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Introduction: Confronted with the emerging threat of antimicrobial resistance, the development of alternative strategies to limit the use of antibiotics or potentiate their effect through synergy with the immune system is urgently needed. Many natural or synthetic biological response modifiers have been investigated in this context. Among them, β-glucans, a type of soluble or insoluble polysaccharide composed of a linear or branched string of glucose molecules produced by various cereals, bacteria, algae, and inferior (yeast) and superior fungi (mushrooms) have garnered interest in the scientific community, with not less than 10,000 publications over the last two decades. Various biological activities of β-glucans have been reported, such as anticancer, antidiabetic and immune-modulating effects. In vitro, yeast β-glucans are known to markedly increase cytokine secretion of monocytes/macrophages during a secondary challenge, a phenomenon called immune training., Methods: Here, we orally delivered β-glucans derived from the yeast S. cerevisiae to mice that were further challenged with Escherichia coli., Results: β-glucan supplementation protected the mice from E. coli intraperitoneal and intra-mammary infections, as shown by a lower bacterial burden and greatly diminished tissue damage. Surprisingly, this was not associated with an increased local immune response. In addition, granulocyte recruitment was transient and limited, as well as local cytokine secretion, arguing for faster resolution of the inflammatory response. Furthermore, ex-vivo evaluation of monocytes/macrophages isolated or differentiated from β-glucan-supplemented mice showed these cells to lack a trained response versus those from control mice., Conclusion: In conclusion, dietary β-glucans can improve the outcome of Escherichia coli infections and dampen tissue damages associated to excessive inflammatory response. The mechanisms associated with such protection are not necessarily linked to immune system hyper-activation or immune training., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Walachowski, Breyne, Secher, Cougoule, Guzylack-Piriou, Meyer, Foucras and Tabouret.)

Background : Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db / db mice. Methods : Female db / db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db / db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results : Vehicle-dosed db / db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db / db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions : Semaglutide improves disease hallmarks in the db / db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of DKD.

The current understanding of molecular mechanisms driving diabetic kidney disease (DKD) is limited, partly due to the complex structure of the kidney. To identify genes and signalling pathways involved in the progression of DKD, we compared kidney cortical versus glomerular transcriptome profiles in uninephrectomized (UNx) db/db mouse models of early-stage (UNx only) and advanced [UNxplus adeno-associated virus-mediated renin-1 overexpression (UNx-Renin)] DKD using RNAseq. Compared to normoglycemic db/m mice, db/db UNx and db/db UNx-Renin mice showed marked changes in their kidney cortical and glomerular gene expression profiles. UNx-Renin mice displayed more marked perturbations in gene components associated with the activation of the immune system and enhanced extracellular matrix remodelling, supporting histological hallmarks of progressive DKD in this model. Single-nucleus RNAseq enabled the linking of transcriptome profiles to specific kidney cell types. In conclusion, integration of RNAseq at the cortical, glomerular and single-nucleus level provides an enhanced resolution of molecular signalling pathways associated with disease progression in preclinical models of DKD, and may thus be advantageous for identifying novel therapeutic targets in DKD., Competing Interests: Competing interests F.E.S., H.M.Æ., J.C.N., T.S., M.V.Ø., K.T.G.R. and L.N.F. are employees of Gubra ApS. N.V. and J.J. are owners of Gubra ApS., (© 2021. Published by The Company of Biologists Ltd.)

