Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 ^−/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.     

肝脏固有免疫系统对肠源性感染的免疫应答与免疫耐受机制

肝特殊的解剖结构及生理特征使其成为暴露肠源性抗原的主要器官。由于肝具有独特的固有免疫系统,在正常情况下,肝分布多种致耐受的抗原提呈细胞,对持续性表达或递呈于肝的肠源性抗原物质,诱发针对该抗原的系统性免疫耐受,避免肝受到不必要的免疫损伤。当炎症发生及肝脏固有免疫系统活化时,则通过免疫效应细胞及免疫效应因子对肠源性病原体发挥强烈地免疫应答以控制感染。该过程形成机制的研究对肝功能的理解及肝性疾病的预防与治疗至关重要。本文就肝固有免疫系统对肠源性感染的免疫应答与免疫耐受形成机制作一综述。     

The Thermodynamic Basis for Viral RNA Detection by the RIG-I Innate Immune Sensor

RIG-I is a cytoplasmic surveillance protein that contributes to the earliest stages of the vertebrate innate immune response. The protein specifically recognizes 5′-triphosphorylated RNA structures that are released into the cell by viruses, such as influenza and hepatitis C. To understand the energetic basis for viral RNA recognition by RIG-I, we studied the binding of RIG-I domain variants to a family of dsRNA ligands. Thermodynamic analysis revealed that the isolated RIG-I domains each make important contributions to affinity and that they interact using different strategies. Covalent linkage between the domains enhances RNA ligand specificity while reducing overall binding affinity, thereby providing a mechanism for discriminating virus from host RNA.     

Adaptation to Host-Specific Bacterial Pathogens Drives Rapid Evolution of a Human Innate Immune Receptor

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The Role of Copper and Zinc Toxicity in Innate Immune Defense against Bacterial Pathogens

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.     

The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance

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Comparative Genomics of the Waterfowl Innate Immune System

Animal species differ considerably in their ability to fight off infections. Finding the genetic basis of these differences is not easy, as the immune response is comprised of a complex network of proteins that interact with one another to defend the body against infection. Here, we used population- and comparative genomics to study the evolutionary forces acting on the innate immune system in natural hosts of the avian influenza virus (AIV). For this purpose, we used a combination of hybrid capture, next- generation sequencing and published genomes to examine genetic diversity, divergence, and signatures of selection in 127 innate immune genes at a micro- and macroevolutionary time scale in 26 species of waterfowl. We show across multiple immune pathways (AIV-, toll-like-, and RIG-I -like receptors signalling pathways) that genes involved genes in pathogen detection (i.e., toll-like receptors) and direct pathogen inhibition (i.e., antimicrobial peptides and interferon-stimulated genes), as well as host proteins targeted by viral antagonist proteins (i.e., mitochondrial antiviral-signaling protein, [MAVS]) are more likely to be polymorphic, genetically divergent, and under positive selection than other innate immune genes. Our results demonstrate that selective forces vary across innate immune signaling signalling pathways in waterfowl, and we present candidate genes that may contribute to differences in susceptibility and resistance to infectious diseases in wild birds, and that may be manipulated by viruses. Our findings improve our understanding of the interplay between host genetics and pathogens, and offer the opportunity for new insights into pathogenesis and potential drug targets.     

Hyperlipidemia Impaired Innate Immune Response to Periodontal Pathogen Porphyromonas gingivalis in Apolipoprotein E Knockout Mice

A finely-tuned innate immune response plays a pivotal role in protecting host against bacterial invasion during periodontal disease progression. Hyperlipidemia has been suggested to exacerbate periodontal health condition. However, the underlying mechanism has not been addressed. In the present study, we investigated the effect of hyperlipidemia on innate immune responses to periodontal pathogen Porphyromonas gingivalis infection. Apolipoprotein E-deficient and wild-type mice at the age of 20 weeks were used for the study. Peritoneal macrophages were isolated and subsequently used for the study of viable P. gingivalis infection. ApoE^−/− mice demonstrated inhibited iNOS production and impaired clearance of P. gingivalis in vitro and in vivo; furthermore, ApoE^−/− mice displayed disrupted cytokine production pattern in response to P. gingivalis, with a decreased production of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein-1. Microarray data demonstrated that Toll-like receptor (TLR) and NOD-like receptor (NLR) pathway were altered in ApoE^−/− mice macrophages; further analysis of pattern recognition receptors (PRRs) demonstrated that expression of triggering receptors on myeloid cells-1 (TREM-1), an amplifier of the TLR and NLR pathway, was decreased in ApoE^−/− mice macrophages, leading to decreased recruitment of NF-κB onto the promoters of the TNF-α and IL-6. Our data suggest that in ApoE^−/− mice hyperlipidemia disrupts the expression of PRRs, and cripples the host’s capability to generate sufficient innate immune response to P. gingivalis, which may facilitate immune evasion, subgingival colonization and establishment of P. gingivalis in the periodontal niche.     

