The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway

The human-restricted pathogens Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis colonize host tissues via carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs). One such receptor, CEACAM3, acts in a host-protective manner by orchestrating the capture and engulfment of invasive bacteria by human neutrophils. Herein, we show that bacterial binding to CEACAM3 causes recruitment of the cytoplasmic tyrosine kinase Syk, resulting in the phosphorylation of both CEACAM3 and Syk. This interaction is specific for the immunoreceptor tyrosine-based activation motif (ITAM) in the CEACAM3 cytoplasmic domain. While dispensable for the phagocytic uptake of single bacteria by CEACAM3, Syk is necessary for internalization when cargo size increases or when the density of CEACAM-binding ligand on the cargo surface is below a critical threshold. Moreover, Syk engagement is required for an effective bacterial killing response, including the neutrophil oxidative burst and degranulation functions in response to N. gonorrhoeae. These data reveal CEACAM3 as a specific innate immune receptor that mediates the opsonin-independent clearance of CEACAM-binding bacteria via Syk, a molecular trigger for functional immunoreceptor responses of both the adaptive (TCR, BCR, FcR) and innate (Dectin-1, CEACAM3) immune systems.     

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

Distinct mechanisms of internalization of Neisseria gonorrhoeae by members of the CEACAM receptor family involving Rac1- and Cdc42-dependent and -independent pathways

Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces.     

MDA‐5 activation by cytoplasmic double‐stranded RNA impairs endothelial function and aggravates atherosclerosis

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA‐5) belongs to the intracellular retinoic acid inducible gene‐I like receptors and its activation promotes pro‐inflammatory mechanisms. Here, we studied the effect of MDA‐5 stimulation in vascular biology. To gain insights into MDA‐5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA‐5 agonist polyIC (long double‐stranded RNA). Human coronary endothelial cell expressed MDA‐5 and reacted with receptor up‐regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro‐inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA‐5 stimulation. To test these effects in vivo, wild‐type mice were transfected with 32.5 μg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC‐treated wild‐type mice, endothelium‐dependent vasodilation and re‐endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA‐5 deficiency in vivo. Finally, chronic MDA‐5 stimulation in Apolipoprotein E/toll‐like receptor 3 (TLR3) double^‐deficient (ApoE^?/?/TLR3^?/?) mice‐enhanced atherosclerotic plaque formation. This study demonstrates that MDA‐5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA‐5.     

CEACAM1 recognition by bacterial pathogens is species-specific

Background  

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, Neisseria gonorrhoeae, N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals.     

Neisseria gonorrhoeae effectively blocks HIV‐1 replication by eliciting a potent TLR9‐dependent interferon‐α response from plasmacytoid dendritic cells

Clinical and epidemiological research provides evidence for a positive correlation between Neisseria gonorrhoeae infection and HIV transmission; however, mechanistic studies examining this relationship have yielded conflicting results. To explore this interaction, we exposed ex vivo cultured peripheral blood cells from acute HIV⁺ individuals to N. gonorrhoeae. Unexpectedly, we observed a profound inhibition in HIV‐1 replication in the ex vivo cultures, and this was recapitulated when peripheral blood mononuclear cells (PBMCs) from healthy donors were co‐infected with HIV‐1 and N. gonorrhoeae. Next, we established that gonococcal‐infected PBMCs liberated a soluble factor that effectively blocked HIV‐1 replication. Cytokine analyses and antibody blocking experiments revealed that the type I interferon, interferon‐α (IFNα), was expressed upon exposure to N. gonorrhoeae and was responsible for the inhibition of HIV‐1. Intracellular staining, TLR9‐blocking and cell depletion‐based studies demonstrated that the IFNα was elicited by plasmacytoid dendritic cells (pDCs) in a TLR9‐dependent manner. The pDC response to N. gonorrhoeae was unexpected given pDCs more established role in innate defence against intracellular pathogens, suggesting this may be a bacterial immune evasion strategy. In the context of HIV, this overcomes the virus's otherwise effective avoidance of the interferon response and represents a previously unrecognized intersection between these two sexually transmitted pathogens.     

Association of Neisseria gonorrhoeae OpaCEA with Dendritic Cells Suppresses Their Ability to Elicit an HIV-1-Specific T Cell Memory Response

Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⁺ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa_CEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa_CEA binding to CEACAM1 reduced the DCs’ capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa_CEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa_CEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.     

