Post translational modifications (PTMs) are exploited by various pathogens in order to escape hos... more Post translational modifications (PTMs) are exploited by various pathogens in order to escape host immune responses. SUMOylation is one of the PTMs which is involved in regulation of a variety of cellular responses. However, the effects of host SUMOylation on pathogenic bacteria largely remain elusive. We, therefore, investigated the role of SUMOylation in regulating defense responses in dendritic cells (DCs) during mycobacterial infection. Dendritic Cells of female BALB/c mice and THP-1 macrophages were used. Western blotting was performed to measure the expression of level of SUMO1, pSTAT1, pp38, pERK, Beclin-1, LC3, Bax and Cytochrome C. For bacterial burden confocal microscopy and CFU (Colony Forming Unit) were used. Flow cytometry was used for ROS and co-stimulatory molecules measurement. Cytokine level were measured using ELISA. We show that stimulation of Bone Marrow Derived Dendritic Cells (BMDCs) with mycobacterial antigen Rv3416 or live infection with Mycobacterium bovis BCG increases the SUMOylation of host proteins. Inhibition of SUMOylation significantly decreased intracellular bacterial loads in DCs. Additionally, inhibiting SUMOylation, induces protective immune responses by increasing oxidative burst, pro-inflammatory cytokine expression and surface expression of T cell costimulatory molecules, and activation of pSTAT1 and Mitogen Activated Protein Kinases (MAPK) proteins-pp38 and pERK. SUMOylation inhibition also increased apoptosis and autophagy in BMDCs. Intriguingly, mycobacteria increased SUMOylation of many of the above molecules. Furthermore, inhibiting SUMOylation in DCs primed T cells that in turn attenuated bacterial burden in infected macrophages. These findings demonstrate that SUMOylation pathway is exploited by mycobacteria to thwart protective host immune responses.
Calcium plays an important role in regulating cell physiology and immune responses to various pat... more Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of costimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance. 2.2. Materials Modulators to various calcium channels, RPMI-1640 medium was procured from Hyclone (GE Healthcare Life Sciences). The modulators used are as follows: BAYK8644 to activate VGCC [7] (50 nM);
Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucl... more Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucleic acids are known ligands of innate inflammatory receptors that trigger multiple inflammatory pathways that may result in acute inflammation and oxidative stress-driven tissue and organ toxicity. When dysregulated, this inflammation may lead to acute toxicity and multiorgan failure. Inflammatory events are often driven by high energy demands and macromolecular biosynthesis. Therefore, we proposed that targeting the metabolism of lipopolysaccharide (LPS)-driven inflammatory events, using an energy restriction approach, can be an effective strategy to prevent the acute or chronic detrimental effects of accidental or seasonal bacterial and other pathogenic exposures. In the present study, we investigated the potential of energy restriction mimetic agent (ERMA) 2-deoxy-D-glucose (2-DG) in targeting the metabolism of inflammatory events during LPS-elicited acute inflammatory response. Mice ...
Pathogen-associated molecular patterns (PAMPs) associated with viral and bacterial infections tri... more Pathogen-associated molecular patterns (PAMPs) associated with viral and bacterial infections trigger multiple inflammatory pathways which may result in oxidative stress driven toxicity, tissue fibrosis organ dysfunction and ageing. Inflammatory events need high energy demands and predominantly depends on the glycolysis. Thus, energy metabolism of the inflammatory events can be targeted to reducing the magnitude of the PAMPs driven inflammation and preventing tissue toxicity. Here we propose that 2-DG, a glycolytic inhibitor, and a potential Energy Restriction Mimetic agent (ERMA) can modulate inflammatory events and can prevent the development of acute as well as chronic pathology. For this study we induced LPS (bacterial PAMP) induced endotoxemia in mice which models infection associated inflammatory acute inflammatory events, tissue damage and organ dysfunction. 2-DG fed mice (0.4% w/v in drinking water) showed reduced LPS driven oxidative stress and capillary damage in lungs. Ad...
We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apo... more We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-cul...
