Full-exon resequencing reveals toll-like receptor variants contribute to human susceptibility to tuberculosis disease

Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Data have accumulated over decades suggesting that variability in human susceptibility to TB disease has a genetic component. Toll-like receptors (TLRs) play a critical role in initiating the innate immune response to many pathogens in mouse models, but little is known about their role in human infections. Human TLRs have been reported to recognize mycobacterial antigens and initiate an immune response. We tested the hypothesis that amino acid-altering polymorphisms in five TLRs were associated with susceptibility to TB disease using a population-based case-control study with 1,312 adult TB patients and controls. Full-coding region sequencing of the five TLR genes in all 1,312 subjects yielded a data set in excess of 16 Mb. Rare nonsynonymous polymorphisms in TLR6-TLR1-TLR10 were significantly overrepresented among African-American TB cases compared with ethnically-matched control subjects. Common nonsynonymous polymorphisms in TLR6-TLR1-TLR10 also were significantly associated with TB disease in certain ethnic groups. Among African Americans, homozygotes for the common-variant haplotype TLR1-248S, TLR1-602I, and TLR6-249S had a significantly increased TB disease risk. A transmission/disequilibrium test on an independent sample found that the TLR1-248S variant was preferentially transmitted to diseased children, thereby confirming disease association. These results are consistent with recent reports implicating TLR1 variants, including TLR1-602, in significantly altered innate immune responses. Also consistent with disease association, rare TLR6 variants were defective in their ability to mediate NF-kappaB signal transduction in transfected human cells. Taken together, the data suggest that variant TLRs contribute to human susceptibility to TB disease. Extensive full-exon resequencing was critical for revealing new information about the role of TLRs in human-pathogen interactions and the genetic basis of innate immune function.     

The genetics and immunopathogenesis of inflammatory bowel disease

Genome-wide association studies efficiently and powerfully assay common genetic variation. The application of these studies to Crohn's disease has provided insight into the immunopathogenesis of this disease, implicating a role for genes of the innate and adaptive immune systems. In this Review, I discuss our current understanding of the genetics and immunopathogenesis of Crohn's disease and ulcerative colitis. Crohn's disease, but not ulcerative colitis, is associated with genetic variation in NOD2 and an autophagy gene, ATG16L1, both of which affect the intracellular processing of bacterial components. By contrast, variation in the gene encoding the interleukin-23 (IL-23) receptor subunit, as well as in the IL12B, STAT3 and NKX2-3 gene regions, is associated with both Crohn's disease and ulcerative colitis. Comparative analyses of gene associations between these two inflammatory bowel diseases reveal common and unique mechanisms of their immunopathogenesis.     

A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-alpha-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease.     

The innate immune system in the intestine

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.     

重组腺相关病毒载体诱导的天然免疫反应及机制

重组腺相关病毒(rAAV)已成为基因治疗领域应用最广泛的载体之一。临床前研究显示其具有很高的安全性,但人体免疫毒性仍是制约其临床疗效的关键,因此有关rAAV免疫机制的研究成为近期热点。尽管天然免疫在获得性免疫反应中发挥重要作用,但与rAAV有关的天然免疫研究过去一直未被重视。直到最近,才确认有至少3种人体细胞(树突状细胞、巨噬细胞和内皮细胞)参与了rAAV的天然免疫,作用机制为可识别载体基因组的TLR9或病毒衣壳TLR2所介导,NF-κB或干扰素调节因子(IRFs)信号通路被激活,导致各种炎性因子及I型干扰素的大量表达。自身互补型rAAV诱导的TLR9依赖性天然免疫较单链rAAV更为强烈。本文重点对近期发现的激活天然免疫反应的宿主与rAAV的相互作用、涉及的信号通路、天然免疫对获得性免疫以及转基因表达的影响进行综述。     

Cutting edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity

Autoimmune diseases can be reduced or even prevented if proinflammatory immune responses are appropriately down-regulated. Receptors (such as CTLA-4), cytokines (such as TGF-beta), and specialized cells (such as CD4+CD25+ T regulatory cells) work together to keep immune responses in check. T cell Ig mucin (Tim) family proteins are key regulators of inflammation, providing an inhibitory signal that dampens proinflammatory responses and thereby reducing autoimmune and allergic responses. We show in this study that reducing Tim-3 signaling during the innate immune response to viral infection in BALB/c mice reduces CD80 costimulatory molecule expression on mast cells and macrophages and reduces innate CTLA-4 levels in CD4+ T cells, resulting in decreased T regulatory cell populations and increased inflammatory heart disease. These results indicate that regulation of inflammation in the heart begins during innate immunity and that Tim-3 signaling on cells of the innate immune system critically influences regulation of the adaptive immune response.     

Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer

Clinical and epidemiologic studies have suggested an association between infectious agents and chronic inflammatory disorders and cancer. Better understanding of microbial pattern-recognition receptors and innate immune signaling pathways of the host is helping to elucidate the connection between microbial infection and chronic disease. We propose that a key aspect of pathogenesis is an aberrant epithelial barrier that can be instigated by microbial toxins, environmental insults, or the genetic predisposition of the host. Loss of epithelial integrity results in activation of resident inflammatory cells by microbial invaders or endogenous ligands. When coupled with a failure of normal control mechanisms that limit leukocyte activation, a cascade is established that induces chronic inflammation and its consequences. Here, we outline this mechanistic framework and briefly review how alteration of innate immune response genes in murine models can provide insights into the potential microbial origins of diverse conditions including Crohn's disease, psoriasis, atherosclerosis, diabetes, and liver cancer.     

Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Self-consuming innate immunity in Arabidopsis

Programmed cell death (PCD) associated with the pathogen-induced hypersensitive response (HR) is a hallmark of plant innate immunity. HR PCD is triggered upon recognition of pathogen effector molecules by host immune receptors either directly or indirectly via effector modulation of host targets. However, it has been unclear by which molecular mechanisms plants execute PCD during innate immune responses. We recently examined HR PCD in autophagy-deficient Arabidopsis knockout mutants (atg) and find that PCD conditioned by one class of plant innate immune receptors is suppressed in atg mutants. Intriguingly, HR triggered by another class of immune receptors with different genetic requirements is not compromised, indicating that only a specific subset of immune receptors engage the autophagy pathway for HR execution. Thus, our work provides a primary example of autophagic cell death associated with innate immune responses in eukaryotes as well as of pro-death functions for the autophagy pathway in plants.     

Does the failure to acquire helminthic parasites predispose to Crohn's disease?

Two polarized patterns (Th1 and Th2) of cytokines regulate inflammatory responses. Each cytokine pattern inhibits production of the opposing pattern. Lymphocytes from inflamed intestine due to Crohn's disease secrete a Th1 pattern of cytokines. Crohn's disease is most prevalent in highly industrialized countries with temperate climates. It occurs rarely in tropical third world countries with poor sanitation. We propose that exposure to an environmental agent predisposes individuals to Crohn's disease. Parasitic worms (helminths) are common in tropical climates and in populations subject to crowding and poor sanitation. Children are most subject to helminthic colonization. Many helminths live within or migrate through the human gut where they interact with the mucosal immune system. The host mounts a mucosal response that includes Th2 cytokine production limiting helminthic colonization. Helminths and their eggs probably are the most potent stimulators of mucosal Th2 responses. The Th2 response provoked by parasitic worms can modulate immune reactions to unrelated parasitic, bacterial, and viral infections. Many people in developed countries now live in increasingly hygienic environments, avoiding exposure to helminths. Perhaps failure to acquire these parasites and experience mucosal Th2 conditioning predisposes to Crohn's disease, which is an overly active Th1 inflammation.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish

Growing numbers of studies have shown that circular RNAs (circRNAs) can function as regulatory factors to regulate the innate immune response, cell proliferation, cell migration, and other important processes in mammals. However, the function and regulatory mechanism of circRNAs in lower vertebrates are still unclear. Here, we discovered a novel circRNA derived from the gene encoding Bcl-2-like protein 1 (BCL2L1) gene, named circBCL2L1, which was related to the innate immune responses in teleost fish. Results indicated that circBCL2L1 played essential roles in host antiviral immunity and antibacterial immunity. Our study also identified a microRNA, miR-30c-3-3p, which could inhibit the innate immune response by targeting inflammatory mediator TRAF6. And TRAF6 is a key signal transduction factor in innate immune response mediated by TLRs. Moreover, we also found that the antiviral and antibacterial effects inhibited by miR-30c-3-3p could be reversed with the expression of circBCL2L1. Our data revealed that circBCL2L1 functioned as a competing endogenous RNA (ceRNA) of TRAF6 by competing for binding with miR-30c-3-3p, leading to activation of the NF-κB/IRF3 inflammatory pathway and then enhancing the innate immune responses. Our results suggest that circRNAs can play an important role in the innate immune response of teleost fish.     

