Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis.

The common bed bug, Cimex lectularius, is an urban pest of global health significance, severely affecting the physical and mental health of humans. In contrast to most other blood-feeding arthropods, bed bugs are not major vectors of pathogens, but the underlying mechanisms for this phenomenon are largely unexplored. Here, we present the first transcriptomics study of bed bugs in response to immune challenges. To study transcriptional variations in bed bugs following ingestion of bacteria, we extracted and processed mRNA from body tissues of adult male bed bugs after ingestion of sterile blood or blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis or the Gram-negative (Gr-) bacterium Escherichia coli. We analyzed mRNA from the bed bugs' midgut (the primary tissue involved in blood ingestion) and from the rest of their bodies (RoB; body minus head and midgut tissues). We show that the midgut exhibits a stronger immune response to ingestion of bacteria than the RoB, as indicated by the expression of genes encoding antimicrobial peptides (AMPs). Both the Toll and Imd signaling pathways, associated with immune responses, were highly activated by the ingestion of bacteria. Bacterial infection in bed bugs further provides evidence for metabolic reconfiguration and resource allocation in the bed bugs' midgut and RoB to promote production of AMPs. Our data suggest that infection with particular pathogens in bed bugs may be associated with altered metabolic pathways within the midgut and RoB that favors immune responses. We further show that multiple established cellular immune responses are preserved and are activated by the presence of specific pathogens. Our study provides a greater understanding of nuances in the immune responses of bed bugs towards pathogens that ultimately might contribute to novel bed bug control tactics.

Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles.

There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

The influence of neonatal BCG vaccination on in vitro cytokine responses to Plasmodium falciparum.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli. RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli. CONCLUSION: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

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.

Exploration of the immune response of grass carp (Ctenopharyngodon idellus) erythrocytes during bacterial infection.

In teleost blood, red blood cells (RBCs) are the most common type of cell, and they differ from mammalian RBCs in having a nucleus and other organelles. As nucleated cells, teleost RBCs contribute to the immune response against pathogens, but their antibacterial mechanism remains unclear. Here, we utilized RNA-Seq to analyze gene expression patterns of grass carp (Ctenopharyngodon idellus) RBCs (GcRBCs) stimulated by Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. Our transcriptomic data showed that bacterial stimulation generated many differentially expressed genes (DEGs). Furthermore, several inflammatory pathways responded to bacterial activation, and the TLR, IL-17, and tumor necrosis factor (TNF) signaling pathways were significantly activated based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, the findings of qRT-PCR showed markedly elevated expression of various cytokines, including IL-1ß, IL4, IL6, IL8, IL12, and TNFα, in GcRBCs after incubation with bacteria. Reactive oxygen species (ROS) production in GcRBCs was markedly increased after the cells were stimulated with the three bacteria, and the expression of superoxide dismutase, glutathione peroxidase, and antioxidant enzymes, including catalase, was altered. Flow cytometry analysis showed that the apoptosis rate of GcRBCs was enhanced after stimulation with the three bacteria for different times. In summary, our findings reveal that bacterial stimulation activates the immune response of GcRBCs by regulating ROS release, cytokine expression, and the antioxidant system, leading to apoptosis of GcRBCs.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

Probiotic-guided CAR-T cells for solid tumor targeting.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

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.

Direct activation of a bacterial innate immune system by a viral capsid protein.

Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity.

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.

Lipofundin mediates major inhibition of intravenous propofol on phorbol myristate acetate and Escherichia coli-induced neutrophil extracellular traps.

BACKGROUND: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. METHODS: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. RESULTS: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. CONCLUSIONS: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.

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.

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.

Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons.

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

Modulation of Dendritic Cells by Microbiota Extracellular Vesicles Influences the Cytokine Profile and Exosome Cargo.

Gut bacteria release extracellular vesicles (BEVs) as an intercellular communication mechanism that primes the host innate immune system. BEVs from E. coli activate dendritic cells (DCs) and subsequent T-cell responses in a strain-specific manner. The specific immunomodulatory effects were, in part, mediated by differential regulation of miRNAs. This study aimed to deepen understanding of the mechanisms of BEVs to drive specific immune responses by analyzing their impact on DC-secreted cytokines and exosomes. DCs were challenged with BEVs from probiotic and commensal E. coli strains. The ability of DC-secreted factors to activate T-cell responses was assessed by cytokine quantification in indirect DCs/naïve CD4+ T-cells co-cultures on Transwell supports. DC-exosomes were characterized in terms of costimulatory molecules and miRNAs cargo. In the absence of direct cellular contacts, DC-secreted factors triggered secretion of effector cytokines by T-cells with the same trend as direct DC/T-cell co-cultures. The main differences between the strains influenced the production of Th1- and Treg-specific cytokines. Exosomes released by BEV-activated DCs were enriched in surface proteins involved in antigen presentation and T-cell activation, but differed in the content of immune-related miRNA, depending on the origin of the BEVs. These differences were consistent with the derived immune responses.

Enhanced antibacterial function of a supramolecular artificial receptor-modified macrophage (SAR-Macrophage).