Staphylococcus aureus(S. aureus) is a gram-positive bacteria, which causes various fatal respirat... more Staphylococcus aureus(S. aureus) is a gram-positive bacteria, which causes various fatal respiratory infections including pneumonia. The emergence of Methicillin-ResistanceStaphylococcus aureus(MRSA) demands a thorough understanding of host-pathogen interactions. Here we report the role of calcium in regulating defence responses ofS. aureusin macrophages. Regulating calcium fluxes in cells by different routes differentially governs the expression of T cell costimulatory molecule CD80 and Th1 promoting IL-12 receptor. Inhibiting calcium influx from extracellular medium increased expression of IFN-γ and IL-10 while blocking calcium release from the intracellular stores inhibited TGF-β levels. Blocking voltage-gated calcium channels (VGCC) inhibited the expression of multiple cytokines. While VGCC regulated the expression of apoptosis protein Bax, extracellular calcium-regulated the expression of Cytochrome-C. Similarly, VGCC regulated the expression of autophagy initiator Beclin-1. Bl...
Calcium plays an important role in regulating cell physiology and immune responses to various pat... more Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of costimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance. 2.2. Materials Modulators to various calcium channels, RPMI-1640 medium was procured from Hyclone (GE Healthcare Life Sciences). The modulators used are as follows: BAYK8644 to activate VGCC [7] (50 nM);
Tuberculosis (TB) is the most common opportunistic infection that makes human immunodeficiency vi... more Tuberculosis (TB) is the most common opportunistic infection that makes human immunodeficiency virus (HIV) infection more complicated. TB-immune reconstitution inflammatory syndrome (TB-IRIS) mainly refers to an excessive immune response among HIV-infected patients. In HIV-infected patients, IRIS occurs after initiation of antiretroviral therapy (ART), irrespective of increased CD4 count and effective suppression of HIV viremia; IRIS may occur at any stage in the progression of immunodeficiency and manifests with weakened immune system. IRIS is associated with various inflammatory processes as the outcome of immunological reaction against a variety of opportunistic infections (OIs). Currently, there is no reliable biological marker available for diagnosis of TB-IRIS. In accordance with current clinical case definition, deterioration of clinical and radiological symptoms of pre-existing TB infection in HIV patients is called "paradoxical TB-IRIS". The appearance of a previously undiagnosed or new TB infection during ART treatment is called "unmasking TB-IRIS". IRIS is a challenging complication for researchers and medical practitioners, as the incidence of IRIS is between 3-40% in patients initiated on ART. The variation in incidence probably reflects the differences in case definitions, patients' population studied, and individual resource-limited settings. The immune pathogenesis of IRIS is poorly understood, and epidemiology partially defined. The complication for clinicians remains challenging in terms of diagnosis and treatment as well as patients' suffering, even though, the mortality is typically low.
Inflammation is a critical component and contributor of carcinogenesis. Toll like receptors (TLRs... more Inflammation is a critical component and contributor of carcinogenesis. Toll like receptors (TLRs) are key regulators of innate immunity and inflammatory signaling. Besides various microbe-associated molecular patterns, TLRs can also bind to endogenously generated damage associated molecular patterns (DAMPS) ligands such as heat shock proteins, surfactant protein A18 and homobility group box-1(HMGB1). While HMGB1, a TLR-4 ligand is over-expressed in several human neoplasms, preclinical studies in mouse tumor models have strengthened the role of TLR-4-HMGB1 axis in promoting carcinogenesis. TLR-4 dependent inflammation and stimulation is accompanied with a switch from respiratory to glycolysis dominated metabolism, which is critical for the differentiation and maturation of myeloid cells thus affecting the outcome of an inflammatory disease. In the present studies we investigated the potential of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) as an energy restriction mimetic agent...
The innate immune system is an integral component of the inflammatory response to pathophysiologi... more The innate immune system is an integral component of the inflammatory response to pathophysiological stimuli. Toll-like receptors (TLRs) and inflammasomes are the major sensors and pattern recognition receptors (PRRs) of the innate immune system that activate stimulus (signal)-specific proinflammatory responses. Chronic activation of PRRs has been found to be associated with the aggressiveness of various cancers and poor prognosis. Involvement of PRRs was earlier considered to be limited to infection- and injury-driven carcinogenesis, where they are activated by pathogenic ligands. With the recognition of damage-associated molecular patterns (DAMPs) as ligands of PRRs, the role of PRRs in carcinogenesis has also been implicated in other non-pathogen-driven neoplasms. Dying (apoptotic or necrotic) cells shed a plethora of DAMPs causing persistent activation of PRRs, leading to chronic inflammation and carcinogenesis. Such chronic activation of TLRs promotes tumor cell proliferation a...