NK and NKT cell functions in immunosenescence

Immunosenescence is defined as the state of dysregulated immune function that contributes to the increased susceptibility to infection, cancer and autoimmune diseases observed in old organisms, including humans. However, dysregulations in the immune functions are normally counterbalanced by continuous adaptation of the body to the deteriorations that occur over time. These adaptive changes are likely to occur in healthy human centenarians. Both innate (natural) and adaptive (acquired) immune responses decline with advancing age. Natural killer (NK) and natural killer T (NKT) cells represent the best model to describe innate and adaptive immune response in aging. NK and NKT cell cytotoxicity decreases in aging as well as interferon-gamma (IFN-gamma) production by both activated cell types. Their innate and acquired immune responses are preserved in very old age. However, NKT cells bearing T-cell receptor (TCR) gammadelta also display an increased cytotoxicity and IFN-gamma production in very old age. This fact suggests that NKT cells bearing TCRgammadelta are more involved in maintaining innate and adaptive immune response in aging leading to successful aging. The role played by the neuroendocrine-immune network and by nutritional factors, such as zinc, in maintaining NK and NKT cell functions in aging is discussed.     

Dominant-negative TLR5 polymorphism reduces adaptive immune response to flagellin and negatively associates with Crohn's disease

Crohn's disease (CD) is associated with elevated adaptive immunity to commensal microbes, with flagellin being a dominant antigen. In light of heightened awareness of the importance of innate immunity in regulating adaptive immunity and ambiguity as to the role of CD-associated immune responses in CD pathophysiology, we sought to determine whether natural acquisition of immune responses to flagellin were regulated by the innate immune flagellin receptor toll-like receptor 5 (TLR5) and determine whether persons carrying a recently defined common dominant-negative TLR5 polymorphism (TLR5-stop) might be protected from developing CD. Carriage rates of a recently defined dominant-negative TLR5 polymorphism (TLR5-stop) and levels of serum immunoreactivity to bacterial products were measured in inflammatory bowel disease patients, first-degree relatives, and unrelated controls. We observed that, in healthy subjects, persons carrying TLR5-stop had significantly lower levels of flagellin-specific IgG and IgA but had similar levels of total and LPS-specific Ig. Moreover, we observed that, among Jewish subjects, the carriage rate of TLR5-stop (in heterozygous state) was significantly less in CD patients, but not ulcerative colitis (UC) patients, compared with unaffected relatives and unrelated controls (5.4, 0.9, 6.0, and 6.5% for unaffected relatives, CD, UC, and unrelated Jewish controls, respectively, n = 296, 215, 185, and 416, respectively; P = 0.037 by likelihood calculation for CD vs. controls), indicating that TLR5-stop can protect persons of Jewish ethnicity against CD. We did not observe a significant association of TLR5-stop with CD in a non-Jewish cohort (11.1, 10.4, and 11.7% for unaffected relatives, CD, and UC, respectively; n = 841, 543, and 300 for unaffected relatives, respectively). These results demonstrate that natural acquisition of immune responses to flagellin are regulated by TLR5 and suggest that immune responses to flagellin are not merely associated with CD but rather promote the pathogenic response.     

Insights into innate immune activation via PS-ASO–protein–TLR9 interactions

Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary—in part—as a function of 2′modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO–protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.     

