The repeated evolution of stripe patterns is correlated with body morphology in the adaptive radiations of East African cichlid fishes.

Color patterns are often linked to the behavioral and morphological characteristics of an animal, contributing to the effectiveness of such patterns as antipredatory strategies. Species-rich adaptive radiations, such as the freshwater fish family Cichlidae, provide an exciting opportunity to study trait correlations at a macroevolutionary scale. Cichlids are also well known for their diversity and repeated evolution of color patterns and body morphology. To study the evolutionary dynamics between color patterns and body morphology, we used an extensive dataset of 461 species. A phylogenetic supertree of these species shows that stripe patterns evolved ~70 times independently and were lost again ~30 times. Moreover, stripe patterns show strong signs of correlated evolution with body elongation, suggesting that the stripes' effectiveness as antipredatory strategy might differ depending on the body shape. Using pedigree-based analyses, we show that stripes and body elongation segregate independently, indicating that the two traits are not genetically linked. Their correlation in nature is therefore likely maintained by correlational selection. Lastly, by performing a mate preference assay using a striped CRISPR-Cas9 mutant of a nonstriped species, we show that females do not differentiate between striped CRISPR mutant males and nonstriped wild-type males, suggesting that these patterns might be less important for species recognition and mate choice. In summary, our study suggests that the massive rates of repeated evolution of stripe patterns are shaped by correlational selection with body elongation, but not by sexual selection.

Of bars and stripes: A Malawi cichlid hybrid cross provides insights into genetic modularity and evolution of modifier loci underlying colour pattern diversification.

Understanding the origins of phenotypic diversity among closely related species remains an important largely unsolved question in evolutionary biology. With over 800 species, Lake Malawi haplochromine cichlid fishes are a prominent example of extremely fast evolution of diversity including variation in colouration. Previously, a single major effect gene, agrp2 (asip2b), has been linked to evolutionary losses and gains of horizontal stripe patterns in cichlids, but it remains unknown what causes more fine-scale variation in the number and continuity of the stripes. Also, the genetic basis of the most common colour pattern in African cichlids, vertical bars, and potential interactions between the two colour patterns remain unknown. Based on a hybrid cross of the horizontally striped Lake Malawi cichlid Pseudotropheus cyaneorhabdos and the vertically barred species Chindongo demasoni we investigated the genetic basis of both colour patterns. The distribution of phenotypes in the F2  generation of the cross indicates that horizontal stripes and vertical bars are independently inherited patterns that are caused by two sets of genetic modules. While horizontal stripes are largely controlled by few major effect loci, vertical bars are a highly polygenic trait. Horizontal stripes show substantial variation in the F2  generation that, interestingly, resemble naturally occurring phenotypes found in other Lake Malawi cichlid species. Quantitative trait loci (QTL) mapping of this cross reveals known (agrp2) and unknown loci underlying horizontal stripe patterns. These findings provide novel insights into the incremental fine-tuning of an adaptive trait that diversified through the evolution of additional modifier loci.

Different Sources of Allelic Variation Drove Repeated Color Pattern Divergence in Cichlid Fishes.

The adaptive radiations of East African cichlid fish in the Great Lakes Victoria, Malawi, and Tanganyika are well known for their diversity and repeatedly evolved phenotypes. Convergent evolution of melanic horizontal stripes has been linked to a single locus harboring the gene agouti-related peptide 2 (agrp2). However, where and when the causal variants underlying this trait evolved and how they drove phenotypic divergence remained unknown. To test the alternative hypotheses of standing genetic variation versus de novo mutations (independently originating in each radiation), we searched for shared signals of genomic divergence at the agrp2 locus. Although we discovered similar signatures of differentiation at the locus level, the haplotypes associated with stripe patterns are surprisingly different. In Lake Malawi, the highest associated alleles are located within and close to the 5' untranslated region of agrp2 and likely evolved through recent de novo mutations. In the younger Lake Victoria radiation, stripes are associated with two intronic regions overlapping with a previously reported cis-regulatory interval. The origin of these segregating haplotypes predates the Lake Victoria radiation because they are also found in more basal riverine and Lake Kivu species. This suggests that both segregating haplotypes were present as standing genetic variation at the onset of the Lake Victoria adaptive radiation with its more than 500 species and drove phenotypic divergence within the species flock. Therefore, both new (Lake Malawi) and ancient (Lake Victoria) allelic variation at the same locus fueled rapid and convergent phenotypic evolution.