Post translational modifications (PTMs) are exploited by various pathogens in order to escape hos... more Post translational modifications (PTMs) are exploited by various pathogens in order to escape host immune responses. SUMOylation is one of the PTMs which is involved in regulation of a variety of cellular responses. However, the effects of host SUMOylation on pathogenic bacteria largely remain elusive. We, therefore, investigated the role of SUMOylation in regulating defense responses in dendritic cells (DCs) during mycobacterial infection. Dendritic Cells of female BALB/c mice and THP-1 macrophages were used. Western blotting was performed to measure the expression of level of SUMO1, pSTAT1, pp38, pERK, Beclin-1, LC3, Bax and Cytochrome C. For bacterial burden confocal microscopy and CFU (Colony Forming Unit) were used. Flow cytometry was used for ROS and co-stimulatory molecules measurement. Cytokine level were measured using ELISA. We show that stimulation of Bone Marrow Derived Dendritic Cells (BMDCs) with mycobacterial antigen Rv3416 or live infection with Mycobacterium bovis BCG increases the SUMOylation of host proteins. Inhibition of SUMOylation significantly decreased intracellular bacterial loads in DCs. Additionally, inhibiting SUMOylation, induces protective immune responses by increasing oxidative burst, pro-inflammatory cytokine expression and surface expression of T cell costimulatory molecules, and activation of pSTAT1 and Mitogen Activated Protein Kinases (MAPK) proteins-pp38 and pERK. SUMOylation inhibition also increased apoptosis and autophagy in BMDCs. Intriguingly, mycobacteria increased SUMOylation of many of the above molecules. Furthermore, inhibiting SUMOylation in DCs primed T cells that in turn attenuated bacterial burden in infected macrophages. These findings demonstrate that SUMOylation pathway is exploited by mycobacteria to thwart protective host immune responses.
Calcium plays an important role in regulating cell physiology and immune responses to various pat... more Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of costimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance. 2.2. Materials Modulators to various calcium channels, RPMI-1640 medium was procured from Hyclone (GE Healthcare Life Sciences). The modulators used are as follows: BAYK8644 to activate VGCC [7] (50 nM);
Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucl... more Pathogen-associated molecular patterns (PAMPs) like bacterial cell wall components and viral nucleic acids are known ligands of innate inflammatory receptors that trigger multiple inflammatory pathways that may result in acute inflammation and oxidative stress-driven tissue and organ toxicity. When dysregulated, this inflammation may lead to acute toxicity and multiorgan failure. Inflammatory events are often driven by high energy demands and macromolecular biosynthesis. Therefore, we proposed that targeting the metabolism of lipopolysaccharide (LPS)-driven inflammatory events, using an energy restriction approach, can be an effective strategy to prevent the acute or chronic detrimental effects of accidental or seasonal bacterial and other pathogenic exposures. In the present study, we investigated the potential of energy restriction mimetic agent (ERMA) 2-deoxy-D-glucose (2-DG) in targeting the metabolism of inflammatory events during LPS-elicited acute inflammatory response. Mice ...
Pathogen-associated molecular patterns (PAMPs) associated with viral and bacterial infections tri... more Pathogen-associated molecular patterns (PAMPs) associated with viral and bacterial infections trigger multiple inflammatory pathways which may result in oxidative stress driven toxicity, tissue fibrosis organ dysfunction and ageing. Inflammatory events need high energy demands and predominantly depends on the glycolysis. Thus, energy metabolism of the inflammatory events can be targeted to reducing the magnitude of the PAMPs driven inflammation and preventing tissue toxicity. Here we propose that 2-DG, a glycolytic inhibitor, and a potential Energy Restriction Mimetic agent (ERMA) can modulate inflammatory events and can prevent the development of acute as well as chronic pathology. For this study we induced LPS (bacterial PAMP) induced endotoxemia in mice which models infection associated inflammatory acute inflammatory events, tissue damage and organ dysfunction. 2-DG fed mice (0.4% w/v in drinking water) showed reduced LPS driven oxidative stress and capillary damage in lungs. Ad...
We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apo... more We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-cul...