Innate immunity to Paracoccidioides brasiliensis infection

Innate immunity is based in pre-existing elements of the immune system that directly interact with all types of microbes leading to their destruction or growth inhibition. Several elements of this early defense mechanism act in concert to control initial pathogen growth and have profound effect on the adaptative immune response that further develops. Although most studies in paracoccidioidomycosis have been dedicated to understand cellular and humoral immune responses, innate immunity remains poorly defined. Hence, the main purpose of this review is to present and discuss some mechanisms of innate immunity developed by resistant and susceptible mice to Paracoccidioides brasiliensis infection, trying to understand how this initial host-pathogen interface interferes with the protective or deleterious adaptative immune response that will dictate disease outcome. An analysis of some mechanisms and mediators of innate immunity such as the activation of complement proteins, the microbicidal activity of natural killer cells and phagocytes, the production of inflammatory eicosanoids, cytokines, and chemokines among others, is presented trying to show the important role played by innate immunity in the host response to P. brasiliensis infection.     

Modeling and Dynamical Analysis of Virus-Triggered Innate Immune Signaling Pathways

The investigation of the dynamics and regulation of virus-triggered innate immune signaling pathways at a system level will enable comprehensive analysis of the complex interactions that maintain the delicate balance between resistance to infection and viral disease. In this study, we developed a delayed mathematical model to describe the virus-induced interferon (IFN) signaling process by considering several key players in the innate immune response. Using dynamic analysis and numerical simulation, we evaluated the following predictions regarding the antiviral responses: (1) When the replication ratio of virus is less than 1, the infectious virus will be eliminated by the immune system’s defenses regardless of how the time delays are changed. (2) The IFN positive feedback regulation enhances the stability of the innate immune response and causes the immune system to present the bistability phenomenon. (3) The appropriate duration of viral replication and IFN feedback processes stabilizes the innate immune response. The predictions from the model were confirmed by monitoring the virus titer and IFN expression in infected cells. The results suggest that the balance between viral replication and IFN-induced feedback regulation coordinates the dynamical behavior of virus-triggered signaling and antiviral responses. This work will help clarify the mechanisms of the virus-induced innate immune response at a system level and provide instruction for further biological experiments.     

Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion

Human cytomegalovirus (CMV) remains the major infectious cause of birth defects as well as an important opportunistic pathogen. Individuals infected with CMV mount a strong immune response that suppresses persistent viral replication and maintains life-long latency. Loss of immune control opens the way to virus reactivation and disease. The large number of immunomodulatory functions encoded by CMV increases the efficiency of infection, dissemination, reactivation and persistent infection in hosts with intact immune systems and could contribute to virulence in immunocompromised hosts. These functions modulate both the innate and adaptive arms of the immune response and appear to target cellular rather than humoral responses preferentially. CMV encodes a diverse arsenal of proteins focused on altering and/or mimicking: (1) classical and non-classical major histocompatibility complex (MHC) protein function; (2) leukocyte migration, activation and cytokine responses; and (3) host cell susceptibility to apoptosis. Evidence that the host evolves mechanisms to counteract virus immune modulation is also accumulating. Although immune evasion is certainly one clear goal of the virus, the pro-inflammatory impact of certain viral functions suggests that increased inflammation benefits viral dissemination. The ability of such viral functions to successfully 'face off' against the host immune system ensures the success of this pathogen in the human population and could provide key insights into disease mechanisms.     

Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus

Microglia are resident central nervous system (CNS) macrophages. Theiler's murine encephalomyelitis virus (TMEV) infection of SJL/J mice causes persistent infection of CNS microglia, leading to the development of a chronic-progressive CD4(+) T-cell-mediated autoimmune demyelinating disease. We asked if TMEV infection of microglia activates their innate immune functions and/or activates their ability to serve as antigen-presenting cells for activation of T-cell responses to virus and endogenous myelin epitopes. The results indicate that microglia lines can be persistently infected with TMEV and that infection significantly upregulates the expression of cytokines involved in innate immunity (tumor necrosis factor alpha, interleukin-6 [IL-6], IL-18, and, most importantly, type I interferons) along with upregulation of major histocompatibility complex class II, IL-12, and various costimulatory molecules (B7-1, B7-2, CD40, and ICAM-1). Most significantly, TMEV-infected microglia were able to efficiently process and present both endogenous virus epitopes and exogenous myelin epitopes to inflammatory CD4(+) Th1 cells. Thus, TMEV infection of microglia activates these cells to initiate an innate immune response which may lead to the activation of naive and memory virus- and myelin-specific adaptive immune responses within the CNS.