Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

IL-22-dependent responses and their role during Citrobacter rodentium infection.

The mouse pathogen Citrobacter rodentium is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and to elucidate mechanisms of mucosal immunity. In response to C. rodentium infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from C. rodentium infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during C. rodentium infection. In line with previous research, mice lacking the IL-22 gene (Il22-/- mice) were highly susceptible to C. rodentium infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: S100A9-/- (calprotectin), Lcn2-/- (lipocalin-2), Reg3b-/- (Reg3ß), Reg3g-/- (Reg3γ), and C3-/- (C3). All knockout mice tested displayed a considerable level of resistance to C. rodentium infection, and none phenocopied the lethality observed in Il22-/- mice. By investigating another arm of the IL-22 response, we observed that C. rodentium-infected Il22-/- mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal C. rodentium infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.

Complement in breast milk modifies offspring gut microbiota to promote infant health.

Breastfeeding offers demonstrable benefits to newborns and infants by providing nourishment and immune protection and by shaping the gut commensal microbiota. Although it has been appreciated for decades that breast milk contains complement components, the physiological relevance of complement in breast milk remains undefined. Here, we demonstrate that weanling mice fostered by complement-deficient dams rapidly succumb when exposed to murine pathogen Citrobacter rodentium (CR), whereas pups fostered on complement-containing milk from wild-type dams can tolerate CR challenge. The complement components in breast milk were shown to directly lyse specific members of gram-positive gut commensal microbiota via a C1-dependent, antibody-independent mechanism, resulting in the deposition of the membrane attack complex and subsequent bacterial lysis. By selectively eliminating members of the commensal gut community, complement components from breast milk shape neonate and infant gut microbial composition to be protective against environmental pathogens such as CR.

Shigella induces epigenetic reprogramming of zebrafish neutrophils.

Trained immunity is a long-term memory of innate immune cells, generating an improved response upon reinfection. Shigella is an important human pathogen and inflammatory paradigm for which there is no effective vaccine. Using zebrafish larvae, we demonstrate that after Shigella training, neutrophils are more efficient at bacterial clearance. We observe that Shigella-induced protection is nonspecific and has differences with training by BCG and ß-glucan. Analysis of histone ChIP-seq on trained neutrophils revealed that Shigella training deposits the active H3K4me3 mark on promoter regions of 1612 genes, dramatically changing the epigenetic landscape of neutrophils toward enhanced microbial recognition and mitochondrial ROS production. Last, we demonstrate that mitochondrial ROS plays a key role in enhanced antimicrobial activity of trained neutrophils. It is envisioned that signals and mechanisms we discover here can be used in other vertebrates, including humans, to suggest new therapeutic strategies involving neutrophils to control bacterial infection.

Trained ILC3 responses promote intestinal defense.

Group 3 innate lymphoid cells (ILC3s) are innate immune effectors that contribute to host defense. Whether ILC3 functions are stably modified after pathogen encounter is unknown. Here, we assess the impact of a time-restricted enterobacterial challenge to long-term ILC3 activation in mice. We found that intestinal ILC3s persist for months in an activated state after exposure to Citrobacter rodentium. Upon rechallenge, these "trained" ILC3s proliferate, display enhanced interleukin-22 (IL-22) responses, and have a superior capacity to control infection compared with naïve ILC3s. Metabolic changes occur in C. rodentium-exposed ILC3s, but only trained ILC3s have an enhanced proliferative capacity that contributes to increased IL-22 production. Accordingly, a limited encounter with a pathogen can promote durable phenotypic and functional changes in intestinal ILC3s that contribute to long-term mucosal defense.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease.

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg-1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg-1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6-8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4-8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6-8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1ß and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.

IRE1α-Driven Inflammation Promotes Clearance of Citrobacter rodentium Infection.

Endoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

Sustained Post-Developmental T-Bet Expression Is Critical for the Maintenance of Type One Innate Lymphoid Cells In Vivo.

