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Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell-specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells. © 2014 Nature America, Inc. All rights reserved.

Background: Gut microbiota dysbiosis significantly contributes to progression of depression. Hypericum perforatum L. (HPL) is traditionally used in Europe for treating depression. However, its mechanism remains largely underexplored., Purpose: This study aims to investigate the pivotal gut microbiota species and microbial signaling metabolites associated with the antidepressant effects of HPL., Methods: Fecal microbiota transplantation was used to assess whether HPL mitigates depression through alterations in gut microbiota. Microbiota and metabolic profiling of control, chronic restraint stress (CRS)-induced depression, and HPL-treated CRS mice were examined using 16S rRNA gene sequencing and metabolomics analysis. The influence of gut microbiota on HPL's antidepressant effects was assessed by metabolite and bacterial intervention experiments., Results: HPL significantly alleviated depression symptoms in a manner dependent on gut microbiota and restored gut microbial composition by enriching Akkermansia muciniphila (AKK). Metabolomic analysis indicated that HPL regulated tryptophan metabolism, reducing kynurenine (KYN) levels derived from microbiota and increasing 5-hydroxytryptophan (5-HTP) levels. Notably, supplementation with KYN activated the NFκB-NLRP2-Caspase1-IL1β pathway and increased proinflammatory IL1β in the hippocampus of mice with depression. Interestingly, mono-colonization with AKK notably increased 5-hydroxytryptamine (5-HT) and decreased KYN levels, ameliorating depression symptoms through modulation of the NFκB-NLRP2-Caspase1-IL1β pathway., Conclusions: The promising therapeutic role of HPL in treating depression is primarily attributed to its regulation of the NFκB-NLRP2-Caspase1-IL1β pathway, specifically by targeting AKK and tryptophan metabolites., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Our previous study revealed that elevated C-C motif chemokine ligand 2 (CCL2) secretion by irradiated cancer cells recruited C-C motif chemokine receptor 2 (CCR2)-positive myeloid cells and polarized M2-type tumor-associated macrophages (TAMs), promoting lung metastasis in an established mouse model. This study investigated the impact of CCL2 and TAMs on adaptive immunity., Methods: We assessed the influence of CCL2 and TAMs on adaptive immunity through two ectopic allograft mouse models constructed with MB49 bladder cancer cells and Lewis lung carcinoma cells. Both models exhibited delayed primary tumor growth following radiation therapy (RT), but RT promoted the development of pulmonary metastases in C57BL/6 mice. Additionally, we employed a direct coculture system to investigate the interaction between macrophages and target cells in the context of adaptive immunity., Results: C-C motif chemokine receptor 4 (CCR4)-positive regulatory T cells (Tregs) were recruited to the postirradiated tumor microenvironment (TME). Utilizing a CCR4 antagonist to inhibit CCL2-CCR4 activation reversed the infiltration of CCR4 + Tregs and reduced the incidence of pulmonary metastases. In addition, a positive feedback loop between M2-type TAMs and Tregs was observed. The combined blockade of the CCL2-CCR4 and CCL2-CCR2 signaling pathways further decreased the risk of RT-promoted lung metastasis., Conclusion: The recruitment of CCR4 + Tregs to the postirradiated TME increases the metastatic potential of tumor cells through increased interactions with M2-type TAMs. A significant reduction in post-RT lung metastases in ectopic mouse models was achieved by disrupting the recruitment of both CCR4 + Tregs and CCR2 + myeloid cells, which are TAM precursors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Type I interferons (IFNs) exert a broad range of biological effects important in coordinating immune responses, which have classically been studied in the context of pathogen clearance. Yet, whether immunomodulatory bacteria operate through IFN pathways to support intestinal immune tolerance remains elusive. Here, we reveal that the commensal bacterium, Bacteroides fragilis, utilizes canonical antiviral pathways to modulate intestinal dendritic cells (DCs) and regulatory T cell (Treg) responses. Specifically, IFN signaling is required for commensal-induced tolerance as IFNAR1-deficient DCs display blunted IL-10 and IL-27 production in response to B. fragilis. We further establish that IFN-driven IL-27 in DCs is critical in shaping the ensuing Foxp3+ Treg via IL-27Rα signaling. Consistent with these findings, single-cell RNA sequencing of gut Tregs demonstrated that colonization with B. fragilis promotes a distinct IFN gene signature in Foxp3+ Tregs during intestinal inflammation. Altogether, our findings demonstrate a critical role of commensal-mediated immune tolerance via tonic type I IFN signaling., (© 2023 Vasquez Ayala et al.)