Hypertension is a critical comorbidity for progression of diabetic kidney disease (DKD). To facilitate the development of novel therapeutic interventions with the potential to control disease progression, there is a need to establish translational animal models that predict treatment effects in human DKD. The present study aimed to characterize renal disease and outcomes of standard of medical care in a model of advanced DKD facilitated by adeno-associated virus (AAV)-mediated renin overexpression in uninephrectomized (UNx) db/db mice. Five weeks after single AAV administration and 4 wk after UNx, female db/db UNx-ReninAAV mice received (PO, QD) vehicle, lisinopril (40 mg/kg), empagliflozin (20 mg/kg), or combination treatment for 12 wk ( n = 17 mice/group). Untreated db /+ mice ( n = 8) and vehicle-dosed db/db UNx-LacZAAV mice ( n = 17) served as controls. End points included plasma, urine, and histomorphometric markers of kidney disease. Total glomerular numbers and individual glomerular volume were evaluated by whole kidney three-dimensional imaging analysis. db/db UNx-ReninAAV mice developed hallmarks of progressive DKD characterized by severe albuminuria, advanced glomerulosclerosis, and glomerular hypertrophy. Lisinopril significantly improved albuminuria, glomerulosclerosis, tubulointerstitial injury, and inflammation. Although empagliflozin alone had no therapeutic effect on renal endpoints, lisinopril and empagliflozin exerted synergistic effects on renal histological outcomes. In conclusion, the db/db UNx-ReninAAV mouse demonstrates good clinical translatability with respect to physiological and histological hallmarks of progressive DKD. The efficacy of standard of care to control hypertension and hyperglycemia provides a proof of concept for testing novel drug therapies in the model. NEW & NOTEWORTHY Translational animal models of diabetic kidney disease (DKD) are important tools in preclinical research and drug discovery. Here, we show that the standard of care to control hypertension (lisinopril) and hyperglycemia (empagliflozin) improves physiological and histopathological hallmarks of kidney disease in a mouse model of hypertension-accelerated progressive DKD. The findings substantiate hypertension and type 2 diabetes as essential factors in driving DKD progression and provide a proof of concept for probing novel drugs for potential nephroprotective efficacy in this model.

Several types of antibodies (Abs) are currently used in non-small cell lung cancer (NSCLC). Anti-angiogenic and immune checkpoint inhibitor (ICI) Abs are the most frequent treatments used alone or with chemotherapy in metastatic NSCLC, for the front line and beyond. Considering the many therapeutic options for locally advanced and metastatic lung cancer and differences in use according to geographic area, we present here a comprehensive review of the marketed ICI and anti-angiogenic Abs approved in the European Union (EU) and the US to treat locally advanced and metastatic NSCLC patients. We briefly describe the different molecules and their development in thoracic oncology and compare pharmacokinetic data, processing decision algorithms and marketing authorizations by the EMA and US Food and Drug Administration (FDA).

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1-deficient mice control chronic P. aeruginosa RP73 infection and IL-1β Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection., (Copyright © 2021 The Authors.)

Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to β-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

Mucosal-associated invariant T (MAIT) cells constitute a highly conserved subset of effector T cells with innate-like recognition of a wide array of bacteria and fungi in humans. Harnessing the potential of these cells could represent a major advance as a new immunotherapy approach to fight difficult-to-treat bacterial infections. However, despite recent advances in the design of potent agonistic ligands for MAIT cells, it has become increasingly evident that adjuvants are required to elicit potent antimicrobial effector functions by these cells, such as IFNγ production and cytotoxicity. Indeed, TCR triggering alone elicits mostly barrier repair functions in MAIT cells, whereas an inflammatory milieu is required to drive the antibacterial functions. Cytokines such as IL-7, IL-12 and IL-18, IL-15 or more recently type 1 IFN all display an apparently similar ability to synergize with TCR stimulation to induce IFNγ production and/or cytotoxic functions in vitro , but their mechanisms of action are not well established. Herein, we show that MAIT cells feature a build-in mechanism to respond to IFNα. We confirm that IFNα acts directly and specifically on MAIT cells and synergizes with TCR/CD3 triggering to induce maximum cytokine production and cytotoxic functions. We provide evidences suggesting that the preferential activation of the Stat4 pathway is involved in the high sensitivity of MAIT cells to IFNα stimulation. Finally, gene expression data confirm the specific responsiveness of MAIT cells to IFNα and pinpoints specific pathways that could be the target of this cytokine. Altogether, these data highlight the potential of IFNα-inducing adjuvants to maximize MAIT cells responsiveness to purified ligands in order to induce potent anti-infectious responses., (Copyright © 2020 Pavlovic, Gross, Chili, Secher and Treiner.)

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN)., Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN., Results: At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification., Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease., Competing Interests: All authors are current employees of Gubra ApS. L. Fink reports other from Novo Nordisk A/S outside the submitted work. J. Jelsing and N. Vrang are owners of Gubra. F. Sembach reports grants from Innovation Foundation outside the submitted work., (Copyright © 2020 by the American Society of Nephrology.)