Staphylococcus aureus(S. aureus) is a gram-positive bacteria, which causes various fatal respirat... more Staphylococcus aureus(S. aureus) is a gram-positive bacteria, which causes various fatal respiratory infections including pneumonia. The emergence of Methicillin-ResistanceStaphylococcus aureus(MRSA) demands a thorough understanding of host-pathogen interactions. Here we report the role of calcium in regulating defence responses ofS. aureusin macrophages. Regulating calcium fluxes in cells by different routes differentially governs the expression of T cell costimulatory molecule CD80 and Th1 promoting IL-12 receptor. Inhibiting calcium influx from extracellular medium increased expression of IFN-γ and IL-10 while blocking calcium release from the intracellular stores inhibited TGF-β levels. Blocking voltage-gated calcium channels (VGCC) inhibited the expression of multiple cytokines. While VGCC regulated the expression of apoptosis protein Bax, extracellular calcium-regulated the expression of Cytochrome-C. Similarly, VGCC regulated the expression of autophagy initiator Beclin-1. Bl...
Calcium plays an important role in regulating cell physiology and immune responses to various pat... more Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of costimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance. 2.2. Materials Modulators to various calcium channels, RPMI-1640 medium was procured from Hyclone (GE Healthcare Life Sciences). The modulators used are as follows: BAYK8644 to activate VGCC [7] (50 nM);
Tuberculosis (TB) is the most common opportunistic infection that makes human immunodeficiency vi... more Tuberculosis (TB) is the most common opportunistic infection that makes human immunodeficiency virus (HIV) infection more complicated. TB-immune reconstitution inflammatory syndrome (TB-IRIS) mainly refers to an excessive immune response among HIV-infected patients. In HIV-infected patients, IRIS occurs after initiation of antiretroviral therapy (ART), irrespective of increased CD4 count and effective suppression of HIV viremia; IRIS may occur at any stage in the progression of immunodeficiency and manifests with weakened immune system. IRIS is associated with various inflammatory processes as the outcome of immunological reaction against a variety of opportunistic infections (OIs). Currently, there is no reliable biological marker available for diagnosis of TB-IRIS. In accordance with current clinical case definition, deterioration of clinical and radiological symptoms of pre-existing TB infection in HIV patients is called "paradoxical TB-IRIS". The appearance of a previously undiagnosed or new TB infection during ART treatment is called "unmasking TB-IRIS". IRIS is a challenging complication for researchers and medical practitioners, as the incidence of IRIS is between 3-40% in patients initiated on ART. The variation in incidence probably reflects the differences in case definitions, patients' population studied, and individual resource-limited settings. The immune pathogenesis of IRIS is poorly understood, and epidemiology partially defined. The complication for clinicians remains challenging in terms of diagnosis and treatment as well as patients' suffering, even though, the mortality is typically low.
Inflammation is a critical component and contributor of carcinogenesis. Toll like receptors (TLRs... more Inflammation is a critical component and contributor of carcinogenesis. Toll like receptors (TLRs) are key regulators of innate immunity and inflammatory signaling. Besides various microbe-associated molecular patterns, TLRs can also bind to endogenously generated damage associated molecular patterns (DAMPS) ligands such as heat shock proteins, surfactant protein A18 and homobility group box-1(HMGB1). While HMGB1, a TLR-4 ligand is over-expressed in several human neoplasms, preclinical studies in mouse tumor models have strengthened the role of TLR-4-HMGB1 axis in promoting carcinogenesis. TLR-4 dependent inflammation and stimulation is accompanied with a switch from respiratory to glycolysis dominated metabolism, which is critical for the differentiation and maturation of myeloid cells thus affecting the outcome of an inflammatory disease. In the present studies we investigated the potential of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) as an energy restriction mimetic agent...
The innate immune system is an integral component of the inflammatory response to pathophysiologi... more The innate immune system is an integral component of the inflammatory response to pathophysiological stimuli. Toll-like receptors (TLRs) and inflammasomes are the major sensors and pattern recognition receptors (PRRs) of the innate immune system that activate stimulus (signal)-specific proinflammatory responses. Chronic activation of PRRs has been found to be associated with the aggressiveness of various cancers and poor prognosis. Involvement of PRRs was earlier considered to be limited to infection- and injury-driven carcinogenesis, where they are activated by pathogenic ligands. With the recognition of damage-associated molecular patterns (DAMPs) as ligands of PRRs, the role of PRRs in carcinogenesis has also been implicated in other non-pathogen-driven neoplasms. Dying (apoptotic or necrotic) cells shed a plethora of DAMPs causing persistent activation of PRRs, leading to chronic inflammation and carcinogenesis. Such chronic activation of TLRs promotes tumor cell proliferation a...
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