Exploiting Gene-Expression Deconvolution to Probe the Genetics of the Immune System

Sequence variation can affect the physiological state of the immune system. Major experimental efforts targeted at understanding the genetic control of the abundance of immune cell subpopulations. However, these studies are typically focused on a limited number of immune cell types, mainly due to the use of relatively low throughput cell-sorting technologies. Here we present an algorithm that can reveal the genetic basis of inter-individual variation in the abundance of immune cell types using only gene expression and genotyping measurements as input. Our algorithm predicts the abundance of immune cell subpopulations based on the RNA levels of informative marker genes within a complex tissue, and then provides the genetic control on these predicted immune traits as output. A key feature of the approach is the integration of predictions from various sets of marker genes and refinement of these sets to avoid spurious signals. Our evaluation of both synthetic and real biological data shows the significant benefits of the new approach. Our method, VoCAL, is implemented in the freely available R package ComICS.     

天然免疫系统新成员Akirin的研究进展

天然免疫系统是多细胞动物抵御细菌感染的第一道防线。Akirin是新近发现于果蝇中的天然免疫系统新成员,它在果蝇免疫缺陷(Imd)通路中发挥重要作用。Akirin同源基因广泛存在于从低等多细胞生物到高等脊椎动物中,进化上高度保守。已有的研究表明:Akirin在果蝇Imd通路和脊椎动物TLR通路下游,与NF-κB家族转录因子形成复合物,参与调控免疫相关靶基因的转录,是天然免疫调控机制中不可或缺的转录因子,其过表达或缺失直接影响动物对细菌的防御能力。近年来,Akirin在相关信号通路中的功能研究取得重大进展。该文对Akirin的结构、参与天然免疫的分子调控机制以及基因进化等方面进行综述。     

Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses

Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.One of the most significant evolutionary features in eukaryotes is the appearance of a membrane system to separate enzymatic reactions and to provide scaffolds for signal transduction. The small genome of viruses requires that they use the host''s cellular machinery, especially host intracellular membranes, to assemble their replication complexes and to complete their replication cycle (1). Therefore, it is not surprising to find that the eukaryotic membrane system is also involved in antiviral responses (2–4).Subcellular organelles with extensive membrane systems include the endoplasmic reticulum (ER)¹, mitochondria, endosomes and peroxisomes. Despite their well-recognized functions in cell metabolism, these organelles and their related membranes have been identified in recent years as important innate immune platforms (5). The ER is a key organelle for maintaining cellular homeostasis. Several recent studies have linked the ER to antiviral immune responses and have elucidated the related mechanisms (6–10). The mitochondria serve mainly as the power plants of eukaryotic cells but also participate in numerous crucial cellular processes, such as calcium homeostasis, apoptosis and aging (11–13). In recent years, the mitochondria and mitochondrial-associated membranes (MAM) have also emerged as fundamental hubs for innate antiviral immunity. Several important antiviral immune-related proteins, such as VISA (also referred to as MAVS/CARDIF/IPS-1) and MITA (also referred to as STING), have been found to localize to the mitochondrial membrane (14–19). Peroxisomes are monolayer-membrane organelles present in nearly all types of human cells, with a particularly high abundance in hepatocytes and nephrocytes, which are involved in various oxidative enzymatic reactions. It has been reported that the peroxisomal-associated protein VISA induces a rapid interferon (IFN)-independent expression that provides short-term protection, whereas the mitochondrial-associated VISA activates an IFN-dependent signaling pathway with delayed kinetics (20). The peroxisomal VISA can activates IRF1-mediated IFN-λ production (21). The endosomes, although known as players in cellular endocytosis and vascular transport, have functions that extend to the antigen presentation of major histocompatibility complex (MHC) class I, MHC class II and CD1 molecules of the adaptive immune system, as well as pattern recognition of innate immune-related receptors, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) (22, 23).Subcellular fractionation is an effective experimental strategy for isolating/enriching specific organelles. Compared with whole-cell-based proteomic analysis, combining the isolation of subcellular components with mass spectrometry-based proteomic analysis is advantageous not only for characterizing low abundance proteins but also for monitoring protein abundance changes at the organelle level. To systematically analyze the role of peroxisomal-related proteins in innate immune responses, we used a modified two-step gradient centrifugation method to enrich the peroxisomes from cells with or without SeV infection, followed by a quantitative proteomic analysis. A total of 2946 proteins were quantified among which 311 proteins were found to be significantly changed by SeV infection. A statistical enrichment test was used to reveal that 13 protein groups were changed significantly (p < 0.05) compared with the entire protein list. Cell cycle-related proteins and membrane attack complex (MAC)-related proteins were down-regulated, which may facilitate virus survival and replication in host cells. Luciferase reporter assays were performed to further screen for the significantly changed proteins that could affect SeV-induced activation of the type I IFN signaling pathway. Not only does our data provide new and unbiased protein-level information regarding viral infection processes, we also provide direct evidence for the involvement of two proteins (CALU and LGALS3BP) as potential negative regulators in the virus-triggered induction of the type I IFNs.     

