Display of FliC131 on the Surface of Lactococcus lactis as a Strategy to Increase its Adjuvanticity for Mucosal Immunization.

Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.

Nasal immunization with H7 flagellin protects mice against hemolytic uremic syndrome secondary to Escherichia coli O157:H7 gastrointestinal infection.

Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections. Materials and methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo. Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo, and surviving mice presented high titres of anti-H7 and Stx antibodies. Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors.

Intestinal Transplant Immunology and Intestinal Graft Rejection: From Basic Mechanisms to Potential Biomarkers.

Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since its inception, it became obvious that intestinal grafts are highly immunogenic, due to their high lymphoid load, the abundance in epithelial cells and constant exposure to external antigens and microbiota. This combination of factors and several redundant effector pathways makes ITx immunobiology unique. To this complex immunologic situation, which leads to the highest rate of rejection among solid organs (>40%), there is added the lack of reliable non-invasive biomarkers, which would allow for frequent, convenient and reliable rejection surveillance. Numerous assays, of which several were previously used in inflammatory bowel disease, have been tested after ITx, but none have shown sufficient sensibility and/or specificity to be used alone for diagnosing acute rejection. Herein, we review and integrate the mechanistic aspects of graft rejection with the current knowledge of ITx immunobiology and summarize the quest for a noninvasive biomarker of rejection.

When less is more: Experimental Bishop-Koop technique for reduction in the use of laboratory animals for intestinal pathophysiological studies.

The use of animals to gain knowledge and understanding of diseases needs to be reduced and refined. In the field of intestinal research, because of the complexity of the gut immune system, living models testing is mandatory. Based on the 3Rs (replacement, reduction and refinement) principles, we aimed to developed and apply the derived-intestinal surgical procedure described by Bishop and Koop (BK) in rats to refine experimental gastrointestinal procedures and reduce the number of animals used for research employing two models of intestinal inflammation: intestinal ischemia-reperfusion injury and chemical-induced colitis. Our results show the feasibility of the application of the BK technique in rodents, with good success after surgical procedure in both small and large intestine (100% survival, clinical recovery and weight regain). A considerable reduction in the use of the number of rats in both intestinal inflammation models (80% in case of intestinal ischemia-reperfusion damage and 66.6% in chemical-induced colitis in our experimental design) was achieved. Compared with conventional experimental models described by various research groups, we report excellent reproducibility of intestinal damage and functionality, survival rate and clinical status of the animals when BK is applied.

Experimental Assessment of Intestinal Damage in Controlled Donation After Circulatory Death for Visceral Transplantation.

There is an urgent need to address the shortage of potential multivisceral grafts in order to reduce the average time in waiting list. Since donation after circulatory death (DCD) has been successfully employed for other solid organs, a thorough evaluation of the use of intestinal grafts from DCD is warranted. Here, we have generated a model of Maastricht III DCD in rodents, focusing on the viability of intestinal and multivisceral grafts at five (DCD5) and twenty (DCD20) minutes of cardiac arrest compared to living and brain death donors. DCD groups exhibited time-dependent damage. DCD20 generated substantial intestinal mucosal injury and decreased number of Goblet cells whereas grafts from DCD5 closely resemble those of brain death and living donors groups in terms intestinal morphology, expression of tight junction proteins and number of Paneth and Globet cells. Upon transplantation, intestines from DCD5 showed increased ischemia/reperfusion damage compared to living donor grafts, however mucosal integrity was recovered 48 h after transplantation. No differences in terms of graft rejection, gene expression and absorptive function between DCD5 and living donor were observed at 7 post-transplant days. Collectively, our results highlight DCD as a possible strategy to increase multivisceral donation and transplantation procedures.

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

OBJECTIVE: We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. SUMMARY BACKGROUND DATA: GVHD is one of the most severe and frequently lethal hematological complications after VT procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. METHODS: Our study included both clinical and experimental data. A total of 108 patients were divided into 3 groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into 2 groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. RESULTS: The patients with native spleen preservation showed a lower rate of GVHD ( P <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). CONCLUSIONS: The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

Lidocaine reinforces the anti-inflammatory action of dexamethasone on myeloid and epithelial cells activated by inflammatory cytokines or SARS-CoV-2 infection.

