Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Cigarette smoke depletes alveolar macrophages and delays clearance of Legionella pneumophila.

Legionella pneumophila is the main etiological agent of Legionnaires' disease, a severe bacterial pneumonia. L. pneumophila is initially engulfed by alveolar macrophages (AMs) and subvert normal cellular functions to establish a replicative vacuole. Cigarette smokers are particularly susceptible to developing Legionnaires' disease and other pulmonary infections; however, little is known about the cellular mechanisms underlying this susceptibility. To investigate this, we used a mouse model of acute cigarette smoke exposure to examine the immune response to cigarette smoke and subsequent L. pneumophila infection. Contrary to previous reports, we show that cigarette smoke exposure alone causes a significant depletion of AMs using enzymatic digestion to extract cells, or via imaging intact lung lobes by light-sheet microscopy. Furthermore, treatment of mice deficient in specific types of cell death with smoke suggests that NLRP3-driven pyroptosis is a contributor to smoke-induced death of AMs. After infection, smoke-exposed mice displayed increased pulmonary L. pneumophila loads and developed more severe disease compared with air-exposed controls. We tested if depletion of AMs was related to this phenotype by directly depleting them with clodronate liposomes and found that this also resulted in increased L. pneumophila loads. In summary, our results showed that cigarette smoke depleted AMs from the lung and that this likely contributed to more severe Legionnaires' disease. Furthermore, the role of AMs in L. pneumophila infection is more nuanced than simply providing a replicative niche, and our studies suggest they play a major role in bacterial clearance.

IL-33 promotes gastric tumour growth in concert with activation and recruitment of inflammatory myeloid cells.

Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130 F/F mouse model of GC. Expression of IL-33 (and it's cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130 F/F /Il33 -/- mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of ß-thalassaemia.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.

The Salmonella Effector SseK3 Targets Small Rab GTPases.

During infection, Salmonella species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, Salmonella exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the Salmonella-containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of Salmonella infection by SseK3, an arginine N-acetylglucosamine (GlcNAc) transferase effector translocated via the Salmonella pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to Salmonella infection by interfering with the activity of key Rab GTPases.

Influence of the MUC1 Cell Surface Mucin on Gastric Mucosal Gene Expression Profiles in Response to Helicobacter pylori Infection in Mice.

The cell surface mucin MUC1 is an important host factor limiting Helicobacter pylori (H. pylori) pathogenesis in both humans and mice by providing a protective barrier and modulating mucosal epithelial and leukocyte responses. The aim of this study was to establish the time-course of molecular events in MUC1-modulated gene expression profiles in response to H. pylori infection in wild type (WT) and MUC1-deficient mice using microarray-determined mRNA expression, gene network analysis and Ingenuity Pathway Analysis (IPA). A time-course over the first 72 h of infection showed significantly higher mucosal loads of bacteria at 8 h of infection in Muc1-/- mice compared with WT, confirming its importance in the early stages of infection (P = 0.0003). Microarray analysis revealed 266 differentially expressed genes at one or more time-points over 72 h in the gastric mucosa of Muc1-/- mice compared with WT control using a threshold of 2-fold change. The SPINK1 pancreatic cancer canonical pathway was strongly inhibited in Muc1-/- mice compared with WT at sham and 8 h infection (P = 6.08E-14 and P = 2.25 E-19, respectively) but potently activated at 24 and 72 h post-infection (P = 1.38E-22 and P = 5.87E-13, respectively). The changes in this pathway are reflective of higher expression of genes mediating digestion and absorption of lipids, carbohydrates, and proteins at sham and 8 h infection in the absence of MUC1, but that this transcriptional signature is highly down regulated as infection progresses in the absence of MUC1. Uninfected Muc1-/- gastric tissue was highly enriched for expression of factors involved in lipid metabolism and 8 h infection further activated this network compared with WT. As infection progressed, a network of antimicrobial and anti-inflammatory response genes was more highly activated in Muc1-/- than WT mice. Key target genes identified by time-course microarrays were independently validated using RT-qPCR. These results highlight the dynamic interplay between the host and H. pylori, and the role of MUC1 in host defense, and provide a general picture of changes in cellular gene expression modulated by MUC1 in a time-dependent manner in response to H. pylori infection.