Innate lymphoid cells (ILC) play a significant role in the intestinal immune response and T-bet+ CD127+ group 1 cells (ILC1) have been linked to the pathogenesis of human inflammatory bowel disease (IBD). However, the functional importance of ILC1 in the context of an intact adaptive immune response has been controversial. In this report we demonstrate that induced depletion of T-bet using a Rosa26-Cre-ERT2 model resulted in the loss of intestinal ILC1, pointing to a post-developmental requirement of T-bet expression for these cells. In contrast, neither colonic lamina propria (cLP) ILC2 nor cLP ILC3 abundance were altered upon induced deletion of T-bet. Mechanistically, we report that STAT1 or STAT4 are not required for intestinal ILC1 development and maintenance. Mice with induced deletion of T-bet and subsequent loss of ILC1 were protected from the induction of severe colitis in vivo. Hence, this study provides support for the clinical development of an IBD treatment based on ILC1 depletion via targeting T-bet or its downstream transcriptional targets.

Aspartate Metabolism Facilitates IL-1ß Production in Inflammatory Macrophages.

Increasing evidence support that cellular amino acid metabolism shapes the fate of immune cells; however, whether aspartate metabolism dictates macrophage function is still enigmatic. Here, we found that the metabolites in aspartate metabolism are depleted in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-stimulated macrophages. Aspartate promotes interleukin-1ß (IL-1ß) secretion in M1 macrophages. Mechanistically, aspartate boosts the activation of hypoxia-inducible factor-1α (HIF-1α) and inflammasome and increases the levels of metabolites in aspartate metabolism, such as asparagine. Interestingly, asparagine also accelerates the activation of cellular signaling pathways and promotes the production of inflammatory cytokines from macrophages. Moreover, aspartate supplementation augments the macrophage-mediated inflammatory responses in mice and piglets. These results uncover a previously uncharacterized role for aspartate metabolism in directing M1 macrophage polarization.

Intimate Adhesion Is Essential for the Pathogen-Specific Inflammatory and Immune Responses in the Gut of Mice Infected with Citrobacter rodentium.

Citrobacter rodentium is a murine pathogenic bacterium that adheres to intestinal epithelial cells, resulting in loss of microvilli and pedestal formation, and alters multiple cellular processes, including actin dynamics. Translocated intimin receptor (Tir), one of its virulence factors, functions as receptor for intimin, a bacterial adhesin, thereby mediating bacterial adhesion to epithelial cells. Although robust immune responses are induced to eliminate pathogenic bacteria in the host, they are suppressed against harmless commensal bacteria. The mechanism(s) underlying such a differentiation remains unclear. This study sought to determine the roles of intimate adhesion in the induction of specific immune responses upon C. rodentium infection. To this end, microbiota-depleted mice were infected with the Tir-F strain expressing full-length Tir or mutant strains expressing the C-terminal truncated Tir that is defective in intimin binding and host cell actin polymerization. There were no differences in the colonization kinetics and Abs responses against C. rodentium LPS among the strains, whereas Abs against the virulence factors were only produced on Tir-F infection. Although there were no differences in the virulence factors mRNA expression levels, colonic hyperplasia, and bacterial translocation to the systemic organs irrespective of the strain, adhesion to colonic epithelial cells was reduced in the mutant strain-infected mice. Furthermore, transcriptomic analysis indicated that robust inflammatory and immune responses were only induced in the Tir-F-infected group and were suppressed in the mutant-infected groups. Taken together, these findings suggest that Tir-mediated intimate adhesion induces inflammatory and immune responses, resulting in the induction of virulence factor-specific Abs.

Hyaluronan-induced alterations of the gut microbiome protects mice against Citrobacter rodentium infection and intestinal inflammation.

Hyaluronan is a glycosaminoglycan polymer that has been shown to play an important role in homeostasis of the gastrointestinal tract. However, its mechanistic significance in gastrointestinal epithelial barrier elements remain unexplored. Here, our results revealed that hyaluronan treatment resulted in significant changes in the gut microbiota in mice. To demonstrate the functional consequences of hyaluronan-treatment and hyaluronan-induced microbiota alterations, Citrobacter rodentium- and DSS-induced colitis models and microbiota transplantation approaches were utilized. We showed that hyaluronan alleviated intestinal inflammation in both pathogen and chemically induced intestinal mucosal damage. The protection in bacterial colitis was associated with enhanced C. rodentium clearance and alleviation of pathogen-induced gut dysbiosis. Microbiota transplantation experiments showed that the hyaluronan-altered microbiota is sufficient to confer protection against C. rodentium infection. Colonization with Akkermansia muciniphila, a commensal bacterium that is greatly enriched by hyaluronan treatment, alleviated C. rodentium-induced bacterial colitis in mice. Additionally, Akkermansia-induced protection was found to be associated with the induction of goblet cells and the production of mucins and epithelial antimicrobial peptides. Collectively, these results provide novel insights into the regulatory role of hyaluronan in modulating the gut microbiota and immunity in enteric infection and inflammation, with therapeutic potential for gut microbiome-targeted immunotherapy.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