细菌病原体突破天然免疫系统的策略

近10年来,天然免疫和病原微生物两个相关领域研究取得了巨大进展。面对功能完备的天然免疫系统,一些病原微生物感染宿主的机制逐步进化了,并且病原体通过此进化避免被识别,促进或避免炎症的产生。事实证明,病原体不仅能在天然免疫反应中幸存,而且还能利用天然免疫反应来提高它的致病性。     

Cyclic Nucleotide Gated Channels and Ca2+-Mediated Signal Transduction During Plant Innate Immune Response to Pathogens

Transitory perturbations in the level of cytosolic Ca²⁺ are well known to be involved in numerous cell signaling pathways in both plant and animal systems. However, not much is known at present about the molecular identity of plant plasma membrane Ca²⁺ conducting ion channels or their specific roles in signal transduction cascades. A recent study employing genetic approaches as well as patch clamp electrophysiological analysis of channel currents has provided the first such direct evidence linking a specific gene product with inward Ca²⁺ currents across the plant cell membrane. This work identified Ca²⁺ permeation through (Arabidopsis) cyclic nucleotide gated channel isoform 2 (CNGC2) as contributing to the plant innate immunity signaling cascade initiated upon perception of a pathogen. Here, we expand on the implications of CNGC2 mediated cytosolic Ca²⁺ elevations associated with plant cell response to pathogen recognition, and propose some additional steps that may be involved in the innate immunity signal cascade.Key Words: calcium, CNGC, hypersensitive response, nitric oxide, plant innate immunity, plant ion channel, reactive oxygen species     

The Genetic and Molecular Basis of Plant Resistance to Pathogens

Plant pathogens have evolved numerous strategies to obtain nutritive materials from their host,and plants in turn have evolved the preformed physical and chemical barriers as well as sophisticated two-tiered immune system to combat pathogen attacks.Genetically, plant resistance to pathogens can be divided into qualitative and quantitative disease resistance,conditioned by major gene(s) and multiple genes with minor effects,respectively.Qualitative disease resistance has been mostly detected in plant defense against biotrophic pathogens,whereas quantitative disease resistance is involved in defense response to all plant pathogens,from biotrophs,hemibiotrophs to necrotrophs.Plant resistance is achieved through interception of pathogen-derived effectors and elicitation of defense response.In recent years,great progress has been made related to the molecular basis underlying host-pathogen interactions.In this review,we would like to provide an update on genetic and molecular aspects of plant resistance to pathogens.     

The Mathematical Basis of Mendelian Genetics

In a climate where increasing numbers of students are encouraged to pursue post-secondary education, the level of preparedness students have for college-level coursework is not far from the minds of all educators, especially high school teachers. Specifically within the biological sciences, introductory biology classes often serve as the gatekeeper or a pre-requisite for subsequent coursework in those fields and pre-professional programmes (eg pre-medicine or pre-veterinarian). Thus, how helpful high school science and mathematics experiences are in preparing students for their introductory biology classes is important and relevant for teachers, science educators and policy makers alike. This quantitative study looked at the association between students' high school science and mathematics experiences with introductory college biology performance. Using a nationally representative sample of US students (n?=?2667) enrolled in 33 introductory college biology courses, a multi-level statistical model was developed to analyse the association between high school educational experiences and the final course grade in introductory biology courses. Advanced high school science and mathematics coursework, an emphasis on a deep conceptual understanding of biology concepts and a prior knowledge of concepts addressed in well-structured laboratory investigations are all positively associated with students' achievement in introductory college biology.     

