Regulatory Innate Lymphoid Cells Control Innate Intestinal Inflammation.

An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-ß1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-ß1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation.

IL-13 secreted by ILC2s promotes the self-renewal of intestinal stem cells through circular RNA circPan3.

Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5+ ISCs showed high expression of the immune cell-associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5+ ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13‒IL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with ß-catenin in rendering its nuclear translocation, which caused activation of the ß-catenin pathway and the maintenance of Lgr5+ ISCs.

Intestinal Tuft-2 cells exert antimicrobial immunity via sensing bacterial metabolite N-undecanoylglycine.

Tuft cells are a type of intestinal epithelial cells that exist in epithelial barriers and play a critical role in immunity against parasite infection. It remains insufficiently clear whether Tuft cells participate in bacterial eradication. Here, we identified Sh2d6 as a signature marker for CD45+ Tuft-2 cells. Depletion of Tuft-2 cells resulted in susceptibility to bacterial infection. Tuft-2 cells quickly expanded in response to bacterial infection and sensed the bacterial metabolite N-undecanoylglycine through vomeronasal receptor Vmn2r26. Mechanistically, Vmn2r26 engaged with N-undecanoylglycine activated G-protein-coupled receptor-phospholipase C gamma2 (GPCR-PLCγ2)-Ca2+ signaling axis, which initiated prostaglandin D2 (PGD2) production. PGD2 enhanced the mucus secretion of goblet cells and induced antibacterial immunity. Moreover, Vmn2r26 signaling also promoted SpiB transcription factor expression, which is responsible for Tuft-2 cell development and expansion in response to bacterial challenge. Our findings reveal an additional function of Tuft-2 cells in immunity against bacterial infection through Vmn2r26-mediated recognition of bacterial metabolites.

The ER membrane adaptor ERAdP senses the bacterial second messenger c-di-AMP and initiates anti-bacterial immunity.

Cyclic diadenylate monophosphate (c-di-AMP) is secreted by bacteria as a secondary messenger. How immune cells detect c-di-AMP and initiate anti-bacterial immunity remains unknown. We found that the endoplasmic reticulum (ER) membrane adaptor ERAdP acts as a direct sensor for c-di-AMP. ERAdP-deficient mice were highly susceptible to Listeria monocytogenes infection and exhibited reduced pro-inflammatory cytokines. Mechanistically, c-di-AMP bound to the C-terminal domain of ERAdP, which in turn led to dimerization of ERAdP, resulting in association with and activation of the kinase TAK1. TAK1 activation consequently initiated activation of the transcription factor NF-κB to induce the production of pro-inflammatory cytokines in innate immune cells. Moreover, double-knockout of ERAdP and TAK1 resulted in heightened susceptibility to L. monocytogenes infection. Thus, ERAdP-mediated production of pro-inflammatory cytokines is critical for controlling bacterial infection.

Long noncoding RNA lncKdm2b is required for ILC3 maintenance by initiation of Zfp292 expression.

Innate lymphoid cells (ILCs) communicate with other hematopoietic and nonhematopoietic cells to regulate immunity, inflammation and tissue homeostasis. How ILC lineages develop and are maintained remains largely unknown. In this study we observed that a divergent long noncoding RNA (lncRNA), lncKdm2b, was expressed at high levels in intestinal group 3 ILCs (ILC3s). LncKdm2b deficiency in the hematopoietic system led to reductions in the number and effector functions of ILC3s. LncKdm2b expression sustained the maintenance of ILC3s by promoting their proliferation through activation of the transcription factor Zfp292. Mechanistically, lncKdm2b recruited the chromatin organizer Satb1 and the nuclear remodeling factor (NURF) complex onto the Zfp292 promoter to initiate its transcription. Deletion of Zfp292 or Bptf also abrogated the maintenance of ILC3s, leading to susceptibility to bacterial infection. Therefore, our findings reveal that lncRNAs may represent an additional layer of regulation of ILC development and function.

Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.

Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

Noncoding RNA circBtnl1 suppresses self-renewal of intestinal stem cells via disruption of Atf4 mRNA stability.

Intestinal stem cells (ISCs) at the crypt base are responsible for the regeneration of the intestinal epithelium. However, how ISC self-renewal is regulated still remains unclear. Here we identified a circular RNA, circBtnl1, that is highly expressed in ISCs. Loss of circBtnl1 in mice enhanced ISC self-renewal capacity and epithelial regeneration, without changes in mRNA and protein levels of its parental gene Btnl1. Mechanistically, circBtnl1 and Atf4 mRNA competitively bound the ATP-dependent RNA helicase Ddx3y to impair the stability of Atf4 mRNA in wild-type ISCs. Furthermore, ATF4 activated Sox9 transcription by binding to its promoter via a unique motif, to enhance the self-renewal capacity and epithelial regeneration of ISCs. In contrast, circBtnl1 knockout promoted Atf4 mRNA stability and enhanced ATF4 expression, which caused Sox9 transcription to potentiate ISC stemness. These data indicate that circBtnl1-mediated Atf4 mRNA decay suppresses Sox9 transcription that negatively modulates self-renewal maintenance of ISCs.