Regulatory T cells (T reg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which T reg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying T reg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal T reg cells to regulate helper T17 cell (T H 17 cell) immunity. Selectively increased intestinal T H 17 cell responses in mice with T reg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83 + CD62L lo T reg cell subset that is distinct from previously characterized intestinal T reg cell populations as the main IL-27 producers. Collectively, our study uncovers a new T reg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific T reg cell-mediated immune regulation., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

Effector regulatory T cells (eTregs) exhibit distinct homeostatic properties and superior suppressor capacities pivotal for controlling immune responses mediated by their conventional T cell counterpart. While the role of microRNAs (miRNAs) in Tregs has been well-established, how miRNAs regulate eTregs remains poorly understood. Here, we demonstrate that miR-15/16 clusters act as key regulators in limiting eTreg responses. Loss of miR-15/16 clusters leads to increased eTreg frequencies with enhanced suppressor function. Consequently, mice with Treg-specific ablation of miR-15/16 clusters display attenuated immune responses during neuroinflammation and upon both infectious and non-infectious challenges. Mechanistically, miR-15/16 clusters exert their regulatory effect in part through repressing IRF4, a transcription factor essential for eTreg differentiation and function. Moreover, miR-15/16 clusters also directly target neuritin, an IRF4-dependent molecule, known for its role in Treg-mediated regulation of plasma cell responses. Together, we identify an miRNA family that controls an important Treg subset and further demonstrate that eTreg responses are tightly regulated at both transcriptional and posttranscriptional levels., (© 2023 Dong et al.)

Peripheral nerve injury is one of the more common forms of peripheral nerve disorders, and the most severe type of peripheral nerve injury is a defect with a gap. Biosynthetic cellulose membrane (BCM) is a commonly used material for repair and ligation of nerve defects with gaps. Meanwhile, exosomes from mesenchymal stem cells can promote cell growth and proliferation. We envision combining exosomes with BCMs to leverage the advantages of both to promote repair of peripheral nerve injury. Prepared exosomes were added to BCMs to form exosome-loaded BCMs (EXO-BCM) that were used for nerve repair in a rat model of sciatic nerve defects with gaps. We evaluated the repair activity using a pawprint experiment, measurement and statistical analyses of sciatica function index and thermal latency of paw withdrawal, and quantitation of the number and diameter of regenerated nerve fibers. Results indicated that EXO-BCM produced comprehensive and durable repair of peripheral nerve defects that were similar to those for autologous nerve transplantation, the gold standard for nerve defect repair. EXO-BCM is not predicted to cause donor site morbidity to the patient, in contrast to autologous nerve transplantation. Together these results indicate that an approach using EXO-BCM represents a promising alternative to autologous nerve transplantation, and could have broad applications for repair of nerve defects., (© 2023 The Authors.)

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo , map2k7 +/- ( map2k7 -/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7 +/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates., Competing Interests: The authors declare no conflict of interest.

