Cheong Hee Chang

Interleukin (IL)-4 inhibits IL-10 to promote IL-12 production by dendritic cells

Interleukin (IL)-4 is known to be the most potent cytokine that can initiate Th2 cell differentiation. Paradoxically, IL-4 instructs dendritic cells (DCs) to promote Th1 cell differentiation. We investigated the mechanisms by which IL-4 directs CD4 T cells toward the Th1 cell lineage. Our study demonstrates that the IL-4–mediated induction of Th1 cell differentiation requires IL-10 production by DCs. IL-4 treatment of DCs in the presence of lipopolysaccharide or CpG resulted in decreased production of IL-10, which was accompanied by enhanced IL-12 production. In IL-10–deficient DCs, the level of IL-12 was greatly elevated and, more importantly, the ability of IL-4 to up-regulate IL-12 was abrogated. Interestingly, IL-4 inhibited IL-10 production by DCs but not by B cells. The down-regulation of IL-10 gene expression by IL-4 depended on Stat6 and was at least partly caused by decreased histone acetylation of the IL-10 promoter. These data indicate that IL-4 plays a key role in inducing Th1 cell differentiation by instructing DCs to produce less IL-10.

ERK and p38 MAPK Signaling Pathways Negatively Regulate CIITA Gene Expression in Dendritic Cells and Macrophages

The CIITA is a master regulator for MHC class II expression, but the signaling events that control CIITA expression remain poorly understood. In this study, we report that both constitutive and IFN-γ-inducible expression of CIITA in mouse bone marrow-derived dendritic cells (DC) and macrophages, respectively, are regulated by MAPK signals. In DC, the inhibitory effect of LPS on CIITA expression was prevented by MyD88 deficiency or pharmacological MAPK inhibitors specific for MEK (U0126) and p38 (SB203580), but not JNK (SP600125). In macrophages, LPS inhibited IFN-γ-inducible CIITA and MHC class II expression without affecting expression of IFN regulatory factor-1 and MHC class I. Blocking ERK and p38 by MAPK inhibitors not only rescued LPS-mediated inhibition, but also augmented IFN-γ induction of CIITA. Moreover, the induction of CIITA by IFN-γ was enhanced by overexpressing MAPK phosphatase-1 that inactivates MAPK. Conversely, CIITA expression was attenuated in the absence of MAPK phosphatase-1. The down-regulation of CIITA gene expression by ERK and p38 was at least partly due to decreased histone acetylation of the CIITA promoter. Our study indicates that both MAPK and phosphatase play an important role for CIITA regulation in DC and macrophages.

Cathepsin E: A Novel Target for Regulation by Class II Transactivator

The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.

Vaccinia Virus Infection Modulates the Hematopoietic Cell Compartments in the Bone Marrow

Successful proliferation and differentiation of hematopoietic progenitor cells in bone marrow (BM) is essential to generate all mature blood cell types, including those involved in the immune response. Although vaccinia virus (VV) is known to induce a strong immune response, the effect of VV infection on hematopoiesis remains largely unknown. Here, we showed that in vivo VV infection results in the expansion of c‐KithiSca‐1+Lin− (KSL) hematopoietic stem cells. The in vivo expansion of the KSL population requires MyD88 that is a critical adaptor for Toll‐like receptor‐mediated signaling. Moreover, in BM of VV‐infected mice, common myeloid progenitors (CMP) was decreased because of the rapid differentiation of CMP to more mature cells. However, the CMP compartment was not affected by VV infection in the absence of MyD88. The common lymphoid progenitor (CLP) cell population was increased regardless of MyD88 status, suggesting the independent regulation of CMP and CLP compartments by VV infection. VV infection also enhanced the potential of progenitors that preferentially induce the programming of dendritic cell (DC) development toward plasmacytoid DC. Therefore, the host immune response is gearing toward antiviral responses as early as at the precursor level upon VV infection.

Enhanced Production of IL-10 by Dendritic Cells Deficient in CIITA

Dendritic cells (DC) are professional APCs that play a critical role in regulating immunity. In DC, maturation-induced changes in MHC class II expression and Ag presentation require transcriptional regulation by CIITA. To study the role of CIITA in DC, we evaluated key cell functions in DC from CIITA-deficient (CIITA−/−) mice. The ability to take up Ag, measured by fluid phase endocytosis, was comparable between CIITA−/− and control DC. Although CIITA−/− DC lack MHC class II, they maintained normal expression of costimulatory molecules CD80, CD86, and CD40. In contrast, CIITA−/− DC activated with LPS or CpG expressed increased IL-10 levels, but normal levels of TNF-α and IL-12 relative to control. Enhanced IL-10 was due to greater IL-10 mRNA in CIITA−/− DC. Aβ−/− DC, which lack MHC class II but express CIITA normally, had exhibited no difference in IL-10 compared with control. When CIITA was cotransfected with an IL-10 promoter-reporter into a mouse monocyte cell line, RAW 264.7, IL-10 promoter activity was decreased. In addition, reintroducing CIITA into CIITA−/− DC reduced production of IL-10. In all, these data suggest that CIITA negatively regulates expression of IL-10, and that CIITA may direct DC function in ways that extend beyond control of MHC class II.

Disruption of MHC Class II-Restricted Antigen Presentation by Vaccinia Virus

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.

IL-4-stimulated NF-κB activity is required for Stat6 DNA binding

IL‐4 is a critical cytokine in the regulation of immune responses. In B lymphocytes, IL‐4 signaling promotes the Stat6‐dependent cell surface expression of several proteins including MHC Class II and CD86. However, the requirement for other transcription factors in IL‐4‐induced B cell gene expression has not been studied extensively. Here, we show that IL‐4 induces NF‐κB p100 processing to NF‐κB p52 in B cells but not in T cells or macrophages. IL‐4 induced NF‐κB p52 production requires PI‐3K activity and correlates with IκB kinase phosphorylation and TNF receptor‐associated factor 3 degradation. Blocking NF‐κB activity eliminates IL‐4‐stimulated gene expression in B cells by reducing IL‐4‐induced DNA binding but not phosphorylation or nuclear localization of Stat6. These results describe a novel role for NF‐κB in IL‐4‐induced signaling and gene expression.

Pulmonary Immunity to Viral Infection: Adenovirus Infection of Lung Dendritic Cells Renders T Cells Nonresponsive to Interleukin-2

ABSTRACT Adenovirus (Ad) infection has been identified as predisposing hosts to the development of pulmonary disease through unknown mechanisms. Lung dendritic cells (DCs) are vital for initiating pulmonary immune responses; however, the effects of Ad infection on primary lung DC have not been studied. In contrast to the effects on bone marrow- and monocyte-derived DCs, the current study shows that Ad infection of murine BALB/c lung DCs in vitro and in vivo suppresses DC-induced T-cell proliferation. The effect of Ad on DCs was not due to a downregulation of major histocompatibility complex or costimulatory molecules. Analysis of the production of interleukin-12 (IL-12), alpha interferon (IFN-α), and IFN-γ by the Ad-infected DCs shows no significant differences over noninfected control lung DCs. Ad-induced suppression was not due to a deficiency of IL-2 or other DC-secreted factors and was dependent on viral protein synthesis, as UV irradiation of Ad abrogated the suppressive effect. Results suggest that Ad-infected DCs induce T cells to be nonresponsive to IL-2 during primary coculture, as the addition of IL-2 in secondary cultures recovered T-cell proliferation. In vivo studies supported in vitro results showing that Ad infection resulted in lung T cells with decreased proliferative ability. This study demonstrates that Ad infection induces local immunoincompetence by altering DC-T-cell interactions.

A Role for Caspases in Controlling IL-4 Expression in T Cells

Although caspase activation is critical for T cell proliferation following activation, the role of caspases in T cell differentiation is unclear. In this study, we have examined the effect of inhibition of caspases on the process of Th1/Th2 differentiation. Naive CD4+ T cells activated under neutral differentiation conditions in the presence of the pan caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (Z-VAD) fluoromethylketone showed increased Th2 cell differentiation concomitant with an up-regulation of GATA-3. Z-VAD induced optimal Th2 differentiation when T cells were stimulated under strong primary activation conditions. Treatment of naive CD4+ T cells with Z-VAD under strong activation conditions led to a 6-fold increase in IL-4 mRNA compared with control-treated T cells. The Z-VAD-induced increase in IL-4 transcription occurred within 24 h of activation and was independent of Stat6. IFN-γ mRNA expression was not affected by Z-VAD at the 24-h time point. Z-VAD did not augment IL-4 expression from a committed Th2 cell, suggesting that caspases regulate IL-4 expression specifically during primary T cell activation. Z-VAD did not augment IL-12-driven Th1 differentiation. Activation of T cells in the presence of Z-VAD led to a specific increase in the expression of the transcription factor c-fos. Lastly, retrovirus-mediated expression of the antiapoptotic protein Bcl-2 resulted in an enhancement of Th2 cytokine expression, suggesting that inhibition of caspase activation by Bcl-2 can also modulate IL-4 expression. These findings reveal a novel regulatory mechanism of cytokine expression by caspases, and may explain how signaling pathways that inhibit apoptosis tend to promote Th2 differentiation.

Protein kinase Cδ is essential to maintain CIITA gene expression in B cells

Expression of MHC class II genes requires CIITA. Although the transactivation function of CIITA is well characterized, the signaling events that regulate CIITA expression are less understood. In this study, we report that CIITA expression in B cells depends on protein kinase Cδ (PKCδ). PKCδ controls CIITA gene transcription mainly via modulating CREB recruitment to the CIITA promoter without affecting CIITA mRNA stability. Inhibition of PKCδ by a pharmacological inhibitor or knocking down of endogenous PKCδ expression by small interfering RNA reduced CREB binding to the CIITA promoter. The decrease of CIITA gene expression in the presence of the PKCδ inhibitor was prevented by ectopically expressing a constitutively active form of CREB. In addition, histone acetylation of the CIITA promoter is regulated by PKCδ since the PKCδ inhibitor treatment or PKCδ small interfering RNA resulted in decreased histone acetylation. Taken together, our study reveals that PKCδ is an important signaling molecule necessary to maintain CIITA and MHC class II expression in B cells.