MXD1 regulates the H9N2 and H1N1 influenza A virus-induced chemokine expression and their replications in human macrophage.

Human infection with influenza A/Hong Kong/156/97 (H5N1) avian influenza virus is associated with a high mortality rate of 60%. This virus is originated from influenza A/Quail/Hong Kong/G1/97 (H9N2/G1) avian influenza virus. Since the 1990s, four lineages of H9N2 viruses have been circulating in poultry and cause occasional infection in humans in different countries. Due to its zoonotic and genetic reassortment potential, H9N2/G1 and H5N1 viruses are believed to be the next pandemic candidates. Previous reports, including ours, showed that the virulence of avian virus strains correlates with their ability to dysregulate cytokine expression, including TNF-α, CXCL10, and related chemokines in the virus-infected cells. However, the transcriptional factors required for this cytokine dysregulation remains undefined. In light of our previous report showing the unconventional role of MYC, an onco-transcriptional factor, for regulating the antibacterial responses, we hypothesize that the influenza virus-induced cytokine productions may be governed by MYC/MAX/MXD1 network members. Here, we demonstrated that the influenza A/Hong Kong/54/98 (H1N1)- or H9N2/G1 virus-induced CXCL10 expressions can be significantly attenuated by knocking down the MXD1 expression in primary human blood macrophages. Indeed, only the MXD1 expression was up-regulated by both H1N1 and H9N2/G1 viruses, but not other MYC/MAX/MXD1 members. The MXD1 expression and the CXCL10 hyperinduction were dependent on MEK1/2 activation. By using EMSAs, we revealed that MXD1 directly binds to the CXCL10 promoter-derived oligonucleotides upon infection of both viruses. Furthermore, silencing of MXD1 decreased the replication of H9N2 but not H1N1 viruses. Our results provide a new insight into the role of MXD1 for the pathogenicity of avian influenza viruses.

Integrin-Linked Kinase Expression in Myeloid Cells Promotes Inflammatory Signaling during Experimental Colitis.

The pathology of inflammatory bowel diseases is driven by the inflammatory signaling pathways associated with mucosal epithelial damage. Myeloid cells are known to play an essential role in mediating epithelial inflammatory responses during injury. However, the precise role of these cells in stimulating intestinal inflammation and the subsequent tissue damage is unclear. In this article, we show that expression of integrin-linked kinase (ILK) in myeloid cells is critical for the epithelial inflammatory signaling during colitis induced by dextran sodium sulfate. Myeloid ILK (M-ILK) deficiency significantly ameliorates the pathology of experimental colitis. In response to dextran sodium sulfate, colonic infiltration of neutrophils and inflammatory cytokine production are impaired in M-ILK-deficient mice, and activation of epithelial NF-κB and PI3K signaling pathways are restricted by the M-ILK deficiency. In contrast, reduced epithelial damage in M-ILK-deficient mice is correlated with elevated levels of epithelial Stat3 activation and proliferation. Moreover, M-ILK-dependent inflammatory signaling in the mucosal epithelium can be therapeutically targeted by the pharmacological inhibition of ILK during experimental colitis. Collectively, these findings identify M-ILK as a critical regulator of epithelial inflammatory signaling pathways during colitis and, as a consequence, targeting M-ILK could provide therapeutic benefit.

The kinase activity of PKR represses inflammasome activity.

The protein kinase R (PKR) functions in the antiviral response by controlling protein translation and inflammatory cell signaling pathways. We generated a transgenic, knock-in mouse in which the endogenous PKR is expressed with a point mutation that ablates its kinase activity. This novel animal allows us to probe the kinase-dependent and -independent functions of PKR. We used this animal together with a previously generated transgenic mouse that is ablated for PKR expression to determine the role of PKR in regulating the activity of the cryopyrin inflammasome. Our data demonstrate that, in contradiction to earlier reports, PKR represses cryopyrin inflammasome activity. We demonstrate that this control is mediated through the established function of PKR to inhibit protein translation of constituents of the inflammasome to prevent initial priming during innate immune signaling. These findings identify an important role for PKR to dampen inflammation during the innate immune response and caution against the previously proposed therapeutic strategy to inhibit PKR to treat inflammation.

BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs.

Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Molecular dynamics reveal a novel kinase-substrate interface that regulates protein translation.

A key control point in gene expression is the initiation of protein translation, with a universal stress response being constituted by inhibitory phosphorylation of the eukaryotic initiation factor 2α (eIF2α). In humans, four kinases sense diverse physiological stresses to regulate eIF2α to control cell differentiation, adaptation, and survival. Here we develop a computational molecular model of eIF2α and one of its kinases, the protein kinase R, to simulate the dynamics of their interaction. Predictions generated by coarse-grained dynamics simulations suggest a novel mode of action. Experimentation substantiates these predictions, identifying a previously unrecognized interface in the protein complex, which is constituted by dynamic residues in both eIF2α and its kinases that are crucial to regulate protein translation. These findings call for a reinterpretation of the current mechanism of action of the eIF2α kinases and demonstrate the value of conducting computational analysis to evaluate protein function.

Protein kinase R and the inflammasome.

Protein kinase R (PKR) was first identified as a mediator of the double-stranded RNA (dsRNA)-mediated inhibition of protein synthesis in extracts from interferon-treated cells. In a physiological context, viral replication results in production of dsRNA, activation of PKR by autophosphorylation, and phosphorylation of the protein synthesis initiation factor eIF2α. Subsequent biochemical, structural, and genetic analyses have identified the dsRNA and kinase domain structure of PKR, and shown that its deletion from the germline of mice results in impaired resistance to infection by many different viruses. These studies have also opened up roles for PKR in different signaling pathways, the most recent being regulation of the inflammasome. Here we review evidence for this newly ascribed function for PKR and discuss roles in inflammasome regulation and associated diseases.

A role for c-Myc in regulating anti-mycobacterial responses.

c-Myc (Myc) is a well known transcription factor that regulates many essential cellular processes; however, its role in modulating immunity is not known. Here, we showed different species of mycobacteria can induce Myc expression via ERK1/2 and JNK activation. Unexpectedly, the induced Myc is localized in the cytoplasm but not in the nucleus. This induced Myc expression is associated with the induction of TNF-α and IL-6 and with the suppression of intracellular mycobacterial growth. To delineate the underlying mechanisms, we demonstrated that Myc enhances IRAK1 degradation, leading to specific activations of ERK1/2 and p38 MAPK but not Akt, and reduces IκBα protein recovery upon degradation. Hence, our findings may provide insights into a potential role for Myc in regulating the antimicrobial responses.

Interleukin-17A differentially modulates BCG induction of cytokine production in human blood macrophages.

The pathogenesis of Mtb depends in part on cytokine cross-regulation between macrophages and T cells in host immunity. Th17 cells produce IL-17A to induce granuloma formation and to restrict mycobacterial dissemination. IL-17A also mediates cytokine responses induced by proinflammatory cytokines such as TNF-α. Our previous results showed that BCG induces IL-6, IL-10, and TNF-α via activity of protein kinases, including dsRNA-activated serine/threonine protein kinase and glycogen synthase kinase-3 in primary human monocytes. Therefore, we investigated whether IL-17A, upon its induction by BCG, plays an additional role to aid the production of downstream proinflammatory cytokines in macrophages. Here, we showed that IL-17A enhanced IL-6 mRNA and protein levels inducible by BCG in a time- and dose-dependent manner, whereas it had no effect on IL-10 and TNF-α production. We also demonstrated that IL-17A activated the phosphorylation of ERK1/2 triggered by BCG. With the use of a specific chemical inhibitor of a MAPK/ERK-activating kinase (MEK1/2), we confirmed the correlation between the enhanced ERK1/2 activation and augmented IL-6 production. Additionally, we revealed that IL-17A acts in concert with BCG-induced TNF-α to enhance the level of IL-6 synthesis. Taken together, our results suggest a significant role of IL-17A to serve as a modulator of cytokine expression in innate immune response during mycobacterial infection.

HIV-1 trans-activator protein dysregulates IFN-γ signaling and contributes to the suppression of autophagy induction.