A Novel microRNA of Japanese Flounder Regulates Antimicrobial Immunity Involving a Bacteria-Binding CSF3.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse biological processes including immunity. In a previous high-throughput RNA sequencing study, a novel miRNA, pol-miR-novel_642, was identified from Japanese flounder (Paralichthys olivaceus), a farmed fish species with important economic value. In this study, we investigated the regulatory mechanism and the function of pol-miR-novel_642 and its target gene. We found that pol-miR-novel_642 targeted, in a sequence-specific manner, a flounder gene encoding an uncharacterized protein that is a structural homologue of murine granulocyte colony stimulating factor 3 (CSF3). The expression of pol-miR-novel_642 and its target gene (named PoCSF3-1) was regulated, in different manners, by the bacterial pathogen Edwardsiella tarda and the viral pathogen megalocytivirus. Overexpression of pol-miR-novel_642 or interference with PoCSF3-1 expression in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoCSF3-1 enhanced bacterial dissemination in flounder tissues but blocked viral replication, whereas in vivo overexpression of PoCSF3-1 inhibited bacterial dissemination and facilitated viral infection. Overexpression/knockdown of PoCSF3-1 and pol-miR-novel_642 also affected the activation of autophagy. Recombinant PoCSF3-1 (rPoCSF3-1) interacted with and inhibited the growth of Gram-negative bacteria in a manner relying on a PoCSF3-1-characteristic structural motif that is absent in mouse CSF3. rPoCSF3-1 also regulated the proliferation, inflammatory response, and immune defense of flounder head kidney leukocytes in a structure-dependent fashion. Together, these results reveal the function of a novel miRNA-CSF3 regulatory system of flounder, and add new insights into the role and mechanism of fish miRNA and CSF3 in antimicrobial immunity.

Zebrafish BID Exerts an Antibacterial Role by Negatively Regulating p53, but in a Caspase-8-Independent Manner.

Bid (BH3-interacting domain death agonist), a member of the Bcl-2 family, plays a crucial role in the initiation of apoptosis. Independent of its apoptotic function, Bid is also involved in the regulation of inflammation and innate immunity. However, the role of Bid during bacterial pathogen infection remains unclear. In the present study, Bid of zebrafish (Dario rerio) was cloned and its functions during Edwardsiella ictaluri infection were investigated. Zebrafish Bid enhances the apoptosis rate of Epithelioma papulosum cyprini (EPC) cells following E. ictaluri infection. Importantly, in vitro and in vivo bacterial invasion assays showed that overexpressed Bid could significantly inhibit the invasion and proliferation of E. ictaluri. Real-time qPCR analysis revealed that p53 gene expression was downregulated in embryos microinjected with Bid-FLAG. Further, in vitro and in vivo bacterial invasion assays showed that overexpressed p53 increased the invasion and proliferation of E. ictaluri. Moreover, the invasion and proliferation of E. ictaluri were inhibited when co-overexpressing Bid and p53 in vivo and in vitro. Further, the numbers of E. ictaluri in larvae treated with Z-IETD-FMK (caspase-8 inhibitor) were higher than those of larvae without Z-IETD-FMK treatment, while the number of E. ictaluri in larvae microinjected with bid-Flag decreased significantly, even if the larvae were treated in advance with Z-IETD-FMK. Collectively, our study demonstrated a novel antibacterial activity of fish Bid, providing evidence for understanding the function of apoptosis associated gene in pathogen infection.

A teleost interleukin-16 is implicated in peripheral blood leukocytes recruitment and anti-bacterial immunity.

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%-67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16.

PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis.

