The crosstalk between enteric nervous system and immune system in intestinal development, homeostasis and diseases.

The gut is the largest digestive and absorptive organ, which is essential for induction of mucosal and systemic immune responses, and maintenance of metabolic-immune homeostasis. The intestinal components contain the epithelium, stromal cells, immune cells, and enteric nervous system (ENS), as well as the outers, such as gut microbiota, metabolites, and nutrients. The dyshomeostasis of intestinal microenvironment induces abnormal intestinal development and functions, even colon diseases including dysplasia, inflammation and tumor. Several recent studies have identified that ENS plays a crucial role in maintaining the immune homeostasis of gastrointestinal (GI) microenvironment. The crosstalk between ENS and immune cells, mainly macrophages, T cells, and innate lymphoid cells (ILCs), has been found to exert important regulatory roles in intestinal tissue programming, homeostasis, function, and inflammation. In this review, we mainly summarize the critical roles of the interactions between ENS and immune cells in intestinal homeostasis during intestinal development and diseases progression, to provide theoretical bases and ideas for the exploration of immunotherapy for gastrointestinal diseases with the ENS as potential novel targets.

Transcription Factor Id1 Plays an Essential Role in Th9 Cell Differentiation by Inhibiting Tcf3 and Tcf4.

T helper type 9 (Th9) cells play important roles in immune responses by producing interleukin-9 (IL-9). Several transcription factors are responsible for Th9 cell differentiation; however, transcriptional regulation of Th9 cells is not fully understood. Here, it is shown that Id1 is an essential transcriptional regulator of Th9 cell differentiation. Id1 is induced by IL-4 and TGF-ß. Id1-deficient naïve CD4 T cells fail to differentiate into Th9 cells, and overexpression of Id1 induce expression of IL-9. Mass spectrometry analysis reveals that Id1 interacts with Tcf3 and Tcf4 in Th9 cells. In addition, RNA-sequencing, chromatin immunoprecipitation, and transient reporter assay reveal that Tcf3 and Tcf4 bind to the promoter region of the Il9 gene to suppress its expression, and that Id1 inhibits their function, leading to Th9 differentiation. Finally, Id1-deficient Th9 cells ameliorate airway inflammation in an animal model of asthma. Thus, Id1 is a transcription factor that plays an essential role in Th9 cell differentiation by inhibiting Tcf3 and Tcf4.

ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m6A modification-mediated RNA stability control.

N6-methyladenosine (m6A) RNA modification controls numerous cellular processes. To what extent these post-transcriptional regulatory mechanisms play a role in hematopoiesis has not been fully elucidated. We here show that the m6A demethylase alkB homolog 5 (ALKBH5) controls mitochondrial ATP production and modulates hematopoietic stem and progenitor cell (HSPC) fitness in an m6A-dependent manner. Loss of ALKBH5 results in increased RNA methylation and instability of oxoglutarate-dehydrogenase (Ogdh) messenger RNA and reduction of OGDH protein levels. Limited OGDH availability slows the tricarboxylic acid (TCA) cycle with accumulation of α-ketoglutarate (α-KG) and conversion of α-KG into L-2-hydroxyglutarate (L-2-HG). L-2-HG inhibits energy production in both murine and human hematopoietic cells in vitro. Impaired mitochondrial energy production confers competitive disadvantage to HSPCs and limits clonogenicity of Mll-AF9-induced leukemia. Our study uncovers a mechanism whereby the RNA m6A demethylase ALKBH5 regulates the stability of metabolic enzyme transcripts, thereby controlling energy metabolism in hematopoiesis and leukemia.

Mettl3-catalyzed m6A regulates histone modifier and modification expression in self-renewing somatic tissue.

N6-methyladenosine (m6A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m6A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m6A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m6A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m6A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m6A in proper epithelial development and self-renewal.

RNA modification-mediated translational control in immune cells.