Sox2 functions as a sequence-specific DNA sensor in neutrophils to initiate innate immunity against microbial infection.

Neutrophils express Toll-like receptors (TLRs) for the recognition of conserved bacterial elements to initiate antimicrobial responses. However, whether other cytosolic DNA sensors are expressed by neutrophils remains elusive. Here we found constitutive expression of the transcription factor Sox2 in the cytoplasm of mouse and human neutrophils. Neutrophil-specific Sox2 deficiency exacerbated bacterial infection. Sox2 directly recognized microbial DNA through its high-mobility-group (HMG) domain. Upon challenge with bacterial DNA, Sox2 dimerization was needed to activate a complex of the kinase TAK1 and its binding partner TAB2, which led to activation of the transcription factors NF-κB and AP-1 in neutrophils. Deficiency in TAK1 or TAB2 impaired Sox2-mediated antibacterial immunity. Overall, we reveal a previously unrecognized role for Sox2 as a cytosolic sequence-specific DNA sensor in neutrophils, which might provide potential therapeutic strategies for the treatment of infectious diseases.

A Circular RNA Protects Dormant Hematopoietic Stem Cells from DNA Sensor cGAS-Mediated Exhaustion.

Disrupting the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) leads to bone marrow failure or hematologic malignancy. However, how HSCs sustain their quiescent state and avoid type I interferon (IFN)-mediated exhaustion remains elusive. Here we defined a circular RNA that we named cia-cGAS that was highly expressed in the nucleus of long-term (LT)-HSCs. Cia-cGAS deficiency in mice caused elevated expression of type I IFNs in bone marrow and led to decreased numbers of dormant LT-HSCs. Under homeostatic conditions, cia-cGAS bound DNA sensor cGAS in the nucleus to block its synthase activity, thereby protecting dormant LT-HSCs from cGAS-mediated exhaustion. Moreover, cia-cGAS harbored a stronger binding affinity to cGAS than self-DNA did and consequently suppressed cGAS-mediated production of type I IFNs in LT-HSCs. Our findings reveal a mechanism by which cia-cGAS inhibits nuclear cGAS by blocking its enzymatic activity and preventing cGAS from recognizing self-DNA to maintain host homeostasis.

Natural Killer-like B Cells Prime Innate Lymphocytes against Microbial Infection.

Natural killer (NK) cells and non-cytotoxic interferon-γ (IFN-γ)-producing group I innate lymphoid cells (ILC1s) produce large amounts of IFN-γ and cause activation of innate and adaptive immunity. However, how NKs and ILC1s are primed during infection remains elusive. Here we have shown that a lymphocyte subpopulation natural killer-like B (NKB) cells existed in spleen and mesenteric lymph nodes (MLNs). NKBs had unique features that differed from T and B cells, and produced interleukin-18 (IL-18) and IL-12 at an early phase of infection. NKB cells played a critical role in eradication of microbial infection via secretion of IL-18 and IL-12. Moreover, IL-18 deficiency abrogated the antibacterial effect of NKBs. Upon bacterial challenge, NKB precursors (NKBPs) rapidly differentiated to NKBs that activated NKs and ILC1s against microbial infection. Our findings suggest that NKBs might be exploited to develop effective therapies for treatment of infectious diseases.

The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells.

Lgr5+ intestinal stem cells (ISCs) exhibit self-renewal and differentiation features under homeostatic conditions, but the mechanisms controlling Lgr5 + ISC self-renewal remain elusive. Here, we show that the chromatin remodeler SRCAP is highly expressed in mouse intestinal epithelium and ISCs. Srcap deletion impairs both self-renewal of ISCs and intestinal epithelial regeneration. Mechanistically, SRCAP recruits the transcriptional regulator REST to the Prdm16 promoter and induces expression of this transcription factor. By activating PPARδ expression, Prdm16 in turn initiates PPARδ signaling, which sustains ISC stemness. Rest or Prdm16 deficiency abrogates the self-renewal capacity of ISCs as well as intestinal epithelial regeneration. Collectively, these data show that the SRCAP-REST-Prdm16-PPARδ axis is required for self-renewal maintenance of Lgr5 + ISCs.

LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling.