MicroRNAs (miRNAs) are evolutionary conserved, non-coding small RNAs that have been shown to regulate diverse biological processes including immunity. In a previous study, a novel miRNA of Japanese flounder (Paralichthys olivaceus), pol-miR-novel_395, was found to be responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. In the present study, we examined the regulation and immune effect of pol-miR-novel_395 and its target gene. We found that pol-miR-novel_395 expression was regulated by E. tarda and megalocytivirus, and pol-miR-novel_395 targeted the gene of PUF60 (poly (U)-binding-splicing factor 60 kDa) of flounder (named PoPUF60). Constitutive expression of PoPUF60 occurred in relatively high levels in the heart and liver of flounder. Bacterial infection upregulated PoPUF60 expression, whereas viral infection downregulated PoPUF60 expression. Interference with PoPUF60 expression or overexpression of pol-miR-novel_395 in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoPUF60 enhanced bacterial dissemination in the tissues of flounder but blocked viral replication, whereas in vivo overexpression of PoPUF60 inhibited bacterial dissemination but facilitated viral replication. Additionally, pol-miR-novel_395 and PoPUF60 were involved in the process of autophagy and apoptosis. Collectively, these results indicated that PoPUF60 and pol-miR-novel_395 play an important role in pathogen infection, autophagy, and apoptosis.

Immune adjuvant effects of interferon-gamma (IFN-γ) of flounder (Paralichthys olivaceus) against Edwardsiella tarda.

IFN-γ plays a key role in T-cell activation and the establishment of the adaptive immune response, which has a potential as a cytokine adjuvant in the context of vaccination. In this study, we evaluated the immune adjuvant effects of two forms of flounder (Paralichthys olivaceus) IFN-γ, including pcDNA3.1-IFN-γ (pcIFN-γ) and recombinant IFN-γ (rIFN-γ), and comparatively analyzed the immune responses of flounder to E. tarda subunit vaccine rOmpV. The results showed that vaccination with rOmpV plus pcIFN-γ or rIFN-γ produced a relative percent survival of 57% and 71%, respectively, which were significantly higher than that of the control groups, rOmpV plus pcN3 (36%) or rHis (40%). Compared with the two control groups, vaccination with rOmpV plus pcIFN-γ or rIFN-γ could induce significantly higher levels of specific serum antibodies and sIg + lymphocytes in peripheral blood, spleen and head kidney, and significantly higher upregulated expressions of CD4-1, CD8α, IgM, MHC Ⅰα, MHC Ⅱα, IL-1ß and TNF-α were also detected in rOmpV plus pcIFN-γ or rIFN-γ vaccinated fish. In addition, compared with pcIFN-γ, rOmpV co-vaccination with rIFN-γ elicited higher levels of sIg + lymphocytes, specific serum antibodies and several immune-related genes expressions in vaccinated flounder. These results demonstrated that rOmpV co-vaccination with rIFN-γ or pcIFN-γ could both boost the immune responses and evoke highly protective effects against E. tarda, indicating that flounder IFN-γ is a promising adjuvant candidate for fish vaccination via an injection administering route.

Functional characterization of cathepsin B and its role in the antimicrobial immune responses in golden pompano (Trachinotus ovatus).

Cathepsin B (CTSB) is one of the typical representatives of cysteine protease family. It has the activity of both exopeptidase and endopeptidase. It plays an important role in antigen presentation, degradation, apoptosis, inflammatory response and physiological process of many diseases. In this study, CTSB of Trachinotus ovatus (TroCTSB) was cloned, and its structure and function were analyzed. The results showed that the coding region of TroCTSB was 993 bp, encoding 330 amino acid residues. The homology analysis showed that the amino acid sequence of TroCTSB was similar to that in other teleosts and mammals (68.69%-88.48%). Under normal physiological conditions, TroCTSB was widely distributed in various tissues with the highest expression level in stomach, followed by liver, and the lowest expression level in blood. The optimal pH and temperature of purified recombinant protein rTroCTSB were 5.5 and 40 °C, respectively. The toxicity test of metal ions showed that Fe2+, Cu2+, Ca2+ and Zn2+ could all inhibit the activity of TroCTSB, with Zn2+ ranking the first. In addition, after Edwardsiella tarda infection, the expression of TroCTSB was significantly up-regulated in liver, spleen and head kidney. The overexpression of TroCTSB significantly inhibited the infection of E. tarda in golden pompano tissues, and the knockdown of TroCTSB remarkably promoted the reproduction of E. tarda in golden pompano tissues in vivo. This study suggests that TroCTSB was involved in the antibacterial immune response of T. ovatus, and provided a reference for further research in elucidating the resistance mechanism of TroCTSB.

TNFRSF13B polymorphisms counter microbial adaptation to enteric IgA.

TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B, corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium, a murine pathogen that models enterohemorrhagic Escherichia coli, and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C. rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.