Siglec-F+ neutrophils in the spleen induce immunosuppression following acute infection.

Background: The mechanisms underlying the increased mortality of secondary infections during the immunosuppressive phase of sepsis remain elusive. Objectives: We sought to investigate the role of Siglec-F+ neutrophils on splenic T lymphocytes in the immunosuppressed phase of sepsis and on secondary infection in PICS mice, and to elucidate the underlying mechanisms. Methods: We established a mouse model of sepsis-induced immunosuppression followed by secondary infection with LPS or E. coli. The main manifestation of immunosuppression is the functional exhaustion of splenic T lymphocytes. Treg depletion reagent Anti-IL-2, IL-10 blocker Anti-IL-10R, macrophage depletion reagent Liposomes, neutrophil depletion reagent Anti-Ly6G, neutrophil migration inhibitor SB225002, Siglec-F depletion reagent Anti-Siglec-F are all used on PICS mice. The function of neutrophil subsets was investigated by adoptive transplantation and the experiments in vitro. Results: Compared to other organs, we observed a significant reduction in pro-inflammatory cytokines in the spleen, accompanied by a marked increase in IL-10 production, primarily by infiltrating neutrophils. These infiltrating neutrophils in the spleen during the immunosuppressive phase of sepsis undergo phenotypic change in the local microenvironment, exhibiting high expression of neutrophil biomarkers such as Siglec-F, Ly6G, and Siglec-E. Depletion of neutrophils or specifically targeting Siglec-F leads to enhance the function of T lymphocytes and a notable improvement in the survival of mice with secondary infections. Conclusions: We identified Siglec-F+ neutrophils as the primary producers of IL-10, which significantly contributed to T lymphocyte suppression represents a novel finding with potential therapeutic implications.

The bone marrow endothelial progenitor cell response to septic infection.

Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24 h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.

A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis.

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1ß maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Goose surfactant protein A inhibits the growth of avian pathogenic Escherichia coli via an aggregation-dependent mechanism that decreases motility and increases membrane permeability.

Pulmonary collectins have been reported to bind carbohydrates on pathogens and inhibit infection by agglutination, neutralization, and opsonization. In this study, surfactant protein A (SP-A) was identified from goose lung and characterized at expression- and agglutination-functional levels. The deduced amino acid sequence of goose surfactant protein A (gSP-A) has two characteristic structures: a shorter, collagen-like region and a carbohydrate recognition domain. The latter contains two conserved motifs in its Ca2+-binding site: EPN (Glu-Pro-Asn) and WND (Trp-Asn-Asp). Expression analysis using qRT-PCR and fluorescence IHC revealed that gSP-A was highly expressed in the air sac and present in several other tissues, including the lung and trachea. We went on to produce recombinant gSP-A (RgSP-A) using a baculovirus/insect cell system and purified using a Ni2+ affinity column. A biological activity assay showed that all bacterial strains tested in this study were aggregated by RgSP-A, but only Escherichia coli AE17 (E. coli AE17, O2) and E. coli AE158 (O78) were susceptible to RgSP-A-mediated growth inhibition at 2-6 h. Moreover, the swarming motility of the two bacterial strains were weakened with increasing RgSP-A concentration, and their membrane permeability was compromised at 3 h, as determined by flow cytometry and laser confocal microscopy. Therefore, RgSP-A is capable of reducing bacterial viability of E. coli O2 and O78 via an aggregation-dependent mechanism which involves decreasing motility and increasing the bacterial membrane permeability. These data will facilitate detailed studies into the role of gSP-A in innate immune defense as well as for development of antibacterial agents.

Protective role for C3aR in experimental chronic pyelonephritis.

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.

Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge.

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

Transcriptome Analysis Reveals the Multiple Functions of pBD2 in IPEC-J2 Cells against E. coli.

