Diverse lectin repertoires in tunicates mediate broad recognition and effector innate immune responses

It is widely recognized that humoral and phagocyte-associated lectins constitute critical components of innate immunity in vertebrates and invertebrates. Their functions include not only self/non-self recognition but also engaging associated effector mechanisms, such as complement-mediated opsonization and killing of potential pathogens. One of the unresolved questions concerns the diversity in recognition capacity of the lectin repertoire, particularly in those organisms lacking adaptive immunity. In this paper, we discuss evidence suggesting that lectin repertoire in invertebrates and protochordates is highly diversified, and includes most of the lectin classes described so far in vertebrate species, as well as associated effector pathways.     

Role of autophagy in innate viral recognition

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes via Toll-like receptors (TLRs) upon endocytosis of virions. Yet, pDC responses to certain single-stranded RNA viruses occur only following live viral infection. In our recent study, we presented evidence that the recognition of such viruses by TLR7 requires autophagy. We speculate that the requirement for autophagy in viral recognition reflects the necessity for transportation of cytosolic viral replication intermediates into the lysosome where TLR7 is activated. In addition, autophagy was found to be required for pDCs to produce type I interferon (IFN) in response to both ssRNA and dsDNA viruses. These results indicated that autophagy plays a key role in mediating virus detection and IFNalpha secretion in pDCs, and suggest that cytosolic replication intermediates of ssRNA viruses serve as pathogen signatures recognized by TLR7.     

Evolutionary perspective on innate immune recognition

Analysis of human and Drosophila genomes demonstrates an ancient origin of innate immunity and the diversity of the mechanisms of innate immune recognition.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.     

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide‐binding leucine‐rich repeat (NLR) protein with a Toll‐like interleukin‐1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely‐related Nicotiana sylvestris and the distantly‐related beet plant (Beta vulgaris). Roq1 was found to co‐immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.     

Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA

RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.     

Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses

LPS binding protein (LBP) is an acute-phase protein synthesized predominantly in the liver of the mammalian host. It was first described to bind LPS of Gram-negative bacteria and transfer it via a CD14-enhanced mechanism to a receptor complex including TLR-4 and MD-2, initiating a signal transduction cascade leading to the release of proinflammatory cytokines. In recent studies, we found that LBP also mediates cytokine induction caused by compounds derived from Gram-positive bacteria, including lipoteichoic acid and peptidoglycan fragments. Lipoproteins and lipopeptides have repeatedly been shown to act as potent cytokine inducers, interacting with TLR-2, in synergy with TLR-1 or -6. In this study, we show that these compounds also interact with LBP and CD14. We used triacylated lipopeptides, corresponding to lipoproteins of Borrelia burgdorferi, mycobacteria, and Escherichia coli, as well as diacylated lipopeptides, corresponding to, e.g., 2-kDa macrophage activating lipopeptide of Mycoplasma spp. Activation of Chinese hamster ovary cells transfected with TLR-2 by both lipopeptides was enhanced by cotransfection of CD14. Responsiveness of human mononuclear cells to these compounds was greatly enhanced in the presence of human LBP. Binding of lipopeptides to LBP as well as competitive inhibition of this interaction by LPS was demonstrated in a microplate assay. Furthermore, we were able to show that LBP transfers lipopeptides to CD14 on human monocytes using FACS analysis. These results support that LBP is a pattern recognition receptor transferring a variety of bacterial ligands including the two major types of lipopeptides to CD14 present in different receptor complexes.     

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.     

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.     

Metabolic enzyme UAP1 mediates IRF3 pyrophosphorylation to facilitate innate immune response

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Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.     

Peptidoglycan recognition proteins of the innate immune system

Peptidoglycan (PGN) is the major component of bacterial cell walls and one of the main microbial products recognized by the innate immune system. PGN recognition is mediated by several families of pattern recognition molecules, including Toll-like receptors, nucleotide-binding oligomerization domain-containing proteins, and peptidoglycan recognition proteins (PGRPs). However, only the interaction of PGN with PGRPs, which are highly conserved from insects to mammals, has so far been characterized at the molecular level. Here, we describe recent structural studies of PGRPs that reveal the basis for PGN recognition and provide insights into the signal transduction and antibacterial activities of these innate immune proteins.     

