Ashok Kumar

The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages.

Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro- and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs −652 and −571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs −652 and −571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

Innate immune responses in hepatitis B virus (HBV) infection

Hepatitis B virus (HBV) infection has a low rate of chronicity compared to HCV infection, but chronic liver inflammation can evolve to life threatening complications. Experimental data from HBV infected chimpanzees and HBV transgenic mice have indicated that cytotoxic T cells are the main cell type responsible for inhibition of viral replication, but also for hepatocyte lysis during chronic HBV infection. Their lower activation and impaired function in later stages of infection was suggested as a possible mechanism that allowed for low levels of viral replication. The lack of an interferon response in these models also indicated the importance of adaptive immunity in clearing the infection. Increased knowledge of the signalling pathways and pathogen associated molecular patterns that govern activation of innate immunity in the early stages of viral infections in general has led to a re-evaluation of the innate immune system in HBV infection. Numerous studies have shown that HBV employs active strategies to evade innate immune responses and induce immunosuppression. Some of the immune components targeted by HBV include dendritic cells, natural killer cells, T regulatory cells and signalling pathways of the interferon response. This review will present the current understanding of innate immunity in HBV infection and of the challenges associated with clearing of the HBV infection.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type I (Th1) (interferon‐gamma (IFN‐γ)) and Th2 (IL‐4, IL‐10) type cytokines in peripheral blood lymphocytes (PBL) from HIV patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) analysis revealed that IFN‐γ mRNA in unstimulated PBL was significantly decreased and IL‐10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n= 30) as compared to patients with > 400 CD4+ T cells/mm3 (n= 6) and normal controls (n= 16). In addition. IL‐10 mRNA levels were inversely associated with IFN‐γ expression. Similar results were obtained by measuring IL‐10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL‐4 and IFN‐7 produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV individuals based on IL‐10 production. PBL from one set of individuals produced low levels of IL‐10 (low IL‐10 producers) whereas the other group produced IL‐10 comparable lo that of normal controls (IL‐10 producers). Production of IL‐4 was significantly reduced in HIV+ individuals with<400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN‐γ by mitogen‐stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulaied and mitogen‐stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Dysregulation of B7.2 (CD86) expression on monocytes of HIV-infected individuals is associated with altered production of IL-2

T helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28–B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen‐presenting cells from HIV+ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV+ individuals compared with HIV− controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV+ individuals compared with HIV− controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL‐10 and tumour necrosis factor‐alpha (TNF‐α), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV+ individuals were distinguished on the basis of the inhibitory effect of IL‐10 and TNF‐α on monocyte B7.2 expression. IL‐10 inhibited B7.2 expression on monocytes from some HIV+ individuals (termed responders) like the HIV− controls. However, in a subset of HIV+ individuals (non‐responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL‐10 was associated with significantly reduced IL‐2 production by phytohaemagglutinin (PHA)‐ stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL‐2 may have implications in HIV immunopathogenesis.

PI3K/Akt regulates survival during differentiation of human macrophages by maintaining NF-κB-dependent expression of antiapoptotic Bcl-xL

Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance are not well understood. We have used M‐CSF‐stimulated primary human monocytes and PMA‐treated THP1 cells to study apoptosis resistance during differentiation of human macrophages. Our results indicate that PI3K/Akt distinctively regulates survival of macrophages during and after differentiation. More specifically, a signaling pathway consisting of PI3K/Akt‐NF‐κB‐Bcl‐xL regulates cell survival during the differentiation process. PI3K/Akt‐mediated activation of NF‐κB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl‐xL expression. With the use of pharmacological inhibitors and siRNA for Akt and Bcl‐xL, we show that in the absence of Akt‐dependent Bcl‐xL expression during differentiation, cells undergo caspase‐mediated apoptosis. In contrast, in differentiated macrophages, Bcl‐xL expression is independent of PI3K/Akt activation. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. Our results also suggest new, potential therapeutic targets to modulate differentiation and survival of this cell type.

STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells

IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK.

Down-regulation of CXCR-4 and CCR-5 expression by interferon-γ is associated with inhibition of chemotaxis and human immunodeficiency virus (HIV) replication but not HIV entry into human monocytes