Phytochemical characterization of different yarrow species (Achillea sp.) and investigations into their antimicrobial activity.

Various Achillea species are rich in bioactive compounds and are important medicinal plants in phytotherapy. In the present study, Achillea millefolium L., Achillea moschata Wulfen, and Achillea atrata L. were compared with respect to their phenolic profile and antibacterial activity against gram-positive bacteria strains (Staphylococcus, Propionibacterium). Particular focus was given to A. atrata, which has hardly been studied so far. Based on the metabolite profile, A. atrata exhibited more similarities to A. moschata than to A. millefolium. The former two only differed in the occurrence of four compounds. The flavonols syringetin-3-O-glucoside and mearnsetin-hexoside, not reported for an Achillea species before, have been detected in A. atrata and A. moschata. All Achillea species reduced growth of the tested bacteria. A. atrata demonstrated highest activity against Propionibacterium acnes and Staphylococcus epidermidis, both being involved in the pathogenesis of acne vulgaris. Furthermore, A. atrata has a pronounced anti-methicillin-resistant Staphylococcus aureus potential. Bioassay-guided fractionation revealed that only the most polar fraction of A. moschata displayed antimicrobial activity, which was attributed to phenolics such as apigenin, centaureidin, and nevadensin, being present in high amounts in A. atrata. Thus, this alpine species shows promising antimicrobial activity and might be a potential source for developing novel dermal/topical drugs.

EVs as Potential New Therapeutic Tool/Target in Gastrointestinal Cancer and HCC.

For more than a decade, extracellular vesicles (EVs) have been in focus of science. Once thought to be an efficient way to eliminate undesirable cell content, EVs are now well-accepted as being an important alternative to cytokines and chemokines in cell-to-cell communication route. With their cargos, mainly consisting of functional proteins, lipids and nucleic acids, they can activate signalling cascades and thus change the phenotype of recipient cells at local and systemic levels. Their substantial role as modulators of various physiological and pathological processes is acknowledged. Importantly, more and more evidence arises that EVs play a pivotal role in many stages of carcinogenesis. Via EV-mediated communication, tumour cells can manipulate cells from host immune system or from the tumour microenvironment, and, ultimately, they promote tumour progression and modulate host immunity towards tumour's favour. Additionally, the role of EVs in modulating resistance to pharmacological and radiological therapy of many cancer types has become evident lately. Our understanding of EV biology and their role in cancer promotion and drug resistance has evolved considerably in recent years. In this review, we specifically discuss the current knowledge on the association between EVs and gastrointestinal (GI) and liver cancers, including their potential for diagnosis and treatment.

Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

BACKGROUND: Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. METHODS: Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. RESULTS: Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. CONCLUSIONS: EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

Evolutionary Dynamics of Structural Variation at a Key Locus for Color Pattern Diversification in Cichlid Fishes.

Color patterns in African cichlid fishes vary spectacularly. Although phylogenetic analysis showed already 30 years ago that many color patterns evolved repeatedly in these adaptive radiations, only recently have we begun to understand the genomic basis of color variation. Horizontal stripe patterns evolved and were lost several times independently across the adaptive radiations of Lake Victoria, Malawi, and Tanganyika and regulatory evolution of agouti-related peptide 2 (agrp2/asip2b) has been linked to this phenotypically labile trait. Here, we asked whether the agrp2 locus exhibits particular characteristics that facilitate divergence in color patterns. Based on comparative genomic analyses, we discovered several recent duplications, insertions, and deletions. Interestingly, one of these events resulted in a tandem duplication of the last exon of agrp2. The duplication likely precedes the East African radiations that started 8-12 Ma, is not fixed within any of the radiations, and is found to vary even within some species. Moreover, we also observed variation in copy number (two to five copies) and secondary loss of the duplication, illustrating a surprising dynamic at this locus that possibly promoted functional divergence of agrp2. Our work suggests that such instances of exon duplications are a neglected mechanism potentially involved in the repeated evolution and diversification that deserves more attention.

