ROR-γt Research Articles

MNAM enhances Blautia abundance and modulates Th17/Treg balance to alleviate diabetes in T2DM mice

This study investigated the therapeutic effects of N1-Methylnicotinamide (MNAM), a metabolic derivative, on T2DM mice induced by a high-fat diet and streptozotocin (STZ), focusing on its impact on the gut microbiome and immune modulation. MNAM significantly reduced hyperglycemia and enhanced insulin secretion, effects that were dependent on the presence of gut microbiota. It also mitigated STZ-induced weight loss and improved islet cell morphology, reducing islet cell mortality and increasing insulin (INS) levels. Flow cytometry analysis showed a decrease in T helper 17 cells (Th17) and an increase in Treg cells after MNAM treatment, corresponding to the upregulation of Treg markers [interleukin (IL)-10, forkhead box P3 (FOXP3)] and downregulation of Th17 markers [IL17A, RAR-related orphan receptor gamma (RORγt)]. Additionally, MNAM raised anti-inflammatory IL-10 levels while reducing pro-inflammatory cytokines [IL-17α, tumor necrosis factor (TNF-α), IL-6]. Microbiome analysis revealed decreased diversity and increased Blautia abundance post-MNAM administration. Treatment with Blautia not only reversed diabetes indicators but also modulated the Th17/Treg balance and reduced inflammation, with its metabolite sodium acetate mimicking these effects through the G protein-coupled receptor 43 (GPR43) pathway. These findings suggest that MNAM’s mitigation of diabetes operates through modulation of the gut microbiota and immune regulation, highlighting Blautia and its metabolite as potential therapeutic agents and providing a theoretical foundation for novel treatment strategies in T2DM.

Transcriptional Activity of Genes Regulating T-Helper Differentiation in the Accidentally Exposed Population of the Southern Urals.

The objective of this work was to study the expression of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes responsible for the regulation of the differentiation of various T-helper subpopulations in individuals chronically exposed to radiation. The object of the study was peripheral blood cells obtained from 120persons chronically exposed to radiation in a wide range of doses on the Techa River. The mean cumulative absorbed dose to red bone marrow in the examined exposed individuals was 742.7 ± 78.6 mGy (dose range, 73.5-3516.1 mGy); in the comparison group, 17.4 ± 2.2 mGy (dose range, 0.0-55.5 mGy). The subpopulation composition of T-helpers (Th1, Th2, and Th17) was analyzed by flow cytofluorometry. The relative mRNA content of the TBX21, RORC, GATA3, NFKB1, MAPK8, and STAT3 genes was estimated by real-time PCR. The study made it possible to note a decrease in the relative number of T-helpers 2 in the populations of T-helpers of the central memory in the group of chronically exposed persons compared to the comparison group. In the population of T-helpers of the central memory, a statistically significant increase in the relative number of T-helpers 1 was shown, depending on the accumulated absorbed dose to red bone marrow. No changes in mRNA expression of the studied genes were observed. The analysis of the correlation between the expression of GATA3, MAPK8, STAT3, RORC, and TBX21 mRNA and the relative number of cells in subpopulations of T-helper types 1, 2, and 17 in the examined people did not reveal statistically significant patterns.

Anti-inflammatory effects of phytocannabinoids and terpenes on inflamed Tregs and Th17 cells in vitro

