Dual-specificity protein phosphatase-1 positively regulates the anti-mycobacterial responses

Posters: abstract no. 214Tuberculosis is still prevalent around world. It is caused by Mycobacterium tuberculosis (Mtb). This microbe stimulates monocytes/macrophages leading to the production of specific cytokines for initiating immune responses. Of these cytokines, tumour necrosis factor-alpha (TNF-α) is crucial for inducing the granuloma formation for restricting Mtb dissemination. Using Bacillus Calmette Guerin (BCG) as a model, we showed ...postprintThe 1st Lorne Infection and Immunity Conference, Lorne, Australia, February 2011. In Abstract Book of the 1st Lorne Infection and Immunity Conference, 2011, p. 111, abstract no. 21

The protein kinase R modifies gut physiology to limit colitis

Here we investigate the function of the innate immune molecule protein kinase R (PKR) in intestinal inflammation. To model a colitogenic role of PKR, we determine the physiological response to dextran sulfate sodium (DSS) of wild-type and two transgenic mice strains mutated to express either a kinase-dead PKR or to ablate expression of the kinase. These experiments recognize kinase-dependent and -independent protection from DSS-induced weight loss and inflammation, against a kinase-dependent increase in the susceptibility to DSS-induced injury. We propose these effects arise through PKR-dependent alteration of gut physiology, evidenced as altered goblet cell function and changes to the gut microbiota at homeostasis that suppresses inflammasome activity by controlling autophagy. These findings establish that PKR functions as both a protein kinase and a signaling molecule in instituting immune homeostasis in the gut

Interleukin-17A differentially modulates mycobacterial-induction of cytokines in human blood macrophage

The 2010 Annual General Meeting and Scientific Meeting of the Hong Kong Society for Immunology (HKSI), Hong Kong, 17 April 2010

Positive role of mitogen-activated protein kinase phosphatase-1 in regulating the anti-mycobacterial immune responses

Cytokine, 2009, v. 48 n. 1-2, p. 61-62 abstract no. PP1-071Poster Presentation ITuberculosis remains one of the major epidemic threats to human. It is caused by Mycobacterium tuberculosis (Mtb). This pathogen is recognized by monocytes/macrophages resulting in the release of specific cytokines for eliciting immune responses to control the infection. Of these, tumour necrosis factor-alpha (TNF-α) plays a key role in establishing granulomas to restrict Mtb dissemination. We previously showed that using Bacillus Calmette Guerin (BCG) as a model, mycobacteria induce TNF-α through the activation of double-stranded RNA-dependent protein kinase (PKR) and mitogen-activated protein kinases (MAPK). To control overactivation of MAPK following Gram-negative bacteria infection, MAPK phosphatase-1 (MKP-1) has been postulated to exert negative regulation on MAPK activation. Therefore, we hypothesize MKP-1 modulates mycobacterium-stimulated immune responses. To examine whether BCG can induce MKP-1, primary human blood monocytes (PBMo) were infected with BCG and MKP-1 expression was assayed by quantitative RT-PCR and Western blot. Our results showed that BCG induced the expression of MKP-1 in a time-dependent manner. This MKP-1 expression was found to be positively controlled by extracellular signal-regulated kinase-1 and -2 (ERK1/2) and p38 MAPK. To investigate the role of MKP-1 in regulating the BCG-induced responses, the cellular level of MKP-1 protein was knocked down by MKP-1-specific siRNA. Consequently, we demonstrated the level of TNF-α expression induced by BCG in PBMo was reduced, but its induction by lipopolysaccharide was further augmented. Moreover, the phosphorylation levels of BCG-activated ERK1/2 and p38 MAPK were decreased by the MKP-1 knockdown. Taken together, our results indicated that apart from its well-known negative role in regulating LPS-induced responses, MKP-1 acts as a positive regulator in modulating the BCG-induced activation of ERK1/2 and p38 MAPK, and consequently the induced expression of TNF-α. These results provide new insights into the regulation of proinflammatory cytokines in the pathogenesis of mycobacterial infections

HIV-1 Tat plays a role in dysregulating lipopolysaccharide-induced cytokine expression: implications for immune defects in AIDs