Anti-HIV-1 Activity of Elafin Depends on Its Nuclear Localization and Altered Innate Immune Activation in Female Genital Epithelial Cells

Elafin (E) and its precursor trappin-2 (Tr) are alarm antiproteases with antimicrobial and immunomodulatory activities. Tr and E (Tr/E) have been associated with HIV-1 resistance. We recently showed that Tr/E reduced IL-8 secretion and NF-κB activation in response to a mimic of viral dsRNA and contributed to anti-HIV activity of cervicovaginal lavage fluid (CVL) of HIV-resistant (HIV-R) commercial sex workers (CSWs). Additionally, Tr, and more so E, were found to inhibit attachment/entry and transcytosis of HIV-1 in human endometrial HEC-1A cells, acting through virus or cells. Given their immunomodulatory activity, we hypothesized that Tr/E could exert anti-HIV-1 activity at multiple levels. Here, using tagged and untagged Tr/E proteins, we comparatively evaluated their protease inhibitory, anti-HIV-1, and immunomodulatory activities, and cellular distribution. E appeared to function as an autocrine/paracrine factor in HEC-1A cells, and anti-HIV-1 activity of E depended on its unmodified N-terminus and altered cellular innate activation, but not its antiprotease activity. Specifically, exogenously added N-terminus-unmodified E was able to enter the nucleus and to reduce viral attachment/entry and transcytosis, preferentially affecting R5-HIV-1_ADA, but not X4-HIV-1_IIIB. Further, anti-HIV-1 activity of E was associated with significantly decreased HIV-1-triggered IL-8 release, attenuated NF-κB/p65 nuclear translocation, and significantly modulated mRNA expression of innate sensors TLR3 and RIG-I in HEC-1A cells. Most importantly, we found that elevated Tr/E in CVLs of HIV-R CSWs were associated with lower mRNA levels of TLRs 2, 3, 4 and RIG-I in the genital ECs from this cohort, suggesting a link between Tr/E, HIV-1 resistance and modulated innate viral recognition in the female genital mucosa. Collectively, our data indicate that unmodified N-terminus is critical for intranuclear localization and anti-HIV-1 activity of E. We also propose that E-mediated altered cellular innate activation most likely contributes to the HIV-R phenotype of these subjects.     

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling.     

Quantitative Analysis of Genera Endemic to China

Chinese flora with many endemic elements is highly important in the world’s flora. According to recent statistics there are about 196 genera of spermatophytes, being 6.5% of total Chinese genera. These endemic genera comprising 377 species belong to 68 families, among which the Gesneriaceae (28 genera), Umbelliferae (13), Compositae (13), Orchidaceae (12) and Labiatae (10) are predominant. The tropical type containing 24 families and 80 genera is dominant. After it follows the temperate type with 23 families and 50 genera. There are also 4 families endemic to China, i.e. Ginkgoaceae, Bretschneideraceae, Eucommiaceae and Davidiaceae. It shows that genera endemic to China are obviously related to the tropical and temperate flora in essence. The endemic monotypic genera (139) and endemic obligotypic genera (48) combined make up more than 95% of the total number of genera endemic to China. Phylogenetically more than half of them are ancient or primitive. The life forms of all endemic genera are also diverse. Herbs, especially perennial herbs, prevail with the proportion of about 62%, and trees and shrubs are the next, with 33%, and the rest are lianas. Based upon the calculated number of genera endemic to China in each province and the similarity coefficents between any two provinces, some conclusions may be drawn as follows: Yunnan and Sichuan Provinces combined are the distribution centre of genera endemic to China and may be their original or differentiation area, because numerous endemic genera, including various groups, exist in these two provinces. The second is Guizhou where there are 62 endemic genera. Others form a declining order, south China, central China and east China. But towards the north China endemic genera decrease gradually, and the Qinling Range is an important distributional limit. The largest simitarity coefficient, over 50%, appears between Shaanxi and Gansu probably because of the Qinling Range linking these two provinces. But between any other two provinces it is less than 30% and it is generaly larger between two south provinces than between two north provinces. These characteristics mentioned above are correlated with topography and climate, and they may be resulted from the diversification in geography and climatic influence for a long time.     

Molecular Population Genetics of Drosophila Immune System Genes

A striking aspect of many vertebrate immune system genes is the exceptionally high level of polymorphism they harbor. A convincing case can be made that this polymorphism is driven by the diversity of pathogens that face selective pressures to evade attack by the host immune system. Different organisms accomplish a defense against diverse pathogens through mechanisms that differ widely in their requirements for specific recognition. It has recently been shown that innate defense mechanisms, which use proteins with broad-spectrum bactericidal properties, are common to both primitive and advanced organisms. In this study we characterize DNA sequence variation in six pathogen defense genes of Drosophila melanogaster and D. mauritiana, including Andropin; cecropin genes CecA1, CecA2, CecB, and CecC; and Diptericin. The necessity for protection against diverse pathogens, which themselves may evolve resistance to insect defenses, motivates a population-level analysis. Estimates of variation levels show that the genes are not exceptionally polymorphic, but Andropin and Diptericin have patterns of variation that differ significantly from neutrality. Patterns of interpopulation and interspecific differentiation also reveal differences among the genes in evolutionary forces.