BACKGROUND: Severe cases of Coronavirus Disease 2019 (COVID-19) that require admission to the Intensive Care Unit (ICU) and mechanical ventilation assistance show a high mortality rate with currently few therapeutic options available. Severe COVID-19 is characterized by a systemic inflammatory condition, also called "cytokine storm", which can lead to various multi-organ complications and ultimately death. Lidocaine, a safe local anesthetic that given intravenously is used to treat arrhythmias, has long been reported to have an anti-inflammatory and pro-homeostatic activity. METHODS: We studied the capacity of lidocaine to modulate cytokine secretion of mouse and human myeloid cell lines activated by different cytokines or Toll Like Receptor (TLR) ligands (flagellin (FliC), Lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (Poly I:C) and N-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl (Pam3Cys-SKKKK)) or by Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection to epithelial cells. Reporter cell lines were used to study modulation of lidocaine of specific signaling pathways. RESULTS: Lidocaine used in combination with dexamethasone, had an additive effect in the modulation of cellular inflammatory response triggered by Tumoral Necrosis Factor alpha (TNFα), Interleukin 1 beta (IL-1ß) as well as different TLR ligands. We also found that lidocaine in combination with dexamethasone modulates the Nuclear factor kappa B (NF-κB) pathway, inflammasome activation as well as interferon gamma receptor (IFNγR) signaling without affecting the type I interferons (Type I IFNs) pathway. Furthermore, we showed that lidocaine and dexamethasone treatment of epithelial cells infected with SARS-CoV-2 modulated the expression of chemokines that contribute to pro-inflammatory effects in severe COVID. CONCLUSIONS: We reported for the first time in vitro anti-inflammatory capacity of lidocaine on SARS-CoV-2 triggered immune pathways. These results indicated the potential of lidocaine to treat COVID-19 patients and add tools to the therapeutic options available for these concerning cases.

COVID-19 Vaccination Responses with Different Vaccine Platforms in Patients with Inborn Errors of Immunity.

Patients with inborn errors of immunity (IEI) in Argentina were encouraged to receive licensed Sputnik, AstraZeneca, Sinopharm, Moderna, and Pfizer vaccines, even though most of the data of humoral and cellular responses combination on available vaccines comes from trials conducted in healthy individuals. We aimed to evaluate the safety and immunogenicity of the different vaccines in IEI patients in Argentina. The study cohort included adults and pediatric IEI patients (n = 118) and age-matched healthy controls (HC) (n = 37). B cell response was evaluated by measuring IgG anti-spike/receptor binding domain (S/RBD) and anti-nucleocapsid(N) antibodies by ELISA. Neutralization antibodies were also assessed with an alpha-S protein-expressing pseudo-virus assay. The T cell response was analyzed by IFN-γ secretion on S- or N-stimulated PBMC by ELISPOT and the frequency of S-specific circulating T follicular-helper cells (TFH) was evaluated by flow cytometry.No moderate/severe vaccine-associated adverse events were observed. Anti-S/RBD titers showed significant differences in both pediatric and adult IEI patients versus the age-matched HC cohort (p < 0.05). Neutralizing antibodies were also significantly lower in the patient cohort than in age-matched HC (p < 0.01). Positive S-specific IFN-γ response was observed in 84.5% of IEI patients and 82.1% presented S-specific TFH cells. Moderna vaccines, which were mainly administered in the pediatric population, elicited a stronger humoral response in IEI patients, both in antibody titer and neutralization capacity, but the cellular immune response was similar between vaccine platforms. No difference in humoral response was observed between vaccinated patients with and without previous SARS-CoV-2 infection.In conclusion, COVID-19 vaccines showed safety in IEI patients and, although immunogenicity was lower than HC, they showed specific anti-S/RBD IgG, neutralizing antibody titers, and T cell-dependent cellular immunity with IFN-γ secreting cells. These findings may guide the recommendation for a vaccination with all the available vaccines in IEI patients to prevent COVID-19 disease.

Luminal Preservation Protects the Small Intestine in a Brain-dead Rat Model.