Defensins play an important role in fighting bacteria, and are a good candidate for bactericidal agents. However, the function and mechanism of defensins in regulating host responses against bacteria is unclear. In this study, transcriptome analysis was used to study the comprehensive functions of pBD2 in IPEC-J2 cells against E. coli. In total, 230 differentially expressed genes (DEGs) were identified in IPEC-J2 cells between the control and E. coli groups, and were found by KEGG analysis to be involved in many signaling pathways related to immunity. Furthermore, 812 DEGs were observed between E. coli and E. coli +pBD2 groups, involved in the ribosome, oxidative phosphorylation, and certain disease pathways. Among these, 94 overlapping DEGs were in the two DEG groups, and 85 DEGs were reverse expression, which is involved in microRNA in cancer, while PTEN and CDC6 were key genes according to PPI net analysis. The results of qRT-PCR verified those of RNA-seq. The results indicated that pBD2 plays an important role against E. coli by acting on the genes related to immune response, cell cycle, ribosomes, oxidative phosphorylation, etc. The results provide new insights into the potential function and mechanism of pBD2 against E. coli. Meanwhile, this study provides a certain theoretical basis for research and the development of novel peptide drugs.

RNA-seq reveals a novel porcine lncRNA MPHOSPH9-OT1 induces CXCL8/IL-8 expression in ETEC infected IPEC-J2 cells.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrhea in piglets, leading to economic losses in the pig industry. In past decades, long non-coding RNAs (lncRNAs) have shown to be widely involved in the regulation of host immunity in porcine infection diseases. In this study, we explored the lncRNAs associated with ETEC F41 infection in IPEC-J2 cells by high-throughput sequencing and bioinformatic analysis. A total of 10150 novel porcine lncRNAs were identified. There were 161 differentially expressed (DE) lncRNAs associated with ETEC F41 infection, of which 65 DE lncRNAs were up-regulated and 96 DE lncRNAs were down-regulated. Functional and KEGG enrichment analysis of predicted target genes of DE lncRNAs indicated they are enriched in cell growth and inflammation-related pathways, such as endocytosis, focal adhesion, TGF-ß signaling pathway, and adherens junctions. We revealed a novel candidate lncRNA MPHOSPH9-OT1 that was up-regulated after ETEC infection. The qRT-PCR validation and ELISA assessment showed the knockdown and overexpression of MPHOSPH9-OT1 resulted in significantly down- and up-regulation of cellular mRNA levels and secreted cytokine levels of CXCL8/IL-8, respectively. Meanwhile, MPHOSPH9-OT1 equilibrium is important to maintain the transepithelial electric resistance value and tight junction protein expression of IPEC-J2 cells. This study provides insights into the functionality of novel porcine lncRNAs in host immune responses to ETEC infection.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

Interferon-ß regulates proresolving lipids to promote the resolution of acute airway inflammation.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-ß (IFN-ß) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli-evoked lung injury, and suppresses production of IFN-ß and the proresolving lipid mediators 15-epi-LXA4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-ß delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-ß accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-ß-mediated resolution. These findings point to a pivotal role of IFN-ß in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-ß-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Glyceryl butyrate attenuates enterotoxigenic Escherichia coli-induced intestinal inflammation in piglets by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota.

The aims of this study were to evaluate whether a diet supplemented with glyceryl butyrate could attenuate the immune-inflammatory response in piglets challenged with enterotoxigenic Escherichia coli (ETEC), and to explore the mechanisms of its regulation. Eighteen weaning piglets were assigned to three diets: basal diet (CON), antibiotics diet (ATB), and 0.5% glyceryl butyrate diet (GB group). Significantly lower concentrations of IL-1ß, IL-6 and TNF-α in the jejunum and IL-6 in the ileum were observed in the GB group than that in the CON group (P < 0.05). Moreover, a decreasing trend of IL-1ß (P = 0.075) and TNF-α (P = 0.070) was observed in the ileum in the GB group. Correspondingly, the GB group had significantly increased mRNA expression of porcine beta defensins (pBDs) in the jejunum (pBD1, pBD2, pBD114 and pBD129) and ileum (pBD2, pBD3, pBD114 and pBD129) (P < 0.05), and protein abundance of Claudin 1, Occludin, and ZO-1 in the jejunum and ileum (P < 0.05). Further research results showed that the improvement of beta defensins and tight junctions in the GB group was related to the decreased phosphorylation of the NFκB/MAPK pathway. In addition, the results of 16S rDNA sequencing showed that glycerol butyrate supplementation altered the ileal microbiota composition of piglets, increasing the relative abundance of Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus agrilis. In summary, glyceryl butyrate attenuated the immune-inflammatory response in piglets challenged with ETEC by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota, and thus improved piglet intestinal health.