Application of a chemical probe to detect neutrophil elastase activation during inflammatory bowel disease.

Neutrophil elastase is a serine protease that has been implicated in the pathogenesis of inflammatory bowel disease. Due to post-translational control of its activation and high expression of its inhibitors in the gut, measurements of total expression poorly reflect the pool of active, functional neutrophil elastase. Fluorogenic substrate probes have been used to measure neutrophil elastase activity, though these tools lack specificity and traceability. PK105 is a recently described fluorescent activity-based probe, which binds to neutrophil elastase in an activity-dependent manner. The irreversible nature of this probe allows for accurate identification of its targets in complex protein mixtures. We describe the reactivity profile of PK105b, a new analogue of PK105, against recombinant serine proteases and in tissue extracts from healthy mice and from models of inflammation induced by oral cancer and Legionella pneumophila infection. We apply PK105b to measure neutrophil elastase activation in an acute model of experimental colitis. Neutrophil elastase activity is detected in inflamed, but not healthy, colons. We corroborate this finding in mucosal biopsies from patients with ulcerative colitis. Thus, PK105b facilitates detection of neutrophil elastase activity in tissue lysates, and we have applied it to demonstrate that this protease is unequivocally activated during colitis.

Effect of MUC1 length polymorphisms on the NLRP3 inflammasome response of human macrophages.

Mucin 1 is a cell-membrane associated mucin, expressed on epithelial and immune cells that helps protect against pathogenic infections. In humans, MUC1 is highly polymorphic, predominantly due to the presence of a variable number tandem repeat (VNTR) region in the extracellular domain that results in MUC1 molecules of typically either short or long length. A genetic link is known between these MUC1 polymorphisms and inflammation-driven diseases, although the mechanism is not fully understood. We previously showed that MUC1 on murine macrophages specifically restricts activation of the NLRP3 inflammasome, thereby repressing inflammation. This study evaluated the effect of MUC1 VNTR polymorphisms on activity of the NLRP3 inflammasome in human macrophages, finding that long MUC1 alleles correlated with increased IL-1ß production following NLRP3 inflammasome activation. This indicates that the length of MUC1 can influence IL-1ß production, thus providing the first evidence of an immune-modulatory role of MUC1 VNTR polymorphisms in human macrophages.

The Mouse as a Model for Pulmonary Legionella Infection.

Infection of C57BL/6 mice with wild-type Legionella pneumophila typically results in very mild disease. However, in mice where the cytosolic recognition of flagellin is impaired by mutation, L. pneumophila infection results in more severe lung inflammation that is reminiscent of Legionnaires' disease. This can be replicated in wild-type mice by using aflagellated mutants of L. pneumophila. These models greatly facilitate the investigation of L. pneumophila virulence factors and the complex pulmonary immune system that is triggered by infection. Here we describe methods for infecting C57BL/6 mice with aflagellated L. pneumophila, the quantification of bacterial load in the lungs and isolation and analysis of invading immune cells. These assays enable the identification of phagocyte subsets and can determine whether phagocytic cells act as a replicative niche for L. pneumophila replication.

Overexpression of IL-11 promotes premalignant gastric epithelial hyperplasia in isolation from germline gp130-JAK-STAT driver mutations.

