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[This corrects the article DOI: 10.1371/journal.ppat.1010026.].

IntroductionCrohn’s disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation.MethodsThe levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used.Results and discussionWe showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

IntroductionTrypanosoma evansi parasite infections cause a chronic animal wasting disease called Surra, and cases of atypical Human Trypanosomosis (aHT). In experimental models, T. evansi infections are hallmarked by the early onset of excessive inflammation. Therefore, balancing the production of inflammatory cytokines by anti-inflammatory IL-10 is crucial for prolonged survival.MethodsTo improve the understanding of trypanosomosis induced immunopathology, we used scRNA-seq data from an experimental chronic T. evansi infection mouse model, resembling natural infection in terms of disease characteristics. Results and discussionFor the first time, obtained results allowed to assess the transcriptomic profile and heterogeneity of splenic CD4+ T cell subsets, during a trypanosome infection. Here, the predominant subpopulation of T cells was represented by Tbx21(T-bet)+Ccr5+ Id2+ type 1 helper T cells (Th1), followed by Icos+ Cxcr5+Follicular T helper cells (Tfh) and very minor fraction of Il2ra(CD25)+Foxp3+ regulatory T cells (Tregs). Interestingly, the profile of Th1 cells shows that besides Ifng, these cells express high levels of Il10 and Il21, coding for anti-inflammatory and immunoregulatory cytokines. This coincides with the elevated expression of key genes involved in IL-10 and IL-21 secretion pathway such as Stat1 and Stat3, as well as the transcriptional factors Prdm1 (Blimp 1), and Maf (c-Maf). In contrast, there is virtually no IL-10 transcription detected in the Treg population. Finally, differential gene expression and gene ontology analysis of infection-induced Ifng+ Il10+ Il21+ Th1 cells highlights their suppressive function on T cell activation, differentiation and INF-γ production itself. This indicates that during trypanosome infections, the Ifng+ Il10+ Il21+ Th1 cells, rather than Tregs, assume an immune regulatory role that is needed for dampening inflammation.

Infection caused by extracellular single-celled trypanosomes triggers a lethal chronic wasting disease in livestock and game animals. Through screening of 10 Trypanosoma evansi field isolates, exhibiting different levels of virulence in mice, the current study identifies an experimental disease model in which infection can last well over 100 days, mimicking the major features of chronic animal trypanosomosis. In this model, despite the well-controlled parasitemia, infection is hallmarked by severe trypanosomosis-associated pathology. An in-depth scRNA-seq analysis of the latter revealed the complexity of the spleen macrophage activation status, highlighting the crucial role of tissue resident macrophages (TRMs) in regulating splenic extramedullary erythropoiesis. These new data show that in the field of experimental trypanosomosis, macrophage activation profiles have so far been oversimplified into a bi-polar paradigm (M1 vs M2). Interestingly, TRMs exert a double-sided effect on erythroid cells. On one hand, these cells express an erythrophagocytosis associated signature. On another hand, TRMs show high levels of Vcam1 expression, known to support their interaction with hematopoietic stem and progenitor cells (HSPCs). During chronic infection, the latter exhibit upregulated expression of Klf1, E2f8, and Gfi1b genes, involved in erythroid differentiation and extramedullary erythropoiesis. This process gives rise to differentiation of stem cells to BFU-e/CFU-e, Pro E, and Baso E subpopulations. However, infection truncates progressing differentiation at the orthochromatic erythrocytes level, as demonstrated by scRNAseq and flow cytometry. As such, these cells are unable to pass to the reticulocyte stage, resulting in reduced number of mature circulating RBCs and the occurrence of chronic anemia. The physiological consequence of these events is the prolonged poor delivery of oxygen to various tissues, triggering lactic acid acidosis and the catabolic breakdown of muscle tissue, reminiscent of the wasting syndrome that is characteristic for the lethal stage of animal trypanosomosis.

Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Salivarian trypanosomes are extracellular protozoan parasites causing infections in a wide range of mammalian hosts, with Trypanosoma evansi having the widest geographic distribution, reaching territories far outside Africa and occasionally even Europe. Besides causing the animal diseases, T. evansi can cause atypical Human Trypanosomosis. The success of this parasite is attributed to its capacity to evade and disable the mammalian defense response. To unravel the latter, we applied here for the first time a scRNA-seq analysis on splenocytes from trypanosome infected mice, at two time points during infection, i.e. just after control of the first parasitemia peak (day 14) and a late chronic time point during infection (day 42). This analysis was combined with flow cytometry and ELISA, revealing that T. evansi induces prompt activation of splenic IgM+CD1d+ Marginal Zone and IgMIntIgD+ Follicular B cells, coinciding with an increase in plasma IgG2c Ab levels. Despite the absence of follicles, a rapid accumulation of Aicda+ GC-like B cells followed first parasitemia peak clearance, accompanied by the occurrence of Xbp1+ expressing CD138+ plasma B cells and Tbx21+ atypical CD11c+ memory B cells. Ablation of immature CD93+ bone marrow and Vpreb3+Ly6d+Ighm+ expressing transitional spleen B cells prevented mature peripheral B cell replenishment. Interestingly, AID-/- mice that lack the capacity to mount anti-parasite IgG responses, exhibited a superior defense level against T. evansi infections. Here, elevated natural IgMs were able to exert in vivo and in vitro trypanocidal activity. Hence, we conclude that in immune competent mice, trypanosomosis associated B cell activation and switched IgG production is rapidly induced by T. evansi, facilitating an escape from the detrimental natural IgM killing activity, and resulting in increased host susceptibility. This unique role of IgM and its anti-trypanosome activity are discussed in the context of the dilemma this causes for the future development of anti-trypanosome vaccines.

Streptomyces species are the most important sources of antibacterial, antifungal, and phytotoxic metabolites. In this study, cycloheximide (CH) and acetoxycycloheximide (ACH) were isolated from the fermentation broth of Streptomyces sp. JCK-6092. The antifungal and phytotoxic activities of the two compounds (CH and ACH) and a cycloheximide derivative, hydroxycycloheximide (HCH), were compared. CH exhibited the strongest antagonistic activity against all the true fungi tested, followed by ACH and HCH. However, both CH and ACH displayed similar mycelial growth inhibitory activities against several phytopathogenic oomycetes, and both were more active than that of HCH. Disparate to antifungal ability, ACH showed the strongest phytotoxic activity against weeds and crops, followed by HCH and CH. ACH caused chlorophyll content loss, leaf electrolytic leakage, and lipid peroxidation in a dose-dependent manner. Its phytotoxicity was stronger than that of glufosinate-ammonium but weaker than that of paraquat in the in vitro experiments. CH and its derivatives are well-known protein synthesis inhibitors; however, the precise differences between their mechanism of action remain undiscovered. A computational study revealed effects of CHs on the protein synthesis of Pythium ultimum (oomycetes), Magnaporthe oryzae (true fungus), and Capsicum annum (plant) and deciphered the differences in their biological activities on different targets. The binding energies and conformation stabilities of each chemical molecule correlated with their biological activities. Thus, molecular docking study supported the experimental results. This is the first comparative study to suggest the ribosomal protein alteration mechanisms of CHs in plants and fungi and to thus show how the protein inhibitory activities of the different derivatives are altered using molecular docking. The correlation of structures features of CHs in respect to bond formation with desired protein was revealed by density functional theory. Overall collective results suggested that CHs can be used as lead molecules in the development of more potent fungicides and herbicides molecules.

