Browsing by Author "Hurdayal, Ramona"

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    Open Access
    An Investigation of the SHIV reservoirs in the lymphoid organs of HIV-vaccinated rhesus monkeys after SHIV challenge
    (2022) Benn, Kealan Timothy; Chapman, Rosamund; Hurdayal, Ramona
    The development of an effective vaccine against HIV-1 is thought to be a key component of combating the current HIV epidemic. This study aimed to investigate viral reservoirs in the blood and lymphoid tissues after HIV vaccination and virus challenge using the rhesus macaque model. In addition, the study investigated the envelope sequences of the virus located in the potential reservoir sites in an attempt to understand viral variability. Cryopreserved peripheral blood mononuclear cells (PBMC) and cells isolated from lymphoid tissues obtained at termination of vaccine group (P4, P11, P25, P47, P52) and control group (P67, P70, P71, P72) macaques were processed for determination of viral RNA and proviral DNA loads, and HIV-1 Env amino acid sequences using single genome amplification (SGA) sequencing method. In addition, cryopreserved plasma obtained at key time points pre- and post- vaccination and SHIV challenge were used to measure the levels of HIV Env and SIV Gag antibodies by Western blotting, and HIV-1 Env gp140 ELISA. RNA viral loads were low for all animals with the median of the averages of the viral load for PBMCs being 3.82 copies/107 cells and a range of < 1 copies/107 cells to 61.15 copies/107 cells. For the control group, only P67 had a detectable viral load in the PBMC. The median of the averages for proviral DNA load for PBMCs was 1903.98 copies/107 cells and a range of 696.93 copies/107 cells to 28 663.88 copies/107 cells. Proviral DNA levels were higher than the RNA viral loads indicating a larger amount of integrated provirus and potentially the presence of reservoirs within the macaques. While the low viral load values could mean there are few actively replicating viruses present in the PBMCs. The median of the averages for the RNA viral loads in the inguinal tissue cells was 58.08 copies/107 cells with a range of 8.85 copies/107 cells to 911.61 copies/107 cells. The median of the average for proviral DNA load in the inguinal tissue cells was 3160.36 copies/107 cells with a range of 604.67 copies/107 cells to 73 140.68 copies/107 cells. Proviral DNA levels in the inguinal tissues were higher than the RNA viral load levels which indicates a larger amount of integrated provirus present in the cells of the inguinal tissues and the presence of a potential reservoir. This also indicates that there is less circulating virus. The Western blots showed that the macaques developed binding antibodies to both HIV Env and SIV Gag, however, non-specific binding of antibodies to proteins other than HIV Env and SIV Gag was also seen. The ELISA effectively showed that both the vaccine group and control group macaques were able to produce binding antibodies. Macaques P4, P47, and P52 had the highest endpoint titre for the vaccine group of 5120. Macaque P71 had the highest overall endpoint titre of 20 480. Macaques P11 and P25 of the vaccine group had an endpoint titre of 1280 which was the same endpoint titre for control group macaques P67 and P70, while macaque P72 had an endpoint titre of 5120. Single genome amplification and sequencing showed that there were very few amino acid changes between sequences found in macaques. There was minimal variation within the vaccine group and between the vaccine and control groups with amino acid changes in the variable region 1 (V1), glycan N276 in the conserved region 2 (C2) and variable region 4 (V4) resulting in potential N-glycosylation sites (PNGS) being shifted as well as new glycosylation sites being formed while other lost. This study demonstrates that RNA viral loads and proviral DNA loads were detectable in both macaque groups with no significant difference being present in the viral RNA and proviral DNA loads between the two macaque groups. The lack of changes seen in the sequences of the inguinal and PBMC tissues are due to the early virus seeding the reservoir. Once the reservoir is seeded active replication does not occur in the reservoir resulting in the viruses having similar sequences.