Diabetic nephropathy (DN) is associated with albuminuria and loss of kidney function and is the leading cause of end-stage renal disease. Despite evidence of sex-associated differences in the progression of DN in human patients, male mice are predominantly being used in preclinical DN research and drug development. Here, we compared renal changes in male and female uninephrectomized (UNx) db/db C57BLKS mice using immunohistochemistry and RNA sequencing. Male and female UNx db/db mice showed similar progression of type 2 diabetes, as assessed by obesity, hyperglycemia, and HbA1c. Progression of DN was also similar between sexes as assessed by kidney and glomerular hypertrophy as well as urine albumin-to-creatinine ratio being increased in UNx db/db compared with control mice. In contrast, kidney collagen III and glomerular collagen IV were increased only in female UNx db/db as compared with respective control mice but showed a similar tendency in male UNx db/db mice. Comparison of renal cortex transcriptomes by RNA sequencing revealed 66 genes differentially expressed (p < .01) in male versus female UNx db/db mice, of which 9 genes were located on the sex chromosomes. In conclusion, male and female UNx db/db mice developed similar hallmarks of DN pathology, suggesting no or weak sex differences in the functional and structural changes during DN progression., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Allergic asthma is characterized by a strong Th2 and Th17 response with inflammatory cell recruitment, airways hyperreactivity and structural changes in the lung. The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with an increasing incidence in developed countries. Recent reports suggest that modulation of the gut microbiota might be one promising therapy for MS. Here, we investigated whether the probiotic Escherichia coli strain Nissle 1917 (ECN) could modulate the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of MS. We evidenced that daily oral treatment with ECN, but not with the archetypal K12 E. coli strain MG1655, reduced the severity of EAE induced by immunization with the MOG 35-55 peptide. This beneficial effect was associated with a decreased secretion of inflammatory cytokines and an increased production of the anti-inflammatory cytokine IL-10 by autoreactive CD4 T cells, both in peripheral lymph nodes and CNS. Interestingly, ECN-treated mice exhibited increased numbers of MOG-specific CD4 + T cells in the periphery contrasting with severely reduced numbers in the CNS, suggesting that ECN might affect T cell migration from the periphery to the CNS through a modulation of their activation and/or differentiation. In addition, we demonstrated that EAE is associated with a profound defect in the intestinal barrier function and that treatment with ECN, but not with MG1655, repaired intestinal permeability dysfunction. Collectively, our data reveal that EAE induces a disruption of the intestinal homeostasis and that ECN protects from disease and restores the intestinal barrier function.

Fibroblast growth factor 23 (FGF23) secreted by osteocytes is a circulating factor essential for phosphate homeostasis. High plasma FGF23 levels are associated with cardiovascular complications and mortality. Increases of plasma FGF23 in uremia antedate high levels of phosphate, suggesting a disrupted feedback regulatory loop or an extra-skeletal source of this phosphatonin. Since induction of FGF23 expression in injured organs has been reported we decided to examine the regulation of FGF23 gene and protein expressions in the kidney and whether kidney-derived FGF23 contributes to the high plasma levels of FGF23 in uremia. FGF23 mRNA was not detected in normal kidneys, but was clearly demonstrated in injured kidneys, already after four hours in obstructive nephropathy and at 8 weeks in the remnant kidney of 5/6 nephrectomized rats. No renal extraction was found in uremic rats in contrast to normal rats. Removal of the remnant kidney had no effect on plasma FGF23 levels. Well-known regulators of FGF23 expression in bone, such as parathyroid hormone, calcitriol, and inhibition of the FGF receptor by PD173074, had no impact on kidney expression of FGF23. Thus, the only direct contribution of the injured kidney to circulating FGF23 levels in uremia appears to be reduced renal extraction of bone-derived FGF23. Kidney-derived FGF23 does not generate high plasma FGF23 levels in uremia and is regulated differently than the corresponding regulation of FGF23 gene expression in bone., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice., Methods: We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor ( Glp1r ), GLP-2 receptor ( Glp2r ), and preproglucagon ( Gcg ) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG)., Results: Comparison of Glp1r , Glp2r , and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r -/- mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology., Conclusion: The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. This study investigates the effect of the food contaminant DON on the genotoxicity of the E. coli strains producing colibactin. In vitro , intestinal epithelial cells were coexposed to DON and E. coli producing colibactin. In vivo , newborn rats colonized at birth with E. coli producing colibactin were fed a DON-contaminated diet. Intestinal DNA damage was estimated by the phosphorylation of histone H2AX. DON exacerbates the genotoxicity of the E. coli producing colibactin in a time- and dose-dependent manner in vitro Although DON had no effect on the composition of the gut microbiota, and especially on the number of E. coli , a significant increase in DNA damage was observed in intestinal epithelial cells of animals colonized by E. coli strains producing colibactin and coexposed to DON compared to animals colonized with E. coli strains unable to produce colibactin or animals exposed only to DON. In conclusion, our data demonstrate that the genotoxicity of E. coli strains producing colibactin, increasingly present in the microbiota of asymptomatic human beings, is modulated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis. IMPORTANCE An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food-especially cereal products-in Europe and North America. Our in vitro and in vivo results demonstrate that the intestinal DNA damage induced by colibactin-producing E. coli strains was exacerbated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis., (Copyright © 2017 Payros et al.)