Viral co-infection has been found in animals; however, the mechanisms of co-infection are unclear. The abundance and diversity of viruses in water make fish highly susceptible to co-infection. Here, we reported a co-infection in fish, which resulted in reduced host lethality and illustrated the intracellular molecular mechanism of viral co-infection. The spring viremia of carp virus (SVCV) is a highly lethal virus that infects Cyprinidae, such as zebrafish. The mortality of SVCV infection was significantly reduced when co-infected with the grass carp reovirus (GCRV). The severity of tissue damage and viral proliferation of SVCV was also reduced in co-infection with GCRV. The transcriptome bioinformatics analysis demonstrated that the effect on the host transcripts in response to SVCV infection was significantly reduced in co-infection. After excluding the extracellular interactions of these two viruses, the intracellular mechanisms were studied. We found that the GCRV NS38 remarkably decreased SVCV infection and viral proliferation. The interaction between GCRV NS38 and SVCV nucleoprotein (N) and phosphoprotein (P) proteins was identified, and NS38 downregulated both N and P proteins. Further analysis demonstrated that the N protein was degraded by NS38 indispensable of the autophagy receptor, sequestosome 1 (p62). Meanwhile, K63-linked ubiquitination of the P protein was reduced by NS38, leading to ubiquitinated degradation of the P protein. These results reveal that the intracellular viral protein interactions are a crucial mechanism of co-infection and influence the host pathology and expand our understanding in intracellular viral interactions co-infection., Competing Interests: Conflict of interest The authors have no financial conflicts of interest., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Fish possess a powerful IFN system to defend against aquatic virus infections. Nevertheless, spring viremia of carp virus (SVCV) causes large-scale mortality in common carp and significant economic losses to aquaculture. Therefore, it is necessary to investigate the strategies used by SVCV to escape the IFN response. In this study, we show that the SVCV nucleoprotein (N protein) negatively regulates cellular IFN production by degrading stimulator of IFN genes (STING) via the autophagy-lysosome-dependent pathway. First, overexpression of N protein inhibited the IFN promoter activation induced by polyinosinic-polycytidylic acid and STING. Second, the N protein associated with STING and experiments using a dominant-negative STING mutant demonstrated that the N-terminal transmembrane domains of STING were indispensable for this interaction. Then, the N protein degraded STING in a dose-dependent and autophagy-lysosome-dependent manner. Intriguingly, in the absence of STING, individual N proteins could not elicit host autophagic flow. Furthermore, the autophagy factor Beclin1 was found to interact with the N protein to attenuate N protein-mediated STING degradation after beclin1 knockdown. Finally, the N protein remarkably weakened STING-enhanced cellular antiviral responses. These findings reveal that SVCV uses the host autophagic process to achieve immune escape, thus broadening our understanding of aquatic virus pathogenesis., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Gut dysbiosis has long been associated with the development of Crohn's disease and other gastrointestinal disorders. Otake-Kasamoto et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20211291) report that dysbiotic microbiota-derived bioactive lipids, lysophosphatidylserines, can promote pathological Th1 cell responses through inducing metabolic reprogramming and epigenetic changes., (© 2022 Lin and Lu.)

Mammalian cyclic GMP-AMP synthase (cGAS) is pivotal for cytosolic DNA-triggered interferon (IFN) response. However, the function of cGAS in fish IFN response remains unclear. Our recent study has reported that cGAS from crucian and grass carps downregulates the IFN response by attenuating the K63-linked ubiquitination of retinoic acid-inducible gene-I (RIG-I) and its interaction with mitochondrial antiviral signaling protein (MAVS). Here, the function of crucian carp cGAS was further investigated. We found that crucian carp cGAS directly binds to poly deoxyadenylic-deoxythymidylic acid (poly (dA:dT)) and exhibits mediator of IFN regulatory factor 3 (IRF3) activation (MITA)-dependent activation of the IFN response, indicating a conserved function of crucian carp cGAS in the MITA-mediated IFN signaling. However, crucian carp cGAS could suppress the IFN activation stimulated by polyinosinic: polycytidylic acid (poly (I:C)) in time- and dose-dependent manners. These data collectively suggest complicated functions of crucian carp cGAS in the IFN antiviral response., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

From insects to mammals, both innate and adaptive immune response are usually higher in females than in males, with the sex chromosome and hormonal differences considered the main reasons. Here, we report that zebrafish cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a), an autosomal gene with female-biased expression, causes female fish to exhibit a lower antiviral response. First, we successfully constructed an infection model by intraperitoneal injection of spring viremia of carp virus (SVCV) into zebrafish (Danio rerio) and Carassius auratus herpesvirus (CaHV) in gibel carp (Carassius gibelio). Specifically, female fish were more vulnerable to viral infection than males, accompanied by a significantly weaker interferon (IFN) expression. After screening several candidates, cyp19a1a, which was highly expressed in female fish tissues, was selected for further analysis. The IFN expression and antiviral response were significantly higher in cyp19a1a-/- than in cyp19a1a+/+. Further investigation of the molecular mechanism revealed that Cyp19a1a targets mediator of IRF3 activation (MITA) for autophagic degradation. Interestingly, in the absence of MITA, Cyp19a1a alone could not elicit an autophagic response. Furthermore, the autophagy factor ATG14 (autophagy-related 14) was found interacted with Cyp19a1a to either promote or attenuate Cyp19a1a-mediated MITA degradation by either being overexpressed or knocked down, respectively. At the cellular level, both the normal and MITA-enhanced cellular antiviral responses were diminished by Cyp19a1a. These findings demonstrated a sex difference in the antiviral response based on a regulation mechanism controlled by a female-biased gene besides sex chromosome and hormonal differences, supplying the current understanding of sex differences in fish., Competing Interests: The authors have declared that no competing interests exist.

