IL-2 amplifies quantitative TCR signalling inputs to drive Th1 and Th2 differentiation.

The activation of CD4+ T-cells in a T cell receptor (TCR)-dependent antigen-specific manner is a central characteristic of the adaptive immune response. In addition to ensuring that CD4+ T-cells recognise their cognate antigen during activation, TCR-mediated signalling can also direct the outcome of differentiation. In both in vivo and in vitro model systems, strong TCR signalling has been demonstrated to drive Th1 differentiation, whereas weak TCR signalling drives Th2 responses. During the process of differentiation, TCR signal strength acts as a quantitative component in combination with the qualitative effects imparted by cytokines to polarise distinct T-helper lineages. Here, we investigated the role of interleukin 2 (IL-2) signalling in determining the outcome of TCR-dependent differentiation. IL-2 production was initiated as an early response to TCR-induced activation and was regulated by the strength of TCR signalling initially received. In the absence of IL-2, TCR dependent differentiation was found to be abolished. However, proliferative responses and early markers of activation were maintained, including the upregulation of GATA3, Tbet and Foxp3 at 24 h post-stimulation. Demonstrating that IL-2 signalling has a key role in stabilising and amplifying lineage-specific transcirption factor expression during differentiation. Further, activation of IL-2-deficient T-cells in the presence of exogenous cytokines was sufficient to restore differentiation whilst maintaining transcriptional signatures imparted during initial TCR signalling. Combined, our data demonstrate that the integration of quantitative TCR-dependent signalling and qualitative IL-2 signalling is essential for determining the fate of CD4+ T-cells during differentiation.

The antioxidant l-Ergothioneine prevents cystine lithiasis in the Slc7a9-/- mouse model of cystinuria.

The high recurrence rate of cystine lithiasis observed in cystinuria patients highlights the need for new therapeutic options to address this chronic disease. There is growing evidence of an antioxidant defect in cystinuria, which has led to test antioxidant molecules as new therapeutic approaches. In this study, the antioxidant l-Ergothioneine was evaluated, at two different doses, as a preventive and long-term treatment for cystinuria in the Slc7a9-/- mouse model. l-Ergothioneine treatments decreased the rate of stone formation by more than 60% and delayed its onset in those mice that still developed calculi. Although there were no differences in metabolic parameters or urinary cystine concentration between control and treated mice, cystine solubility was increased by 50% in the urines of treated mice. We also demonstrate that l-Ergothioneine needs to be internalized by its transporter OCTN1 (Slc22a4) to be effective, as when administrated to the double mutant Slc7a9-/-Slc22a4-/- mouse model, no effect on the lithiasis phenotype was observed. In kidneys, we detected a decrease in GSH levels and an impairment of maximal mitochondrial respiratory capacity in cystinuric mice that l-Ergothioneine treatment was able to restore. Thus, l-Ergothioneine administration prevented cystine lithiasis in the Slc7a9-/- mouse model by increasing urinary cystine solubility and recovered renal GSH metabolism and mitochondrial function. These results support the need for clinical trials to test l-Ergothioneine as a new treatment for cystinuria.

Applications of Genome-Editing Technologies for Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing pancreatic ß-cells by the immune system. Although conventional therapeutic modalities, such as insulin injection, remain a mainstay, recent years have witnessed the emergence of novel treatment approaches encompassing immunomodulatory therapies, such as stem cell and ß-cell transplantation, along with revolutionary gene-editing techniques. Notably, recent research endeavors have enabled the reshaping of the T-cell repertoire, leading to the prevention of T1D development. Furthermore, CRISPR-Cas9 technology has demonstrated remarkable potential in targeting endogenous gene activation, ushering in a promising avenue for the precise guidance of mesenchymal stem cells (MSCs) toward differentiation into insulin-producing cells. This innovative approach holds substantial promise for the treatment of T1D. In this review, we focus on studies that have developed T1D models and treatments using gene-editing systems.

Gut microbial influences on the adaptive immune system and the development of cow milk allergy.

Allergic diseases constitute significant health and economic issues in both developed and developing nations, with epidemiological studies demonstrating a rapid increase in the global prevalence of food allergy among the pediatric population. Cow milk protein allergy (CMPA), one of the most common forms of food allergies observed in early childhood, affects between 2%-6% of infants and children under 3 years of age. CMPA can present as either an IgE-mediated atopic allergy or a non-IgE mediated allergic response. Antigen-specific T cells play a pivotal role in directing the type of inflammatory immune response that occurs as well as in the formation of immunological memory. IgE-mediated CMPA is thought to develop because of an abnormal expansion of allergen-specific type-2 helper T (Th2) cells and a corresponding deficiency in immune regulation by regulatory T cells (Tregs), thereby altering the Th2/Treg balance. The gut microbiota, established very early during childhood through host-microbe interactions, can influence the incidence of allergic diseases. In this study, we aimed to analyze both the microbiome composition and CD4+T cell differentiation patterns in pediatric patients with and without cow milk allergy to establish the association between these factors. Using 16S rRNA sequencing, we analyzed the microbiome composition in stool samples of allergic and non-allergic pediatric patients aged between 1-4 years and identified the microbial species abundant in IgE and non-IgE mediated cow milk allergies. To assess the CD4+T cell differentiation patterns, peripheral blood mononuclear cells (PBMCs) from these patients were re-stimulated with cow milk antigen, and T cell subsets were assessed using flow cytometry. Antigen-specific CD4+T cells were identified and sorted for high throughput sequencing and subsequent gene expression analysis. The CD4+T cell differentiation patterns of the total and antigen-specific T cells were analyzed and statistically compared with controls. The identification of the correlation between the CD4+T cell differentiation patterns and species-specific microbial abundance in IgE and non-IgE mediated cow milk allergies can help in determining how the gut microbiome influences the CD4+T cell immune compartment development, ultimately leading to the development of cow milk allergy in pediatric patients.