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    Open Access
    Characterization of interleukin-4 induced gene-1 (IL-4I1) as a host immunoregulator to cutaneous leishmaniasis in cell based models
    (2022) Fouad, Aya; Hurdayal, Ramona
    Macrophage activation can be split into two distinct groups: Classically-Activated (M1) and Alternatively-Activated (M2) macrophages. Each are associated with a certain immune response; in the context of cutaneous leishmaniasis caused by infection with Leishmania major. Specifically, M1 macrophages allow for disease control through stimulation of cytokines such as IFN-g and release of nitric oxide for killing of intracellular parasites. Contrastingly, M2 macrophages allow for parasite survival through stimulation of cytokines such as IL-4 and IL-13 and release of urea that supports parasite growth. Candidate genes responsible for promoting M1 or M2 macrophages are being investigated as the macrophage is the sight of infection for leishmaniasis. One such candidate is IL-4i1, which has been shown to be responsible for M2 polarization. Notably, IL-4i1 catalyses phenylalanine, water, and oxygen to produce phenylpyruvate, ammonia and hydrogen peroxide. Interestingly, high levels of hydrogen peroxide create a toxic cellular environment and has been shown to lead to parasite destruction. Due to these contrasting roles of IL-4i1, IL-4i1 may be pivotal in modulating macrophage activation and host response to infection. Accordingly, we investigated IL-4i1 in a murine and human cutaneous leishmaniasis (CL) cell-based model using bone marrow-derived macrophages (BMDMs) derived from BALB/c IL-4i1+/+ and IL-4i1-/- mice and silencing of IL-4i1 in THP-1-derived macrophages. THP-1 monocytes were differentiated into macrophages via phorbol 12-myristate 13-acetate (PMA). Following macrophage verification, THP-1-derived macrophages were silenced with IL-4i1 siRNA constructs using the Silencer SelectTM siRNA Construction kit and Lipofectamine 3000 transfection agent. In IL-4 and IFN- �-stimulated IL-4i1-/- BMDM infected with L. major, an increase in parasite burden was found (p< 0.05, 24 hr PI). Immunologically, nitrite was reduced, and levels of IL-12 were below detection limit whilst IL-10 was increased upon IL-4 stimulation, compared to IL-4i1+/+. Thus, the presence of IL-4i1 may play a role in controlling parasite replication and mediating M1 polarization. After 24h of infection with L. major, IL-4i1- silenced THP-1-derived macrophages showed increases in production of nitrite (p< 0.05), hence the absence of IL-4i1 induced M1 activation. Collectively, these data indicate that IL-4i may indeed favour M2 activation in the context of L. major infection as absence thereof promoted a detrimental environment for parasite burden. Altogether, this underscores IL-4i1 as a potential susceptibility factor to CL. Understanding how Leishmania parasites can modify host immune response could lead to identifying novel therapeutics such as host-directed therapies.
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    Open Access
    Cysteinyl leukotriene receptor 1 as a host determinant for susceptibility to cutaneous leishmaniasis in a mouse model
    (2023) Cele, Zama; Hurdayal, Ramona
    Leishmaniasis is a vector-borne neglected tropical disease caused by Leishmania parasites. Approximately one million Leishmania infections are reported annually with one billion people residing in 98 countries at risk of infection. Cutaneous leishmaniasis (CL), characterized by skin lesions caused by Leishmania major and other species including Leishmania tropica and Leishmania aethiopica,is the most common form of the disease. With no effective vaccine, drug treatment is fraught with complications hence leishmaniasis is a major global public health burden. Interestingly, not all infected people develop active disease showing that an in-depth understanding of immune mechanisms could allow clear criteria for developing alternate host- derived therapies. Leukotrienes (LTs), produced by macrophages and other phagocytic cells including neutrophils, mast cells and dendritic cells during arachidonic acid metabolism, are hypothesized to induce a protective response during cutaneous leishmaniasis. However, as LTs consist of cysteinyl leukotrienes (CysLTs- LTC4, LTD4, LTE4) and LTB4, it is unclear which LT subclass is responsible for this protection. Recent studies suggested treatment of cutaneous leishmaniasis infected animals with cysteinyl leukotrienes induce host protection. To investigate this further, we targeted CysLTs via one of its receptors, cysteinyl leukotriene receptor 1 (CysLTR1), for its role in resistance or susceptibility to L. major-induced CL. In male and female BALB/c mice homozygous deficient for CysLTR1, we found that disease progression, parasite burdens, antibody and cytokine responses showed no significant alteration when compared to control animals with normal CysLTR1 expression. Importantly, expression of CysLTR2 did not appear to compensate for lack of CysLTR1 in CysLTR1-/- mice. Overall, these findings indicate that CysLTR1, as a host factor, is dispensable to the non- healing response associated with CL caused by L. major in mice.