AimsPhytocannabinoids and terpenes from Cannabis sativa have demonstrated limited anti-inflammatory and analgesic effects in several inflammatory conditions. In the current study, we test the hypothesis that phytocannabinoids exert immunomodulatory effects in vitro by decreasing inflammatory cytokine expression and activation. Key methodsCD3/CD28 and lipopolysaccharide activated peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 6) were treated with phytocannabinoid compounds and terpenes in vitro. Flow cytometry was used to determine regulatory T cell (Treg) and T helper 17 (Th17) cell responses to treatments. Cell pellets were harvested for qRT-PCR gene expression analysis of cytokines, cell activation markers, and inflammation-related receptors. Cell culture supernatants were analysed by ELISA to quantify IL-6, TNF-α and IL-10 secretion. Main findingsIn an initial screen of 20 μM cannabinoids and terpenes which were coded to blind investigators, cannabigerol (GL4a), caryophyllene oxide (GL5a) and gamma-terpinene (GL6a) significantly reduced cytotoxicity and gene expression levels of IL6, IL10, TNF, TRPV1, CNR1, HTR1A, FOXP3, RORC and NFKΒ1. Tetrahydrocannabinol (GL7a) suppression of T cell activation was associated with downregulation of RORC and NFKΒ1 gene expression and reduced IL-6 (p < 0.0001) and IL10 (p < 0.01) secretion. Cannabidiol (GL1b) significantly suppressed activation of Tregs (p < 0.05) and Th17 cells (p < 0.05) in a follow-on in vitro dose-response study. IL-6 (p < 0.01) and IL-10 (p < 0.01) secretion was significantly reduced with 50 μM cannabidiol. SignificanceThe study provides the first evidence that cannabidiol and tetrahydrocannabinol suppress extracellular expression of both anti- and pro-inflammatory cytokines in an in vitro PBMC model of inflammation.

OLFM4 modulates intestinal inflammation by promoting IL-22+ILC3 in the gut

Group 3 innate lymphoid cells (ILC3s) play key roles in intestinal inflammation. Olfactomedin 4 (OLFM4) is highly expressed in the colon and has a potential role in dextran sodium sulfate-induced colitis. However, the detailed mechanisms underlying the effects of OLFM4 on ILC3-mediated colitis remain unclear. In this study, we identify OLFM4 as a positive regulator of IL-22+ILC3. OLFM4 expression in colonic ILC3s increases substantially during intestinal inflammation in humans and mice. Compared to littermate controls, OLFM4-deficient (OLFM4−/−) mice are more susceptible to bacterial infection and display greater resistance to anti-CD40 induced innate colitis, together with impaired IL-22 production by ILC3, and ILC3s from OLFM4−/−mice are defective in pathogen resistance. Besides, mice with OLFM4 deficiency in the RORγt compartment exhibit the same trend as in OLFM4−/−mice, including colonic inflammation and IL-22 production. Mechanistically, the decrease in IL-22+ILC3 caused by OLFM4 deficiency involves the apoptosis signal-regulating kinase 1 (ASK1)- p38 MAPK signaling-dependent downregulation of RAR-related orphan receptor gamma (RORγt) protein. The OLFM4-metadherin (MTDH) complex upregulates p38/RORγt signaling, which is necessary for IL-22+ILC3 activation. The findings indicate that OLFM4 is a novel regulator of IL-22+ILC3 and essential for modulating intestinal inflammation and tissue homeostasis.

RORγt-dependent antigen-presenting cells direct regulatory T cell-mediated tolerance to food antigen.

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While RORγt+ antigen-presenting cells (RORγt-APCs) were shown to program gut microbiota-specific pTregs5-7, understanding of their characteristics remains incomplete, and the APC subset responsible for food tolerance has remained elusive. Here, we demonstrate that RORγt-APCs are similarly required for differentiation of food antigen-specific pTregs and establishment of oral tolerance. The ability of these cells to direct both food and microbiota-specific pTreg cell differentiation is contingent on expression of RORγt and on a unique cis-regulatory element within the Rorc gene locus (Rorc(t) +7kb). Absent this +7kb element, there was a notable increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTregs, leading to compromised tolerance in a mouse asthma model. By employing single-cell analyses across these models, as well as freshly resected mesenteric lymph nodes from a human organ donor, we identified a rare subset of evolutionarily conserved APCs that are dependent on RORγt, uniquely express the Prdm16 transcription factor, and are endowed with essential mediators for inducing pTreg cell differentiation. Our findings suggest that a better understanding of how RORγt-APCs develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.

A systematic search for RNA structural switches across the human transcriptome

RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3ʹ untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

PD-1 regulation of pathogenic IL-17-secreting γδ T cells in experimental autoimmune encephalomyelitis.