In the viral infection process, host gene function is usually reported as either defending the host or assaulting the virus. In this study, we demonstrated that zebrafish ceramide kinase-like (CERKL) mediates protection against viral infection via two distinct mechanisms: stabilization of TANK-binding kinase 1 (TBK1) through impairing K48-linked ubiquitination and degradation of spring viremia of carp virus (SVCV) P protein by dampening K63-linked ubiquitination, resulting in an improvement of the host immune response and a decline in viral activity in epithelioma papulosum cyprini (EPC) cells. On SVCV infection, ifn φ 1 expression was increased or blunted by CERKL overexpression or knockdown, respectively. Subsequently, we found that CERKL localized in the cytoplasm, where it interacted with TBK1 and enhanced its stability by impeding the K48-linked polyubiquitination; meanwhile, the antiviral capacity of TBK1 was significantly potentiated by CERKL. In contrast, CERKL also interacted with and degraded SVCV P protein to disrupt its function in viral proliferation. Further mechanism analysis revealed K63-linked deubiquitination is the primary means of CERKL-mediated SVCV P protein degradation. Taken together, our study reveals a novel mechanism of fish defense against viral infection: the single gene cerkl is both a shield for the host and a spear against the virus, which strengthens resistance., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Since emerged in the late 1990s, cyprinid herpesvirus 3 (CyHV-3) has caused huge economic losses in common and koi carp culture worldwide. Accumulating evidences suggest that teleost fish microRNA (miRNA), a class of non-coding RNA of ∼22 nucleotides, can participate in many cellular processes, especially in host antiviral defenses. However, the roles of miRNAs in CyHV-3 infection are still unclear. Here, using high-throughput miRNA sequencing and quantitative real-time PCR (qRT-PCR) verification, we found that miR-155 was significantly upregulated in common carp brain (CCB) cells upon CyHV-3 infection. Overexpression of miR-155 effectively inhibited CyHV-3 replication in CCB cells and promoted type I interferon (IFN-I) expression. Further study revealed that miR-155 targeted the 3' untranslated region (UTR) of the mRNA of 5'AMP-activated protein kinase (AMPK), and that AMPK could interact with and degrade the mitochondrial antiviral signaling protein (MAVS), resulting in the reduction of interferon (IFN) expression. Collectively, our results show that miR-155, induced by CyHV-3 infection, exhibits anti-CyHV-3 activity via regulating AMPK-MAVS-IFN axis, which will help design anti-CyHV-3 drugs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A novel Gram-stain-positive, strictly anaerobic, elliptical, non-motile and non-flagellated bacterium, designed LZLJ-2 T , was isolated from the mud in a fermentation cellar used for the production of Chinese Luzhou-flavour Baijiu. Growth occurred at 28-45 °C (optimum, 37 °C), at pH 6.0-7.0 (optimum, pH 6.0) and with concentrations of NaCl up to 2 % (w/v; optimum, 0 %). On the basis of 16S rRNA gene sequence similarity, strain LZLJ-2 T belonged to the genus Thermophilibacter and was most closely related to Thermophilibacter mediterraneus Marseille-P3256 T (similarity 96.9 %), Olsenella gallinarum ClaCZ62 T (similarity 96.6 %) and Thermophilibacter provencensis Marseille-P2912 T (similarity 96.4 %). In addition, strain LZLJ-2 T had high similarity to the genus Olsenella , including Olsenella profusa DSM 13989 T (similarity 94.9 %), Olsenella umbonata DSM 22620 T (similarity 94.9 %), Olsenella uli ATCC 49627 T (similarity 94.22 %), Tractidigestivibacter scatoligenes DSM 28304 T (similarity 93.9 %) and Paratractidigestivibacter faecalis KCTC 15699 T (similarity 93.25 %). Comparative genome analysis showed that orthoANI values between strain LZLJ-2 T and Thermophilibacter mediterraneus Marseille-P3256 T , Olsenella gallinarum ClaCZ62 T , Thermophilibacter provencensis Marseille-P2912 T , Olsenella profusa DSM 13989 T , Olsenella umbonata DSM 22620 T , Olsenella uli ATCC 49627 T , Tractidigestivibacter scatoligenes DSM 28304 T and Paratractidigestivibacter faecalis KCTC 15699 T were 78.68, 78.99, 78.29, 73.40, 74.00, 74.30, 75.08 and 77.23 %, and the genome-to-genome distance values were respectively 22.3, 22.5, 22.4, 19.6, 20.5, 19.7, 20.5 and 21.5 %. The genomic DNA G+C content of strain LZLJ-2 T was 65.21 mol%. The predominant cellular fatty acids (>10 %) of strain LZLJ-2 T were C 18 : 1 cis 9 (33.7 %), C 14 : 0 (22.0 %) and C 18 : 1 cis 9 DMA (13.5 %). d-Glucose, sucrose, mannose, maltose, lactose (weak), salicin, glycerol (weak), cellobiose and trehalose (weak) could be used by strain LZLJ-2 T as sole carbon sources. Enzyme activity results showed positive reactions with valine arylamidase, leucine arylamidase, crystine arylamidase, acid phosphatase, alkaline phosphatase, esterase (C4) (weakly positive), naphthol-AS-BI-phosphohydrolase, α -glucosidase and β -glucosidase. The major end products of glucose fermentation were lactic acid and acetic acid. It produced skatole from indole acetic acid, and produced p -cresol from modified peptone-yeast extract medium with glucose. Based on the 16S rRNA gene trees as well as the genome core gene tree, it is suggested that Olsenella gallinarum are transferred to genus Thermophilibacter as Thermophilibacter gallinarum comb. nov. Based on phenotypic, genotypic and phylogenetic data, strain LZLJ-2 T is considered to represent a novel species of the genus Thermophilibacter , for which the name Thermophilibacter immobilis sp. nov. is proposed. The type strain is LZLJ-2 T (=KCTC 25162 T =JCM 34224 T ).

Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 ( ptpn6 ) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp ( Carassius gibelio ) ptpn6 homeologs ( Cgptpn6-A and Cgptpn6-B ), each of which had three alleles with high identities. Then, relative to Cgptpn6-B , dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that Cg SHP1-A (encoded by the Cgptpn6-A gene) and Cg SHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of Ca TBK1-induced phosphorylation of Ca MITA shared by Cg SHP1-A and Cg SHP1-B, and the autophagic degradation of Ca MITA exclusively by Cg SHP1-A. Meanwhile, the data support that Cg SHP1-A and Cg SHP1-B have sub-functionalized and that Cg SHP1-A overwhelmingly dominates Cg SHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tong, Zhou, Li, Lu, Li, Li, Gan, Mou, Zhang, Wang, Zhang, Wang and Gui.)

IL-27 controls a diverse range of immune responses in many disease settings. Here, we identify intestinal epithelial cells (IECs) as one of the major IL-27 cellular sources in the gut-associated tissue. Unlike IL-27 secreted by innate immune cells, gut epithelial IL-27 is dispensable for T-bet+ regulatory T cell (T reg cell) differentiation or IL-10 induction. Rather, IEC-derived IL-27 specifically promotes a distinct CD8αα+CD4+ intraepithelial lymphocyte (IEL) population that acquires their functional differentiation at the intestinal epithelium. Loss of IL-27 in IECs leads to a selective defect in CD8αα+CD4+ IELs over time. Consequently, mice with IEC-specific IL-27 ablation exhibited elevated pathogen burden during parasitic infection, and this could be rescued by transfer of exogenous CD8αα+CD4+ IELs. Collectively, our data reveal that in addition to its known regulatory properties in preventing immune hyperactivity, gut epithelial IL-27 confers barrier immunity by inducing a specific IEL subset and further suggest that IL-27 produced by different cell types plays distinct roles in maintaining intestinal homeostasis., Competing Interests: Disclosures: L.-F. Lu is a scientific advisor for Elixiron Immunotherapeutics. No other disclosures were reported., (© 2021 Lin et al.)