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    Open Access
    Deletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection
    (2013) Hurdayal, Ramona; Brombacher, Frank; Revaz-Breton, Melanie
    In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4α ). While IL-4 is the main inducer of Th2 responses, paradoxically it has been shown that exogenously administered IL-4 can promote dendritic cell IL-12 production and enhance Th1 development if given early during infection.
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    Deletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection
    (Public Library of Science, 2013) Hurdayal, Ramona; Nieuwenhuizen, Natalie E; Revaz-Breton, Mélanie; Smith, Liezel; Hoving, Jennifer C; Parihar, Suraj P; Reizis, Boris; Brombacher, Frank
    In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.
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    Open Access
    Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice
    (Public Library of Science, 2012) Masic, Anita; Hurdayal, Ramona; Nieuwenhuizen, Natalie E; Brombacher, Frank; Moll, Heidrun
    Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.
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    Investigating the role of IL-4/IL-13 signalling through the IL-4 receptor alpha (IL-4Rα) on keratinocytes in murine models of Leishmania major and Schistosoma mansoni
    (2017) Govender, Melissa; Brombacher, Frank; Hurdayal, Ramona; Guler, Reto
    Keratinocytes represent the major cell type in the skin. During cutaneous leishmaniasis (CL) and schistosomiasis, the skin is important during the parasite life cycle. While Th1 immunity is required to control CL, protection during schistosomiasis requires Th2 immunity. Paradoxically, Th2 characteristic IL-4 secreted early during L. major infection in mice, can drive a Th1 response by instructing dendritic cells to produce IL-12. Additionally, keratinocytes at the site of L. major infection in C57BL/6 mice, were postulated to be the source of the IL-4. We investigated if IL-4/IL-13 signalling via the IL-4Rα on keratinocytes contributed to early immunity during CL and schistosomiasis. Keratinocyte-specific IL-4Rα deficient (KRT14creIL-4Rα-/lox) BALB/c and C57BL/6 mice were generated by gene targeting and site-specific recombination (cre/loxP) under control of the KRT14 locus. In the L. major footpad model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, and cytokine and antibody secretion similar to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice had decreased footpad swelling, low parasite burdens, a dominant Th1 cytokine response, and low type 1 and 2 antibody titres, similar to littermate control and resistant C57BL/6. In the L major ear model, KRT14creIL-4Rα-/lox BALB/c mice developed increased swelling, high parasite burdens, Th1 and Th2 cytokines, and high antibody titres, similar to littermate controls. L. major LV39-infected KRT14creIL-4Rα-/lox BALB/c mice showed significantly decreased parasite burdens in the ear, compared to littermate controls. L. major-infected KRT14creIL-4Rα-/lox C57BL/6 mice in the ear model, had decreased swelling, low parasite burdens, a dominant Th1 immune response, and low type 1 and 2 antibody titres, similar to littermate control and C57BL/6 mice. In the Schistosoma model, survival of S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice was similar to littermate controls during mortality studies. During acute infection, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice showed gut pathology, hepatosplenomegaly, cytokine production, low type 1 and high type 2 antibodies, similar to littermate controls. In comparison to littermate controls, S. mansoni-infected KRT14creIL-4Rα-/lox BALB/c mice had smaller granulomas. Collectively, our results indicate that IL-4/IL-13 signalling through the IL-4Rα on keratinocytes is not required for control during CL or acute schistosomiasis, but does contribute to efficient granuloma formation during acute schistosomiasis.
