Immune metabolism programming in bovine innate cells

International audienc

Do human digestive physicochemical parameters contribute to children higher susceptibility to cryptosporidiosis ?

National audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. However, the impact of the gastrointestinal route the parasite takes before reaching its site of infection on the severity of the disease, has never been investigated. The in vitro computer-controlled TNO gastrointestinal model (TIM) was used for a comparative study of C. parvum survival and virulence under adult and child digestive conditions. Parasite survival and excystation kinetics in the in vitro digestive tract were determined by flow cytometry analysis while virulence was assessed after reinoculation of sporozoites onto HCT-8 cells. A luciferase reporter gene was also used to follow sporozoite activity throughout the digestive process. A global transcriptome analysis by RNA-Seq will be performed to identify differentially expressed parasite genes. Preliminary data show that the excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM. However, a higher amount of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. After three hours of digestion, the luciferase activity expressed by released sporozoites is significantly higher in the distal intestinal compartments of child compared to adult. Differences in digestive physicochemical parameters may partially explain why children are more susceptible to cryptosporidiosis than adults. This study is the first one exploring the impact of various digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model

Impact of early-life exposure to <em>Cryptosporidium parvum</em> infection on intestinal homeostasis at adulthood

National audienceNumerous studies recently describe the relationship between influences during early-life period and later-life health and disease. Cryptosporidium parvum is a zoonotic parasite responsible for a diarrheal disease that affects mostly children under 5 and immunocompromised patients (AIDS or organ transplant patients). Epidemiological studies have reported that after resolution of C. parvum infection, patients still suffer for abdominal pain. In this context, by using a neonatal mouse model of cryptosporidiosis, we tried to decipher the intestinal consequences at adulthood of the neonatal infection by analyzing the composition of the microbiota, the composition of immune cells in the intestine but also consequences on visceral sensitivity and the susceptibility to an unrelated intestinal infection. We observed that adult mice infected by C. parvum during the neonatal period display a modification of microbiota and of the composition of immune cells. These intestinal modifications were associated with an increased viscero-sensitivity and with a higher sensitivity to Salmonella infection. Altogether these results clearly demonstrate that an infection by C. parvum during the neonatal period induces intestinal imprinting that can be responsible for abdominal pains and increased susceptibility to another intestinal infection, way after the resolution of C. parvum infection

The Intracellular Ion flux mediated by TMEM176A and TMEM176B is involved in the regulation of Dendritic Cell Functions

International audienceThe regulation of immune cells by intracellular ion channels remains poorly explored. Here we investigated the role of two cation channels encoded by Tmem176a and Tmem176b that are highly expressed in myeloid cells in the immune system. To avoid any functional compensation, we generated double knock-out (DKO) mice, allowing simultaneous deletion of these two highly redundant and coregulated genes using the CRISPR-Cas9 system. The absence of Tmem176a/b significantly impacted antigen processing and presentation to CD4+ T cells in vivo and selectively altered cytokine production by dendritic cells (DCs). Using a novel real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. Thus, these results highlight the importance of TMEM176A/B-mediated cation flux for the fine regulation of DC biology

The Intracellular Ion flux mediated by TMEM176A and TMEM176B is involved in the regulation of Dendritic Cell Functions

International audienceThe regulation of immune cells by intracellular ion channels remains poorly explored. Here we investigated the role of two cation channels encoded by Tmem176a and Tmem176b that are highly expressed in myeloid cells in the immune system. To avoid any functional compensation, we generated double knock-out (DKO) mice, allowing simultaneous deletion of these two highly redundant and coregulated genes using the CRISPR-Cas9 system. The absence of Tmem176a/b significantly impacted antigen processing and presentation to CD4+ T cells in vivo and selectively altered cytokine production by dendritic cells (DCs). Using a novel real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. Thus, these results highlight the importance of TMEM176A/B-mediated cation flux for the fine regulation of DC biology

Role of the intracellular ion channels TMEM176A and TMEM176B in the immune system