Hepatocellular carcinoma (HCC) is the most prevalent liver cancer, characterized by a high rate of recurrence and heterogeneity. Liver cancer stem cells (CSCs) may well contribute to both of these pathological properties, but the mechanism underlying their self-renewal maintenance is poorly understood. Here, we identified a long noncoding RNA (lncRNA) termed HAND2-AS1 that is highly expressed in liver CSCs. Human HAND2-AS1 and its mouse ortholog lncHand2 display a high level of conservation. HAND2-AS1 is required for the self-renewal maintenance of liver CSCs to initiate HCC development. Mechanistically, HAND2-AS1 recruits the INO80 chromatin-remodeling complex to the promoter of BMPR1A, thereby inducing its expression and leading to the activation of BMP signaling. Importantly, interfering with expression of HAND2-AS1 by antisense oligonucleotides (ASOs) and BMPR1A by siRNAs has synergistic anti-tumorigenic effects on humanized HCC models. Moreover, knockout of lncHand2 or Bmpr1a in mouse hepatocytes impairs BMP signaling and suppresses the initiation of liver cancer. Our findings reveal that HAND2-AS1 promotes the self-renewal of liver CSCs and drives liver oncogenesis, offering a potential new target for HCC therapy.

LncKdm2b controls self-renewal of embryonic stem cells via activating expression of transcription factor Zbtb3.

Divergent long noncoding RNAs (lncRNAs) represent a major lncRNA biotype in mouse and human genomes. The biological and molecular functions of the divergent lncRNAs remain largely unknown. Here, we show that lncKdm2b, a divergent lncRNA for Kdm2b gene, is conserved among five mammalian species and highly expressed in embryonic stem cells (ESCs) and early embryos. LncKdm2b knockout impairs ESC self-renewal and causes early embryonic lethality. LncKdm2b can activate Zbtb3 by promoting the assembly and ATPase activity of Snf2-related CREBBP activator protein (SRCAP) complex in trans Zbtb3 potentiates the ESC self-renewal in a Nanog-dependent manner. Finally, Zbtb3 deficiency impairs the ESC self-renewal and early embryonic development. Therefore, our findings reveal that lncRNAs may represent an additional layer of the regulation of ESC self-renewal and early embryogenesis.

A higher-order configuration of the heterodimeric DOT1L-AF10 coiled-coil domains potentiates their leukemogenenic activity.

Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1-AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10's octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1-AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L-AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L-AF10 complex is essential for MLL1-AF10's leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.

Circular RNA circIPO11 drives self-renewal of liver cancer initiating cells via Hedgehog signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most intractable tumors in the world due to its high rate of recurrence and heterogeneity. Liver cancer initiating cells also called cancer stem cells (CSCs) play a critical role in resistance against typical therapy and high tumor-initiating potential. However, the role of the novel circular RNA (circRNA) circIPO11 in the maintenance of liver cancer initiating cells remains elusive. METHODS: CircRNAs highly conserved in humans and mice were identified from 3 primary HCC samples by circRNA array. The expression and function of circIPO11 were further evaluated by Northern blot, limiting dilution xenograft analysis, chromatin isolation by RNA purification-PCR assay (ChIRP) and HCC patient-derived tumor cells (PDC) models. CircIpo11 knockout (KO) mice were generated by a CRISPR/Cas9 technology. RESULTS: CircIPO11 is highly expressed in HCC tumor tissues and liver CSCs. CircIPO11 is required for the self-renewal maintenance of liver CSCs to initiate HCC development. Mechanistically, circIPO11 recruits TOP1 to GLI1 promoter to trigger its transcription, leading to the activation of Hedgehog signaling. Moreover, GLI1 is also highly expressed in HCC tumor tissues and liver CSCs, and TOP1 expression levels positively correlate with the metastasis, recurrence and survival of HCC patients. Additionally, circIPO11 knockout in mice suppresses the progression of chemically induced liver cancer development. CONCLUSION: Our findings reveal that circIPO11 drives the self-renewal of liver CSCs and promotes the propagation of HCC via activating Hedgehog signaling pathway. Antisense oligonucleotides (ASOs) against circIPO11 combined with TOP1 inhibitor camptothecin (CPT) exert synergistic antitumor effect. Therefore, circIPO11 and the Hedgehog signaling pathway may provide new potential targets for the treatment of HCC patients.

The long non-coding RNA LncHDAC2 drives the self-renewal of liver cancer stem cells via activation of Hedgehog signaling.

