Evasion of innate and adaptive immunity by flaviviruses

After a virus infects an animal, antiviral responses are generated that attempt to prevent dissemination. Interferons, antibody, complement, T and natural killer cells all contribute to the control and eradication of viral infections. Most flaviviruses, with the exception of some of the encephalitic viruses, cause acute disease and do not establish persistent infection. The outcome of flavivirus infection in an animal is determined by a balance between the speed of viral replication and spread, and the immune system response. Although many of the mechanistic details require further elucidation, flaviviruses have evolved specific tactics to evade the innate and adaptive immune response. A more thorough understanding of these principles could lead to improved models for viral pathogenesis and to strategies for the development of novel antiviral agents.     

Innate antifungal immunity of human eosinophils mediated by a beta 2 integrin, CD11b

Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile beta(2) integrin molecule, CD11b, to adhere to a major cell wall component, beta-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils' interaction with beta-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.     

Bacterial proteases and bacterial resistance against human innate immunity factors

The molecular and cell-mediated mechanisms that are developed by certain opportunistic and pathogenic bacteria and were obtained over the course of evolution to preserve resistance against principal components of human body innate immunity are summarized.     

Dendritic cells are host cells for mycobacteria in vivo that trigger innate and acquired immunity.

In the present study, we investigated in vivo the infection and APC functions of dendritic cells (DC) and macrophages (Mphi) after administration of live mycobacteria to mice. Experiments were conducted with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or a rBCG expressing a reporter Ag. Following infection of mice, DC and Mphi were purified and the presence of immunogenic peptide/MHC class II complexes was detected ex vivo on sorted cells, as was the secretion of IL-12 p40. We show in this study that DC is a host cell for mycobacteria, and we provide an in vivo detailed picture of the role of Mphi and DC in the mobilization of immunity during the early stages of a bacterial infection. Strikingly, BCG bacilli survive but remain stable in number in the DC leukocyte subset during the first 2 wk of infection. As Ag presentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria.     

CD11b is a calcium-dependent epitope in human neutrophils.

Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.     

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF‐κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC–Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.     

Evasion of human innate immunity without antagonizing TLR4 by mutant Salmonella enterica serovar Typhimurium having penta-acylated lipid A

Modification of a lipid A moiety in Gram-negative bacterial LPS to a less acylated form is thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. The contribution of less-acylated lipid A to interactions of whole bacterial cells with host cells (especially in humans) remains unclear. Mutant strains of Salmonella enterica serovar Typhimurium with fewer acylated groups were generated. The major lipid A form in wild-type (WT) and the mutant KCS237 strain is hexa-acylated; in mutant strains KCS311 and KCS324 it is penta-acylated; and in KCS369 it is tetra-acylated. WT and KCS237 formalin-killed and live bacteria, as well as their LPS, strongly stimulated production of pro-inflammatory cytokines in human U937 cells; this stimulation was suppressed by TLR4 suppressors. LPS of other mutants produced no agonistic activity, but strong antagonistic activity, while their formalin-killed and live bacteria preparations had weak agonistic and no antagonistic activity. Moreover, these less-acylated mutants had increased resistance to phagocytosis by U937 cells. Our results indicate that a decrease of one acyl group (from six to five) is enough to allow Salmonella to evade human innate immunity and that the antagonistic activity of less-acylated lipid A is not utilized for this evasion.     

NAIPs: building an innate immune barrier against bacterial pathogens. NAIPs function as sensors that initiate innate immunity by detection of bacterial proteins in the host cell cytosol

The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

CD11b and CD11c antigens are rapidly increased on human natural killer cells upon activation.

A brief incubation with PMA or other secretagogues has been reported to enhance the expression of C3 receptors on myeloid cells. We now observed increases up to threefold in the expression of the CD11b/CD18 Ag (CR3) and the CD11c/CD18 (CR4, p150,95) Ag after 30-min incubation with PMA on a subpopulation of PBL. The majority of these cells was CD56+ and CD16+. Isolated NK cells retained their ability to respond to PMA with increased CD11b and CD11c membrane Ag expression. Preincubation of the cells with cycloheximide did not abrogate the effects of PMA. Other membrane molecules on lymphocytes (CD11a, CD35, CD45, CD45R0, CD56) were not modulated by PMA. Purified C5a, FMLP, or LPS increased CR3 on myeloid cells but not on lymphocytes. In contrast, cell activation by K562 cells led to an augmentation of the CD11b Ag expression on CD56+ lymphocytes but not on other lymphocytes or monocytes. This increase was inhibitable by CD11a mAb. Rapid increases of CD11b and CD11c Ag on the membrane of NK cells may be of biologic significance because many functions have been attributed to these molecules.     

