m6A mRNA modification potentiates Th17 functions to inflame autoimmunity.

N6-methyladenosine (m6A), the most common and abundant epigenetic RNA modification, governs mRNA metabolism to determine cell differentiation, proliferation and response to stimulation. m6A methyltransferase METTL3 has been reported to control T cell homeostasis and sustain the suppressive function of regulatory T cells (Tregs). However, the role of m6A methyltransferase in other subtypes of T cells remains unknown. T helper cells 17 (Th17) play a pivotal role in host defense and autoimmunity. Here, we found that the loss of METTL3 in T cells caused serious defect of Th17 cell differentiation, and impeded the development of experimental autoimmune encephalomyelitis (EAE). We generated Mettl3f/fIl17aCre mice and observed that METTL3 deficiency in Th17 cells significantly suppressed the development of EAE and displayed less Th17 cell infiltration into central nervous system (CNS). Importantly, we demonstrated that depletion of METTL3 attenuated IL-17A and CCR5 expression by facilitating SOCS3 mRNA stability in Th17 cells, leading to disrupted Th17 cell differentiation and infiltration, and eventually attenuating the process of EAE. Collectively, our results highlight that m6A modification sustains Th17 cell function, which provides new insights into the regulatory network of Th17 cells, and also implies a potential therapeutic target for Th17 cell mediated autoimmune disease.

m6A mRNA modification maintains colonic epithelial cell homeostasis via NF-κB-mediated antiapoptotic pathway.

Colonic mucosal barrier dysfunction is one of the major causes of inflammatory bowel disease (IBD). However, the mechanisms underlying mucosal barrier dysfunction are poorly understood. N6-methyladenosine (m6A) mRNA modification is an important modulator of epitranscriptional regulation of gene expression, participating in multiple physiological and pathological processes. However, the function of m6A modification in colonic epithelial cells and stem cells is unknown. Here, we show that m6A modification is essential for maintaining the homeostatic self-renewal in colonic stem cells. Specific deletion of the methyltransferase 14 (Mettl14) gene in mouse colon resulted in colonic stem cell apoptosis, causing mucosal barrier dysfunction and severe colitis. Mechanistically, we revealed that Mettl14 restricted colonic epithelial cell death by regulating the stability of Nfkbia mRNA and modulating the NF-κB pathway. Our results identified a previously unidentified role for m6A modification in colonic epithelial cells and stem cells, suggesting that m6A modification may be a potential therapeutic target for IBD.

Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation.

m6A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m6A "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3 flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m6A modification. METTL3 deficiency led to the loss of m6A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling-mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m6A modification in innate immune responses and implicate the m6A machinery as a potential cancer immunotherapy target.

A single-cell survey of the human glomerulonephritis.

Glomerulonephritis is the one of the major causes of the end-stage kidney disease, whereas the pathological process of glomerulonephritis is still not completely understood. Single-cell RNA sequencing (scRNA-seq) emerges to be a powerful tool to evaluate the full heterogeneity of kidney diseases. To reveal cellular gene expression profiles of glomerulonephritis, we performed scRNA-seq of 2 human kidney transplantation donor samples, 4 human glomerulonephritis samples, 1 human malignant hypertension (MH) sample and 1 human chronic interstitial nephritis (CIN) sample, all tissues were taken from the biopsy. After filtering the cells with < 200 genes and > 10% mitochondria (MT) genes, the resulting 14 932 cells can be divided into 20 cell clusters, consistently with the previous report, in disease samples dramatic immune cells infiltration was found, among which a proximal tubule (PT) subset characterized by wnt-ß catenin activation and a natural killer T (NKT) subset high expressing LTB were found. Furthermore, in the cluster of the podocyte, three glomerulonephritis related genes named FXYD5, CD74 and B2M were found. Compared with the mesangial of donor, the gene CLIC1 and RPS26 were up-regulated in mesangial of IgA nephropathy(IgAN), whereas the gene JUNB was up-regulated in podocyte of IgAN in comparison with that of donor. Meanwhile, some membranous nephropathy (MN) high expressed genes such as HLA-DRB5, HLA-DQA2, IFNG, CCL2 and NR4A2, which involve in highest enrichment pathway, display the cellular-specific expression style, whereas monocyte marker of lupus nephritis (LN) named TNFSF13B was also found and interferon alpha/beta signalling pathway was enriched in B and NKT of LN comparing with donor. By scRNA-seq, we first defined the podocyte markers of glomerulonephritis and specific markers in IgA, MN and LN were found at cellular level. Furthermore, the critical role of interferon alpha/beta signalling pathway was enriched in B and NKT of LN was declared.

Identification of two IL-4/13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes/macrophages.

Mammalian interleukin-4 (IL-4) and -13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1ß and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-ß and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Construction of the High-Density Genetic Linkage Map and Chromosome Map of Large Yellow Croaker (Larimichthys crocea).

High-density genetic maps are essential for genome assembly, comparative genomic analysis and fine mapping of complex traits. In this study, 31,191 single nucleotide polymorphisms (SNPs) evenly distributed across the large yellow croaker (Larimichthys crocea) genome were identified using restriction-site associated DNA sequencing (RAD-seq). Among them, 10,150 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 5451.3 cM with an average distance of 0.54 cM between loci. This represents the densest genetic map currently reported for large yellow croaker. Using 2889 SNPs to target specific scaffolds, we assigned 533 scaffolds, comprising 421.44 Mb (62.04%) of the large yellow croaker assembled sequence, to the 24 linkage groups. The mapped assembly scaffolds in large yellow croaker were used for genome synteny analyses against the stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes). Greater synteny was observed between large yellow croaker and stickleback. This supports the hypothesis that large yellow croaker is more closely related to stickleback than to medaka. Moreover, 1274 immunity-related genes and 195 hypoxia-related genes were mapped to the 24 chromosomes of large yellow croaker. The integration of the high-resolution genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits in large yellow croaker.