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.

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.

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.

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.

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.

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.

The Protective Effects of Lactobacillus plantarum KLDS 1.0344 on LPS-Induced Mastitis In Vitro and In Vivo.

Cow mastitis, which significantly lowers milk quality, is mainly caused by pathogenic bacteria such as E. coli. Previous studies have suggested that lactic acid bacteria can have antagonistic effects on pathogenic bacteria that cause mastitis. In the current study, we evaluated the in vitro and in vivo alleviative effects of L. plantarum KLDS 1.0344 in mastitis treatment. In vitro antibacterial experiments were performed using bovine mammary epithelial cell (bMEC), followed by in vivo studies involving mastitis mouse models. In vitro results indicate that lactic acid was the primary substance inhibiting the E. coli pathogen. Meanwhile, treatment with L. plantarum KLDS 1.0344 can reduce cytokines' mRNA expression levels in the inflammatory response of bMEC induced by LPS. In vivo, the use of this strain reduced the secretion of inflammatory factors IL-6, IL-1ß, and TNF-α, and decreased the activity of myeloperoxidase (MPO), and inhibited the secretion of p-p65 and p-IκBα. These results indicate that L. plantarum KLDS 1.0344 pretreatment can reduce the expression of inflammatory factors by inhibiting the activation of NF-κB signaling pathway, thus exerting prevent the occurrence of inflammation in vivo. Our findings show that L. plantarum KLDS 1.0344 has excellent properties as an alternative to antibiotics and can be developed into lactic acid bacteria preparation to prevent mastitis disease.

O-Acetylation of Capsular Polysialic Acid Enables Escherichia coli K1 Escaping from Siglec-Mediated Innate Immunity and Lysosomal Degradation of E. coli-Containing Vacuoles in Macrophage-Like Cells.

Escherichia coli K1 causes bacteremia and meningitis in human neonates. The K1 capsule, an α2,8-linked polysialic acid (PSA) homopolymer, is its essential virulence factor. PSA is usually partially modified by O-acetyl groups. It is known that O-acetylation alters the antigenicity of PSA, but its impact on the interactions between E. coli K1 and host cells is unclear. In this study, a phase variant was obtained by passage of E. coli K1 parent strain, which expressed a capsule with 44% O-acetylation whereas the capsule of the parent strain has only 3%. The variant strain showed significantly reduced adherence and invasion to macrophage-like cells in comparison to the parent strain. Furthermore, we found that O-acetylation of PSA enhanced the modulation of trafficking of E. coli-containing vacuoles (ECV), enabling them to avoid fusing with lysosomes in these cells. Intriguingly, by using quartz crystal microbalance, we demonstrated that the PSA purified from the parent strain interacted with human sialic acid-binding immunoglobulin-like lectins (Siglecs), including Siglec-5, Siglec-7, Siglec-11, and Siglec-14. However, O-acetylated PSA from the variant interacted much less and also suppressed the production of Siglec-mediated proinflammatory cytokines. The adherence of the parent strain to human macrophage-like cells was significantly blocked by monoclonal antibodies against Siglec-11 and Siglec-14. Furthermore, the variant strain caused increased bacteremia and higher lethality in neonatal mice compared to the parent strain. These data elucidate that O-acetylation of K1 capsule enables E. coli to escape from Siglec-mediated innate immunity and lysosomal degradation; therefore, it is a strategy used by E. coli K1 to regulate its virulence. IMPORTANCE Escherichia coli K1 is a leading cause of neonatal meningitis. The mortality and morbidity of this disease remain significantly high despite antibiotic therapy. One major limitation on advances in prevention and therapy for meningitis is an incomplete understanding of its pathogenesis. E. coli K1 is surrounded by PSA, which is observed to have high-frequency variation of O-acetyl modification. Here, we present an in-depth study of the function of O-acetylation in PSA at each stage of host-pathogen interaction. We found that a high level of O-acetylation significantly interfered with Siglec-mediated bacterial adherence to macrophage-like cells, and blunted the proinflammatory response. Furthermore, the O-acetylation of PSA modulated the trafficking of ECVs to prevent them from fusing with lysosomes, enabling them to escape degradation by lysozymes within these cells. Elucidating how subtle modification of the capsule enhances bacterial defenses against host innate immunity will enable the future development of effective drugs or vaccines against infection by E. coli K1.

Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem.

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coli Assisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag-Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein-protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

Transcriptional Profiling of Mouse Eosinophils Identifies Distinct Gene Signatures Following Cellular Activation.