Alterations in the expression of CXCR4 and CCR5, the co‐receptors for HIV entry, may be associated with susceptibility of monocytic cells to HIV infection. Interferon (IFN)‐γ has been shown to inhibit HIV replication in monocytic cells, but the molecular mechanism involved is not well understood. To determine if IFN‐γ regulates HIV replication by altering CXCR‐4/CCR‐5 expression and hence virus entry into monocytic cells, we investigated the effects of IFN‐γ on CXCR‐4 and CCR‐5 expression and its biological implications with respect to HIV entry, replication and chemotaxis towards the CXCR‐4 and CCR‐5 ligands SDF‐1 and MIP‐1α, respectively. IFN‐γ decreased CXCR‐4 and CCR‐5 expression on monocytes derived from HIV‐negative adults, HIV‐positive adults and HIV‐negative cord blood. This down‐regulation of chemokine receptor expression did not result in a corresponding change in mRNA expression but was associated with elevated levels of the endogenously produced chemokines SDF‐1 and RANTES. Furthermore, IFN‐γ inhibited chemotaxis in response to SDF‐1 and MIP‐1α, inhibited HIV replication, but failed to inhibit virus entry in monocytic cells. These results suggest that although IFN‐γ‐induced down‐regulation of CXCR‐4 and CCR‐5 expression is associated with an inhibition of SDF‐1‐/MIP‐1α‐mediated chemotaxis, IFN‐γ‐induced inhibition of HIV replication may be mediated at levels subsequent to the virus entry.

Characterization of In vitro Generated Human Polarized Macrophages

Objective: Contact with invading pathogens and/or tissue injury leads to the polarization of macrophages into either a M1 or a M2 state which is further divided into M2a, M2b and M2c subsets. The human macrophage subsets have been poorly characterized. The present study was undertaken to characterize macrophage polarization using a non-exhaustive panel of surface markers with respect to M1, M2a, M2b and M2c macrophages and production of pro- and anti-inflammatory cytokines in response to various toll-like receptors (TLR), ligands.Methods: We generated various macrophage subsets by treating monocyte-derived macrophages (MDMs) with IFNγ (M1), IL-4 (M2a), LPS and IL-1β (M2b) or IL-10 (M2c) followed by stimulation with toll-like receptor (TLR)- 2, TLR-3 and TLR-4 agonists and analysis of surface marker and cytokines expression was carried out by flow cytometry and ELISA, respectively.Results: M2a subset was characterized by CD14low, CD163low and TLR4low phenotype and produced high levels of IL-10. M2b subset was characterized by CD14high, CD80high and CD200Rlow phenotype and produced IL-6 prior to stimulation. M2c subset displayed a CD86low, CD163high phenotype and produced high levels of IL-10. M1 subset was characterized by CD80high, CD86high, CD163low and TLR4high phenotype and produced high levels of proinflammatory IFN-g, IL-12, TNFα and IL-23 following stimulation.Conclusion: This study characterizes all four polarization states in human macrophages. Each polarization state demonstrated a unique cell surface marker profile and cytokine profile. These phenotypic markers can be used to characterize macrophage populations in tissue inflammatory disease conditions in vivo to further understand disease pathogenesis.

Delivery of immunogens to mucosal immune system using an oral inactivated cholera vaccine: A new approach for development of oral vaccines

Oral vaccines have several attractive features; however, due to several challenges, to date, only a limited number of oral vaccines are licensed. Over the past two decades, several oral vehicle delivery systems have been developed to address these challenges and deliver antigens to the target cells in the mucosal immune system. While the size of vehicle delivery systems, the quantity of components in the vehicle formulation, the dose of administration, and even the type of animals species, are important aspects in development of a suitable oral vaccine, our results showed that entrapment of inactivated Vibrio cholera, a component in the structure of Dukoral vaccine into oral vehicle delivery systems, is able to induce a more rigorous humoral immune response in the systemic compartment. We further investigated the mechanism of Dukoral vaccine as a potential stimulator in induction of immune response by immunizing TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. We are hopeful that these findings will lead to development of more precisely-designed oral vaccines in the future.

Bacterial DNA Protects Monocytic Cells against HIV-Vpr–Induced Mitochondrial Membrane Depolarization

Monocytes and macrophages are important HIV reservoirs, as they exhibit marked resistance to apoptosis upon infection. However, the mechanism underlying resistance to apoptosis in these cells is poorly understood. Using HIV–viral protein R-52–96 aa peptide (Vpr), we show that primary monocytes and THP-1 cells treated with Vpr are highly susceptible to mitochondrial depolarization, but develop resistance following stimulation with bacterial DNA or CpG oligodeoxynucleotide. We have shown that Vpr-induced mitochondrial depolarization is mediated by TNFR-associated factor-1 (TRAF-1) and TRAF-2 degradation and subsequent activation of caspase-8, Bid, and Bax. To provide the mechanism governing such resistance to mitochondrial depolarization, our results show that prior stimulation with CpG oligodeoxynucleotide or Escherichia coli DNA prevented: 1) TRAF-1/2 downregulation; 2) activation of caspase-8, Bid, and Bax; and 3) subsequent mitochondrial depolarization and release of apoptosis-inducing factor and cytochrome c. Furthermore, this protection was mediated by upregulation of antiapoptotic protein (c-IAP-2) through calmodulin-dependent kinase-II activation. Thus, c-IAP-2 may prevent Vpr-mediated mitochondrial depolarization through stabilizing TRAF-1/2 expression and sequential inhibition of caspase-8, Bid, and Bax.