T-bet Regulates Ion Channels and Transporters and Induces Apoptosis in Intestinal Epithelial Cells.

T-bet, encoded by TBX21, is extensively expressed across various immune cell types, and orchestrates critical functions in their development, survival, and physiological activities. However, the role of T-bet in non-immune compartments, notably the epithelial cells, remains obscure. Herein, a Tet-O-T-bet transgenic mouse strain is generated for doxycycline-inducible T-bet expression in adult animals. Unexpectedly, ubiquitous T-bet overexpression causes acute diarrhea, intestinal damage, and rapid mortality. Cell-type-specific analyses reveal that T-bet-driven pathology is not attributable to its overexpression in CD4+ T cells or myeloid lineages. Instead, inducible T-bet overexpression in the intestinal epithelial cells is the critical determinant of the observed lethal phenotype. Mechanistically, T-bet overexpression modulates ion channel and transporter profiles in gut epithelial cells, triggering profound fluid secretion and subsequent lethal dehydration. Furthermore, ectopic T-bet expression enhances gut epithelial cell apoptosis and markedly suppresses colon cancer development in xenograft models. Collectively, the findings unveil a previously unrecognized role of T-bet in intestinal epithelial cells for inducing apoptosis, diarrhea, and local inflammation, thus implicating its potential as a therapeutic target for the treatment of cancer and inflammatory diseases.

ACE2-dependent and -independent SARS-CoV-2 entries dictate viral replication and inflammatory response during infection.

Excessive inflammation is the primary cause of mortality in patients with severe COVID-19, yet the underlying mechanisms remain poorly understood. Our study reveals that ACE2-dependent and -independent entries of SARS-CoV-2 in epithelial cells versus myeloid cells dictate viral replication and inflammatory responses. Mechanistically, SARS-CoV-2 NSP14 potently enhances NF-κB signalling by promoting IKK phosphorylation, while SARS-CoV-2 ORF6 exerts an opposing effect. In epithelial cells, ACE2-dependent SARS-CoV-2 entry enables viral replication, with translated ORF6 suppressing NF-κB signalling. In contrast, in myeloid cells, ACE2-independent entry blocks the translation of ORF6 and other viral structural proteins due to inefficient subgenomic RNA transcription, but NSP14 could be directly translated from genomic RNA, resulting in an abortive replication but hyperactivation of the NF-κB signalling pathway for proinflammatory cytokine production. Importantly, we identified TLR1 as a critical factor responsible for viral entry and subsequent inflammatory response through interaction with E and M proteins, which could be blocked by the small-molecule inhibitor Cu-CPT22. Collectively, our findings provide molecular insights into the mechanisms by which strong viral replication but scarce inflammatory response during the early (ACE2-dependent) infection stage, followed by low viral replication and potent inflammatory response in the late (ACE2-independent) infection stage, may contribute to COVID-19 progression.

USP3 plays a critical role in the induction of innate immune tolerance.

Microbial products, such as lipopolysaccharide (LPS), can elicit efficient innate immune responses against invading pathogens. However, priming with LPS can induce a form of innate immune memory, termed innate immune "tolerance", which blunts subsequent NF-κB signaling. Although epigenetic and transcriptional reprogramming has been shown to play a role in innate immune memory, the involvement of post-translational regulation remains unclear. Here, we report that ubiquitin-specific protease 3 (USP3) participates in establishing "tolerance" innate immune memory through non-transcriptional feedback. Upon NF-κB signaling activation, USP3 is stabilized and exits the nucleus. The cytoplasmic USP3 specifically removes the K63-linked polyubiquitin chains on MyD88, thus negatively regulating TLR/IL1ß-induced inflammatory signaling activation. Importantly, cytoplasmic translocation is a prerequisite step for USP3 to deubiquitinate MyD88. Additionally, LPS priming could induce cytoplasmic retention and faster and stronger cytoplasmic translocation of USP3, enabling it to quickly shut down NF-κB signaling upon the second LPS challenge. This work identifies a previously unrecognized post-translational feedback loop in the MyD88-USP3 axis, which is critical for inducing normal "tolerance" innate immune memory.

Corrigendum: PHF20 promotes glioblastoma cell malignancies through a WISP1/BGN-dependent pathway.

[This corrects the article DOI: 10.3389/fonc.2020.573318.].