Genome of the Malawi golden cichlid fish (Melanochromis auratus) reveals exon loss of oca2 in an amelanistic morph.

The tropical freshwater fish family Cichlidae is famous for its record-breaking rates of speciation and diversity in colors and color patterns. Here, we sequenced the genome of the Lake Malawi cichlid Melanochromis auratus to study the genetic basis of an amelanistic morph of this species that lacks the typical melanic stripes and markings. Genome sequencing of the amelanistic and wild-type morph revealed the loss of the second exon of the known pigmentation gene oculocutaneous albinism II (oca2), also known as p(ink-eyed dilution) gene or melanocyte-specific transporter gene. Additional genotyping confirms the complete association with this recessive Mendelian phenotype. The deletion results in a shorter transcript, lacking an acidic di-leucine domain that is crucial for trafficking of the Oca2 protein to melanosomes. The fact that oca2 is involved in a wide range of amelanistic morphs across vertebrates demonstrates its highly conserved function.

Diagnostic and prognostic biomarkers in cholangiocarcinoma.

The high mortality rate of cholangiocarcinoma (CCA) is due, in part, to the lack of non-invasive approaches able to accurately detect this silent tumour at early stages, when therapeutic options can be potentially curative or may at least increase the overall survival of patients. The fact that the majority of CCA tumours are not linked to any known aetiological factor highly compromises the monitoring of patients at risk for tumour development and also their early diagnosis. Combination of clinical/biochemical features, imaging techniques and analysis of non-specific tumour biomarkers in serum are commonly used to help in the diagnosis of CCA, but tumour biopsy is usually required to confirm the diagnosis. Moreover, no prognostic biomarkers are currently used in the clinical setting, deserving more innovative research, and international validation and consensus. Important efforts have been made in the last few years to identify accurate non-invasive biomarkers, by using innovative techniques and high-throughput omics technologies. This review summarizes and discusses the advances in the investigation of novel diagnostic and prognostic biomarkers in CCA and envisions the future directions in this field of research.

Extracellular Vesicles in Liver Diseases: Diagnostic, Prognostic, and Therapeutic Application.

Extracellular vesicles, comprising exosomes, microvesicles, and apoptotic bodies, represent an emerging field in disease diagnostics and prognosis. They can be isolated from peripheral blood of patients as well as from other body fluids and can therefore be considered a minimally invasive liquid biopsy screening tool. Especially their surface antigen composition can reveal information about disease backgrounds. For several liver diseases, including fatal hepatocellular and cholangiocellular carcinoma as well as other nonmalignant liver disorders such as nonalcoholic fatty liver disease, alcoholic hepatitis, or acute liver failure, it has been shown that extracellular vesicle (EV) surface profiling can be useful for disease diagnosis and prognosis. This review focuses on latest advances in these areas to improve liver disorder detection and management. Additionally, the authors will discuss possible therapeutic applications of EVs in liver diseases, which might be a potent treatment option in the future.

Transmaternal Helicobacter pylori exposure reduces allergic airway inflammation in offspring through regulatory T cells.

BACKGROUND: Transmaternal exposure to tobacco, microbes, nutrients, and other environmental factors shapes the fetal immune system through epigenetic processes. The gastric microbe Helicobacter pylori represents an ancestral constituent of the human microbiota that causes gastric disorders on the one hand and is inversely associated with allergies and chronic inflammatory conditions on the other. OBJECTIVE: Here we investigate the consequences of transmaternal exposure to H pylori in utero and/or during lactation for susceptibility to viral and bacterial infection, predisposition to allergic airway inflammation, and development of immune cell populations in the lungs and lymphoid organs. METHODS: We use experimental models of house dust mite- or ovalbumin-induced airway inflammation and influenza A virus or Citrobacter rodentium infection along with metagenomics analyses, multicolor flow cytometry, and bisulfite pyrosequencing, to study the effects of H pylori on allergy severity and immunologic and microbiome correlates thereof. RESULTS: Perinatal exposure to H pylori extract or its immunomodulator vacuolating cytotoxin confers robust protective effects against allergic airway inflammation not only in first- but also second-generation offspring but does not increase susceptibility to viral or bacterial infection. Immune correlates of allergy protection include skewing of regulatory over effector T cells, expansion of regulatory T-cell subsets expressing CXCR3 or retinoic acid-related orphan receptor γt, and demethylation of the forkhead box P3 (FOXP3) locus. The composition and diversity of the gastrointestinal microbiota is measurably affected by perinatal H pylori exposure. CONCLUSION: We conclude that exposure to H pylori has consequences not only for the carrier but also for subsequent generations that can be exploited for interventional purposes.

