infection, antigen-presenting cells release cytokines and eicosanoids (including leukotrienes, epoxyeicosatrienoic acids, and prostanoids) to activate adaptive immunity. Leukotrienes (LTs), an eicosanoids subset, are produced from arachidonic acid via 5- lipoxygenase metabolism, resulting in LTB4 and cysteinyl LTs (cysLTs; LTC4, LTD4 and LTE4). CysLTs, which are pro-inflammatory lipid mediators, play a role in asthma and interact with three G-protein-coupled receptors (CysLTR1, CysLTR2, and GPR99). While the role of CysLTR1 and its ligand has been studied in asthmatic and allergic responses, its function during bacterial infections remains unclear. Our study aims to elucidate the role of CysLTR1 in disease progression using C57BL/6 and Balb/c mice infected either with Listeria monocytogenes (Lm) or with Mycobacterium tuberculosis (Mtb). Using CysLTR1 knockout (Cysltr1-/-) mice, we conducted time course and survival studies on both C57BL/6 and Balb/C genetic backgrounds. Our findings revealed that the function of CysLTR1 in bacterial infections is influenced by the host's genetic background and gender. Interestingly, CysLTR1 deletion did not impact survival or tissue pathology in C57BL/6 mice during Lm and Mtb infections. However, in Lm infection, CysLTR1 deletion led to increased recruitment of neutrophils to the liver and spleen, while in female Cysltr1-/- mice during Mtb infection, lung neutrophil recruitment was elevated. In Balb/C mice, CysLTR1 deletion during Lm infection affected survival in a sex-dependent manner, providing protection to females by reducing neutrophil recruitment. Conversely, Balb/C male mice were more susceptible to Lm infection. Notably, during Mtb infection, CysLTR1 deletion in Balb/C mice resulted in improved disease outcomes due to reduced lung neutrophils and pathology. In summary, CysLTR1 signalling significantly influences neutrophil recruitment and activation during intracellular bacterial infections, with its impact varying based on gender and genetic background.
  • Thumbnail Image
    Item
    Open Access
    The role of Cysteinyl leukotriene type 1 receptor (CysLTR1) during Listeria monocytogenes infection in mice
    (2020) Poswayo, Sibongiseni Kwakho Luntukazi; Parihar, Suraj; Ozturk, Mumin; Brombacher, Frank
    South Africa recently experienced a Listeriosis outbreak, which was responsible for over 180 deaths, caused by an intracellular, rod-shaped bacilli called Listeria monocytogenes (LM). LM can infect both phagocytic and non-phagocytic cell types and induces its uptake by expressing internalin A and B, then secretes listeriolysin O (LLO), a virulence factor forming pores on the phagosome membrane to escape into the cytosol. Macrophages can phagocytose invading pathogens and induce innate inflammatory responses. Production of cytokines and eicosanoids by antigen presenting cells activates the adaptive immunity. Eicosanoids (epoxyeicosatreinoic acids, prostanoids and leukotrienes) are generated from metabolites of 20-carbon chained polyunsaturated fatty acids and arachidonic acid. Leukotrienes (LTs) are generated from 5- lipoxygenase-metabolism of arachidonic acid to LTB4 and cysteinyl LTs (cysLTs). CysLTs are pro-inflammatory lipids that have pathobiological functions in asthma. CysLTs function through three G-protein coupled receptors (CysLTR1, CysLTR2 and GPR99). The CysLTR1 and its ligands function has been well elucidated in asthmatic and allergic responses however, its role in bacterial infections is unknown. The aim of our study was to elucidate the role of CysLTR1 on disease progression in mice and macrophages infected with LM. In this study, we showed that CysLTR1 mRNA expression is upregulated by LM infection in WT macrophages and mice. Mice deficient of CysLTR1 had no defects at homeostasis. During time kinetic experiments with LM, CysLTR1 knockout mice displayed increased neutrophil recruitment and decreased lymphocyte cells at 3dpi, however, bacterial burdens were comparable to wild-type mice. In addition, macrophages deficient of CysLTR1 have no effect on the intracellular growth of LM. In conclusion, CysLTR1 signalling plays a role in lymphoid cell activation and neutrophilic recruitment during early LM infection, however, further studies are required to better understand the role of CysLTR1 during inflammatory responses.
  • Thumbnail Image
    Item
    Open Access
    The Role of IL-4 Receptor Alpha signalling on Foxp3 T Regulatory cells in Listeriosis and Tuberculosis
    (2020) Chia, Julius Ebua; Brombacher, Frank; Parihar, Suraj
    T regulatory cells are critical in the maintenance of self-tolerance, immune homeostasis and regulation of the immune system. Cytokine signalling is a dominant component of environmental signals which controls the function of Forkhead box P3 (Foxp3) regulatory T cells. This thesis addressed the hypothesis that interleukin-4 receptor alpha (IL-4Rα) signalling on T regulatory cells (T reg) play a role in the stability of T reg cells. Loss of IL-4Rα signalling on T reg cells may shift the immune balance from a Foxp3+ T reg to a Th1 effector function essential for Th1 disease outcome. Regulatory cells have a major function to dampen cytokine production; however, this role can be detrimental for host-protective immune responses in diseases such as tuberculosis. Here, we used two Th1 models of intracellular pathogens Listeria monocytogenes (Lm) and Mycobacterium tuberculosis (Mtb), to understand the role of IL-4Rα signalling on Foxp3+ T regulatory cells. Infection studies with L. monocytogenes demonstrated an impairment of T reg responses, with a decreased bacterial burden and diminished pathology both in the liver and spleen at 7 days post-infection, ultimately translated in better survival. Mechanistically, enhanced Th1 signature with the characteristic T-bet transcriptional factor and increased effector T cells producing IFN-γ, IL-2 following ex-vivo stimulation with PMA/Ionomycin, and heat-killed Lm (HKLM) were observed in Foxp3creIL-4Rα-/lox mice. Furthermore, CD8+ T cells of Foxp3creIL-4Rα-/lox mice showed increased cytotoxicity (Granzyme-B secretion) with higher proliferation capacity (Ki-67), better survival (Bcl-2) and decreased apoptosis (activated caspase3), suggesting contribution towards the observed protection against listeriosis. Subsequently, we investigated the role of IL-4Rα on Foxp3 T reg cells in Mycobacterium tuberculosis infection. To our surprise, in contrast to Lm infection, survival Survival of Mtb-infected Foxp3creIL-4Rα-/lox mice was similar to littermate control following infection with an intermediate dose of Mtb (H37Rv). We observed no differences in acute and chronic stages of infection in bacterial burden and histopathological scores in Foxp3creIL-4Rα-/lox mice when compared to littermate control animals in acute and chronic stages of infection. Importantly, Mtb infected FoxP3creIL-4Rα-/lox mice, exhibited significantly enhanced CD4+ T effector functions with increased pro-inflammatory cytokine secretion upon stimulation ex-vivo.