International audienceIon channels represent attractive targets for the development of new therapies but still remain poorly studied in the immune system, in particular intracellular ion channels. In this work, we explored the role of two highly redundant ion channels named TMEM176A and TMEM176B that are intriguingly strongly expressed both in RORγt+ cells (ILC3, Th17) and immature dendritic cells. To investigate the role of these homologs and avoid any compensation effect, we generated a double KO (DKO) mouse using CRISPR-Cas9. Surprisingly, Tmem176a/b appeared dispensable for the function of RORγt+ cells and in the protective function of type 17 immunity during chemically-induced or infectious colitis. In contrast, antigen presentation by dendritic cells to CD4+ T cells through MHC II was selectively impaired in DKO mice. Using a real-time fluorescence-based system to analyze intracellular trafficking we found that both channels co-localized in highly dynamic post-Golgi vesicles preferentially interacting with, but not accumulating in, acidic organelles. These results indicate that TMEM176A/B ion channels play a predominant role in adaptive immunity as new intracellular components of the intracellular MHC II machinery

KiT-GENIE, the French genetic biobank of kidney transplantation

International audienceKiT-GENIE is a monocentric DNA biobank set up to consolidate the very rich and homogeneous DIVAT French cohort of kidney donors and recipients (D/R) in order to explore the molecular factors involved in kidney transplantation outcomes. We collected DNA samples for kidney transplantations performed in Nantes, and we leveraged GWAS genotyping data for securing high-quality genetic data with deep SNP and HLA annotations through imputations and for inferring D/R genetic ancestry. Overall, the biobank included 4217 individuals (n = 1945 D + 2,272 R, including 1969 D/R pairs), 7.4 M SNPs and over 200 clinical variables. KiT-GENIE represents an accurate snapshot of kidney transplantation clinical practice in Nantes between 2002 and 2018, with an enrichment in living kidney donors (17%) and recipients with focal segmental glomerulosclerosis (4%). Recipients were predominantly male (63%), of European ancestry (93%), with a mean age of 51yo and 86% experienced their first graft over the study period. D/R pairs were 93% from European ancestry, and 95% pairs exhibited at least one HLA allelic mismatch. The mean follow-up time was 6.7 years with a hindsight up to 25 years. Recipients experienced biopsy-proven rejection and graft loss for 16.6% and 21.3%, respectively. KiT-GENIE constitutes one of the largest kidney transplantation genetic cohorts worldwide to date. It includes homogeneous high-quality clinical and genetic data for donors and recipients, hence offering a unique opportunity to investigate immunogenetic and genetic factors, as well as donor-recipient interactions and mismatches involved in rejection, graft survival, primary disease recurrence and other comorbidities

Dendritic Cells Require TMEM176A/B Ion Channels for Optimal MHC Class II Antigen Presentation to Naive CD4 + T Cells

International audienceIntracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming

Performance of Repeated Measures of (1–3)-β-D-Glucan, Mannan Antigen, and Antimannan Antibodies for the Diagnosis of Invasive Candidiasis in ICU Patients: A Preplanned Ancillary Analysis of the EMPIRICUS Randomized Clinical Trial

International audienceBackground. We aimed to assess the prognostic value of repeated measurements of serum (1-3)-β-D-glucan (BDG), mannanantigen (mannan-Ag), and antimannan antibodies (antimannan-Ab) for the occurrence of invasive candidiasis (IC) in a high-risk nonimmunocompromised population. Methods. This was a preplanned ancillary analysis of the EMPIRICUS Randomized Clinical Trial, including nonimmunocompromised critically ill patients with intensive care unit-acquired sepsis, multiple Candida colonization, and multiple organ failure who were exposed to broad-spectrum antibacterial agents. BDG (>80 and >250 pg/mL), mannan-Ag (>125 pg/ mL), and antimannan-Ab (>10 AU) were collected repeatedly. We used cause-specific hazard models. Biomarkers were assessed at baseline in the whole cohort (cohort 1). Baseline covariates and/or repeated measurements and/or increased biomarkers were then studied in the subgroup of patients who were still alive at day 3 and free of IC (cohort 2). Results. Two hundred thirty-four patients were included, and 215 were still alive and free of IC at day 3. IC developed in 27 patients (11.5%), and day 28 mortality was 29.1%. Finally, BDG >80 pg/mL at inclusion was associated with an increased risk of IC (CSHR[IC], 4.67; 95% CI, 1.61-13.5) but not death (CSHR[death], 1.20; 95% CI, 0.71-2.02). Conclusions. Among high-risk patients, a first measurement of BDG >80 pg/mL was strongly associated with the occurrence of IC. Neither a cutoff of 250 pg/mL nor repeated measurements of fungal biomarkers seemed to be useful to predict the occurrence of IC. The cumulative risk of IC in the placebo group if BDG >80 pg/mL was 25.39%, which calls into question the efficacy of empirical therapy in this subgroup