The PD-1-PD-L1 immune checkpoint helps to maintain self-tolerance and prevent the development of autoimmune diseases. Immune checkpoint inhibitors are successful immunotherapeutics for several cancers, but responding patients can develop immune-mediated adverse events. It is well established that PD-1 regulates CD4 and CD8 T-cell responses, but its role in controlling the activation of pathogenic γδ T cells is less clear. Here we examined the role of PD-1 in regulating γδ T cells in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that PD-1 was highly expressed on CD27- Vγ4 γδ T cells in the lymph node (LN) and CNS of mice with EAE. Treatment of mice with anti-PD-1 significantly augmented IL-17A-producing CD27- Vγ4 γδ T cells in the LN and CNS and enhanced the severity of EAE. The exacerbating effect of anti-PD-1 on EAE was lost in Tcrd-/- mice. Conversely, ligation of PD-1 suppressed Il17a and Rorc gene expression and IL-17A production by purified Vγ4 γδ T cells stimulated via the TCR, but not with IL-1β and IL-23. Our study demonstrates that PD-1 regulates TCR-activated CD27- Vγ4 γδ T cells, but that cytokine-activated IL-17A producing γδ T cells escape the regulatory effects of the PD-1-PD-L1 pathway.

Th17 pathway‐related immune signatures in the pathobiology of myasthenia gravis: Integrating the roles of regulatory/effector cytokines and transcription factors

AbstractObjectivesMyasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptor (AChR), muscle specific kinase (MuSK), and other antigens in the neuromuscular junction. Antibody production is influenced by T lymphocytes. The T helper 17 (Th17) subset, an inflammatory lineage of Th cells, is associated with several autoimmune diseases. Functional interactions between T lymphocytes and pathogenic antibody responses including aberrant Th17 cell responses have been reported in MG. However, the precise mechanism(s) underlying the activation and/or pathogenic transformation of Th17 cells are not clearly known. The current study aimed at simultaneously exploring the roles of the inducers, master regulator transcription factors, and effectors of Th17 cells in patients with MG.MethodsIn this cross‐sectional study, quantification of gene expression of IL6, IL17A, STAT3, and RORC in the peripheral mononuclear cells by quantitative polymerase chain reaction (qPCR) as well as estimation of plasma levels of IL‐1β, IL‐6, and IL‐17A cytokines by multiplex suspension assay were carried out in 59 patients with MG and 61 healthy controls.ResultsGene expression levels of IL17A were significantly upregulated in patients as compared to healthy controls. The plasma levels of IL‐1β, IL‐6, and IL‐17A were significantly elevated in patients compared to healthy controls. IL17A as well as RORC gene expressions correlated with the clinical features. IL17A gene expression levels were higher in AChR‐MG patients.ConclusionThe present study supports the crucial role of the Th17 pathway in the pathobiology of MG, including its potential influence on disease severity.

Immunomodulation of adipose-derived mesenchymal stem cells on peripheral blood mononuclear cells in colorectal cancer patients with COVID-19.

Accumulating evidence has shown that adipose tissue-derived mesenchymal stem cells (ADSCs) are an effective therapeutic approach for managing coronavirus disease 2019 (COVID-19); however, further elucidation is required to determine their underlying immunomodulatory effect on the mRNA expression of T helper cell-related transcription factors (TFs) and cytokine release in peripheral blood mononuclear cells (PBMCs). To investigate the impact of ADSCs on the mRNA expression of TFs and cytokine release in PBMCs from colorectal cancer (CRC) patients with severe COVID-19 (CRC+ patients). PBMCs from CRC+ patients (PBMCs-C+) and age-matched CRC patients (PBMCs-C) were stimulated and cultured in the presence/absence of ADSCs. The mRNA levels of T-box TF TBX21 (T-bet), GATA binding protein 3 (GATA-3), RAR-related orphan receptor C (RORC), and forkhead box P3 (FoxP3) in the PBMCs were determined by reverse transcriptase-polymerase chain reaction. Culture supernatants were evaluated for levels of interferon gamma (IFN-γ), interleukin 4 (IL-4), IL-17A, and transforming growth factor beta 1 (TGF-β1) using an enzyme-linked immunosorbent assay. Compared with PBMCs-C, PBMCs-C+ exhibited higher mRNA levels of T-bet and RORC, and increased levels of IFN-γ and IL-17A. Additionally, a significant decrease in FoxP3 mRNA and TGF-β1, as well as an increase in T-bet/GATA-3, RORC/FoxP3, IFN-γ/IL-4, and IL-17A/TGF-β1 ratios were observed in PBMCs-C+. Furthermore, ADSCs significantly induced a functional regulatory T cell (Treg) subset, as evidenced by an increase in FoxP3 mRNA and TGF-β1 release levels. This was accompanied by a significant decrease in the mRNA levels of T-bet and RORC, release of IFN-γ and IL-17A, and T-bet/GATA-3, RORC/FoxP3, IFN-γ/IL-4, and IL-17A/TGF-β1 ratios, compared with the PBMCs-C+alone. The present in vitro studies showed that ADSCs contributed to the immunosuppressive effects on PBMCs-C+, favoring Treg responses. Thus, ADSC-based cell therapy could be a beneficial approach for patients with severe COVID-19 who fail to respond to conventional therapies.