Intestinal transplantation depends on donation after brain death (DBD). Luminal preservation (LP) has been beneficial against preservation injury in previous studies in animal models, but none include DBD. This study aims to investigate whether these benefits occur also with DBD. Methods: Wistar rats (male, N = 9) underwent brain death for 2 h. Thereafter, vascular perfusion was done with University of Wisconsin solution (UW). The small intestine was then explanted and randomized into 3 groups: control (empty segment), LP+PEG (with polyethylene glycol 3350 solution), or LP+UW (with UW), treated and tied shut. Ice-cold UW was used for cold storage. Samples were taken at procurement and after 4 (t = 4) and 8 h (t = 8) of preservation. Histopathological scorings were performed for intestinal preservation injury, subepithelial space, absence of epithelial lining, and hemeoxygenase-1 expression. Results: There was low-level mucosal injury (median intestinal preservation injury score 2) at procurement. At t = 4, bowels treated without LP had more damage than LP-treated samples (control score 4, LP+PEG 2 and LP+UW 2, P < 0.001 control versus LP+UW). At t = 8, no benefit of LP was observed (control 2, LP+PEG 3, LP+UW 2). Subepithelial space increased with time and the presence of LP; epithelial lining was better conserved in LP-treated samples. Hemeoxygenase-1 staining showed increased intensity with increased damage, irrespective of treatment. Conclusions: Luminal perfusion of the small intestine with UW or PEG protects the mucosa in brain-dead rats for up to 4 h. Fewer benefits of LP were found than previously described in non-DBD models. To mimic the clinical situation, DBD should be included in future animal studies on intestinal preservation.

Few Amino Acid Mutations in H6 Influenza A Virus From South American Lineage Increase Viral Replication Efficiency in Poultry.

In chickens, infections due to influenza A virus (IAV) can be mild to severe and lethal. The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passaged in chickens 20 times, and the immunopathological effects of the last passage virus, 20Ch557/H6N2, in chickens were compared to those of the parental strain. A comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild-type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naïve chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from the ileum revealed a significant increase in the percentages of CD3+TCRγδ+ IELs in chickens inoculated with the 20Ch557/H6N2 strain compared to those inoculated with the WT557/H6N2 strain.

Experimental study to assess the impact of vasopressors administered during maintenance of the brain-dead donation in the quality of the intestinal graft.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3+ counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3+ mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE (p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver (p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

BACKGROUND: The hemodynamic maintenance of brain-dead donors will influence the quality of the organs procured for transplantation, including the intestine. Although norepinephrine (NE) and dopamine (DA) are commonly used to sustain mean arterial pressure in humans, there are no standardized protocols for their use during maintenance of brain-dead donors. Our aim was to compare the effects of each drug, in the intestinal graft quality using a rat brain-dead donation model. METHODS: Wistar rats (N = 17) underwent brain death (BD) for 2 hours with NE (NE group) or with DA (DA group) administration; the control group was mechanically ventilated for 2 hours without BD. Jejunum biopsies were obtained at the end of the maintenance period. Histological damage was evaluated using Park-Chiu scale. Villi/crypt ratio, mucosal thickness, Goblet cell count, and villi density were evaluated using ImageJ software (US National Institutes of Health, Bethesda, MD). Barrier damage was assessed by bacterial translocation culture counting on liver samples. The inflammatory status of the intestine was evaluated by CD3 + counting by immunohistochemistry and gene expression analysis of interleukin (IL)-6, IL-22, and CXCL10. RESULTS: Norepinephrine-treated donors had higher focal ischemic injury in the intestinal mucosa without a substantial modification of morphometrical parameters compared with DA-treated donors. CD3 + mucosal infiltration was greater in intestines procured from brain-dead donors, being highest in NE ( p ˂ 0.001). Local inflammatory mediators were affected in BD: DA and NE groups showed a trend to lower expression of IL-22, whereas CXCL10 expression was higher in NE versus control group. Brain death promoted intestinal bacterial translocation, but the use of NE resulted in the highest bacterial counting in the liver ( p ˂ 0.01). CONCLUSION: Our results favor the use of DA instead of NE as main vasoactive drug to manage BD-associated hemodynamic instability. Dopamine may contribute to improve the quality of the intestinal graft, by better preserving barrier function and lowering immune cell infiltration.

Induction Versus Maintenance Immunosuppression After Intestinal Transplant: Determining Which Treatment Most Impacts Long-Term Patient And Graft Survival.