Eosinophils are multifunctional, evolutionary conserved leukocytes that are involved in a plethora of responses ranging from regulation of tissue homeostasis to host defense and cancer. Eosinophils have been studied mostly in the context of Type 2 inflammatory responses such as those found in allergy. Nonetheless, it is now evident that they participate in Type 1 inflammatory responses and can respond to Type 1 cytokines such as IFN-γ. Recent data suggest that the pleotropic roles of eosinophils are due to heterogeneous responses to environmental cues. Despite this, the activation profile of eosinophils, in response to various stimuli is yet to be defined. To better understand the transcriptional spectrum of eosinophil activation, we exposed eosinophils to Type 1 (e.g. IFN-γ, E. coli) vs. Type 2 (e.g. IL-4) conditions and subjected them to global RNA sequencing. Our analyses show that IL-4, IFN-γ, E. coli and IFN-γ in the presence of E. coli (IFN-γ/E. coli)-stimulated eosinophils acquire distinct transcriptional profiles, which polarize them towards what we termed Type 1 and Type 2 eosinophils. Bioinformatics analyses using Gene Ontology based on biological processes revealed that different stimuli induced distinct pathways in eosinophils. These pathways were confirmed using functional assays by assessing cytokine/chemokine release (i.e. CXCL9, CCL24, TNF-α and IL-6) from eosinophils following activation. In addition, analysis of cell surface markers highlighted CD101 and CD274 as potential cell surface markers that distinguish between Type 1 and Type 2 eosinophils, respectively. Finally, the transcriptome signature of Type 1 eosinophils resembled that of eosinophils that were obtained from mice with experimental colitis whereas the transcriptome signature of Type 2 eosinophils resembled that of eosinophils from experimental asthma. Our data demonstrate that eosinophils are polarized to distinct "Type 1" and "Type 2" phenotypes following distinct stimulations. These findings provide fundamental knowledge regarding the heterogeneity of eosinophils and support the presence of transcriptional differences between Type 1 and Type 2 cells that are likely reflected by their pleotropic activities in diverse disease settings.

Cordyceps militaris Immunomodulatory Protein Promotes the Phagocytic Ability of Macrophages through the TLR4-NF-κB Pathway.

Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p < 0.05), but also enhanced the bactericidal activity (p < 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p < 0.01), accompanied by an increase in the F-actin expression (p < 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p < 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p < 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

Psychological stress impairs IL22-driven protective gut mucosal immunity against colonising pathobionts.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45+CD90+ cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis.

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti.

BACKGROUND: Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies-CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE-based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. METHODS: Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. RESULTS: We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. CONCLUSIONS: Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.

Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling.

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

Faster and less invasive tools to identify patients with ileal colonization by adherent-invasive E. coli in Crohn's disease.

BACKGROUND AND AIMS: The identification of Crohn's disease (CD)-associated adherent and invasive Escherichia coli (AIEC) is time-consuming and requires ileal biopsies. We aimed to identify a faster and less invasive methods to detect ileal colonization by AIEC in CD patients. METHODS: CD patients requiring ileo-colonoscopy were consecutively enrolled in this prospective multicenter study. Samples from saliva, serum, stools, and ileal biopsies of CD patients were collected. RESULTS: Among 102 CD patients, the prevalence of AIEC on ileal biopsies was 24.5%. The abundance and global invasive ability of ileal-associated total E. coli were respectively ten-fold (p = 0.0065) and two-fold (p = 0.0007) higher in AIEC-positive (vs. AIEC-negative), while abundance of total E. coli in the feces was not correlated with AIEC status in the ileum. The best threshold of ileal total E. coli was 60 cfu/biopsy to detect AIEC-positive patients, with high negative predictive value (NPV) (94.1%[80.3-99.3]), while the global invasive ability (>9000 internalized bacteria) was able to detect the presence of AIEC with high positive predictive value (80.0% [55.2-100.0]). Overall, 78.1% of the AIEC + patients were colonized by two or less different AIEC strains. The level of serum anti-total E. coli antibodies (AEcAb) was higher in AIEC-positive patients (p = 0.038) with a very high negative predictive value (96.6% [89.9-100.0]) (p = 0.038) for a cut-off value > 1.9 × 10-3 . CONCLUSIONS: More than two thirds of AIEC-positive CD patients were colonized by two or less AIEC strains. While stools samples are not accurate to screen AIEC status, the AEcAb level appears to be an attractive, rapid and easier biomarker to identify patients with Crohn's disease harboring AIEC.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis.

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing ß-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines - interleukin (IL) 6, IL-1ß and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, "opsonisation" of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.