Séverine Remy

The non-canonical NF-κB pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c- terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2 ^p.Tyr867* ). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5 ⁺ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4 ⁺ and CD8 ⁺ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4 ⁺ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM ⁺ IgD ⁻ transitional cells and a marked reduction of IgD ⁺ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

To investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. Animals from a new transgenic rat line expressing the green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received a single oral bolus of miR-320-3p or miR-375-3p embedded in DiOleyl-Succinyl-Paromomycin (DOSP) on D-12. The pups were weaned early (D-15), or regularly (D-30). The expression of relevant miRNA, mRNAs, chromatin complexes, and duodenal cell density were assessed at 8 h post-inoculation and on D-45. The miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p < 0.05). On regular weaning, on D-45, miR-320-3p and 375-3p were found to be downregulated in the stomach and upregulated in the hypothalamus (p < 0.001), whereas miR-320-3p was upregulated in the duodenum. After early weaning, miR-320-3p and miR-375-3p were downregulated in the stomach and the duodenum, but upregulated in the hypothalamus and the hippocampus. Combination of miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the female brain stem, miR-320-3p, miR-504, and miR-16-5p levels were all upregulated. Investigating the oral miRNA-320-3p loads in the duodenal cell lineage paved the way for designing new therapeutics to avoid unexpected long-term impacts on the brain.

Background: CD4 ⁺ and CD8 ⁺ regulatory T cells (Treg) in diverse species include different subsets from different origins. In all species, CD8 ⁺ Treg have been poorly characterized. CD4 ⁺ and CD8 ⁺ Treg in rats have only partially been characterized and there is no rat model in which FOXP3 ⁺ Treg are genetically tagged. Results: We generated a rat transgenic line using the CRISPR/Cas9 system in which EGFP was inserted in frame on the 3’ end of the Foxp3 gene using a 2A self-cleaving peptide. EGFP was exclusively expressed by CD4 ⁺ and CD8 ⁺ T cells in similar proportion as observed with anti-FOXP3 antibodies. CD4 ⁺ EGFP ⁺ Treg were 5-10 times more frequent than CD8 ⁺ EGFP ⁺ Treg. CD4 ⁺ and CD8 ⁺ EGFP ⁺ Treg expressed both the CD25 ^high CD127 ^low CD45RC ^low/- markers. The suppressive activity of CD4 ⁺ and CD8 ⁺ Treg was largely confined to EGFP ⁺ cells. RNAseq analyses showed similarities but also differences among CD4 ⁺ and CD8 ⁺ EGFP ⁺ cells and provided the first description of the natural FOXP3 ⁺ CD8 ⁺ Treg transcriptome. In vitro culture of CD4 ⁺ and CD8 ⁺ EGFP ^- cells with TGFbeta and IL-2 resulted in the induction of EGFP ⁺ Treg. Preferential expansion of CD4 ⁺ and CD8 ⁺ EGFP ⁺ Treg could be detected upon in vivo administration of a low dose of IL-2. Conclusions: This new and unique Foxp3-EGFP rat line constitutes a useful model to identify and isolate viable natural and induced CD4 ⁺ and CD8 ⁺ Treg. Additionally, it allows to identify new molecules expressed in CD8 ⁺ Treg that may allow to better define their phenotype and function not only in rats but also in other species.

Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal sodium retention leading to edema. This sodium retention is usually attributed to epithelial sodium channel (ENaC) activation after plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped (CC) rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We then identified a truncated variant of acid-sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when coexpressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that the expression of the truncated ASIC2b in the kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but not in any of the patients with other renal diseases. We have therefore identified a variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.

Intracellular 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. Key Points Germline and conditional double KO mice target Tmem176a and Tmem176b. Tmem176a/b appear dispensable in RORγt⁺ cells. Tmem176a/b are required in DCs for optimal MHC class II–mediated antigen presentation.

Summary: Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. Here we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in RORγt ⁺ cells and conventional dendritic cells (cDCs) using germline and conditional double knock-out (DKO) mice. While Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells (ILC3s) in the intestinal mucosa, we found that they were required in cDCs for optimal antigen 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. Together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC II compartment (MIIC) of DCs for the fine regulation of antigen presentation and naive CD4 ⁺ T cell priming.

Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (CS) that decrease inflammation and immune responses present in patients and animal models. CS have however limited efficacy and important and numerous side effects. Therefore, there is a need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease doses of CS. We first assessed the status of immune system of dystrophin-deficient rats ( Dmd ^mdx ) that closely reproduce the phenotype of DMD patients. Dmd ^mdx rats showed increased leukocyte infiltration in skeletal and cardiac muscles, containing mostly macrophages but also T cells, and increased expression of several cytokines. Anti-CD45RC Monoclonal antibody (Mab) treatment induced immune tolerance in models of organ transplantation and GVHD (Graft Versus Host Disease). We observed that muscles and blood of DMD patients contained T CD4 ⁺ and CD8 ⁺ expressing high levels of CD45RC ^high cells. Treatment of young Dmd ^mdx rats with anti-CD45RC MAb corrected skeletal muscle strength associated to a depletion of effectors CD45RC ^high T cells with no obvious side-effects. Prednisolone treatment of Dmd ^mdx rats similarly increased skeletal muscle strength and was also associated to a depletion of effectors CD45RC ^high cells but resulted in severe weight loss. Overall, Dmd ^mdx rats display important immune inflammatory response and thus represent a useful model to analyze new anti-inflammatory and tolerogenic treatments for DMD. As an example, a new treatment with anti-CD45RC antibodies improved muscle strength in Dmd ^mdx rats as prednisolone did but without side effects. Anti-CD45RC therapy could complement other therapies in DMD patients.

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25–100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.