Majie Cataplasm Alleviates Asthma by Regulating Th1/Th2/Treg/Th17 Balance

Introduction: T cells play a critical role in inflammatory diseases. The aim of the present study was to investigate the effects of Majie cataplasm (MJC) on asthma and to propose a possible mechanism involved in this process. Methods: Airway inflammation, infiltration of inflammatory cells, levels of interleukin (IL)-4, IL-10, IL-17, and interferon (IFN)-γ, levels of Th2, Treg, Th17, and Th1 cells, and the expressions of IL-4, IL-10, IL-17, IFN-γ, GATA binding protein 3 (GATA-3), Foxp3, RAR-related orphan receptor gamma (RORγt), and T-bet were detected. Result: MJC treatment reduced lung airway resistance and inflammatory infiltration in lung tissues. MJC treatment also reduced the numbers of eosinophils and neutrophils in the blood and bronchoalveolar lavage fluid (BALF). The levels of IL-4 and IL-17 in the blood, BALF, and lungs were suppressed by MJC, and IFN-γ and IL-10 were increased. Furthermore, MJC suppressed the percentage of Th2 and Th17 and increased the percentage of Th1 and Treg in spleen cells. In addition, MJC can inhibit asthma by increasing expressions of IFN-γ, IL-10, T-bet, and Foxp3, as well as decreasing expressions of IL-4, IL-17, GATA-3, and RORγt. Conclusion: MJC may improve airway hyperresponsiveness and inflammation by regulating Th1/Th2/Treg/Th17 balance in ovalbumin-induced rats. And MJC may be a new source of anti-asthma drugs.

Downregulation of Mirlet7 miRNA family promotes Tc17 differentiation and emphysema via de-repression of RORγt.

Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 microRNA (Mirlet7 miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the Mirlet7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the Mirlet7 clusters, Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the Mirlet7b/Mirlet7c2 cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the Mirlet7b/Mirlet7c2 cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing Mirlet7g in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of Mirlet7 in T cells. Overall, our findings shed light on the Mirlet7/RORγt axis with Mirlet7 acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

Downregulation of Mirlet7 miRNA family promotes Tc17 differentiation and emphysema via de-repression of RORγt

Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 microRNA (Mirlet7 miRNA) family is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the Mirlet7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here, we show that overall expression of the Mirlet7 clusters, Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the Mirlet7b/Mirlet7c2 cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the Mirlet7b/Mirlet7c2 cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing Mirlet7g in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of Mirlet7 in T cells. Overall, our findings shed light on the Mirlet7/RORγt axis with Mirlet7 acting as a molecular brake in the generation of Tc17 cells and suggest a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

Characteristic features of in vitro differentiation of human naïve CD4+ T cells to induced regulatory T cells (iTreg) and T helper (Th) 17 cells: Sharing of lineage-specific markers

In vitro induced regulatory T cells (iTreg) and IL-17 producing T cells (Th17-like cells) can be generated in culture from native CD4+ T cells in peripheral blood by different sets of cytokines. In the presence of transforming growth factor (TGF)-β plus interleukin (IL)-2, cells differentiate into Treg cells with increased expression of the forkhead box P3 (FOXP3). In the presence of TGF-β, IL-6, IL-1β and IL-23, cells differentiate into Th17 cells that produce IL-17A. However, protocols for the generation of human iTreg and Th17 are still controversial. In this study, we characterized the biological features of iTreg and Th17 cells differentiated from peripheral blood naïve CD4+ T cells in vitro using the established protocols. We showed that cells obtained from Treg or Th17 culture conditions shared some phenotypic markers. Cells under Treg conditions had an up-regulated FOXP3 gene and a down-regulated RAR-related orphan receptor C (RORC) gene. Cells derived from the Th17 condition exhibited a down-regulated FOXP3 gene and had significantly higher RORC gene expression than Treg cells. Both resulting cells showed intracellular production of IL-17A and IL-10. Th17 condition-cultured cells exhibited more glycolytic activity and glucose uptake compared to the Treg cells. The findings suggest that cells obtained from established protocols for the differentiation of iTreg and Th17 cells in vitro are possibly in the intermediate stage of differentiation or may be two different types of cells that share a lineage-specific differentiation program.