OBJECTIVES: Immunosuppressive strategies for intestinal transplant have changed over time. However, specific intestinal transplant-oriented protocols and reports on long-term maintenance regimens are scarce. Our objective was to evaluate the impact of 2 different initial immunosuppressive protocols based on thymoglobulin (group A) and basiliximab (anti-interleukin 2 antibody) (group B) and of changes to maintenance immunosuppression over long-term follow-up in intestinal transplant recipients. MATERIALS AND METHODS: We performed a retrospective analysis of a prospectively established protocol for intestinal transplant immunosuppression, conducted between May 2006 and December 2020. We analyzed 51 intestinal transplant recipients, with 6 patients excluded because of early death or graft loss. Acute cellular rejection frequency and grade, number of acute cellular rejection episodes, time to the first acute cellular rejection episode, response to treatment, number of patients who progressed to chronic allograft rejection, kidney function, infections, incidence of posttransplant lymphoproliferative disorder and graft-versus-host disease, and patient and graft survival were analyzed. RESULTS: In the study groups, there were 87 acute cellular rejection episodes in 45 patients (33 in group A and 54 in group B). We found degree of acute cellular rejection to be mild in 45 patients, moderate in 18, and severe in 24 (not significant between groups). Our comparison of induction therapy (thymoglobulin [group A] vs interleukin 2 antibody [group B]) did not show any statistical difference during clinical followup. Long-term review showed that all patients were on tacrolimus. Five-year patient and graft survival rates were 62% and 45% for group A and 54% and 46% for group B, respectively (not significant). CONCLUSIONS: Long-term patient and graft outcomes reflected the use of an individualized follow-up with adjustments and changes in immunosuppressive medications according to the patient's clinical course and complications rather than based on the induction immunosuppressive protocol used.

A biorefinery concept for the production of fuel ethanol, probiotic yeast, and whey protein from a by-product of the cheese industry.

Agroindustrial by-products and residues can be transformed into valuable compounds in biorefineries. Here, we present a new concept: production of fuel ethanol, whey protein, and probiotic yeast from cheese whey. An initial screening under industrially relevant conditions, involving thirty Kluyveromyces marxianus strains, was carried out using spot assays to evaluate their capacity to grow on cheese whey or on whey permeate (100 g lactose/L), under aerobic or anaerobic conditions, in the absence or presence of 5% ethanol, at pH 5.8 or pH 2.5. The four best growing K. marxianus strains were selected and further evaluated in a miniaturized industrial fermentation process using reconstituted whey permeate (100 g lactose/L) with cell recycling (involving sulfuric acid treatment). After five consecutive fermentation cycles, the ethanol yield on sugar reached 90% of the theoretical maximum in the best cases, with 90% cell viability. Cells harvested at this point displayed probiotic properties such as the capacity to survive the passage through the gastrointestinal tract and capacity to modulate the innate immune response of intestinal epithelium, both in vitro. Furthermore, the CIDCA 9121 strain was able to protect against histopathological damage in an animal model of acute colitis. Our findings demonstrate that K. marxianus CIDCA 9121 is capable of efficiently fermenting the lactose present in whey permeate to ethanol and that the remaining yeast biomass has probiotic properties, enabling an integrated process for the obtainment of whey protein (WP), fuel ethanol, and probiotics from cheese whey.Key points• K. marxianus-selected strains ferment whey permeate with 90% ethanol yield.• Industrial fermentation conditions do not affect selected yeast probiotic capacity.• Whey permeate, fuel ethanol, and probiotic biomass can be obtained in a biorefinery.

Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

"Spexin improves adipose tissue inflammation and macrophage recruitment in obese mice".

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 µg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1ß and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C- populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C- macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.

Acute cellular rejection in small-bowel transplantation impairs NCR+ innate lymphoid cell subpopulation 3/interleukin 22 axis.

Acute cellular rejection (ACR) remains as one of the main causes of graft loss and death in intestinal transplant (ITx) patients. ACR promotes intestinal injury, disruption of the mucosal barrier, bacterial translocation, and organ dysfunction. As epithelial regeneration is critical in reversing these consequences, the functional axis between the innate lymphoid cell subpopulation 3 (ILC3) and interleukin 22 plays an essential role in that process. Natural-cytotoxic-receptor-positive (NCR+) ILC3 cells have been demonstrated to induce intestinal-stem-cell proliferation along with an IL-22-dependent expansion of that population in several intestinal pathologies, though thus far not after ITx. Therefore, we intended to determine the impact of chronic immunosuppression and ACR on ILC3 cells and interleukin-22 (IL-22) production in the lamina propria after that intervention. MATERIALS AND METHODS: We compared biopsies from healthy volunteers with biopsies from ITx recipients without or with mild-to-moderate ACR, using flow cytometry and the quantitative-PCR. RESULTS: NCR+ ILC3 cells were found to be unaffected by immunosuppression at different time points posttransplant when patients did not experience ACR, but were diminished upon the occurrence of ACR independently of the post-ITx time. Moreover, IL-22-expression levels were notably reduced in ACR. CONCLUSION: The NCR+-ILC3/IL-22 axis is impaired during ACR contributing to a delay in or lack of a complete and efficient epithelial regeneration. Thus, our findings reveal that IL-22 analogues could potentially be used as a new complementary therapeutic approach, in conjunction with immunosuppressant drugs, in order to promote mucosal regeneration upon ACR.