ORCID Profile
0000-0001-8267-1446
Current Organisation
Mater Research Institute The University of Queensland
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Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.MOLIMM.2012.10.042
Abstract: Extracellular high mobility group box 1 (HMGB1) protein and nitric oxide (NO) has been credited with multiple inflammatory functions using in vivo and in vitro systems. Therefore, delineating their regulation may be an important therapeutic strategy for the treatment of sepsis. In the present study, it is demonstrated that recombinant HMGB1 (rHMGB1) synergizes with sub threshold concentration of TLR2 agonist (PGN 1 μg/ml) as well as with TLR4 agonist (LPS 1 ng/ml) to induce NO release in mouse peritoneal macrophages. The enhanced iNOS expression was also observed at the transcription and translational level. Co-incubation of macrophages with rHMGB1 with either PGN or LPS showed enhanced expression of TLR2, TLR4 and RAGE. TLR2, TLR4 or RAGE knockdown macrophages effectively inhibited the rHMGB1+PGN or LPS induced NO synergy. It was further observed that the JNK MAPK inhibitor SP600125 attenuated the PGN+rHMGB1 induced iNOS/NO synergy whereas p38 MAPK inhibitor SB908912 inhibited iNOS/NO synergy induced by LPS+rHMGB1. It was also observed that the activation of NF-κB is essential for the synergy as the pharmacological inhibition or siRNA knockdown of NF-κB (cRel) significantly reduced the rHMGB1+PGN or rHMGB1+LPS induced enhanced iNOS/NO expression. Altogether, the data suggests that the co-incubation of macrophages with rHMGB1 with either LPS or PGN induces the synergistic effect on iNOS expression and NO release by the upregulation of surface receptors (TLR2, TLR4 and RAGE) which in turn lifies the MAPKs (p38 and JNK) and NF-κB activation and results in enhanced iNOS expression and NO production.
Publisher: Frontiers Media SA
Date: 06-04-2023
DOI: 10.3389/FIMMU.2023.1127896
Abstract: Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4 + and CD8 + T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models.
Publisher: Wiley
Date: 06-12-2017
DOI: 10.1111/EXD.13436
Abstract: The molecular links between sterile inflammation and induction of adaptive immunity have not been fully identified. Here, we examine how damage-associated molecular patterns (DAMPs), as opposed to pathogen-associated molecules (PAMPs), regulate the immune response to non-self-antigens presented at the site of a physical injury. Heat applied briefly to the skin invokes sterile inflammation, characterized by local cell death and caspase-1 activation without demonstrably disrupting skin integrity. Co-delivery of ovalbumin (OVA) with heat injury induces OVA-specific CD8
Publisher: American Society for Microbiology
Date: 06-2011
DOI: 10.1128/CVI.00541-10
Abstract: The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo . PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mapk in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity.
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.CYTO.2012.07.036
Abstract: The peroxisome proliferator-activated receptor (PPAR) γ plays an important role in macrophage inflammatory homeostasis. Here we investigate the cross talk between PPARγ and TLR2 signaling pathway in mouse peritoneal macrophages. Real time RT-PCR and immunoblot analysis revealed that peptidoglycan (PGN) treatment of macrophages leads to biphasic effect on PPARγ expression i.e. an early upregulation and a late suppression. Inhibition of ERK MAP kinase by PD98059 abolished the early and rapid induction of PPARγ, while the inhibition of JNK MAP kinase by SP600125 nullifies the late inhibitory effect on the PPARγ expression in a dose-dependent manner. Furthermore, PPARγ knockdown macrophages showed enhanced NF-κB activity after PGN treatment. PGN treatment also enhances PPARγ interaction with p65 as observed by immunoprecipitation. This interaction may inhibit NF-κB (p65) activity as increased nuclear localization of p65 was observed in PPARγ knockdown macrophages after PGN treatment. PPARγ knockdown also increased the PGN-induced inflammatory cytokines (TNF-α, IL-1β, IL-12p40) production. Thus, our observations suggest that PGN induces PPARγ expression which is regulated by MAPKs activation and this enhanced PPARγ in turn attenuate NF-κB activity probably via enhancing p65 nuclear export. These results provide insight into how these pathways could be modulated in inflammatory diseases.
Publisher: Elsevier BV
Date: 02-2012
DOI: 10.1016/J.IMBIO.2012.02.006
Abstract: High mobility group box 1 (HMGB1) protein is a unique non histone nuclear protein that acts extracellularly as a mediator of delayed inflammation. Sub lethal dose of UVB triggers the release of cytokines from macrophages (MΦs). Adding to the panoply of UVB induced cytokines it is reported that UVB induces HMGB1 release from mouse peritoneal MΦs in time and partially dose dependent manner, independent of TNF-α. UVB also enhanced the transcription of HMGB1 gene and expression of cellular protein, which influences its subsequent release. HMGB1 is secreted by an unconventional secretion pathway of unknown mechanism. Caspase-1 has been shown to function as a general regulator of stress induced unconventional secretion for a number of cytokines. In the present study, we have observed that pharmacological inhibitors specific for caspase-1 (ZVAD and YVAD) abrogated UVB induced HMGB1 release from MΦs. This effect was most likely mediated via physical interaction between HMGB1 and active caspase-1 (p10 and p20) as demonstrated by immunoprecipitation. In addition, it was found that HMGB1 and active caspase-1 p20 release depends on UVB mediated enhancement of intracellular Ca(2+). Thus our data suggests that optimal dose of UVB (50 mJ/cm(2)) induces HMGB1 upregulation and active release from mouse peritoneal MΦs which is mediated by caspase-1 in a Ca(2+) dependent manner.
Location: Australia
No related grants have been discovered for Rituparna Bhatt.