Transcriptome analysis reveals that gga-miR-2954 inhibits the inflammatory response against Eimeria tenella infection

Coccidiosis is an important parasitic protozoan disease in poultry farming, causing huge economic losses in the global poultry industry every year. MicroRNAs (miRNAs) are a class of RNA macromolecules that play important roles in the immune response to pathogens. However, the expression profiles and functions of miRNAs during Eimeria tenella (E. tenella) infection in chickens remain mostly uncharacterized. In this study, high-throughput sequencing of cecal tissues of control (JC), resistant (JR), and susceptible (JS) chickens led to the identification of 35 differentially expressed miRNAs among the three groups. Functional enrichment analysis showed that the differentially expressed miRNAs were mainly associated with the TGF-beta, NF-kB, and Jak-STAT signaling pathways. Notably, gga-miR-2954 was found to be significantly upregulated after coccidial infection. Functional analysis showed that gga-miR-2954 inhibited the production of the inflammatory cytokines IL-6, IL-1β, TNF-α, and IL-8 in sporozoite-stimulated DF-1 cells. Mechanistically, we found that gga-miR-2954 targeted the RORC gene and that RORC promoted the inflammatory response in sporozoite-stimulated DF-1 cells. In conclusion, our study was the first to identify differentially expressed miRNAs in chicken cecal tissue during E. tenella infection and found that gga-miR-2954 regulates the host immune response to coccidial infection in chickens by targeting the RORC gene.

Aryl hydrocarbon receptor gene expression in ankylosing spondylitis and its correlation with interleukin-17, RAR-related orphan receptor gamma t expression, and disease activity indices.

Considering the role of T helper (Th)17 cells in the pathogenesis of ankylosing spondylitis (AS), the aim of this study was to determine the correlation between aryl hydrocarbon receptor (AHR) gene expression and the expression of Th17-related genes including interleukin (IL)-17 and RAR-related orphan receptor gamma t (RORγt) transcription factor. Thirty patients with AS (26 males, 4 females; mean age: 36.1±8.1 years) and 30 age- and sex-matched healthy individuals (26 males, 4 females; mean age: 36.2±14.6 years) were recruited for the case-control study between June 2021 and January 2022. Ribonucleic acid (RNA) was extracted from peripheral blood cells and expression levels of AHR, IL-17, RORγt, and AHR repressor (AHRR) genes were evaluated using real-time polymerase chain reaction technique. The serum level of IL-17 was evaluated with enzyme-linked immunosorbent assay. The results showed a nonsignificant elevation of AHR, IL-17, and RORγt gene expression in the patient group compared to the control. There was a direct correlation between AHR gene expression and IL-17 and RORγt genes and a negative correlation between AHR and AHRR expression. Moreover, AHR gene expression showed a weak correlation with disease activity indices, including Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, Bath Ankylosing Spondylitis Metrology Index, Bath Ankylosing Spondylitis Global Score, and Ankylosing Spondylitis Quality of Life. Moreover, the serum level of IL-17 was higher in AS patients compared to the healthy group (p=0.02). Upregulated expression of the AHR gene in ankylosing spondylitis and its correlation with IL-17 and ROR-γ t gene expression suggests that it could be a potential diagnostic and therapeutic target for AS.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep

BackgroundThe integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control.MethodsEggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content.ResultsThe heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep.ConclusionsThe present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.Graphical

Core fucosylation of maternal milk N-glycans imparts early-life immune tolerance through gut microbiota-dependent regulation in RORγt+ Treg cells.

Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.

Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5–1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors invitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.