BACKGROUND & AIMS: Liver cancer is the second leading cause of cancer death worldwide. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults. The aim of this study was to define the role of the long non-coding RNA lncHDAC2 in the tumorigenesis of HCC. METHODS: CD13+CD133+ cells (hereafter called liver cancer stem cells [CSCs]) and CD13-CD133- cells (referred to as non-CSCs) were sorted from 3 primary HCC tumor tissues and followed by transcriptome microarray. The expression and function of lncHDAC2 were further assessed by northern blot, sphere formation and xenograft tumor models. RESULTS: LncHDAC2 is highly expressed in HCC tumors and liver CSCs. LncHDAC2 promotes the self-renewal of liver CSCs and tumor propagation. In liver CSCs, lncHDAC2 recruits the NuRD complex onto the promoter of PTCH1 to inhibit its expression, leading to activation of Hedgehog signaling. Moreover, HDAC2 expression levels are positively related to HCC severity and PTCH1 levels are negatively related to HCC severity. Additionally, the Smo inhibitor cyclopamine was shown to impair the self-renewal of liver CSCs and suppress tumor propagation. CONCLUSION: Our findings reveal that lncHDAC2 promotes the self-renewal of liver CSCs and tumor propagation by activating the Hedgehog signaling pathway. Downregulating lncHDAC2 is a promising antitumor strategy in HCC. LAY SUMMARY: Liver cancer stem cells harbor high tumor-initiating potential and confer resistance to typical therapies, but the mechanism underlying their self-renewal remains elusive. LncHDAC2 augments the self-renewal of these cells, promoting tumor propagation. In liver cancer stem cells, lncHDAC2 activates Hedgehog signaling to initiate liver tumorigenesis. Therefore, lncHDAC2 and the Hedgehog signaling pathway may serve as biomarkers and potential drug targets for hepatocellular carcinoma.

Long noncoding RNA lncHand2 promotes liver repopulation via c-Met signaling.

BACKGROUND & AIMS: Long noncoding RNAs (lncRNAs) play important roles in various biological processes, regulating gene expression by diverse mechanisms. However, how lncRNAs regulate liver repopulation is unknown. Herein, we aimed to identify lncRNAs that regulate liver repopulation and elucidate the signaling pathways involved. METHODS: Herein, we performed 70% partial hepatectomy in wild-type and gene knockout mice. We then performed transcriptomic analyses to identify a divergent lncRNA termed lncHand2 that is highly expressed during liver regeneration. RESULTS: LncHand2 is constitutively expressed in the nuclei of pericentral hepatocytes in mouse and human livers. LncHand2 knockout abrogates liver regeneration and repopulation capacity. Mechanistically, lncHand2 recruits the Ino80 remodeling complex to initiate expression of Nkx1-2 in trans, which triggers c-Met (Met) expression in hepatocytes. Finally, knockout of both Nkx1-2 and c-Met causes more severe liver injury and poorer repopulation ability. Thus, lncHand2 promotes liver repopulation via initiating Nkx1-2-induced c-Met signaling. CONCLUSIONS: Our findings reveal that lncHand2 acts as a critical mediator regulating liver repopulation. It does this by inducing Nkx1-2 expression, which in turn triggers c-Met signaling. LAY SUMMARY: Long noncoding RNAs play important roles in various biological processes. While long noncoding RNAs do not directly code proteins, they can regulate gene expression by diverse mechanisms. We identified the long noncoding RNA, termed lncHand2 because of its proximity to the gene Hand2, to be an important determinant of liver regeneration through c-Met signaling.

TRIM25 Is Required for the Antiviral Activity of Zinc Finger Antiviral Protein.

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mRNAs and repressing the translation and/or promoting the degradation of target mRNA. In addition, ZAP regulates the expression of certain cellular genes. Here, we report that tripartite motif-containing protein 25 (TRIM25), a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 abolished ZAP's antiviral activity. The E3 ligase activity of TRIM25 is required for this regulation. TRIM25 mediated ZAP ubiquitination, but the ubiquitination of ZAP itself did not seem to be required for its antiviral activity. Downregulation of endogenous ubiquitin or overexpression of the deubiquitinase OTUB1 impaired ZAP's activity. We provide evidence indicating that TRIM25 modulates the target RNA binding activity of ZAP. These results uncover a mechanism by which the antiviral activity of ZAP is regulated.IMPORTANCE ZAP is a host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1, Sindbis virus, and Ebola virus. ZAP binds directly to target mRNA, and it represses the translation and promotes the degradation of target mRNA. While the mechanisms by which ZAP posttranscriptionally inhibits target RNA expression have been extensively studied, how its antiviral activity is regulated is not very clear. Here, we report that TRIM25, a ubiquitin E3 ligase, is required for the antiviral activity of ZAP. Downregulation of endogenous TRIM25 remarkably abolished ZAP's activity. TRIM25 is required for ZAP optimal binding to target mRNA. These results help us to better understand how the antiviral activity of ZAP is regulated.