Interaction between microbiota and immunity and its implication in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Besides genetic causes, colonic inflammation is one of the major risk factors for CRC development, which is synergistically regulated by multiple components, including innate and adaptive immune cells, cytokine signaling, and microbiota. The complex interaction between CRC and the gut microbiome has emerged as an important area of current CRC research. Metagenomic profiling has identified a number of prominent CRC-associated bacteria that are enriched in CRC patients, linking the microbiota composition to colitis and cancer development. Some microbiota species have been reported to promote colitis and CRC development in preclinical models, while a few others are identified as immune modulators to induce potent protective immunity against colitis and CRC. Mechanistically, microbiota regulates the activation of different immune cell populations, inflammation, and CRC via crosstalk between innate and adaptive immune signaling pathways, including nuclear factor kappa B (NF-κB), type I interferon, and inflammasome. In this review, we provide an overview of the potential interactions between gut microbiota and host immunity and how their crosstalk could synergistically regulate inflammation and CRC, thus highlighting the potential roles and mechanisms of gut microbiota in the development of microbiota-based therapies to prevent or alleviate colitis and CRC.

Activation of cGAS-STING by Lethal Malaria N67C Dictates Immunity and Mortality through Induction of CD11b+ Ly6Chi Proinflammatory Monocytes.

Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) play critical roles in the innate immunity against infectious diseases and are required to link pathogen DNA sensing to immune responses. However, the mechanisms by which cGAS-STING-induced cytokines suppress the adaptive immune response against malaria infections remain poorly understood. Here, cGAS-STING signaling is identified to play a detrimental role in regulating anti-malaria immunity. cGAS or STING deficiency in mice markedly prolongs mouse survival during lethal malaria Plasmodium yoelii nigeriensis N67C infections by reducing late interleukin (IL)-6 production. Mechanistically, cGAS/STING recruits myeloid differentiation factor 88 (MyD88) and specifically induces the p38-dependent signaling pathway for late IL-6 production, which, in turn, expands CD11b+ Ly6Chi proinflammatory monocytes to inhibit immunity. Moreover, the blockage or ablation of the cGAS-STING-MyD88-p38-IL-6 signaling axis or the depletion of CD11b+ Ly6Chi proinflammatory monocytes provides mice a significant survival benefit during N67C and other lethal malaria-strain infections. Taken together, these findings identify a previously unrecognized detrimental role of cGAS-STING-MyD88-p38 axis in infectious diseases through triggering the late IL-6 production and proinflammatory monocyte expansion and provide insight into how targeting the DNA sensing pathway, dysregulated cytokines, and proinflammatory monocytes enhances immunity against infection.

Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations.

The extent to which neurogenesis occurs in adult primates remains controversial. In this study, using an optimized single-cell RNA sequencing pipeline, we profiled 207,785 cells from the adult macaque hippocampus and identified 34 cell populations comprising all major hippocampal cell types. Analysis of their gene expression, specification trajectories and gene regulatory networks revealed the presence of all key neurogenic precursor cell populations, including a heterogeneous pool of radial glia-like cells (RGLs), intermediate progenitor cells (IPCs) and neuroblasts. We identified HMGB2 as a novel IPC marker. Comparison with mouse single-cell transcriptomic data revealed differences in neurogenic processes between species. We confirmed that neurogenesis is recapitulated in ex vivo neurosphere cultures from adult primates, further supporting the existence of neural precursor cells (NPCs) that are able to proliferate and differentiate. Our large-scale dataset provides a comprehensive adult neurogenesis atlas for primates.

Toll-Like Receptor Signaling and Its Role in Cell-Mediated Immunity.

Innate immunity is the first defense system against invading pathogens. Toll-like receptors (TLRs) are well-defined pattern recognition receptors responsible for pathogen recognition and induction of innate immune responses. Since their discovery, TLRs have revolutionized the field of immunology by filling the gap between the initial recognition of pathogens by innate immune cells and the activation of the adaptive immune response. TLRs critically link innate immunity to adaptive immunity by regulating the activation of antigen-presenting cells and key cytokines. Furthermore, recent studies also have shown that TLR signaling can directly regulate the T cell activation, growth, differentiation, development, and function under diverse physiological conditions. This review provides an overview of TLR signaling pathways and their regulators and discusses how TLR signaling, directly and indirectly, regulates cell-mediated immunity. In addition, we also discuss how TLR signaling is critically important in the host's defense against infectious diseases, autoimmune diseases, and cancer.

Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy.