Expression of the cytokine IL-11 is elevated in human Helicobacter pylori infection and progressively increases with worsening gastric pathology. Additionally, IL-11 is required for tumor development in STAT3-dependent murine models of gastric cancer (GC) and, when administered acutely, causes resolving atrophic gastritis. However, it is unclear whether locally elevated IL-11 ligand expression can, in isolation from oncogenic gp130-JAK-STAT pathway mutations, initiate GC pathogenesis. Here we developed a transgenic mouse model of stomach-specific (keratin 19 promoter) IL-11 ligand overexpression. Keratin 19 promoter-IL-11 transgenic ( K19-IL11Tg) mice showed specific IL-11 overexpression in gastric corpus and antrum but not elsewhere in the gastrointestinal tract or in other tissues. K19-IL11Tg mice developed spontaneous premalignant disease of the gastric epithelium, progressing from atrophic gastritis to TFF2-positive metaplasia and severe epithelial hyperplasia, including adenoma-like lesions in a subset of older (1 yr old) animals. Although locally advanced, the hyperplastic lesions remained noninvasive. H. pylori infection in K19-IL11Tg mice accelerated some aspects of the premalignant phenotype. Finally, K19-IL11Tg mice had splenomegaly in association with elevated serum IL-11, with spleens showing an expanded myeloid compartment. Our results provide direct in vivo functional evidence that stomach-specific overexpression of IL-11, in isolation from germline gp130-JAK-STAT3 genetic drivers, is sufficient for premalignant progression. These findings have important functional implications for human GC, in which frequent IL-11 overexpression occurs in the reported absence of somatic mutations in gp130 signaling components. NEW & NOTEWORTHY We provide direct in vivo functional evidence that stomach-specific overexpression of the cytokine IL-11, in isolation from gp130-JAK-STAT3 pathway mutations, can trigger spontaneous atrophic gastritis progressing to locally advanced epithelial hyperplasia (but not dysplasia or carcinoma), which does not require, but may be accelerated by, concomitant Helicobacter pylori infection.

NOD1 is required for Helicobacter pylori induction of IL-33 responses in gastric epithelial cells.

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL-33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL-33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide-Binding Oligomerisation Domain-Containing Protein 1 (NOD1) and its adaptor protein receptor-interacting serine-threonine Kinase 2, to promote production of both full-length and processed IL-33 in gastric epithelial cells. Furthermore, IL-33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL-33 and splenic IL-13 responses, but decreased IFN-γ responses, when compared with Nod1-/- animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL-33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.

Superoxide dismutase from Helicobacter pylori suppresses the production of pro-inflammatory cytokines during in vivo infection.

BACKGROUND: Helicobacter pylori has undergone considerable adaptation to allow chronic persistence within the gastric environment. While H. pylori-associated diseases are driven by an excessive inflammation, severe gastritis is detrimental to colonization by this pathogen. Hence, H. pylori has developed strategies to minimize the severity of gastritis it triggers in its host. Superoxide dismutase (SOD) is well known for its role in protecting against oxidative attack; less recognized is its ability to inhibit immunity, shown for SOD from mammalian sources and those of some bacterial species. This study examined whether H. pylori SOD (HpSOD) has the ability to inhibit the host immune response to these bacteria. MATERIALS AND METHODS: The ability of recombinant HpSOD to modify the response to LPS was measured using mouse macrophages. A monoclonal antibody against HpSOD was generated and injected into H. pylori-infected mice. RESULTS: Addition of HpSOD to cultures of mouse macrophages significantly inhibited the pro-inflammatory cytokine response to LPS stimulation. A monoclonal antibody was generated that was specific for SOD from H. pylori. When injected into mice infected with H. pylori for 3 months, this antibody was readily detected in both sera and gastric tissues 5 days later. While treatment with anti-HpSOD had no effect on H. pylori colonization at this time point, it significantly increased the levels of a range of pro-inflammatory cytokines in the gastric tissues. This did not occur with antibodies against other antioxidant enzymes. CONCLUSIONS: SOD from H. pylori can inhibit the production of pro-inflammatory cytokine during in vivo infection.