T cells are crucial to generate an effective response against numerous invading microbial pathogens and play a pivotal role in tumor surveillance and elimination. However, unwanted T cell activation can also lead to deleterious immune-mediated inflammation and tissue damage. To ensure that an optimal T cell response can be established, each step, beginning from T cell development in the thymus to their activation and function in the periphery, is tightly regulated by many transcription factors and epigenetic regulators including microRNAs (miRNAs). Here, we first summarize recent progress in identifying major immune regulatory miRNAs in controlling the differentiation and function of distinct T cell subsets. Moreover, as emerging evidence has demonstrated that miRNAs can impact T cell immunity through targeting both immune- and non-immune cell populations that T cells closely interact with, the T cell-extrinsic role of miRNAs in regulating different aspects of T cell biology is also addressed. Finally, we discuss the complex nature of miRNA-mediated control of T cell immunity and highlight important questions that remain to be further investigated., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3 T , was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3 T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3 T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1 T (91.7 %) and Blautia faecicola KGMB01111 T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3 T and B. stercoris GAM6-1 T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3 T and B. faecicola KGMB01111 T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3 T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3 T were C 16 : 0 FAME (27.9 %), C 14 : 0 FAME (17.6 %) and C 16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3 T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α -galactosidase, β -galactosidase, α -glucosidase and β -glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3 T is considered to represent a novel species of Blautia , for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3 T (=KCTC 25163 T =CGMCC 1.5299 T =JCM 34225 T ).

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs ( Cgviperin-A and Cgviperin-B ) in auto-allo-hexaploid gibel carp ( Carassius gibelio ). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both Cg Viperin-A and Cg Viperin-B were found to interact with crucian carp ( C. auratus ) herpesvirus ( Ca HV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, Cg Viperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in Cg Viperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of Cg Viperin-A and Cg Viperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of Cg Viperin-A and Cg Viperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mou, Li, Lu, Wang, Yu, Li, Tong, Zhang, Wang, Zhang, Wang, Zhou and Gui.)

Objective: To investigate the effect and safety of low-intensity pulsed ultrasound (LIPUS) versus low-energy shock wave (LESW) in the treatment of neurogenic penile ED in male SD rats., Methods: Twenty-four 12-week-old male SD rats were randomly divided into four groups of an equal number: sham operation, bilateral cavernous nerves injury (BCNI), LIPUS (300 mW /cm2, 3 times a week for 2 weeks), and LESW (300 strokes once, 3 times a week for 2 weeks). At 28 days after intervention, the erectile function of the rats was assessed by comparing the ratio of maximum intracavernous pressure to mean arterial pressure (ICPmax/MAP), and the histopathological changes in the corpus cavernosum of the penis were observed by immunohistochemistry, immunofluorescence staining, and Masson trichromatic staining., Results: After treatment, the LIPUS and LESW groups, compared with the BCNI group, showed significantly increased ICPmax/MAP ratio (0.56 ± 0.13 and 0.55 ± 0.10 versus 0.35 ± 0.14, P = 0.017 and P = 0.013), improved smooth muscle/collagen value (0.08 ± 0.01 and 0.08 ± 0.02 versus 0.06 ± 0.02, P = 0.017 and P = 0.019), and elevated proportion of smooth muscle to cavernosum (0.20 ± 0.05 and 0.21 ± 0.03 versus 0.15 ± 0.02, P = 0.046 and P = 0.020), with no statistically significant difference between the LIPUS and LESW groups. No obvious adverse reactions were observed in the LIPUS or LESW group., Conclusions: Both LIPUS and LESW can effectively improve penile erectile function and repair histopathological injury in the animal model of neurogenic ED.