Agouti-related peptide 2 facilitates convergent evolution of stripe patterns across cichlid fish radiations.

The color patterns of African cichlid fishes provide notable examples of phenotypic convergence. Across the more than 1200 East African rift lake species, melanic horizontal stripes have evolved numerous times. We discovered that regulatory changes of the gene agouti-related peptide 2 (agrp2) act as molecular switches controlling this evolutionarily labile phenotype. Reduced agrp2 expression is convergently associated with the presence of stripe patterns across species flocks. However, cis-regulatory mutations are not predictive of stripes across radiations, suggesting independent regulatory mechanisms. Genetic mapping confirms the link between the agrp2 locus and stripe patterns. The crucial role of agrp2 is further supported by a CRISPR-Cas9 knockout that reconstitutes stripes in a nonstriped cichlid. Thus, we unveil how a single gene affects the convergent evolution of a complex color pattern.

Large Extracellular Vesicles: Have We Found the Holy Grail of Inflammation?

The terms microparticles (MPs) and microvesicles (MVs) refer to large extracellular vesicles (EVs) generated from a broad spectrum of cells upon its activation or death by apoptosis. The unique surface antigens of MPs/MVs allow for the identification of their cellular origin as well as its functional characterization. Two basic aspects of MP/MV functions in physiology and pathological conditions are widely considered. Firstly, it has become evident that large EVs have strong procoagulant properties. Secondly, experimental and clinical studies have shown that MPs/MVs play a crucial role in the pathophysiology of inflammation-associated disorders. A cardinal feature of these disorders is an enhanced generation of platelets-, endothelial-, and leukocyte-derived EVs. Nevertheless, anti-inflammatory effects of miscellaneous EV types have also been described, which provided important new insights into the large EV-inflammation axis. Advances in understanding the biology of MPs/MVs have led to the preparation of this review article aimed at discussing the association between large EVs and inflammation, depending on their cellular origin.

NLRP3 Controls the Development of Gastrointestinal CD11b+ Dendritic Cells in the Steady State and during Chronic Bacterial Infection.

The gastric lamina propria is largely uncharted immunological territory. Here we describe the evolution and composition of the gastric, small intestinal, and colonic lamina propria mononuclear phagocyte system during the steady state and infection with the gastric pathogen Helicobacter pylori. We show that monocytes, CX3CR1hi macrophages, and CD11b+ dendritic cells are recruited to the infected stomach in a CCR2-dependent manner. All three populations, but not BATF3-dependent CD103+ DCs, sample red fluorescent protein (RFP)+Helicobacter pylori (H. pylori). Mice reconstituted with human hematopoietic stem cells recapitulate several features of the myeloid cell-H. pylori interaction. The differentiation in and/or recruitment to gastrointestinal, lung, and lymphoid tissues of CD11b+ DCs requires NLRP3, but not apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) or caspase-1, during steady-state and chronic infection. NLRP3-/- mice fail to generate Treg responses to H. pylori and control the infection more effectively than wild-type mice. The results demonstrate a non-canonical inflammasome-independent function of NLRP3 in DC development and immune regulation.

Multi-Surface Antigen Staining of Larger Extracellular Vesicles.

Larger extracellular vesicles, microparticles (MPs) or microvesicles (MVs), especially their acquisition and characterization by flow cytometry (FACS), is increasingly in focus of clinical/translational research efforts. Several laboratories have shown that MPs/MVs might be suitable for the diagnosis and predicting prognosis in various diseases including cancer. However, FACS staining of larger extracellular vesicles (EVs) can be difficult and results potentially in false positive and inconsistent data interpretation. Despite that FACS equipment is well maintained and the operators have ample experience, a reliable and for larger EVs optimized staining protocol is missing. Here, we aim to close that gap and provide a working multi-antibody staining protocol for larger EVs isolated from human serum samples. We describe in detail the needed steps as currently done in our laboratory. Staining is demonstrated exemplarily for multi-antibody mix including CD147 , a potential cancer marker if applied in combination with other MP/MV surface markers.

Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma.

BACKGROUND & AIMS: Large extracellular vesicles, specifically AnnexinV+ EpCAM+ CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+ EpCAM+ CD147+ taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+ EpCAM+ ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled. RESULTS: The results indicate that AnnexinV+ EpCAM+ CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+ EpCAM+ ASGPR1+ CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+ EpCAM+ ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11-15mm. AnnexinV+ EpCAM+ ASGPR1+ taMPs decreased at 7days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+ EpCAM+ ASGPR1+ taMPs. CONCLUSION: These data provide strong evidence that AnnexinV+ EpCAM+ ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis.

Lymphotoxin ß receptor signalling executes Helicobacter pylori-driven gastric inflammation in a T4SS-dependent manner.

OBJECTIVE: Lymphotoxin ß receptor (LTßR) signalling has been implicated in inflammation-associated tumour development in different tissues. We have analysed the role of LTßR and alternative NF-κB signalling in Helicobacter pylori-mediated gastric inflammation and pathology. DESIGN: We analysed several ligands and receptors of the alternative NF-κB pathway, RelB, p52 nuclear translocation and target genes in tissue samples of H. pylori-infected patients with different degrees of gastritis or early gastric tumours by in situ hybridisation, immunohistochemistry, Western blot and real-time PCR analyses. Molecular mechanisms involved in LTßR activation by H. pylori were assessed in vitro using human gastric cancer cell lines and distinct H. pylori isolates. The effects of blocking or agonistically activating LTßR on gastric pathology during challenge with a human pathogenic H. pylori strain were studied in a mouse model. RESULTS: Among the tested candidates, LT was significantly increased and activated alternative NF-κB signalling was observed in the gastric mucosa of H. pylori-infected patients. H. pyloriinduced LTßR-ligand expression in a type IV secretion system-dependent but CagA-independent manner, resulting in activation of the alternative NF-κB pathway, which was further enhanced by blocking canonical NF-κB during infection. Blocking LTßR signalling in vivo suppressed H. pylori-driven gastritis, whereas LTßR activation in gastric epithelial cells of infected mice induced a broadened pro-inflammatory chemokine milieu, resulting in exacerbated pathology. CONCLUSIONS: LTßR-triggered activation of alternative NF-κB signalling in gastric epithelial cells executes H. pylori-induced chronic gastritis, representing a novel target to restrict gastric inflammation and pathology elicited by H. pylori, while exclusively targeting canonical NF-κB may aggravate pathology by enhancing the alternative pathway.

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

Helicobacter urease-induced activation of the TLR2/NLRP3/IL-18 axis protects against asthma.

Inflammasome activation and caspase-1-dependent (CASP1-dependent) processing and secretion of IL-1ß and IL-18 are critical events at the interface of the bacterial pathogen Helicobacter pylori with its host. Whereas IL-1ß promotes Th1 and Th17 responses and gastric immunopathology, IL-18 is required for Treg differentiation, H. pylori persistence, and protection against allergic asthma, which is a hallmark of H. pylori-infected mice and humans. Here, we show that inflammasome activation in DCs requires the cytoplasmic sensor NLRP3 as well as induction of TLR2 signaling by H. pylori. Screening of an H. pylori transposon mutant library revealed that pro-IL-1ß expression is induced by LPS from H. pylori, while the urease B subunit (UreB) is required for NLRP3 inflammasome licensing. UreB activates the TLR2-dependent expression of NLRP3, which represents a rate-limiting step in NLRP3 inflammasome assembly. ureB-deficient H. pylori mutants were defective for CASP1 activation in murine bone marrow-derived DCs, splenic DCs, and human blood-derived DCs. Despite colonizing the murine stomach, ureB mutants failed to induce IL-1ß and IL-18 secretion and to promote Treg responses. Unlike WT H. pylori, ureB mutants were incapable of conferring protection against allergen-induced asthma in murine models. Together, these results indicate that the TLR2/NLRP3/CASP1/IL-18 axis is critical to H. pylori-specific immune regulation.