Bacterial infection has become a global concern owing to the significant morbidity and mortality. Although the phagocytosis of bacteria by immune cells acts as the front line to protect human body from invading pathogens, the relatively slow encounter and insufficient capture of bacteria by immune cells often lead to an inefficient clearance of pathogens. Herein, a supramolecular artificial receptor-modified macrophage (SAR-Macrophage) was developed to enhance the recognition and latch of bacteria in the systemic circulation, mediated via strong and multipoint host-guest interactions between the artificial receptors (cucurbit[7]uril) on the macrophage and the guest ligands (adamantane) selectively anchored on Escherichia coli (E. coli). As a result, the SAR-Macrophage could significantly accelerate the recognition of E. coli, catch and internalize more pathogens, which subsequently induced the M1 polarization of macrophages to generate ROS and effectively kill the intracellular bacteria. Therefore, the SAR-Macrophage represents a simple, yet powerful anti-bacterial approach.

E. coli and the etiology of human PBC: Antimitochondrial antibodies and spreading determinants.

BACKGROUND AND AIMS: The increased frequency of urinary tract infections in patients with primary biliary cholangitis (PBC) and the cross-reactivity between the lipoyl domains (LD) of human pyruvate dehydrogenase complex (hPDC-E2) and Escherichia coli PDC-E2 (ePDC-E2) have long suggested a role of E. coli in causality of PBC. This issue, however, has remained speculative. We hypothesized that by generating specific constructs of human and E. coli PDC-E2, we would be able to assess the specificity of autoantibody responses and define whether exposure to E. coli in susceptible hosts is the basis for the antimitochondrial antibody (AMA) response. APPROACH AND RESULTS: Importantly, the reactivity of hPDC-E2 LD (hPDC-E2LD) affinity-purified antibodies against hPDC-E2LD could only be removed by prior absorption with hPDC-E2LD and not ePDC-E2, suggesting the presence of unique human PDC-E2 epitopes distinct from E. coli PDC-E2. To identify the autoepitope(s) present in hPDC-E2LD, a more detailed study using a variety of PDC-E2 constructs was tested, including the effect of lipoic acid (LA) on ePDC-E2 conformation and AMA recognition. Individual recombinant ePDCE2 LD domains LD1, LD2 and LD3 did not react with either AMA or antibodies to LA (anti-LA), but in contrast, anti-LA was readily reactive against purified recombinant LD1, LD2, and LD3 expressed in tandem (LP); such reactivity increased when LP was precultured with LA. Moreover, when the three LD (LD1, LD2, LD3) domains were expressed in tandem in pET28a or when LD1 was expressed in another plasmid pGEX, they were lipoylated and reactive to PBC sera. CONCLUSIONS: In conclusion, our data are consistent with an exposure to E. coli that elicits specific antibody to ePDC-E2 resulting in determinant spreading and the classic autoantibody to hPDC-E2LD. We argue this is the first step to development of human PBC.

Vitamin C, Hydrocortisone, and the Combination Thereof Significantly Inhibited Two of Nine Inflammatory Markers Induced by Escherichia Coli But Not by Staphylococcus Aureus - When Incubated in Human Whole Blood.

ABSTRACT: Vitamin C combined with hydrocortisone is increasingly being used to treat septic patients, even though this treatment regimen is based on questionable evidence. When used, a marked effect on key players of innate immunity would be expected, as sepsis is featured by a dysregulated immune response.Here, we explored the effect of vitamin C and hydrocortisone alone and combined, in an ex vivo human whole-blood model of Escherichia coli- or Staphylococcus aureus-induced inflammation. Inflammatory markers for activation of complement (terminal C5b-9 complement complex [TCC]), granulocytes (myeloperoxidase), platelets (ß-thromboglobulin), cytokines (tumor necrosis factor [TNF], IL-1ß, IL6, and IL-8), and leukocytes (CD11b and oxidative burst) were quantified, by enzyme-linked immunosorbent assay, multiplex technology, and flow cytometry.In E. coli- and S. aureus-stimulated whole blood, a broad dose-titration of vitamin C and hydrocortisone alone did not lead to dose-response effects for the central innate immune mediators TCC and IL-6. Hence, the clinically relevant doses were used further. Compared to the untreated control sample, two of the nine biomarkers induced by E. coli were reduced by hydrocortisone and/or vitamin C. TNF was reduced by hydrocortisone alone (19%, P = 0.01) and by the combination (31%, P = 0.01). The oxidative burst of monocytes and granulocytes was reduced for both drugs alone and their combination, (ranging 8-19%, P < 0.05). Using S. aureus, neither of the drugs, alone nor in combination, had any effects on the nine biomarkers.In conclusion, despite the limitation of the ex vivo model, the effect of vitamin C and hydrocortisone on bacteria-induced inflammatory response in human whole blood is limited and following the clinical data.

Recombinant Escherichia coli Cell Lysates as a Low-Cost Alternative for Vaccines Against Veterinary Clostridial Diseases.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.