BACKGROUND: The current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NY-ESO-1, a cancer-testis antigen, to develop a TCR-like fully human IgG1 antibody and its derivative, CAR-T cells, for cancer immunotherapy. METHODS: Human single-chain variable antibody fragment (scFv) phage library (~10∧11) was screened against HLA-A2/NY-ESO-1 (peptide 157-165) complex to obtain target-specific antibodies. The specificity and affinity of those antibodies were characterized by flow cytometry, ELISA, biolayer interferometry, and confocal imaging. The biological functions of CAR-T cells were evaluated against target tumor cells in vitro. In vivo antitumor activity was investigated in a triple-negative breast cancer (TNBC) model and primary melanoma tumor model in immunocompromised mice. RESULTS: Monoclonal antibody 2D2 identified from phage-displayed library specifically bound to NY-ESO-1157-165 in the context of human leukocyte antigen HLA-A*02:01 but not to non-A2 or NY-ESO-1 negative cells. The second-generation CAR-T cells engineered from 2D2 specifically recognized and eliminated A2+/NY-ESO-1+tumor cells in vitro, inhibited tumor growth, and prolonged the overall survival of mice in TNBC and primary melanoma tumor model in vivo. CONCLUSIONS: This study showed the specificity of the antibody identified from human scFv phage library and demonstrated the potential antitumor activity by TCR-like CAR-T cells both in vitro and in vivo, warranting further preclinical and clinical evaluation of the TCR-like antibody in patients. The generation of TCR-like antibody and its CAR-T cells provides the state-of-the-art platform and proof-of-concept validation to broaden the scope of target antigen recognition and sheds light on the development of novel therapeutics for cancer immunotherapy.

Microstructure Evolution and Properties Induced by Multi-Pass Drawing of Graphene/Copper Nanocomposite.

The influence of multi-pass cold drawing on the evolution of microstructure, texture, and properties of Cu matrix composite, reinforced by in situ grown graphene, has been systematically investigated. Under continuous and severe plastic deformation, the grains in the composite were continuously refined to nanoscale. In addition, graphene in the composite could be gradually refined, exfoliated, and redispersed. Interestingly, dynamic recrystallization of the composite was formed after 80% drawing reduction and its formation mechanism was discussed. The texture of the as-drawn composite comprised a mixture of fiber textures with dominated <111> and minor <100> orientation after 99.7% severe drawing reduction. The tensile properties and electrical conductivity of the as-drawn composites were also investigated. This work provides a better guideline on the plastic deformation behavior of the advanced graphene/metal nanocomposite.

BECN2 (beclin 2) Negatively Regulates Inflammasome Sensors Through ATG9A-Dependent but ATG16L1- and LC3-Independent Non-Canonical Autophagy.

Macroautophagy/autophagy-related proteins regulate infectious and inflammatory diseases in autophagy-dependent or -independent manner. However, the role of a newly identified mammalian-specific autophagy protein-BECN2 (beclin 2) in innate immune regulation is largely unknown. Here we showed that loss of BECN2 enhanced the activities of NLRP3, AIM2, NLRP1, and NLRC4 inflammasomes upon ligand stimulations. Mechanistically, BECN2 interacted with inflammasome sensors and mediated their degradation through a ULK1- and ATG9A-dependent, but BECN1-WIPI2-ATG16L1-LC3-independent, non-canonical autophagic pathway. BECN2 recruited inflammasome sensors on ATG9A+ vesicles to form a complex (BECN2-ATG9A-sensors) upon ULK1 activation. Three soluble NSF attachment protein receptor (SNARE) proteins (SEC22A, STX5, and STX6) were further shown to mediate the BECN2-ATG9A-dependent inflammasome sensor degradation. Loss of BECN2 promoted alum-induced peritonitis, which could be rescued by the ablation of CASP1 in Becn2-deficient mice. Hence, BECN2 negatively regulated inflammasome activation to control inflammation, serving as a potential therapeutic target for the treatment of infectious and inflammatory diseases.Abbreviations: AIM2: absent in melanoma 2; ATG: autophagy related; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cells; BMDM: bone marrow-derived macrophages; CASP1: caspase 1; CQ: chloroquine; gMDSC: granulocytic myeloid-derived suppressor cells; IL: interleukin; LPS: lipopolysaccharide; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; mMDSC: monocytic myeloid-derived suppressor cells; NLRC4: NLR family CARD domain containing 4; NLRP1: NLR family pyrin domain containing 1; NLRP3: NLR family pyrin domain containing 3; PECs: peritoneal exudate cells; PYCARD/ASC: apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; SNAREs: soluble NSF attachment protein receptors; STX5: syntaxin 5; STX6: syntaxin 6; ULK1: unc-51 like autophagy activating kinase 1; WIPI: WD repeat domain, phosphoinositide interacting.