Alveolar macrophage-derived NRP2 curtails lung injury while boosting host defense in bacterial pneumonia.

Clearance of airway intruders by immune cells is required to resolve infectious pneumonia. However, the molecular mechanisms underlying this process remain elusive. Here, we demonstrated that alveolar macrophage (AM)-derived neuropilin 2 (NRP2) plays an essential role in controlling severe pneumonia by enhancing microbial clearance. Mice with conditional deletion of the NRP2 gene in AM had persistent bacteria, uncontrolled neutrophil influx, and decreased survival during Escherichia coli-induced pneumonia. In vitro assays demonstrated that NRP2 could bind to CD11b+ Ly6Glo/+ neutrophils and promote their capacities in phagocytosis and killing of bacteria, which is partially contributed to the increased expression of TLR4 and TNF-a. These findings collectively revealed that AM-derived NRP2 protects the lungs from unwanted injury by promoting the clearance of invading pathogens. This study might provide a promising diagnostic biomarker and therapeutic target for severe pneumonia.

Mitogen-activated protein kinase phosphatase-1 controls PD-L1 expression by regulating type I interferon during systemic Escherichia coli infection.

Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.

The role of caspase-1, caspase-4 and NLRP3 in regulating the host cell response evoked by uropathogenic Escherichia coli.

The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.

IL-22 initiates an IL-18-dependent epithelial response circuit to enforce intestinal host defence.

IL-18 is emerging as an IL-22-induced and epithelium-derived cytokine which contributes to host defence against intestinal infection and inflammation. In contrast to its known role in Goblet cells, regulation of barrier function at the molecular level by IL-18 is much less explored. Here we show that IL-18 is a bona fide IL-22-regulated gate keeper for intestinal epithelial barrier. IL-22 promotes crypt immunity both via induction of phospho-Stat3 binding to the Il-18 gene promoter and via Il-18 independent mechanisms. In organoid culture, while IL-22 primarily increases organoid size and inhibits expression of stem cell genes, IL-18 preferentially promotes organoid budding and induces signature genes of Lgr5+ stem cells via Akt-Tcf4 signalling. During adherent-invasive E. coli (AIEC) infection, systemic administration of IL-18 corrects compromised T-cell IFNγ production and restores Lysozyme+ Paneth cells in Il-22-/- mice, but IL-22 administration fails to restore these parameters in Il-18-/- mice, thereby placing IL-22-Stat3 signalling upstream of the IL-18-mediated barrier defence function. IL-18 in return regulates Stat3-mediated anti-microbial response in Paneth cells, Akt-Tcf4-triggered expansion of Lgr5+ stem cells to facilitate tissue repair, and AIEC clearance by promoting IFNγ+ T cells.

Lactic Acid Bacteria Mixture Isolated From Wild Pig Alleviated the Gut Inflammation of Mice Challenged by Escherichia coli.

Wild pigs usually showed high tolerance and resistance to several diseases in the wild environment, suggesting that the gut bacteria of wild pigs could be a good source for discovering potential probiotic strains. In our study, wild pig feces were sequenced and showed a higher relative abundance of the genus Lactobacillus (43.61% vs. 2.01%) than that in the domestic pig. A total of 11 lactic acid bacteria (LAB) strains including two L. rhamnosus, six L. mucosae, one L. fermentum, one L. delbrueckii, and one Enterococcus faecalis species were isolated. To investigate the synergistic effects of mixed probiotics strains, the mixture of 11 LAB strains from an intestinal ecology system was orally administrated in mice for 3 weeks, then the mice were challenged with Escherichia coli ATCC 25922 (2 × 109 CFU) and euthanized after challenge. Mice administrated with LAB strains showed higher (p < 0.05) LAB counts in feces and ileum. Moreover, alterations of specific bacterial genera occurred, including the higher (p < 0.05) relative abundance of Butyricicoccus and Clostridium IV and the lower (p < 0.05) abundance of Enterorhabdus in mice fed with mixed LAB strains. Mice challenged with Escherichia coli showed vacuolization of the liver, lower GSH in serum, and lower villus to the crypt proportion and Claudin-3 level in the gut. In contrast, administration of mixed LAB strains attenuated inflammation of the liver and gut, especially the lowered IL-6 and IL-1ß levels (p < 0.05) in the gut. Our study highlighted the importance of gut bacterial diversity and the immunomodulation effects of LAB strains mixture from wild pig in gut health.

Effect of biannual azithromycin distribution on antibody responses to malaria, bacterial, and protozoan pathogens in Niger.

The MORDOR trial in Niger, Malawi, and Tanzania found that biannual mass distribution of azithromycin to children younger than 5 years led to a 13.5% reduction in all-cause mortality (NCT02048007). To help elucidate the mechanism for mortality reduction, we report IgG responses to 11 malaria, bacterial, and protozoan pathogens using a multiplex bead assay in pre-specified substudy of 30 communities in the rural Niger placebo-controlled trial over a three-year period (n = 5642 blood specimens, n = 3814 children ages 1-59 months). Mass azithromycin reduces Campylobacter spp. force of infection by 29% (hazard ratio = 0.71, 95% CI: 0.56, 0.89; P = 0.004) but serological measures show no significant differences between groups for other pathogens against a backdrop of high transmission. Results align with a recent microbiome study in the communities. Given significant sequelae of Campylobacter infection among preschool aged children, our results support an important mechanism through which biannual mass distribution of azithromycin likely reduces mortality in Niger.

ß-glucan-coupled superparamagnetic iron oxide nanoparticles induce trained immunity to protect mice against sepsis.

Background: Innate immune memory, also termed "trained immunity", is thought to protect against experimental models of infection, including sepsis. Trained immunity via reprogramming monocytes/macrophages has been reported to result in enhanced inflammatory status and antimicrobial activity against infection in sepsis. However, a safe and efficient way to induce trained immunity remains unclear. Methods: ß-glucan is a prototypical agonist for inducing trained immunity. Ferumoxytol, superparamagnetic iron oxide (SPIO) with low cytotoxicity, has been approved by FDA for clinical use. We synthesized novel nanoparticles BSNPs by coupling ß-glucan with SPIO. BSNPs were further conjugated with fluorescein for quantitative analysis and trace detection of ß-glucan on BSNPs. Inflammatory cytokine levels were measured by ELISA and qRT-PCR, and the phagocytosis of macrophages was detected by flow cytometry and confocal microscopy. The therapeutic effect of BSNPs was evaluated on the well-established sepsis mouse model induced by both clinical Escherichia coli (E. coli) and cecal ligation and puncture (CLP). Results: BSNPs were synthesized successfully with a 3:20 mass ratio of ß-glucan and SPIO on BSNPs, which were mainly internalized by macrophages and accumulated in the lungs and livers of mice. BSNPs effectively reprogrammed macrophages to enhance the production of trained immunity markers and phagocytosis toward bacteria. BSNP-induced trained immunity protected mice against sepsis caused by E. coli and CLP and also against secondary infection. We found that BSNP treatment elevated Akt, S6, and 4EBP phosphorylation, while mTOR inhibitors decreased the trained immunity markers and phagocytosis enhanced by BSNPs. Furthermore, the PCR Array analysis revealed Igf1, Sesn1, Vegfa, and Rps6ka5 as possible key regulators of mTOR signaling during trained immunity. BSNP-induced trained immunity mainly regulated cellular signal transduction, protein modification, and cell cycle by modulating ATP binding and the kinase activity. Our results indicated that BSNPs induced trained immunity in an mTOR-dependent manner. Conclusion: Our data highlight that the trained immunity of macrophages is an effective strategy against sepsis and suggest that BSNPs are a powerful tool for inducing trained immunity to prevent and treat sepsis and secondary infections.