In mammals, cyclic GMP-AMP synthase (cGAS) recognizes cytosolic dsDNA to induce the type I IFN response. However, the functional role of cGAS in the IFN response of fish remains unclear or controversial. In this study, we report that cGAS orthologs from crucian carp Carassius auratus ( Ca cGAS) and grass carp Ctenopharyngodon idellus ( Ci cGAS) target the dsRNA sensor retinoic acid-inducible gene I (RIG-I) for negative regulation of the IFN response. First, poly(deoxyadenylic-deoxythymidylic) acid-, polyinosinic-polycytidylic acid-, and spring viremia of carp virus-induced IFN responses were impaired by overexpression of Ca cGAS and Ci cGAS. Then, Ca cGAS and Ci cGAS interacted with Ci RIG-I and Ci MAVS and inhibited Ci RIG-I- and Ci MAVS-mediated IFN induction. Moreover, the K63-linked ubiquitination of Ci RIG-I and the interaction between Ci RIG-I and Ci MAVS were attenuated by Ca cGAS and Ci cGAS. Finally, Ca cGAS and Ci cGAS decreased Ci RIG-I-mediated the cellular antiviral response and facilitated viral replication. Taken together, data in this study identify Ca cGAS and Ci cGAS as negative regulators in RIG-I-like receptor signaling, which extends the current knowledge regarding the role of fish cGAS in the innate antiviral response., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Fish IFN regulatory factor 3 (IRF3) is a crucial transcription factor in the IFN activation signaling pathway, which leads to IFN production and a positive cycle. Unrestricted IFN expression results in hyperimmune responses and therefore, IFN must be tightly regulated. In the current study, we found that zebrafish Ub-activating enzyme (Uba1) negatively regulated IRF3 via the K-48 ubiquitin proteasome degradation of IRF3. First, ifn expression stimulated by spring viraemia of carp virus infection was blunted by the overexpression of Uba1 and enhanced by Uba1 knockdown. Afterward, we found that Uba1 was localized in the cytoplasm, where it interacted with and degraded IRF3. Functional domains analysis revealed that the C-terminal ubiquitin-fold domain was necessary for IRF3 degradation by Uba1 and the N-terminal DNA-binding domain of IRF3 was indispensable for the degradation by Uba1.The degradation of IRF3 was subsequently impaired by treatment with MG132, a ubiquitin proteasome inhibitor. Further mechanism analysis revealed that Uba1 induced the K48-linked Ub-proteasomal degradation of IRF3. Finally, the antiviral capacity of IRF3 was significantly attenuated by Uba1. Taken together, our study reveals that zebrafish Uba1 interacts with and activates the ubiquitinated degradation of IRF3, providing evidence of the IFN immune balance mechanism in fish., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Zhang, Li, Zhou, Jiang, Chen, Zhang and Li.)

The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process., Competing Interests: The authors have declared that no competing interests exist.

In mammals, cyclic GMP-AMP synthase (cGAS) is a crucial cytosolic DNA sensor responsible for activating the interferon (IFN) response. A cGAS-like (cGASL) gene was previously identified from grass carp Ctenopharyngodon idellus, which is evolutionarily closest to cGAS but not a true ortholog of cGAS. Here, we found that grass carp cGASL targets mitochondrial antiviral signaling protein (MAVS) for autophagic degradation to negatively regulate fish IFN response. Firstly, the transcriptional level of cellular cgasl was upregulated by poly I:C stimulation, and overexpression of cGASL significantly decreased poly I:C- and MAVS-induced promoter activities and transcriptional levels of IFN and IFN-stimulated genes (ISGs). In addition, cGASL associated with MAVS and prompted autophagic degradation of MAVS in a dose-dependent manner. Finally, overexpression of cGASL attenuated MAVS-mediated cellular antiviral response. These results collectively indicate that cGASL negatively regulates fish IFN response by triggering autophagic degradation of MAVS., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

During thymocyte development, medullary thymic epithelial cells (mTECs) provide appropriate instructive cues in the thymic microenvironment for not only negative selection but also the generation of regulatory T (T reg) cells. Here, we identify that miR-155, a microRNA whose expression in T reg cells has previously been shown to be crucial for their development and homeostasis, also contributes to thymic T reg (tT reg) cell differentiation by promoting mTEC maturation. Mechanistically, we show that RANKL stimulation induces expression of miR-155 to safeguard the thymic medulla through targeting multiple known and previously uncharacterized molecules within the TGFβ signaling pathway, which is recognized for its role in restricting the maturation and expansion of mTECs. Our work uncovers a miR-155-TGFβ axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tT reg cells and suggests that miR-155 ensures proper tT reg cell development in both cell-intrinsic and -extrinsic manners., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Dong et al.)

Background: Hirschsprung disease (HSCR) is a hereditary defect, which is characterized by the absence of enteric ganglia and is frequently concurrent with Hirschsprung-associated enterocolitis (HAEC). However, the pathogenesis for HSCR is complicated and remains unclear. Recent studies have shown that pro-inflammatory cytokines such as interleukin-11 (IL-11) are involved in the enteric nervous system's progress. It was found that IL-11 SNPs (rs8104023 and rs4252546) are associated with HSCR in the Korean population waiting for replication in an independent cohort. This study evaluated the relationship between IL-11 and the susceptibility of patients to HSCR by performing subphenotype interaction examination, HAEC pre-/post-surgical patient-only association analysis, and independence testing., Methods: In this study, a cohort consisting of children from Southern China, comprising 1470 cases and 1473 controls, was chosen to examine the relationship between two polymorphisms (rs8104023 and rs4252546 in IL-11) and susceptibility to HSCR by replication research, subphenotype association analysis, and independence testing., Results: The results showed that IL-11 gene polymorphisms (rs8104023 and rs4252546) are not associated with the risk of HSCR in the Chinese population. The results of both short-segment and long-segment (S-HSCR and L-HSCR) surgery (3.34 ≤ OR ≤ 4.05, 0.02 ≤ P ≤ 0.04) showed that single nucleotide polymorphisms (SNP) rs8104023 is associated with susceptibility to HAEC., Conclusions: This study explored the relationship between genetic polymorphisms and susceptibility to HAEC in HSCR subtypes for the first time. These findings should be replicated in a larger and multicentre study.