RNA modifications play a vital role in multiple pathways of mRNA metabolism, and translational regulation is essential for immune cells to promptly respond to stimuli and adapt to the microenvironment. N6-methyladenosine (m6A) methylation, which is the most abundant mRNA modification in eukaryotes, primarily functions in the regulation of RNA splicing and degradation. However, the role of m6Amethylation in translational control and its underlying mechanism remain controversial. The role of m6A methylation in translation regulation in immune cells has received relatively limited attention. In this review, we aim to provide a comprehensive summary of current studies on the translational regulation of m6A modifications and recent advances in understanding the translational control regulated by RNA modifications during the immune response. Furthermore, we envision the possible pathways through which m6A modifications may be involved in the regulation of immune cell function via translational control.

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.

A Treg-specific long noncoding RNA maintains immune-metabolic homeostasis in aging liver.

Regulatory T (Treg) cells modulate several aging-related liver diseases. However, the molecular mechanisms regulating Treg function in this context are unknown. Here we identified a long noncoding RNA, Altre (aging liver Treg-expressed non-protein-coding RNA), which was specifically expressed in the nucleus of Treg cells and increased with aging. Treg-specific deletion of Altre did not affect Treg homeostasis and function in young mice but caused Treg metabolic dysfunction, inflammatory liver microenvironment, liver fibrosis and liver cancer in aged mice. Depletion of Altre reduced Treg mitochondrial integrity and respiratory capacity, and induced reactive oxygen species accumulation, thus increasing intrahepatic Treg apoptosis in aged mice. Moreover, lipidomic analysis identified a specific lipid species driving Treg aging and apoptosis in the aging liver microenvironment. Mechanistically, Altre interacts with Yin Yang 1 to orchestrate its occupation on chromatin, thereby regulating the expression of a group of mitochondrial genes, and maintaining optimal mitochondrial function and Treg fitness in the liver of aged mice. In conclusion, the Treg-specific nuclear long noncoding RNA Altre maintains the immune-metabolic homeostasis of the aged liver through Yin Yang 1-regulated optimal mitochondrial function and the Treg-sustained liver immune microenvironment. Thus, Altre is a potential therapeutic target for the treatment of liver diseases affecting older adults.

A lncRNA from an inflammatory bowel disease risk locus maintains intestinal host-commensal homeostasis.

Inflammatory bowel diseases (IBD) are known to have complex, genetically influenced etiologies, involving dysfunctional interactions between the intestinal immune system and the microbiome. Here, we characterized how the RNA transcript from an IBD-associated long non-coding RNA locus ("CARINH-Colitis Associated IRF1 antisense Regulator of Intestinal Homeostasis") protects against IBD. We show that CARINH and its neighboring gene coding for the transcription factor IRF1 together form a feedforward loop in host myeloid cells. The loop activation is sustained by microbial factors, and functions to maintain the intestinal host-commensal homeostasis via the induction of the anti-inflammatory factor IL-18BP and anti-microbial factors called guanylate-binding proteins (GBPs). Extending these mechanistic insights back to humans, we demonstrate that the function of the CARINH/IRF1 loop is conserved between mice and humans. Genetically, the T allele of rs2188962, the most probable causal variant of IBD within the CARINH locus from the human genetics study, impairs the inducible expression of the CARINH/IRF1 loop and thus increases genetic predisposition to IBD. Our study thus illustrates how an IBD-associated lncRNA maintains intestinal homeostasis and protects the host against colitis.

N6 - Methyladenosine defines a new checkpoint in γδ T cell development.

T cells, which are derived from hematopoietic stem cells (HSCs), are the most important components of adaptive immune system. Based on the expression of αß and γδ receptors, T cells are mainly divided into αß and γδ T cells. In the thymus, they share common progenitor cells, while undergoing a series of well-characterized and different developmental processes. N6 -Methyladenosine (m6 A), one of the most abundant modifications in mRNAs, plays critical roles in cell development and maintenance of function. Recently, we have demonstrated that the depletion of m6 A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells through the regulation of Jag1/Notch2 signaling, but not αß T cells, indicating a checkpoint role of ALKBH5 and m6 A modification in the early development of γδ T cells. Based on previous studies, many key pathway molecules, which exert dominant roles in γδ T cell fate determination, have been identified as the targets regulated by m6 A modification. In this review, we mainly summarize the potential regulation between m6 A modification and these key signaling molecules in the γδ T cell lineage commitment, to provide new perspectives in the checkpoint of γδ T cell development.

tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis.

Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.

RNA m6A demethylase ALKBH5 regulates the development of γδ T cells.

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

Diverging regulation of Bach2 protein and RNA expression determine cell fate in early B cell response.

During the early phase of primary humoral responses, activated B cells can differentiate into different types of effector cells, dependent on B cell receptor affinity for antigen. However, the pivotal transcription factors governing these processes remain to be elucidated. Here, we show that transcription factor Bach2 protein in activated B cells is transiently induced by affinity-related signals and mechanistic target of rapamycin complex 1 (mTORC1)-dependent translation to restrain their expansion and differentiation into plasma cells while promoting memory and germinal center (GC) B cell fates. Affinity-related signals also downregulate Bach2 mRNA expression in activated B cells and their descendant memory B cells. Sustained and higher concentrations of Bach2 antagonize the GC fate. Repression of Bach2 in memory B cells predisposes their cell-fate choices upon memory recall. Our study reveals that differential dynamics of Bach2 protein and transcripts in activated B cells control their cell-fate outcomes and imprint the fates of their descendant effector cells.

DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion.

RNA-binding proteins (RBPs) can recognize thousands of RNAs that help to maintain cell homeostasis, and RBP dysfunction is frequently observed in various cancers. However, whether specific RBPs are involved in tumor immune evasion by regulating programmed death ligand-1 (PD-L1) is unclear. Here, we perform targeted RBP CRISPR/Cas9 screening and identify density regulated re-initiation and release factor (DENR) as a PD-L1 regulator. DENR-depleted cancer cells exhibit reduced PD-L1 expression in vitro and in vivo. DENR depletion significantly suppresses tumor growth and enhances the tumor-killing activity of CD8+ T cells. Mechanistically, DENR antagonizes the translational repression of three consecutive upstream open reading frames (uORFs) upstream of Janus kinase 2 (Jak2); thus, DENR deficiency impairs JAK2 translation and the IFNγ-JAK-STAT signaling pathway, resulting in reduced PD-L1 expression in tumors. Overall, we discover an RBP DENR that could regulate PD-L1 expression for tumor immune evasion, and highlight the potential of DENR as a therapeutic target for immunotherapy.

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.

m6A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity.

N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m6A "writer" protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m6A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m6A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity.

METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells.

Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A "writer" METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells.

Altered topographical organization of grey matter structural network in early-onset schizophrenia.

Schizophrenia is characterized by both disrupted neurodevelopmental processes and abnormal brain connectivity. However, few studies have examined the atypical features of brain network topography associated with schizophrenia during childhood and adolescence. We used graph theory to compare the grey matter structural networks of individuals (aged 10-15 years) with early-onset schizophrenia (EOS) (n = 25) and a typically-developing (TD) comparison group (n = 31). Compared with the TD group, EOS patients showed significantly increased clustering and local efficiency across a range of network densities (0.3 - 0.4). The network of EOS patients also had more modules (6 modules in EOS vs. 3 modules in controls), indicating a more segregated network at the cost of functional integration. Although our results were preliminary and failed to survive corrections for multiple comparisons, EOS patients might be characterized by altered nodal centrality in several higher-order associative regions including the prefrontal cortex, the hippocampus and the cerebellum. The EOS structural network also lacked the typical left-hemispheric-dominant hub distribution compared with the TD group. These findings suggest that brain structural network was not only globally but also regionally altered in EOS patients.

m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity.

N6-methyladenosine (m6A) modification is dynamically regulated by "writer" and "eraser" enzymes. m6A "writers" have been shown to ensure the homeostasis of CD4+ T cells, but the "erasers" functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naïve CD4+ T cells to induce adoptive transfer colitis. In addition, T cell-specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

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.