Dendritic cell activation and screening for key molecular signatures required for the induction of allergic responses.

The chain of events that leads to the sensitization of the immune system to environmental antigens, resulting in the onset of allergic disease, has been studied in great detail over the past 30 years. However, during this time, the rate of allergic diseases has increased exponentially, indicating the need to concentrate our studies on host-environmental factors that contribute to the onset of disease. Monocyte-derived dendritic cells (DCs) play a key role in driving localized and systemic immune responses. In this study, we developed a platform for screening the molecular signature and phenotypic profile of DCs activated by allergenic stimuli, including TSLP, IL-25, IL-33, IL-1a, Vit-D3 (1α,25-Dihydroxyvitamin D3), PAR1-AP Peptide, Papain, and recombinant human DerP1 protein to induce a type II associated inflammatory signature. Following activation with allergenic stimuli, modulated DCs are subjected to deep phenotyping via flow cytometry for surface and intracellular markers to detect and/or validate immunomodulatory properties. RNA sequencing is further used to compare the gene expression profiles of DCs responding to either allergenic or microbial stimuli, including the TLR3 agonist dsRNA Poly I:C and TLR4 agonist LPS. In our study, we aimed to identify key molecular signatures of DCs involved in the development of asthma and allergy based on their comparative activation with this broad panel of allergens. We expect to determine central control modules of transcription factors in DCs associated with Th2 induction.

Asthma and the Missing Heritability Problem: Necessity for Multiomics Approaches in Determining Accurate Risk Profiles.

Asthma is ranked among the most common chronic conditions and has become a significant public health issue due to the recent and rapid increase in its prevalence. Investigations into the underlying genetic factors predict a heritable component for its incidence, estimated between 35% and 90% of causation. Despite the application of large-scale genome-wide association studies (GWAS) and admixture mapping approaches, the proportion of variants identified accounts for less than 15% of the observed heritability of the disease. The discrepancy between the predicted heritable component of disease and the proportion of heritability mapped to the currently identified susceptibility loci has been termed the 'missing heritability problem.' Here, we examine recent studies involving both the analysis of genetically encoded features that contribute to asthma and also the role of non-encoded heritable characteristics, including epigenetic, environmental, and developmental aspects of disease. The importance of vertical maternal microbiome transfer and the influence of maternal immune factors on fetal conditioning in the inheritance of disease are also discussed. In order to highlight the broad array of biological inputs that contribute to the sum of heritable risk factors associated with allergic disease incidence that, together, contribute to the induction of a pro-atopic state. Currently, there is a need to develop in-depth models of asthma risk factors to overcome the limitations encountered in the interpretation of GWAS results in isolation, which have resulted in the missing heritability problem. Hence, multiomics analyses need to be established considering genetic, epigenetic, and functional data to create a true systems biology-based approach for analyzing the regulatory pathways that underlie the inheritance of asthma and to develop accurate risk profiles for disease.

Functional Characterization of the MYO6 Variant p.E60Q in Non-Syndromic Hearing Loss Patients.

Hereditary hearing loss (HHL) is a common genetic disorder accounting for at least 60% of pre-lingual deafness in children, of which 70% is inherited in an autosomal recessive pattern. The long tradition of consanguinity among the Qatari population has increased the prevalence of HHL, which negatively impacts the quality of life. Here, we functionally validated the pathogenicity of the c.178G>C, p.E60Q mutation in the MYO6 gene, which was detected previously in a Qatari HHL family, using cellular and animal models. In vitro analysis was conducted in HeLa cells transiently transfected with plasmids carrying MYO6WT or MYO6p.E60Q, and a zebrafish model was generated to characterize the in vivo phenotype. Cells transfected with MYO6WT showed higher expression of MYO6 in the plasma membrane and increased ATPase activity. Modeling the human MYO6 variants in zebrafish resulted in severe otic defects. At 72 h post-injection, MYO6p.E60Q embryos demonstrated alterations in the sizes of the saccule and utricle. Additionally, zebrafish with MYO6p.E60Q displayed super-coiled and bent hair bundles in otic hair cells when compared to control and MYO6WT embryos. In conclusion, our cellular and animal models add support to the in silico prediction that the p.E60Q missense variant is pathogenic and damaging to the protein. Since the c.178G>C MYO6 variant has a 0.5% allele frequency in the Qatari population, about 400 times higher than in other populations, it could contribute to explaining the high prevalence of hearing impairment in Qatar.

Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease.

Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y+LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7-/-). The Slc7a7-/- model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7-/- mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7-/- model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations.