Genhong Cheng

Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species

Bacteria of the genus Clostridium promote the induction of suppressor T cells in the colons of mice. CD4+ T regulatory cells (Tregs), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor–β and affected Foxp3+ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

MicroRNA-155 is induced during the macrophage inflammatory response

The mammalian inflammatory response to infection involves the induction of several hundred genes, a process that must be carefully regulated to achieve pathogen clearance and prevent the consequences of unregulated expression, such as cancer. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that has also been linked to cancer. However, the relationship between inflammation, innate immunity, and miRNA expression is just beginning to be explored. In the present study, we use microarray technology to identify miRNAs induced in primary murine macrophages after exposure to polyriboinosinic:polyribocytidylic acid or the cytokine IFN-β. miR-155 was the only miRNA of those tested that was substantially up-regulated by both stimuli. It also was induced by several Toll-like receptor ligands through myeloid differentiation factor 88- or TRIF-dependent pathways, whereas up-regulation by IFNs was shown to involve TNF-α autocrine signaling. Pharmacological inhibition of the kinase JNK blocked induction of miR-155 in response to either polyriboinosinic:polyribocytidylic acid or TNF-α, suggesting that miR-155-inducing signals use the JNK pathway. Together, these findings characterize miR-155 as a common target of a broad range of inflammatory mediators. Importantly, because miR-155 is known to function as an oncogene, these observations identify a potential link between inflammation and cancer.

IRF3 Mediates a TLR3/TLR4-Specific Antiviral Gene Program

We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-ε, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.

Involvement of CRAF1, a relative of TRAF, in CD40 signaling

CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-alpha (TNF-alpha) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-alpha receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.

LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

The signaling adaptors and pathways activated by TNF superfamily

Members of the TNF receptor superfamily play pivotal roles in numerous biological events in metazoan organisms. Ligand-mediated trimerization by corresponding homo- or heterotrimeric ligands, the TNF family ligands, causes recruitment of several intracellular adaptors, which activate multiple signal transduction pathways. While recruitment of death domain (DD) containing adaptors such as Fas associated death domain (FADD) and TNFR associated DD (TRADD) can lead to the activation of a signal transduction pathway that induces apoptosis, recruitment of TRAF family proteins can lead to the activation of transcription factors such as, NF-kappaB and JNK thereby promoting cell survival and differentiation as well as immune and inflammatory responses. Individual TNF receptors are expressed in different cell types and have a range of affinities for various intracellular adaptors, which provide tremendous signaling and biological specificities. In addition, numerous signaling modulators are involved in regulating activities of signal transduction pathways downstream of receptors in this superfamily. Most of the TNF receptor superfamily members as well as many of their signaling mediators, have been uncovered in the last two decades. However, much remains unknown about how individual signal transduction pathways are regulated upon activation by any particular TNF receptor, under physiological conditions.

Type I Interferon Production Enhances Susceptibility to Listeria monocytogenes Infection

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes–induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.

Activation of noncanonical NF-κB requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2, TRAF3 and the kinase NIK

Recent studies suggest that nuclear factor κB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-κB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway.

The triggering molecular mechanism of ischemia-reperfusion injury (IRI), which in clinical settings results in excessive and detrimental inflammatory responses, remains unclear. This study analyzes the role of the TLR system in an established murine model of liver warm ischemia followed by reperfusion. By contrasting in parallel TLR knockout mice with their wild-type counterparts, we found that TLR4, but not TLR2, was specifically required in initiating the IRI cascade, as manifested by liver function (serum alanine aminotransferase levels), pathology, and local induction of proinflammatory cytokines/chemokines (TNF-α, IL-6, IFN-inducible protein 10). We then investigated the downstream signaling pathway of TLR4 activation. Our results show that IFN regulatory factor 3, but not MyD88, mediated IRI-induced TLR4 activation leading to liver inflammation and hepatocellular damage. This study documents the selective usage of TLR in a clinically relevant noninfectious disease model, and identifies a triggering molecular mechanism in the pathophysiology of liver IRI.