TANK-binding kinase 1 (TBK1) plays a vital role in activating interferon (IFN) production and positively regulating antiviral response in mammals. Research on more species of fish is necessary to clarify whether the function of fish TBK1 is conserved compared to that in mammals. Here, a cyprinid fish (Ancherythroculter nigrocauda) TBK1 (AnTBK1) was functionally identified and characterized. The full-length open reading frame (ORF) of AnTBK1 consists of 2184 nucleotides encoding 727 amino acids and contains a conserved Serine/Threonine protein kinase catalytic domain (S_TKc) in the N-terminal, similar to TBK1 in other species. The transcripts of AnTBK1 were found in all the tissues evaluated and the cellular distribution indicated that AnTBK1 was localized in the cytoplasm. In terms of functional identification, AnTBK1 induced a variety of IFN promoter activities as well as the expression of downstream IFN-stimulated genes (ISGs). In addition, AnTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3 (IRF3), exhibiting the canonical kinase activity of TBK1. Finally, AnTBK1 presented strong antiviral activity against spring viremia of carp virus (SVCV) infection. Taken together, our research on the features and functions of AnTBK1 demonstrated that AnTBK1 plays a central role in IFN induction against SVCV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Viruses typically target host RIG-I-like receptors (RLRs), a group of key factors involved in interferon (IFN) production, to enhance viral infection. To date, though immune evasion methods to contradict IFN production have been characterized for a series of terrestrial viruses, the strategies employed by fish viruses remain unclear. Here, we report that all grass carp reovirus (GCRV) proteins encoded by segments S1 to S11 suppress mitochondrial antiviral signaling protein (MAVS)-mediated IFN expression. First, the GCRV viral proteins blunted the MAVS-induced expression of IFN, and impair MAVS antiviral capacity significantly. Interestingly, subsequent co-immunoprecipitation experiments demonstrated that all GCRV viral proteins interacted with several RLR cascades, especially with TANK-binding kinase 1 (TBK1) which was the downstream factor of MAVS. To further illustrate the mechanisms of these interactions between GCRV viral proteins and host RLRs, two of the viral proteins, NS79 (S4) and VP3 (S3), were selected as representative proteins for two distinguished mechanisms. The obtained data demonstrated that NS79 was phosphorylated by gcTBK1, leading to the reduction of host substrate gcIRF3/7 phosphorylation. On the other hand, VP3 degraded gcMAVS and the degradation was significantly reversed by 3-MA. The biological effects of both NS79 and VP3 were consistently found to be related to the suppression of IFN expression and the promotion of viral evasion. Our findings shed light on the special evasion mechanism utilized by fish virus through IFN regulation, which might differ between fish and mammals., (Copyright © 2020 Lu, Li, Zhang, Zhou, Zhou, Jiang, Chen, Li and Zhang.)

In this study, we investigated the biophysical interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80. CTLA-4 is a key molecule in immunosuppression, and CD80 is a costimulatory receptor promoting T cell activation. We observed that after cell-cell contact was established between breast cancer cells and antigen presenting cells (APCs), CTLA-4 expressed on the breast cancer cells bind to CD80 expressed on the APCs, and underwent trans-endocytosis to deplete CD80. Force measurement and live cell imaging revealed that upon binding to CD80, forces generated by breast cancer cells and transmitted via CTLA-4 were sufficiently strong to displace CD80 from the surface of APCs to be internalized by breast cancer cells. We further demonstrated that because of the force-dependent trans-endocytosis of CD80, the capacity of APCs to activate T cells was significantly attenuated. Furthermore, inhibiting force generation in cancer cells would increase the T cell activating capacity of APCs. Our results provide a possible mechanism behind the immunosuppression commonly seen in breast cancer patients, and may lead to a new strategy to restore anti-tumor immunity by inhibiting pathways of force-generation., (Copyright © 2020 Elsevier B.V. All rights reserved.)