PACAP enhances the expression of CD11b,CD66b and CD63 in human neutrophils

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.     

Staurosporine inhibits neutrophil phagocytosis but not iC3b binding mediated by CR3 (CD11b/CD18).

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data.     

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust

Key message

The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat.

Abstract

The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.     

Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils.

The CD11b/CD18 integrin is a major cell adhesion molecule of myelomonocytic cells. Exposure of human neutrophils in suspension to CD11b or CD18 monoclonal antibodies (mAbs)2 does not affect the resting level of cytosolic free Ca2+ in these cells; however, a subsequent cross-linking of either of these antibodies triggers a prompt and significant cytosolic-free Ca2+ transient lasting about 10 min. The rise in cytosolic-free Ca2+ (from 130 +/- 2 to 414 +/- 12 nM or 111 +/- 12 to 331 +/- 22 nM caused by cross-linking of CD11b or CD18 subunits, respectively) is due to both mobilization of Ca2+ from intracellular stores and influx of Ca2+ across the plasma membrane. Cross-linking of the common leukocyte antigen (CD45) did not alter the basal level of cytosolic free Ca2+. In accordance with other adherence-induced phenomena and with CD11/CD18-mediated phagocytosis, these Ca2+ signals were only modestly affected by pertussis toxin. Thus, the present data clearly indicate that the CD11b/CD18 integrin on human neutrophils is capable of inducing a prompt cytosolic-free Ca2+ signal. These findings directly support the recent suggestion that the CD11b/CD18 integrin is responsible for the "spontaneous oscillations" of cytosolic-free Ca2+ observed in adherent neutrophils and, at least partially, also explain how integrin-mediated adherence can modify the functional responsiveness of neutrophils to a subsequent agonist stimulation.     

The crystal structure of human CD1b with a bound bacterial glycolipid

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains. The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.     

Interleukin-17 as an effector molecule of innate and acquired immunity against infections

Interleukin (IL)-17 is a proinflammatory cytokine which induces differentiation and migration of neutrophils through induction of cytokines and chemokines including granulocyte-colony stimulating factor and CXCL8/IL-8. IL-17-producing CD4(+) T cells (Th17) have pivotal role in pathogenesis of autoimmune diseases. IL-17 is also involved in protective immunity against various infections. IL-17 has important role in induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumoniae and Candida albicans. Importance of IL-17 in protection against intracellular pathogens including Mycobacterium has also been reported. Interestingly, not only CD4(+) T cells but atypical CD4(-)CD8(-) T cells expressing T cell receptor (TCR) gammadelta produce IL-17, and IL-17 producing cells participate in both innate and acquired immune response to infections. Furthermore, neutrophil induction may not be the only mechanism of IL-17-mediated protective immunity. IL-17 seems to participate in host defense through regulation of cell-mediated immunity or induction of antimicrobial peptides such as beta-defensins. In this review, we summarize recent progress on the role of IL-17 in immune response against infections, and discuss possible application of IL-17 in prevention and treatment of infectious diseases.     

Evasion of innate and adaptive immune responses by influenza A virus

Host organisms have developed sophisticated antiviral responses in order to defeat emerging influenza A viruses (IAVs). At the same time IAVs have evolved immune evasion strategies. The immune system of mammals provides several lines of defence to neutralize invading pathogens or limit their replication. Here, we summarize the mammalian innate and adaptive immune mechanisms involved in host defence against viral infection and review strategies by which IAVs avoid, circumvent or subvert these mechanisms. We highlight well‐characterized, as well as recently described features of this intriguing virus‐host molecular battle.     

Intraepithelial gammadelta T cells may bridge a gap between innate immunity and acquired immunity to herpes simplex virus type 2

Mice depleted of gammadelta T cells by in vivo administration of anti-TCRgammadelta monoclonal antibodies showed susceptibility against an intravaginal infection with herpes simplex virus type 2 (HSV-2). The systemic Th1 response was impaired in the gammadelta T-cell-depleted mice. Mice deficient in the Vdelta1 T subset were susceptible to an intravaginal infection with HSV-2. Intraepithelial gammadelta T cells bearing Vdelta1 may help protect against intravaginal infection with HSV-2 through promoting the systemic Th1 response.     

Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils

The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.