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The intestinal microbiota exerts vital biological processes throughout the human lifetime, and imbalances in its composition have been implicated in both health and disease status. Upon birth, the neonatal gut moves from a barely sterile to a massively colonized environment. The development of the intestinal microbiota during the first year of life is characterized by rapid and important changes in microbial composition, diversity, and magnitude. The pioneer bacteria colonizing the postnatal intestinal tract profoundly contribute to the establishment of the host-microbe symbiosis, which is essential for health throughout life. Escherichia coli is one of the first colonizers of the gut after birth. E. coli is a versatile population including harmless commensal, probiotic strains as well as frequently deadly pathogens. The prevalence of the specific phylogenetic B2 group, which encompasses both commensal and extra- or intraintestinal pathogenic E. coli strains, is increasing among E. coli strains colonizing infants quickly after birth. Fifty percent of the B2 group strains carry in their genome the pks gene cluster encoding the synthesis of a nonribosomal peptide-polyketide hybrid genotoxin named colibactin. In this review, we summarize both clinical and experimental evidence associating the recently emerging neonatal B2 E. coli population with several pathology and discuss how the expression of colibactin by both normal inhabitants of intestinal microflora and virulent strains may darken the borderline between commensalism and pathogenicity., (Copyright © 2016 the American Physiological Society.)

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

The intestinal barrier controls the balance between tolerance and immunity to luminal antigens. When this finely tuned equilibrium is deregulated, inflammatory disorders can occur. There is a concomitant increase, in urban populations of developed countries, of immune-mediated diseases along with a shift in Escherichia coli population from the declining phylogenetic group A to the newly dominant group B2, including commensal strains producing a genotoxin called colibactin that massively colonized the gut of neonates. Here, we showed that mother-to-offspring early gut colonization by colibactin-producing E. coli impairs intestinal permeability and enhances the transepithelial passage of luminal antigen, leading to an increased immune activation. Functionally, this was accompanied by a dramatic increase in local and systemic immune responses against a fed antigen, decreased regulatory T cell population, tolerogenic dendritic cells, and enhanced mucosal delayed-type hypersensitivity response. Conversely, the abolition of colibactin expression by mutagenesis abrogates the alteration of oral tolerance induced by neonatal colonization by E. coli. In conclusion, the vertical colonization by E. coli producing the genotoxin colibactin enhances intestinal translocation and subsequently alters oral tolerance. Thus, early colonization by E. coli from the newly dominant phylogenetic group B2, which produces colibactin, may represent a risk factor for the development of immune-mediated diseases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or β-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67(+) cells in the exocrine compartment; however, pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 phosphorylation, and mediated through the GLP-1R. Rapamycin abrogated the GLP-1R-dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3β gene expression. Mass spectrometry analysis identified GLP-1R-dependent upregulation of Reg family members, as well as proteins important for translation and export, including Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis, leading to increased pancreatic mass, independent of changes in DNA content or cell proliferation in mice., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB) and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal) activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.

Pseudomonas aeruginosa (P. aeruginosa) infections are associated with considerable morbidity and mortality in immunocompromised patients due to antibiotic resistance. Therefore, we investigated the efficacy of the anti-P. aeruginosa serotype O11 lipopolysaccharide monoclonal antibody Panobacumab in a clinically relevant murine model of neutropenia induced by cyclophosphamide and in combination with meropenem in susceptible and meropenem resistant P. aeruginosa induced pneumonia. We observed that P. aeruginosa induced pneumonia was dramatically increased in neutropenic mice compared to immunocompetent mice. First, Panobacumab significantly reduced lung inflammation and enhanced bacterial clearance from the lung of neutropenic host. Secondly, combination of Panobacumab and meropenem had an additive effect. Third, Panobacumab retained activity on a meropenem resistant P. aeruginosa strain. In conclusion, the present data established that Panobacumab contributes to the clearance of P. aeruginosa in neutropenic hosts as well as in combination with antibiotics in immunocompetent hosts. This suggests beneficial effects of co-treatment even in immunocompromised individuals, suffering most of the morbidity and mortality of P. aeruginosa infections.

