Host-microbe interactions: innate pattern recognition of fungal pathogens

The recognition of fungi is mediated by germline pattern recognition receptors (PRRs) such as Toll-like receptors and lectin receptors that interact with conserved structures of the microorganisms, the pathogen-associated molecular patterns (PAMPs). Subsequently, PRRs activate intracellular signals that collaborate for the efficient activation of the host defense. The specificity of these responses is achieved through the activation of a particular mosaic of PRRs, that is determined by the available fungal PAMPs and the innate immune cells involved. This will determine a divergence of the final type of reaction, and in this way the innate host defense has the capability to deliver tailored responses to each pathogen.     

Salivary non‐apoptotic tumoral microvesicles: A potential progressive marker in oral cancer patients

Tumour cell‐secreted microvesicles (MVs) contribute immensely to tumour progression. However, the role of tumoral salivary MVs in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we elucidated the role of non‐apoptotic salivary tumoral MVs in OSCC development, especially relating to the migration ability. We purified and compared non‐apoptotic salivary tumoral MVs from 63 OSCC patients and orthotopic OSCC mice model. Next, we compared the protein difference between apoptotic and non‐apoptotic MVs by Western blot, proteomics and flow cytometry from saliva and CAL27 cells. Finally, we collected the non‐apoptotic MVs and co‐cultured with normal oral epithelial cells, the migration ability was examined by wound healing assay and Western blot assay. Our results indicated that the levels of non‐apoptotic tumoral S‐MVs were significantly higher in OSCC patients with T3 to T4 stages than in patients with T1 to T2 stages or healthy donors. In OSCC mice model, we found elevations of non‐apoptotic tumoral MVs associated with tumoral volume. EGFR overexpression increased the generation of non‐apoptotic tumoral MVs which could significantly promote normal epithelial cell migration. In conclusion, elevated levels of non‐apoptotic tumoral S‐MVs are associated with clinicopathologic features of OSCC patients, implying that non‐apoptotic tumoral S‐MVs are a potential progressive marker of OSCC.     

Innate immunity and cardiomyocytes in ischemic heart disease

Myocardial ischemia/reperfusion (I/R) is the most common cause of myocardial inflammation, which is primarily a manifestation of the innate immune responses. Innate immunity is activated when pattern recognition receptors (PRRs) respond to molecular patterns common to microbes and to danger signals expressed by injured or infected cells, so called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The expression of various PRRs in cardiomyocytes and the release of DAMPs from cardiomyocytes subjected to I/R injury, through active mechanisms as well as passive processes, enable cardiomyocytes to generate innate immune responses. Studies in isolated heart and cardiomyocytes have confirmed the inflammatory and functional effects of cardiac PRRs especially Toll-like receptors in response to I/R-derived DAMPs, such as heat shock proteins. This review addresses the active role of cardiomyocytes in mediating innate inflammatory responses to myocardial I/R. We propose that cardiomyocytes act as innate immune cells in myocardial I/R injury.     

Innate recognition of microbial-derived signals in immunity and inflammation

Microbes generate a vast array of different types of conserved structural components called pathogen-associated molecular patterns(PAMPs),which canbe recognized by cells of the innate immune system.This recognition of "nonself" signatures occurs through host pattern recognition receptors(PRRs),suggesting that microbial-derived signals are good targets for innate immunity to discriminate between self- and nonself.Such PAMP-PRR interactions trigger multiple but distinct downstream signaling cascades,subsequently leading to production of proinflammatory cytokines and interferons that tailor immune responses to particular microbes.Aberrant PRR signals have been associated with various inflammatory diseases and fine regulation of PRR signaling is essential for avoiding excessive inflammatory immune responses and maintaining immune homeostasis.In this review we summarize the ligands and signal transduction pathways of PRRs and highlight recent progress of the mechanisms involved in microbe-specific innate immune recognition during immune responses and inflammation,which may provide new targets for therapeutic intervention to the inflammatory disorders.     

Plant pattern-recognition receptors controlling innate immunity

Plants are exposed to numerous potential pathogenic microbes. To counter the threat, plants have evolved diverse patternrecognition receptors(PRRs), which are receptor kinases(RKs) and receptor proteins(RPs) specialized to detect conserved pathogen/microbe-associated molecular patterns(PAMPs/MAMPs). Although only a handful of RKs and RPs are known PRRs,they belong to the receptor-like kinase(RLK) and receptor-like protein(RLP) superfamilies that undergo lineage-specific expansion, suggesting that many of these RLKs and RLPs are potential PRRs. Analyses of existing PRRs have uncovered ligand-induced RLK-RK or RLK-RP oligomerization as a common mechanism for immune activation. PRRs can recruit additional components to form dynamic receptor complexes, which mediate specific cellular responses. Detailed analyses of these components are shedding light on molecular mechanisms underlying the regulation of PRR activity and downstream signaling.     

Erratum to: Plant pattern-recognition receptors controlling innate immunity

Plants are exposed to numerous potential pathogenic microbes. To counter the threat, plants have evolved diverse patternrecognition receptors(PRRs), which are receptor kinases(RKs) and receptor proteins(RPs) specialized to detect conserved pathogen/microbe-associated molecular patterns(PAMPs/MAMPs). Although only a handful of RKs and RPs are known PRRs, they belong to the receptor-like kinase(RLK) and receptor-like protein(RLP) superfamilies that undergo lineage- specific expansion, suggesting that many of these RLKs and RLPs are potential PRRs. Analyses of existing PRRs have uncovered ligand-induced RLK-RK or RLK-RP oligomerization as a common mechanism for immune activation. PRRs can recruit additional components to form dynamic receptor complexes, which mediate specific cellular responses. Detailed analyses of these components are shedding light on molecular mechanisms underlying the regulation of PRR activity and downstream signaling.     