Mucin 1 protects against severe Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a bacterial pathogen that commonly resides in the human nasopharynx, typically without causing any disease. However, in some cases these bacteria migrate from the nasopharynx to other sites of the body such as the lungs and bloodstream causing pneumonia and sepsis, respectively. This study used a mouse model of infection to investigate the potential role of Mucin 1 (MUC1), a cell membrane-associated glycoprotein known for playing a key barrier role at mucosal surfaces, in regulating this process. Wildtype (WT) and MUC1-deficient (Muc1-/-) mice were infected intranasally with an invasive strain of S. pneumoniae and bacterial loads in the nasopharynx, lungs, and blood were analyzed. Lungs were graded histologically for inflammation and cytokine profiles in the lungs analyzed by ELISA. While there was no difference in pneumococcal colonization of the nasopharynx between WT and Muc1-/- mice, infected Muc1-/- mice showed high pneumococcal loads in their lungs 16 hours post-infection, as well as bacteremia. In contrast, infected WT mice cleared the pneumococci from their lungs and remained asymptomatic. Infection in Muc1-/- mice was associated with an elevation in lung inflammation, with cellular recruitment especially of monocytes/macrophages. While MUC1-deficiency has been shown to increase phagocytosis of Pseudomonas aeruginosa, macrophages from Muc1-/- mice exhibited a reduced capacity to phagocytose S. pneumoniae indicating diverse and bacterial-specific effects. In conclusion, these findings indicate that MUC1 plays an important role in protection against severe pneumococcal disease, potentially mediated by facilitating macrophage phagocytosis.

Protease-activated Receptor 1 Plays a Proinflammatory Role in Colitis by Promoting Th17-related Immunity.

BACKGROUND: Proteolytic cleavage of protease-activated receptor 1 (PAR1) can result in potent downstream regulatory effects on inflammation. Although PAR1 is expressed throughout the gastrointestinal tract and activating proteases are increased in inflammatory bowel disease, the effect of PAR1 activation on colitis remains poorly understood, and has not previously been studied in pediatric disease. METHODS: Expression of PAR1 and inflammatory cytokines in colonic biopsies from pediatric patients with Crohn's disease exhibiting active moderate to severe colitis was measured by quantitative PCR. The functional relevance of these clinical data was further studied in a mouse model of Citrobacter rodentium-induced colitis. RESULTS: PAR1 expression was significantly upregulated in the inflamed colons of pediatric patients with Crohn's disease, with expression levels directly correlating to disease severity. In patients with severe colitis, PAR1 expression uniquely correlated with Th17-related (IL17A, IL22, and IL23A) cytokines. Infection of PAR1-deficient (PAR1) and wildtype mice with colitogenic C. rodentium revealed that disease severity and colonic pathology were strongly attenuated in mice lacking PAR1. Furthermore, Th17-type immune response was completely abolished in the colons of infected PAR1 but not wildtype mice. Finally, PAR1 was shown to be essential for secretion of the Th17-driving cytokine IL-23 by C. rodentium-stimulated macrophages. CONCLUSIONS: This study demonstrates a strong link between PAR1 expression, Th17-type immunity, and disease severity in both pediatric patients with Crohn's disease and C. rodentium-induced colitis in mice. The data presented suggest PAR1 exerts a proinflammatory role in colitis in both humans and mice by promoting a Th17-type immune response, potentially by supporting the production of IL-23.

No evidence of a role for mitochondrial complex I in Helicobacter pylori pathogenesis.

BACKGROUND: Complex I is the first enzyme complex in the mitochondrial respiratory chain, responsible for generating a large fraction of energy during oxidative phosphorylation. Recently, it has been identified that complex I deficiency can result in increased inflammation due to the generation of reactive oxygen species by innate immune cells. As a reduction in complex I activity has been demonstrated in human stomachs with atrophic gastritis, we investigated whether complex I deficiency could influence Helicobacter pylori pathogenesis. MATERIALS AND METHODS: Ndufs6gt/gt mice have a partial complex I deficiency. Complex I activity was quantified in the stomachs and immune cells of Ndufs6gt/gt mice by spectrophotometric assays. Ndufs6gt/gt mice were infected with H. pylori and bacterial colonization assessed by colony-forming assay, gastritis assessed histologically, and H. pylori -specific humoral response quantified by ELISA. RESULTS: The immune cells and stomachs of Ndufs6gt/gt mice were found to have significantly decreased complex I activity, validating the model for assessing the effects of complex I deficiency in H. pylori infection. However, there was no observable effect of complex I deficiency on either H. pylori colonization, the resulting gastritis, or the humoral response. CONCLUSIONS: Although complex I activity is described to suppress innate immune responses and is decreased during atrophic gastritis in humans, our data suggest it does not affect H. pylori pathogenesis.