Introduction: During bacterial infection, macrophages/ monocytes are activated by lipopolysaccharides (LPS), the bacterial cell wall-associated endotoxins, to trigger innate and adaptive immune responses. However, these responses are impaired in HIV-infected patients especially in children, and such immune defects may contribute to the higher incidence of secondary bacterial infections and rapid progression of AIDS. The mechanisms on how HIV impairs these immune responses are not fully understood. Previous reports including ours indicated that Tat, the transactivator for transcriptional activation of the HIV genome, is partly responsible for mediating the retrovirus-induced subversion of immunity and enhancement of HIV replication. Therefore, we hypothesise that Tat plays a role in the dysregulation of the LPS-induced immune responses, thereby contributing to the pathogenesis of AIDS. Methods: Primary human blood monocytes were pretreated with recombinant Tat protein prior to LPS addition. Expression levels of specific cytokines were assayed by Q-RTPCR and ELISA. Levels of signalling kinases and nuclear factors were examined by Western analysis. Results: Our results demonstrated that Tat differentially suppresses the LPS-induction of IFN-β but enhances the induction of IL-6. On the contrary, Tat was shown to have a slight enhancing effect on the LPS-induction of TNF-α. To investigate the underlying mechanisms of Tat in cytokine dysregulation, we showed that the HIV protein inhibits LPSinduced activation of ERK1/2 but not p38 MAP kinase. We also demonstrated that Tat suppresses LPS-induced degradation of IκBα, resulting in the release and activation of NFκB for subsequent transcription of downstream cytokines and targeted genes. Conclusion: Taken together, these results imply that Tat may suppress the host anti-viral responses due to IFN-β suppression and yet promote HIV replication via the enhancement of IL-6 expression. Hence, during Gramnegative bacterial infection in AIDS patients, Tat may play a role in dysregulating the immune responses induced by the bacteria for providing a favourable environment for HIV survival and replication. Conflict of Interest Statements: There is no conflict of interest. Parts of the results were presented in Annual Scientific Meeting 2008, Hong Kong Society for Immunology. (Supported in part by HK Research Grants Council, HKU7408/04M and HKU7594/06M

Cellular signaling events in cytokines and sulphamethoxasole interactions

Introduction: The use of sulphonamides as antimicrobial agents has been practiced for decades. It is frequently used with another antimicrobial agent trimethoprim to provide a more effective antimicrobial spectrum in treating bacterial and protozoan infections in immunocompromised patients. During bacterial infection, proinflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12 as well as anti-inflammatory cytokines including IL-4 and IL-10 are produced by macrophages. The complexity of cytokine interactions provides a favorable environment for the host immune system to fight against pathogens. In previous studies, the metabolites of sulphamethoxazole have been demonstrated to have a role in the antimicrobial effects but without the details on molecular mechanisms. In the present study, we delineated the mechanisms and effects of sulphamethoxazole metabolites on cytokine production. Methods: With the use of primary human differentiated blood macrophages as our model, the cells were treated for 30 minutes with the drug metabolites and followed by the addition of bacterial endotoxin (lipopolysaccharide LPS) for 15 minutes to 3 hours. Cytokine mRNA and protein productions were measured by Quantitative Real-time PCR and ELISA, respectively. For the investigation of signaling events including the role of kinases and transcription factors, cellular and nuclear protein fractions were collected and analysed by specific Western blot assays. Results: The results showed that the LPS-induced cytokines including TNF-α, IL-6 and IL-10 were downregulated by the sulphamethoxazole metabolites both at the transcription and translation levels. Since the expression of cytokines is mediated by the action of signaling kinases such as mitogen-activated protein kinases (MAPK) and transcription factors, we measured the activity status of MAPK in the sulfphamethoxazole metabolites-treated macrophages. The results showed that sulphamethoxazole metabolites abrogated the LPS-induced MAPK phosphorylation, concomitant with their effects on cytokine downregulation. Furthermore, the activation of nuclear factor-κB (NF-κB) induced by LPS was also suppressed by the metabolites. Conclusion: In conclusion, our data elucidated that in addition to their antimicrobial effects, sulphamethoxazole and its metabolites may play a role in limiting the propagation of uncontrolled inflammation, via the suppression of MAPK and NF-κB activities, in microbial infections