Salivarian trypanosomes comprise a group of extracellular anthroponotic and zoonotic parasites. The only sustainable method for global control of these infection is through vaccination of livestock animals. Despite multiple reports describing promising laboratory results, no single field-applicable solution has been successful so far. Conventionally, vaccine research focusses mostly on exposed immunogenic antigens, or the structural molecular knowledge of surface exposed invariant immunogens. Unfortunately, extracellular parasites (or parasites with extracellular life stages) have devised efficient defense systems against host antibody attacks, so they can deal with the mammalian humoral immune response. In the case of trypanosomes, it appears that these mechanisms have been perfected, leading to vaccine failure in natural hosts. Here, we provide two examples of potential vaccine candidates that, despite being immunogenic and accessible to the immune system, failed to induce a functionally protective memory response. First, trypanosomal enolase was tested as a vaccine candidate, as it was recently characterized as a highly conserved enzyme that is readily recognized during infection by the host antibody response. Secondly, we re-addressed a vaccine approach towards the Invariant Surface Glycoprotein ISG75, and showed that despite being highly immunogenic, trypanosomes can avoid anti-ISG75 mediated parasitemia control.

Salivarian trypanosomes are extracellular parasites causing anthroponotic and zoonotic infections. Anti-parasite vaccination is considered the only sustainable method for global trypanosomosis control. Unfortunately, no single field applicable vaccine solution has been successful so far. The active destruction of the host’s adaptive immune system by trypanosomes is believed to contribute to this problem. Here, we show that Trypanosome brucei brucei infection results in the lasting obliteration of immunological memory, including vaccine-induced memory against non-related pathogens. Using the well-established DTPa vaccine model in combination with a T. b. brucei infection and a diminazene diaceturate anti-parasite treatment scheme, our results demonstrate that while the latter ensured full recovery from the T. b. brucei infection, it failed to restore an efficacious anti-B. pertussis vaccine recall response. The DTPa vaccine failure coincided with a shift in the IgG1/IgG2a anti-B. pertussis antibody ratio in favor of IgG2a, and a striking impact on all of the spleen immune cell populations. Interestingly, an increased plasma IFNγ level in DTPa-vaccinated trypanosome-infected mice coincided with a temporary antibody-independent improvement in early-stage trypanosomosis control. In conclusion, our results are the first to show that trypanosome-inflicted immune damage is not restored by successful anti-parasite treatment.

Background: Escherichia coli producing the genotoxin colibactin (CoPEC or colibactin-producing E. coli) abnormally colonize the colonic mucosa of colorectal cancer (CRC) patients. We previously showed that deficiency of autophagy in intestinal epithelial cells (IECs) enhances CoPEC-induced colorectal carcinogenesis in ApcMin/+ mice. Here, we tested if CoPEC trigger tumorigenesis in a mouse model lacking genetic susceptibility or the use of carcinogen. Methods: Mice with autophagy deficiency in IECs (Atg16l1∆IEC) or wild-type mice (Atg16l1flox/flox) were infected with the CoPEC 11G5 strain or the mutant 11G5∆clbQ incapable of producing colibactin and subjected to 12 cycles of DSS treatment to induce chronic colitis. Mouse colons were used for histological assessment, immunohistochemical and immunoblot analyses for DNA damage marker. Results: 11G5 or 11G5∆clbQ infection increased clinical and histological inflammation scores, and these were further enhanced by IEC-specific autophagy deficiency. 11G5 infection, but not 11G5∆clbQ infection, triggered the formation of invasive carcinomas, and this was further increased by autophagy deficiency. The increase in invasive carcinomas was correlated with enhanced DNA damage and independent of inflammation. Conclusions: CoPEC induce colorectal carcinogenesis in a CRC mouse model lacking genetic susceptibility and carcinogen. This work highlights the role of (i) CoPEC as a driver of CRC development, and (ii) autophagy in inhibiting the carcinogenic properties of CoPEC.

Background: Adherent-invasive Escherichia coli (AIEC) have been implicated in the etiology of Crohn’s disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of Yersinia spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection. Methods: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression. Results: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells. Conclusion: Our study highlights a novel role for yersiniabactin siderophore in AIEC–host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.