The gut flora is composed of a huge number of diverse, well-adapted symbionts that interact with epithelial lining throughout the host's entire life. Not all commensals have the same ability to maintain quiescent, protective inflammation. Importantly, instability in the composition of gut microbial communities (referred to as dysbiosis) has been linked to loss of gut barrier in the context of common human illnesses with increasing socio-economic impacts, such as Crohn disease and colorectal cancer. Our recent findings suggest that disease-predisposing dysbiosis can now be intentionally manipulated by targeting the major Crohn disease-predisposing NOD2 gene. That knowledge will not only add a new dimension to the often overlooked microbiology of Crohn disease and colorectal cancer, but will also have a broad impact on biomedical sciences worldwide.

Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn's disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Chronic intestinal inflammation has been linked to the development of colorectal cancer. Recent studies suggest that during chronic inflammation, the innate immune system may facilitate colon tumorigenesis in genetically predisposed individuals in response to certain indigenous microorganisms and/or cell remnants. In these individuals, therapeutic approaches that reroute the innate immune system's inflammatory and antimicrobial activities might help prevent colon tumorigenesis and metastasis.

There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF(-/-), IFN-gamma(-/-), IL-12(-/-) and RAG-1(-/-) malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses.

Toll-like receptor 2 (TLR2) signaling has been shown to contribute to resistance to Listeria monocytogenes infection, as TLR2-deficient mice have a heightened susceptibility to infection with this organism. Because CD14 may associate with TLR2, we investigated the role of CD14 in Listeria responses. In both CD14-deficient and TLR2-deficient macrophages, nuclear factor kappaB translocation; CD40 and CD86; and the production of interleukin (IL)-12, IL-6, tumor necrosis factor, and nitric oxide are reduced. The absence of CD14 augmented susceptibility to Listeria infection, reduced survival, and diminished bacterial clearance, as observed in TLR2-deficient mice. Compared with C57BL/6 control mice, CD14-deficient mice were observed to have a greater number of hepatic microabscesses containing abundant neutrophils, these abscesses were larger in size, and there was reduced inducible nitric oxide synthase expression. Further, mice that are both CD14 deficient and TLR2 deficient display susceptibility to infection that is comparable to that of mice deficient in either CD14 or TLR2 alone. Therefore, the present data demonstrate the role of CD14 and TLR2 in the recognition and control of Listeria infection and host resistance.

Recent studies on endotoxin/lipopolysaccharide (LPS)-induced acute inflammatory response in the lung are reviewed. The acute airway inflammatory response to inhaled endotoxin is mediated through Toll-like receptor 4 (TLR4) and CD14 signalling as mice deficient for TLR4 or CD14 are unresponsive to endotoxin. Acute bronchoconstriction, tumour necrosis factor (TNF), interleukin (IL)-12 and keratinocyte-derived chemokine (KC) production, protein leak and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adaptor protein (TIRAP), but independent of TIR-domain-containing adaptor-inducing interferon-beta (TRIF). In particular, LPS-induced TNF is required for bronchoconstriction, but dispensable for inflammatory cell recruitment. Lipopolysaccharide induces activation of the p38 mitogen-activated protein kinase (MAPK). Inhibition of pulmonary MAPK activity abrogates LPS-induced TNF production, bronchoconstriction, neutrophil recruitment into the lungs and broncho-alveolar space. In conclusion, TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin are dependent on TLR4/CD14/MD2 expression using the adapter proteins TIRAP and MyD88, while TRIF, IL-1R1 or IL-18R signalling pathways are dispensable. Further downstream in this axis of signalling, TNF blockade reduces only acute bronchoconstriction, while MAPK inhibition abrogates completely endotoxin-induced inflammation.

The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH(2). Here, we have cloned an A-type allatostatin receptor from the silkworm Bombyx mori (BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the first Drosophila allatostatin receptor (DAR-1), and 48% identity with the second Drosophila allatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned a Bombyx allatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 x 10(-9)M Bombyx A-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest that Bombyx has at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.