OBJECTIVE AND DESIGN: HIV-1 transactivator protein, Tat, has been identified as an activator of HIV-1 replication. It also dysregulates cytokine production and apoptosis in T-cells. Of the various cell death processes, autophagy is a self-digestion and degradation mechanism that recycles the contents of the cytosol, including macromolecules and cellular organelles, resulting in self-repair and conservation for survival. Recent reports demonstrated that autophagosomes can be activated by interferon-γ (IFN-γ) to participate in immune defence by processing foreign antigens for the recognition and killing of intracellular pathogens. As we previously showed that HIV-1 Tat perturbs IFN-γ signaling through the suppression of STAT1 phosphorylation and consequently inhibits major histocompatibility complex class-II antigen expression, we postulate that Tat plays a role in regulating autophagy. METHODS: The role of STAT1 in IFN-γ-induced autophagy in primary human blood macrophages was examined using a small molecule inhibitor or siRNA specific for STAT1. The effect of HIV-1 Tat on autophagy was investigated by pretreating the macrophages with HIV-1 Tat and followed by IFN-γ stimulation. The expressions of autophagy-associated genes and their effects on engulfing mycobacteria were examined. RESULTS: The activation of STAT1 resulted in IFN-γ-induced LC3B protein expression and autophagosome formation. As postulated, HIV-1 Tat protein suppressed IFN-γ-induced autophagy processes, including LC3B expression. Additionally, HIV-1 Tat restricted the capturing of mycobacteria by autophagosomes. CONCLUSION: HIV-1 Tat suppressed the induction of autophagy-associated genes and inhibited the formation of autophagosomes. Perturbation of such cellular processes by HIV-1 would impair the effective containment of invading pathogens, thereby providing a favorable environment for opportunistic microbes in HIV-infected individuals.

A novel anti-mycobacterial function of mitogen-activated protein kinase phosphatase-1.

BACKGROUND: Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-alpha (TNF-alpha), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-alpha via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-alpha inducible by BCG. CONCLUSIONS: Since TNF-alpha is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity.

HIV-1 Tat dysregulation of lipopolysaccharide-induced cytokine responses: microbial interactions in HIV infection.

OBJECTIVE: To examine whether the HIV-1 Tat protein impairs the lipopolysaccharide (LPS)-induced cytokine responses. DESIGN: Concurrent infections with pathogens including bacteria and viruses are common in AIDS patients. However, cytokine and interferon responses during infection with or translocation from the gut of these pathogens in HIV-infected patients are not well studied. As HIV-1 Tat contributes partly to the HIV-induced immune dysregulation, we investigated whether the protein may play a role in perturbing the LPS-induced cytokine responses. METHODS: Expression levels of cytokines in human primary blood monocytes/macrophages were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Expression level of the cell surface Toll-like receptor 4 was examined by flow cytometry. Activations of signaling molecules were assayed by western blot and immunofluorescence. RESULTS: We demonstrated that HIV-1 Tat downregulated the LPS-induction of IFN-beta and concomitantly upregulated IL-6 expression in primary blood monocytes/macrophages, whereas the viral protein had no significant effects on TNF-alpha expression. To delineate the underlying mechanism, we showed that Tat inhibited the LPS-activation of ERK1/2 but not the p38 mitogen-activated protein kinases. The viral protein suppressed the LPS-induced activation of NFkappaB p65 via its induction of IkappaBalpha expression, which resulted in retention of NFkappaB p65 in the cytosol. CONCLUSION: These findings suggest that Tat may play a role in modulating the immune responses triggered by other coinfecting pathogens and thus providing a permissive environment for both HIV and other opportunistic microbes.

Role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. Since its associated morbidity and mortality have been postulated to be due to immune dysregulation, we investigated which of the viral proteins is responsible for chemokine overexpression. To delineate the viral and cellular factor interactions, the role of four SARS coronavirus proteins, including nonstructural protein 1 (nsp-1), nsp-5, envelope, and membrane, were examined in terms of cytokine induction. Our results showed that the SARS coronavirus nsp-1 plays an important role in CCL5, CXCL10, and CCL3 expression in human lung epithelial cells via the activation of NF-kappaB.