Pharmacological inhibition of fatty acid synthesis blocks SARS-CoV-2 replication.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 is a virus-induced inflammatory disease of the airways and lungs that leads to severe multi-organ damage and death. Here we show that cellular lipid synthesis is required for SARS-CoV-2 replication and offers an opportunity for pharmacological intervention. Screening a short-hairpin RNA sublibrary that targets metabolic genes, we identified genes that either inhibit or promote SARS-CoV-2 viral infection, including two key candidate genes, ACACA and FASN, which operate in the same lipid synthesis pathway. We further screened and identified several potent inhibitors of fatty acid synthase (encoded by FASN), including the US Food and Drug Administration-approved anti-obesity drug orlistat, and found that it inhibits in vitro replication of SARS-CoV-2 variants, including more contagious new variants, such as Delta. In a mouse model of SARS-CoV-2 infection (K18-hACE2 transgenic mice), injections of orlistat resulted in lower SARS-CoV-2 viral levels in the lung, reduced lung pathology and increased mouse survival. Our findings identify fatty acid synthase inhibitors as drug candidates for the prevention and treatment of COVID-19 by inhibiting SARS-CoV-2 replication. Clinical trials are needed to evaluate the efficacy of repurposing fatty acid synthase inhibitors for severe COVID-19 in humans.

Microbiota regulate innate immune signaling and protective immunity against cancer.

Microbiota play critical roles in regulating colitis and colorectal cancer (CRC). However, it is unclear how the microbiota generate protective immunity against these disease states. Here, we find that loss of the innate and adaptive immune signaling molecule, TAK1, in myeloid cells (Tak1ΔM/ΔM) yields complete resistance to chemical-induced colitis and CRC through microbiome alterations that drive protective immunity. Tak1ΔM/ΔM mice exhibit altered microbiota that are critical for resistance, with antibiotic-mediated disruption ablating protection and Tak1ΔM/ΔM microbiota transfer conferring protection against colitis or CRC. The altered microbiota of Tak1ΔM/ΔM mice promote IL-1ß and IL-6 signaling pathways, which are required for induction of protective intestinal Th17 cells and resistance. Specifically, Odoribacter splanchnicus is abundant in Tak1ΔM/ΔM mice and sufficient to induce intestinal Th17 cell development and confer resistance against colitis and CRC in wild-type mice. These findings identify specific microbiota strains and immune mechanisms that protect against colitis and CRC.

Clinicopathological and prognostic significance of osteopontin expression in patients with prostate cancer: a systematic review and meta-analysis.

BACKGROUND: Evaluation of the feasibility for osteopontin (OPN) to serve as a biomarker in the prognosis and clinical-pathological features of prostate cancer (PCA) patients. METHODS: The original publications related to OPN and PCA were comprehensively searched in the online databases, including PubMed, Embase, Cochrane Library, Web of Science, Medline, Wanfang and China National Knowledge Infrastructure up to August 2019. Results were analyzed by Revman 5.3 and Stata 12.0. RESULTS: A total of 21 studies were included in the analysis and the result showed that the positive OPN expression group had a lower overall survival than the negative expression group (univariate: hazards ratio (HR) = 2.32, 95% confidence interval (95% CI) [1.74, 3.10], multivariate: HR = 2.41, 95% CI [1.63, 3.57]) and a lower biochemical relapse-free survival than the negative group (univariate: HR = 1.42, 95% CI [0.92, 2.17], multivariate: HR = 1.61, 95% CI [1.39, 1.87]). In addition, there was a higher expression level of OPN in PCA tissues than in normal prostate tissues (OR = 46.55, 95% CI [12.85, 168.59], P<0.00001) and benign prostatic hyperplasia (BPH) tissues (OR = 11.07, 95% CI [3.43, 35.75], P<0.0001). Moreover, OPN positive expression was also related to high Gleason score (OR = 2.64, 95% CI [1.49, 4.70], P=0.0009), high TNM stage (OR = 3.15, 95% CI [1.60, 6.20, P=0.0009), high Whitmore-Jewett stage (OR = 2.53, 95% CI [1.06, 6.03], P=0.04), high lymph node (OR = 3.69, 95% CI [1.88, 7.23], P=0.0001), and distant metastasis (OR = 8.10, 95% CI [2.94, 22.35], P=0.01). There was no difference observed in the differentiation of PCA (OR = 1.79, 95% CI [0.39, 8.33], P=0.46). CONCLUSION: OPN could be recognized as a promising diagnostic and prognostic biomarker for PCA patients.