Innate immunity: structure and function of TLRs

The innate immune system provides the first line of defence against infection. Through a limited number of germline-encoded receptors called pattern recognition receptors (PRRs), innate cells recognize and are activated by highly conserved structures expressed by large group of microorganisms called pathogen-associated molecular patterns (PAMPs). PRRs are involved either in recognition (scavenger receptors, C-type lectins) or in cell activation (Toll-like receptors or TLR, helicases and NOD molecules). TLRs play a pivotal role in cell activation in response to PAMPs. TLR are type I transmembrane proteins characterized by an intracellular Toll/IL 1 receptor homology domain that are expressed by innate immune cells (dendritic cells, macrophages, NK cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). In all the cell types analyzed, TLR agonists, alone or in combination with costimulatory molecules, induce cell activation. The crucial role played by TLR in immune cell activation has been detailed in dendritic cells. A TLR-dependent activation of dendritic cells is required to induce their maturation and migration to regional lymph nodes and to activate na?ve T cells. The ability of different cell types to respond to TLR agonists is related to the pattern of expression of the TLRs and its regulation as well as their intracellular localization. Recent studies suggest that the nature of the endocytic and signaling receptors engaged by PAMPs may determine the nature of the immune response generated against the microbial molecules, highlighting the role of TLRs as molecular interfaces between innate and adaptive immunity. In this review are summarized the main biological properties of the TLR molecules.     

New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.     

Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs)

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

Toll样受体识别机制及其生物学意义

Toll样受体介导的信号转导通路在对抗外来病原体的天然免疫应答中起重要作用。Toll样受体是一个天然模板识别受体家族,能识别固有性模板(微生物和哺乳动物所共有的病原相联的分子模板PAMPs)。Toll样受体通过巨噬细胞和其他免疫细胞来识别,其中TLR4识别内毒素、TLR2识别肽聚糖、TLR9识别细菌DNA、TLR5识别鞭毛蛋白、TLR3识别双链RNA等。本探讨了多种Toll受体家族成员在动物体内识别机理及功能,概述了其应用研究进展。     

抗病毒感染固有免疫应答的信号转导

入侵病毒的探知和适应性免疫应答启动均依靠固有免疫系统。三种模式识别受体(PRRs)在宿主防御系统第一线占据极其重要地位:Toll样受体、维甲酸诱导基因I样受体、核苷酸结合寡聚化结构域样受体。PRRs识别病原相关分子模式(PAMP)或危险信号分子模式(DAMPs)启动和调节固有免疫和适应性免疫应答。每种PRR都有单独的识别配体和细胞定位。激活的PRRs将信号分子传递给其配体分子(MyD88,TRIF,IRAK,IPS-1),配体活化后作为信使激活信号途径下游激酶(IKK复合物,MAPKs,TBK1,RIP-1)和转录因子(NF-κB,AP-1,IRF3),最终产生细胞因子、趋化因子、促炎细胞因子和I型干扰素。本文重点讨论PRRs信号通路及该领域取得的成果,以期为人类健康和免疫疾病防治提供策略。     

Receptor-like cytoplasmic kinases are pivotal components in pattern recognition receptor-mediated signaling in plant immunity

Innate immunity is generally initiated with recognition of conserved pathogen-associated molecular patterns (PAMPs). PAMPs are perceived by pattern recognition receptors (PRRs), leading to activation of a series of immune responses, including the expression of defense genes, ROS production and activation of MAP kinase. Recent progress has indicated that receptor-like cytoplasmic kinases (RLCKs) are directly activated by ligand- activated PRRs and initiate pattern -triggered immunity (PTI) in both Arabidopsis and rice. To suppress PTI, pathogens inhibit the RLCKs by many types of effectors, including AvrAC, AvrPphB and Xoo1488. In this review, we summarize recent advances in RLCK-mediated PTI in plants.     

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples.     

SGT1 is not required for plant LRR-RLK-mediated immunity

Plant immune signalling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by pattern-recognition receptors (PRRs) and nucleotide-binding and leucine-rich repeat domain-containing receptors (NLRs), which often share cellular components and downstream responses. Many PRRs are leucine-rich repeat receptor-like kinases (LRR-RLKs), which mostly perceive proteinaceous PAMPs. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for immune responses mediated by several LRR-RLKs in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or antibacterial immunity. We therefore conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.     

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase ‘eat me'' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp–annexin A5, RGD–anxA5) that has gained the function to interact with α_vβ₃ receptors of the phagocyte. We describe design and characterization of RGD–anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD–anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD–anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD–anxA5 is a promising therapeutic agent.     

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14⁺ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P₃ antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP⁺ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP⁺ cells represented more significant populations in tissues of Cd14^−/− than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14⁺ phagocytes.Subject terms: Phospholipids, Cell death and immune response     

Macrophage recognition of ICAM-3 on apoptotic leukocytes.

Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.     

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.