How host regulation of Helicobacter pylori-induced gastritis protects against peptic ulcer disease and gastric cancer.

The bacterial pathogen Helicobacter pylori is the etiological agent of a range of gastrointestinal pathologies including peptic ulcer disease and the major killer, gastric adenocarcinoma. Infection with this bacterium induces a chronic inflammatory response in the gastric mucosa (gastritis). It is this gastritis that, over decades, eventually drives the development of H. pylori-associated disease in some individuals. The majority of studies investigating H. pylori pathogenesis have focused on factors that promote disease development in infected individuals. However, an estimated 85% of those infected with H. pylori remain completely asymptomatic, despite the presence of pathogenic bacteria that drive a chronic gastritis that lasts many decades. This indicates the presence of highly effective regulatory processes in the host that, in most cases, keeps a check on inflammation and protect against disease. In this minireview we discuss such known host factors and how they prevent the development of H. pylori-associated pathologies.

Activation of TRIF-dependent and independent immune responses by neisserial heat shock protein complex vaccines.

ASBTRACT Heat shock protein Complex (HspC) vaccines are composed of Hsp purified from pathogenic bacteria along with their chaperoned protein cargo. Mouse studies have shown that HspC vaccines can induce a strong immune response against pathogenic bacteria without addition of an exogenous adjuvant. These vaccines are now entering clinical trials. It was predicted, but not previously tested, that HspC vaccines induce an immune response due to the activation of Toll-Like Receptors (TLR) by their component Hsp. Recently we tested this supposition and found that while this held true for the cellular response to neisserial HspC vaccines, strong antigen-specific antibody responses were surprisingly generated in mice deficient in MyD88 and thus most TLR signaling. This suggested an unidentified mechanism by which HspC vaccines induce an antibody response. We have now examined the antigenic profile of this response and found no evidence that this is due to the induction of T-independent antibodies. Examination of the MyD88-dependent signaling pathways involved in the cellular response to neisserial HspC showed that both TRIF-dependent and TRIF-independent pathways are activated, each resulting in the secretion of different cytokines. Hence the mechanism of action of HspC vaccines is clearly more complicated than originally thought.

Loss of gastrokine-2 drives premalignant gastric inflammation and tumor progression.

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.

Heat shock protein complex vaccines induce antibodies against Neisseria meningitidis via a MyD88-independent mechanism.

BACKGROUND: Neisseria meningitidis are common colonizers of the human nasopharynx. In some circumstances, N. meningitidis becomes an opportunistic pathogen that invades tissues and causes meningitis. While a vaccine against a number of serogroups has been in effective use for many years, a vaccine against N. meningitidis group B has not yet been universally adopted. Bacterial heat shock protein complex (HSPC) vaccines comprise bacterial HSPs, purified with their chaperoned protein cargo. HSPC vaccines use the intrinsic adjuvant activity of their HSP, thought to act via Toll-like receptors (TLR), to induce an immune response against their cargo antigens. This study evaluated HSPC vaccines from N. meningitidis and the closely related commensal N. lactamica. RESULTS: The protein composition of N. lactamica and N. meningitidis HSPCs were similar. Using human HEK293 cells we found that both HSPCs can induce an innate immune response via activation of TLR2. However, stimulation of TLR2 or TLR4 deficient murine splenocytes revealed that HSPCs can activate an innate immune response via multiple receptors. Vaccination of wildtype mice with the Neisseria HSPC induced a strong antibody response and a Th1-restricted T helper response. However, vaccination of mice deficient in the major TLR adaptor protein, MyD88, revealed that while the Th1 response to Neisseria HSPC requires MyD88, these vaccines unexpectedly induced an antigen-specific antibody response via a MyD88-independent mechanism. CONCLUSIONS: N. lactamica and N. meningitidis HSPC vaccines both have potential utility for immunising against neisserial meningitis without the requirement for an exogenous adjuvant. The mode of action of these vaccines is highly complex, with HSPCs inducing immune responses via both MyD88-dependent and -independent mechanisms. In particular, these HSPC vaccines induced an antibody response without detectable T cell help.

The MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome.

OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1ß and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1ß, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.