In this paper, we report on an international collaborative project designed to address the professional development needs of Vietnamese teachers for the implementation of Content and Language Integrated Learning (CLIL). We collected data on a group of CLIL Vietnamese teachers and leaders through online interviews. Drawing on Cultural-Historical Activity Theory (CHAT), the paper illuminates how CLIL teachers, as the subject of the professional learning (PL) system, utilized tools to develop their capacity for CLIL teaching against the backdrop of various sociocultural factors that regulate this learning process. The analysis helped us to identify various challenges facing CLIL teachers' PL. These challenges arose from an absence of an effective learning system mostly related to contextual obstacles and widespread misconceptions, even among the leaders and CLIL trainers, about the nature of CLIL. This study contributes suggestions to enhance the quality of PL for CLIL teachers, which is a pressing issue for the successful implementation of CLIL in the context of Vietnam and beyond.

Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn’s disease (CD) patients, are able to invade intestinal epithelial cells (IECs) and translocate through M cells overlying Peyer’s patches. The levels of microRNA (miRNA) and gene expression in IECs and M cells upon AIEC infection have not been investigated. Here, we used human intestinal epithelial Caco-2 monolayers and an in vitro M-cell model of AIEC translocation to analyze comprehensive miRNA and gene profiling under basal condition and upon infection with the reference AIEC LF82 strain. Our results showed that AIEC LF82 translocated through M cells but not Caco-2 monolayers. Both differential gene expression and miRNA profile in M cells compared to Caco-2 cells were obtained. In addition, AIEC infection induces changes in gene and miRNA profiles in both Caco-2 and M cells. In silico analysis showed that certain genes dysregulated upon AIEC infection were potential targets of AIEC-dysregulated miRNAs, suggesting a miRNA-mediated regulation of gene expression during AIEC infection in Caco-2, as well as M cells. This study facilitates the discovery of M cell-specific and AIEC response-specific gene-miRNA signature and enhances the molecular understanding of M cell biology under basal condition and in response to infection with CD-associated AIEC.

To communicate with each other, cells release exosomes that transfer their composition, including lipids, proteins and nucleic acids, to neighboring cells, thus playing a role in various pathophysiological processes. During an infection with pathogenic bacteria, such as adherent-invasive E. coli (AIEC) associated with Crohn disease, exosomes secreted by infected cells can have an impact on the innate immune responses of surrounding cells to infection. Furthermore, inflammation can be amplified via the exosomal shuttle during infection with pathogenic bacteria, which could contribute to the development of the associated disease. Since these vesicles can be released in various biological fluids, changes in exosomal content may provide a means for the identification of non-invasive biomarkers for infectious and inflammatory bowel diseases. Moreover, evidence suggests that exosomes could be used as vaccines to prime the immune system to recognize and kill invading pathogens, and as therapeutic components relieving intestinal inflammation. Here, we summarize the current knowledge on the role of exosomes in bacterial infections and highlight their potential use as biomarkers, vaccines and conveyers of therapeutic molecules in inflammatory bowel diseases.

Vietnam is one of the countries most affected worldwide by the highly pathogenic avian influenza (HPAI) virus, which caused enormous economic loss and posed threats to public health. Over nearly two decades, with the antigenic changes in the diversified H5Ny viruses, the limited protective efficacy of the available vaccines was encountered. Therefore, it is necessary to approach a technology platform for the country to accelerate vaccine production that enables quick response to new influenza subtypes. This study utilized a powerful reverse genetics technique to successfully generate a recombinant H5N1 vaccine strain (designated as IBT-RG02) containing two surface proteins (haemagglutinin (HA) and neuraminidase (NA)) from the HPAI H5N1 (A/duck/Vietnam/HT2/2014(H5N1)) of the dominant clade 2.3.2.1c in Vietnam during 2012–2014. Importantly, the IBT-RG02 vaccine candidate has elicited high antibody titres in chickens (geometric mean titre (GMT) of 6.42 and 6.92, log2 on day 14 and day 28 p.i., respectively). To test the efficacy, immunized chickens were challenged with the circulating virulent strains. As results, there was a high protection rate of 91.6% chickens against the virulent A/DK/VN/Bacninh/NCVD-17A384/2017 of the same clade and a cross-protection of 83.3% against A/duck/TG/NAVET(3)/2013 virus of clade 1.1. Our promising results showed that we can independently master the reverse genetics technology for generation of highly immunogenic vaccine candidates, and henceforth, it is a timely manner to reformulate avian influenza virus vaccines against variable H5 clade HPAI viruses in Vietnam.