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    Investigating the role of IL-4Rα mediated signalling on Foxp3⁺ T regulatory cells during cutaneous leishmaniasis
    (University of Cape Town, 2020) Maine, Rebeng; Hurdayal, Ramona; Brombacher, Frank
    In a murine model of Leishmania major infection, susceptible BALB/c mice develop a detrimental Type 2 immune response characterized by the production of interleukin (IL)-4 and IL-13, which single through a common receptor, the IL-4 receptor alpha chain (IL-4Rα). Forkhead box P3 (Foxp3⁺) Regulatory T (Treg) cells are an unique subset of CD4⁺ T cells that play important immunomodulatory roles maintaining a balance between Type 1 and Type 2 immune responses. During L. major-induced cutaneous leishmaniasis, Treg cells accumulation at the site of infection has been implicated in suppressing a detrimental Type 2 immune response by modulating early interleukin (IL)-4 production, however it remains unclear if IL- 4Rα mediated signalling on Treg cells play a significant role in this process. To investigate this further, a novel BALB/c model was utilized in which the IL-4Rα chain was conditionally knocked out on Treg cells (Foxp3ᶜʳᵉIL-4Rα⁻/ˡᵒˣ mice). We demonstrated that the differential IL- 4Rα deletion efficiency in male (approximately 102 %) and female (approximately 32%) was maintained during L. major infection. Foxp3ᶜʳᵉIL-4Rα⁻/ˡᵒˣ male mice, which had a greater degree of IL-4Rα deletion on Foxp3⁺ Treg cells, developed significant footpad swellings and ear swellings, increased parasitic burdens at the site of infection and within draining lymph nodes. This hypersusceptible phenotype observed in Foxp3ᶜʳᵉIL-4Rα⁻/ˡᵒˣ BALB/c male mice was accompanied with an increased Treg cell activity and amplified Type-2 immune response with an increase in IL-4, IL-10 from L. major-infected lymph node samples and IgE antibody secretion in L. major infected serum samples. Flow cytometry analysis revealed that a L. major-induced Indoleamine 2,3 dioxygenase (IDO)-mechanism could allow for increased Leishmania replication. Collectively, these data suggest a protective role for IL-4Rα signalling on Treg cells in suppressing a detrimental Type 2 during cutaneous leishmaniasis.
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    Nterleukin-4 responsive dendritic, macrophage/neutrophil cells are dispensable for host resistance against Leishmania Mexicana infection in mice
    (2022) Ong'ondo, Bernard Osero; Hurdayal, Ramona
    Studies have shown that the protection against L. mexicana infection is potentiated by a type 1 response, driven by IL-12 production by dendritic cells. In contrast, IL-4 and IL-13 cytokines are associated with susceptibility to L. mexicana causing cutaneous leishmaniasis. This has been demonstrated in studies involving BALB/c mice deficient in IL-4, IL-13, and IL-4Rα infected with L. mexicana. To determine the specific cell in IL-4Rα-/- BALB/c mice that contribute to the control of L. mexicana infections, studies on cell-specific IL-4Rα deficient mice need to be investigated. IL4Rα-CD4+T deficient mice revealed sex-dependent protection from L. mexicana infection, suggesting the critical role of non-lymphocyte cells in conferring protection against L. mexicana amastigote infection. Macrophage/neutrophil-specific IL-4Rα deficient mice are protected from L. major infection in the footpad. Surprisingly, this mouse strain infected in the base of the tail failed to control L. mexicana amastigote infection. Nonetheless, IL-4Rα-DC deficient mice were hyper-susceptible to L. major infection. This conundrum suggests that different Leishmania species, site of infection, and developmental stages of parasite dictate the outcome of the disease. Here, mice with a deficiency of IL-4Rα signaling on DCs and macrophage/ neutrophil cells were subcutaneously infected with L. mexicana promastigotes in the footpad, and skin lesion progression was measured, and the clinical phenotype was evaluated by investigating both humoral and cellular immune responses. Mouse strains had similar footpad lesion progression, parasite loads, humoral responses, expansion of CD4+ and CD8+ T cells, their activation, memory phenotypes, and infiltration of DCs, macrophages, and neutrophils into the lymph nodes compared to their littermate IL-4Rα-/lox controls. Interestingly, IL‐12p70 and IL‐10 produced by BMDCs and BMDMs were similar. Nevertheless, nitrite/urea production was not affected. Together, this study suggests that, unlike L. major, IL-4Rα signaling on DCs and macrophage/ neutrophil cells does not contribute to the susceptibility or resistance to BALB/c mice to infection with L. mexicana.