Galectin‐3 deficiency protects pancreatic islet cells from cytokine‐triggered apoptosis in vitro

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3^?/?) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3^?/? islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013. © 2012 Wiley Periodicals, Inc.     

A critical role for CARD9 in pneumocystis pneumonia host defence

Caspase recruitment domains‐containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C‐type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4‐depleted CARD9^?/? and immunocompetent hosts. Card9 gene‐disrupted (CARD9^?/?) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild‐type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9^?/? macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin‐1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9^?/? animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9^?/? animals during PCP, T‐helper cell cytokines were normal in immunocompetent CARD9^?/? animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.     

Two lytic transglycosylases in Neisseria gonorrhoeae impart resistance to killing by lysozyme and human neutrophils

Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil‐rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram‐negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild‐type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule‐localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation.     

Polyaniline/carbon nanotubes platform for sexually transmitted disease detection

Polyaniline/carbon nanotubes composite (PANI‐CNT) electrochemically deposited onto indium‐tin‐oxide (ITO) coated glass plate has been utilized for Neisseria gonorrhoeae detection by immobilizing 5′‐amino‐labeled Neisseria gonorrhoeae probe (aDNA) using glutaraldehyde as a cross‐linker. PANI‐CNT/ITO and aDNA‐Glu‐PANI‐CNT/ITO electrodes have been characterized using scanning electron microscopy (SEM), Fourier Transform Infrared (FT‐IR) spectroscopy, cyclic voltammetry (CV), and differential pulse voltammetry (DPV). This bioelectrode can be used to detect N. gonorrhoeae using methylene blue (MB) as redox indicator with response time of 60 s and stability of about 75 days when stored under refrigerated conditions. DPV studies reveal that this bioelectrode can detect complementary DNA concentration from 1 × 10^?6 M to 1 × 10^?17 M with detection limit of 1.2 × 10^?17 M. Further, this bioelectrode (aDNA‐Glu‐PANI‐CNT/ITO) exhibits specificity toward N. gonorrhoeae species and shows negative response with non‐Neisseria gonorrhoeae Neisseria species (NgNS) and other gram negative bacteria (GNB). Copyright © 2010 John Wiley & Sons, Ltd.     

Opa proteins and CEACAMs: pathways of immune engagement for pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae are globally important pathogens, which in part owe their success to their ability to successfully evade human immune responses over long periods. The phase-variable opacity-associated (Opa) adhesin proteins are a major surface component of these organisms, and are responsible for bacterial adherence and entry into host cells and interactions with the immune system. Most immune interactions are mediated via binding to members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family. These Opa variants are able to bind to different receptors of the CEACAM family on epithelial cells, neutrophils, and T and B lymphocytes, influencing the innate and adaptive immune responses. Increased epithelial cell adhesion creates the potential for prolonged infection, invasion and dissemination. Furthermore, Opa proteins may inhibit T-lymphocyte activation and proliferation, B-cell antibody production, and innate inflammatory responses by infected epithelia, in addition to conferring increased resistance to antibody-dependent, complement-mediated killing. While vaccines containing Opa proteins could induce adhesion-blocking and bactericidal antibodies, the consequence of CEACAM binding by a candidate Opa-containing vaccine requires further investigation. This review summarizes current knowledge of the immunological consequences of the interaction between meningococcal and gonococcal Opa proteins and human CEACAMs, considering the implications for pathogenesis and vaccine development.     

Moraxella catarrhalis induces CEACAM3‐Syk‐CARD9‐dependent activation of human granulocytes

The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria‐induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte‐specific carcinoembryonic antigen‐related cell adhesion molecule (CEACAM)‐3 is linked to NF‐κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme‐linked immunosorbent assay for chemokines. NF‐κB activation was determined using a luciferase reporter gene assay and chromatin‐immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro‐inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF‐κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM‐like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis.     

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson–Gilford Progeria Syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle‐derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24^?/? (Z24^?/?) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin‐induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F‐actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei‐induced cGAS‐Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24^?/? mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.     

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet‐induced obesity

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high‐fat diet‐induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil‐ and macrophage‐based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi‐infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high‐fat diet, toll‐like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow‐derived macrophages from obese, B. burgdorferi‐infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.