Colorectal cancer, the fourth leading cause of cancer-related death worldwide, is a multifactorial disease involving genetic, environmental and lifestyle risk factors. In addition, increased evidence has established a role for the intestinal microbiota in the development of colorectal cancer. Indeed, changes in the intestinal microbiota composition in colorectal cancer patients compared to control subjects have been reported. Several bacterial species have been shown to exhibit the pro-inflammatory and pro-carcinogenic properties, which could consequently have an impact on colorectal carcinogenesis. This review will summarize the current knowledge about the potential links between the intestinal microbiota and colorectal cancer, with a focus on the pro-carcinogenic properties of bacterial microbiota such as induction of inflammation, the biosynthesis of genotoxins that interfere with cell cycle regulation and the production of toxic metabolites. Finally, we will describe the potential therapeutic strategies based on intestinal microbiota manipulation for colorectal cancer treatment.

Feedback is widely viewed as a powerful tool, yet a challenging professional undertaking, in initial teacher education. This paper explored how feedback was used to mediate pre-service teachers' (PSTs') learning, in the form of a qualitative study that illuminates both the perspectives of HEI tutors, school mentors and PSTs and the actual feedback provision process taking place in an EFL practicum in Vietnam. Data revealed a diversity of ways through which feedback was provided by mentors. Most prominent seemed to be that of 'detailed' but 'authoritative' feedback, the latter denoting a transmission approach whereby mentors were positioned as 'knowledge givers' and PSTs as 'knowledge receivers'. Drawing on the reflective practitioner perspective, the paper, however, argues that in a Confucian-influenced culture where PSTs might be accustomed to the 'listener' role like Vietnam, the monological feedback could be an effective supplement to the dialogical, reflective feedback widely advocated in Western contexts.

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3'-untranslated regions (3'-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3'-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-kappaB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-kappaB and Sp1 to bind to their binding sites. Knock-down of either NF-kappaB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-kappaB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.

BackgroundPepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte.ResultsWe found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles.ConclusionsCollectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation. [ABSTRACT FROM AUTHOR]

Infection caused by extracellular single-celled trypanosomes triggers a lethal chronic wasting disease in livestock and game animals. Through screening of 10 Trypanosoma evansi field isolates, exhibiting different levels of virulence in mice, the current study identifies an experimental disease model in which infection can last well over 100 days, mimicking the major features of chronic animal trypanosomosis. In this model, despite the well-controlled parasitemia, infection is hallmarked by severe trypanosomosis-associated pathology. An indepth scRNA-seq analysis of the latter revealed the complexity of the spleen macrophage activation status, highlighting the crucial role of tissue resident macrophages (TRMs) in regulating splenic extramedullary erythropoiesis. These new data show that in the field of experimental trypanosomosis, macrophage activation profiles have so far been oversimplified into a bi-polar paradigm (M1 vs M2). Interestingly, TRMs exert a double-sided effect on erythroid cells. On one hand, these cells express an erythrophagocytosis associated signature. On another hand, TRMs show high levels of Vcam1 expression, known to support their interaction with hematopoietic stem and progenitor cells (HSPCs). During chronic infection, the latter exhibit upregulated expression of Klf1, E2f8, and Gfi1b genes, involved in erythroid differentiation and extramedullary erythropoiesis. This process gives rise to differentiation of stem cells to BFUe/CFU-e, Pro E, and Baso E subpopulations. However, infection truncates progressing differentiation at the orthochromatic erythrocytes level, as demonstrated by scRNAseq and flow cytometry. As such, these cells are unable to pass to the reticulocyte stage, resulting in reduced number of mature circulating RBCs and the occurrence of chronic anemia. The physiological consequence of these events is the prolonged poor delivery of oxygen to various tissues, triggering lactic acid acidosis and the catabolic breakdown of muscle tissue, reminiscent of the wasting syndrome that is characteristic for the lethal stage of animal trypanosomosis. [ABSTRACT FROM AUTHOR]