Innate immune recognition of viral infection

Toll-like receptors (TLRs) are key molecules of the innate immune systems, which detect conserved structures found in a broad range of pathogens and triggers innate immune responses. A subset of TLRs recognize viral components and induce antiviral responses by producing type I interferons. Whereas TLR2 and TLR4 recognize viral components at the cell surface, TLR3, TLR7, TLR8 and TLR9 are exclusively expressed in endosomal compartments. After phagocytes internalize viruses or virus-infected apoptotic cells, viral nucleic acids are released in phagolysosomes and are recognized by these TLRs. Recent reports have shown that hosts also have a mechanism to detect replicating viruses in the cytoplasm in a TLR-independent manner. In this review, we focus on the viral recognition by innate immunity and the signaling pathways.     

Novel paradigms of innate immune sensing of viral infections

According to the existing paradigm, cellular recognition of viral infection is mediated by molecular patterns within the virus particle or produced during virus replication. However, there are various physical cellular changes indicative of infection that could also trigger innate antiviral responses. The type-I interferon response is rapidly engaged to limit viral infection and a number of studies have shown that the interferon response, or components of it, are induced by general perturbations to cellular processes. Virus entry requires membrane and cytoskeletal perturbation, and both membrane fusion or actin depolymerising agents alone are able to activate antiviral genes. Viruses cause cellular stress and change the cellular environment, and oxidative stress or endoplasmic reticulum stress will amplify antiviral signaling. Many of these responses converge on interferon regulatory factor 3, suggesting that it plays a crucial role in determining the degree to which the cell responds. This review highlights novel paradigms of viral recognition and speculates that viral infection is sensed as a danger signal.     

Vascular adhesion protein 1 mediates binding of immunotherapeutic effector cells to tumor endothelium

Tumor-infiltrating lymphocytes (TIL) can be used as an immunotherapeutic tool to treat cancer. Success of this therapy depends on the homing and killing capacity of in vitro-activated and -expanded TIL. Vascular adhesion protein 1 (VAP-1) is an endothelial molecule that mediates binding of lymphocytes to vessels of inflamed tissue. Here, we studied whether VAP-1 is involved in binding of TIL, lymphokine-activated killer (LAK) cells, and NK cells to vasculature of the cancer tissue. We demonstrated that VAP-1 is expressed on the endothelium of cancer vasculature. The intensity and number of positive vessels varied greatly between the individual specimens, but it did not correlate with the histological grade of the cancer. Using an in vitro adhesion assay we showed that VAP-1 mediates adhesion of TIL, LAK, and NK cells to cancer vasculature. Treatment of the tumor sections with anti-VAP-1 Abs diminished the number of adhesive cells by 60%. When binding of different effector cell types was compared, it was evident that different cancer tissues supported the adhesion of TIL to a variable extent and LAK cells were more adhesive than TIL and NK cells to tumor vasculature. These data suggest that VAP-1 is an important interplayer in the antitumor response. Thus, by up-regulating the expression of VAP-1 in tumor vasculature, it can be possible to improve the effectiveness of TIL therapy.     

Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin

Tracheal cytotoxin (TCT) was originally described as the minimal effector that was able to reproduce the cytotoxic response of Bordetella pertussis on ciliated epithelial cells. This molecule triggers pleiotropic effects such as immune stimulation or slow-wave sleep modulation. Further characterization identified TCT as a specific diaminopimelic acid (DAP)-containing muropeptide, GlcNAc-(anhydro)MurNAc-L-Ala-D-Glu-mesoDAP-D-Ala. Here, we show that the biological activity of TCT depends on Nod1, an intracellular sensor of bacterial peptidoglycan. However, Nod1-dependent detection of TCT was found to be host specific, as human Nod1 (hNod1) poorly detected TCT, whereas mouse Nod1 (mNod1) did so efficiently. More generally, hNod1 required a tripeptide (L-Ala-D-Glu-mesoDAP) for efficient sensing of peptidoglycan, whereas mNod1 detected a tetrapeptide structure (L-Ala-D-Glu-mesoDAP-D-Ala). In murine macrophages, TCT stimulated cytokine secretion and NO production through Nod1. Finally, in vivo, injection of the tetrapeptide structure in mice triggered a transient yet strong release of cytokines into the bloodstream and the maturation of macrophages, in a Nod1-dependent manner. This study thereby identifies Nod1 as the long sought after sensor of TCT in mammals.