PHF20 Promotes Glioblastoma Cell Malignancies Through a WISP1/BGN-Dependent Pathway.

Glioblastoma (GBM) stem cells are resistant to cancer therapy, and therefore responsible for tumor progression and recurrence after conventional therapy. However, the molecular mechanisms driving the maintenance of stemness and dedifferentiation are poorly understood. In this study, we identified plant homeodomain finger-containing protein 20 (PHF20) as a crucial epigenetic regulator for sustaining the stem cell-like phenotype of GBM. It is highly expressed in GBM and tightly associated with high levels of aggressiveness of tumors and potential poor prognosis in GBM patients. Knockout of PHF20 inhibits GBM cell proliferation, as well as its invasiveness and stem cell-like traits. Mechanistically, PHF20 interacts with WDR5 and binds to the promoter regions of WISP1 for its expression. Subsequently, WISP1 and BGN act in concert to regulate the degradation of ß-Catenin. Our findings have identified PHF20 as a key driver of GBM malignant behaviors, and provided a potential target for developing prognosis and therapy.

BECN2 (beclin 2)-mediated non-canonical autophagy in innate immune signaling and tumor development.

BECN2 (beclin 2) is a newly identified mammalian-specific macroautophagy/autophagy family member, and plays a critical role in the control of obesity and insulin sensitivity. However, its role in innate immune signaling and inflammation remains elusive. In our recent study, we show that BECN2 functions as a negative regulator in innate immune signaling and tumor development through non-canonical autophagy. Loss of Becn2 causes splenomegaly, lymphadenopathy, elevated proinflammatory cytokine production and spontaneous lymphoma development in mice. Mechanistically, BECN2 mediates the degradation of MAP3K7/TAK1 and MAP3K3/MEKK3 through an ATG9A- and ULK1-dependent but ATG16L1-BECN1-MAP1LC3B/LC3B-independent autophagy pathway to control systemic inflammation. BECN2 interacts with MAP3K7 and MAP3K3 through the engagement of ATG9A+ vesicles upon ULK1 activation, and promotes the fusion of MAP3K3- or MAP3K7-associated ATG9A+ vesicles with phagophores for subsequent degradation. Our findings have identified a previously unrecognized role of BECN2 in innate immune signaling and tumor development through non-canonical autophagy, thus providing a potential target for inflammatory disease and cancer therapy.

Beclin 2 negatively regulates innate immune signaling and tumor development.

Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.

The risk of rheumatoid arthritis among patients with inflammatory bowel disease: a systematic review and meta-analysis.

BACKGROUND: Studies have suggested that patients with inflammatory bowel disease (IBD) have an increased risk of rheumatoid arthritis (RA). However, the available data on this association are inconsistent. This meta-analysis aimed to determine the association between IBD and the risk of RA. METHODS: Observational studies investigating the RA risk among patients with IBD (Crohn disease (CD) and/or ulcerative colitis (UC)) were searched in PubMed, Embase, and Web of Science from the date of inception to December 2019. The methodological quality of the included studies was assessed using the Newcastle-Ottawa Scale. Relative risks (RRs) and corresponding 95% confidential intervals (CIs) were pooled with a random-effects model. Heterogeneity was evaluated using I2 statistics while publication bias was determined using Begg's and Egger's tests. Subgroup and sensitivity analyses were performed. RESULTS: A total of three cohort studies, three cross-sectional studies, and two case-control studies were included in the meta-analyses. Compared to the non-IBD control or general population, there was a significantly higher risk of RA among patients with IBD (RR = 2.59; 95% CI: 1.93-3.48). Moreover, both CD (RR = 3.14; 95% CI: 2.46-4.01) and UC (RR = 2.29; 95% CI: 1.76-2.97) were associated with a significantly increased risk of RA. However, heterogeneity was substantial across studies and the subgroup analyses failed to identify the potential source of heterogeneity. CONCLUSIONS: Patients with IBD have a greater risk of developing RA. Rheumatologists should be consulted when patients with IBD present with undifferentiated joint complaints. However, more prospective cohort studies are needed to validate these results.