Eosinophils promote CD8+ T cell memory generation to potentiate anti-bacterial immunity.

Memory CD8+ T cell generation is crucial for pathogen elimination and effective vaccination against infection. The cellular and molecular circuitry that underlies the generation of memory CD8+ T cells remains elusive. Eosinophils can modulate inflammatory allergic responses and interact with lymphocytes to regulate their functions in immune defense. Here we report that eosinophils are required for the generation of memory CD8+ T cells by inhibiting CD8+ T cell apoptosis. Eosinophil-deficient mice display significantly impaired memory CD8+ T cell response and weakened resistance against Listeria monocytogenes (L.m.) infection. Mechanistically, eosinophils secrete interleukin-4 (IL-4) to inhibit JNK/Caspase-3 dependent apoptosis of CD8+ T cells upon L.m. infection in vitro. Furthermore, active eosinophils are recruited into the spleen and secrete more IL-4 to suppress CD8+ T cell apoptosis during early stage of L.m. infection in vivo. Adoptive transfer of wild-type (WT) eosinophils but not IL-4-deficient eosinophils into eosinophil-deficient mice could rescue the impaired CD8+ T cell memory responses. Together, our findings suggest that eosinophil-derived IL-4 promotes the generation of CD8+ T cell memory and enhances immune defense against L.m. infection. Our study reveals a new adjuvant role of eosinophils in memory T cell generation and provides clues for enhancing the vaccine potency via targeting eosinophils and related cytokines.

EFHD2 suppresses intestinal inflammation by blocking intestinal epithelial cell TNFR1 internalization and cell death.

TNF acts as one pathogenic driver for inducing intestinal epithelial cell (IEC) death and substantial intestinal inflammation. How the IEC death is regulated to physiologically prevent intestinal inflammation needs further investigation. Here, we report that EF-hand domain-containing protein D2 (EFHD2), highly expressed in normal intestine tissues but decreased in intestinal biopsy samples of ulcerative colitis patients, protects intestinal epithelium from TNF-induced IEC apoptosis. EFHD2 inhibits TNF-induced apoptosis in primary IECs and intestinal organoids (enteroids). Mice deficient of Efhd2 in IECs exhibit excessive IEC death and exacerbated experimental colitis. Mechanistically, EFHD2 interacts with Cofilin and suppresses Cofilin phosphorylation, thus blocking TNF receptor I (TNFR1) internalization to inhibit IEC apoptosis and consequently protecting intestine from inflammation. Our findings deepen the understanding of EFHD2 as the key regulator of membrane receptor trafficking, providing insight into death receptor signals and autoinflammatory diseases.

Nuclear RPSA senses viral nucleic acids to promote the innate inflammatory response.

Innate sensors initiate the production of type I interferons (IFN-I) and proinflammatory cytokines to protect host from viral infection. Several innate nuclear sensors that mainly induce IFN-I production have been identified. Whether there exist innate nuclear sensors that mainly induce proinflammatory cytokine production remains to be determined. By functional screening, we identify 40 S ribosomal protein SA (RPSA) as a nuclear protein that recognizes viral nucleic acids and predominantly promotes proinflammatory cytokine gene expression in antiviral innate immunity. Myeloid-specific Rpsa-deficient mice exhibit less innate inflammatory response against infection with Herpes simplex virus-1 (HSV-1) and Influenza A virus (IAV), the viruses replicating in nucleus. Mechanistically, nucleus-localized RPSA is phosphorylated at Tyr204 upon infection, then recruits ISWI complex catalytic subunit SMARCA5 to increase chromatin accessibility of NF-κB to target gene promotors without affecting innate signaling. Our results add mechanistic insights to an intra-nuclear way of initiating proinflammatory cytokine expression in antiviral innate defense.

Lysine methyltransferase SMYD2 inhibits antiviral innate immunity by promoting IRF3 dephosphorylation.

Phosphorylation of IRF3 is critical to induce type I interferon (IFN-I) production in antiviral innate response. Here we report that lysine methyltransferase SMYD2 inhibits the expressions of IFN-I and proinflammatory cytokines in macrophages upon viral infections. The Smyd2-deficient mice are more resistant to viral infection by producing more IFN-I and proinflammatory cytokines. Mechanistically, SMYD2 inhibits IRF3 phosphorylation in macrophages in response to viral infection independent of its methyltransferase activity. We found that SMYD2 interacts with the DNA-binding domain (DBD) and IRF association domain (IAD) domains of IRF3 by its insertion SET domain (SETi) and could recruit phosphatase PP1α to enhance its interaction with IRF3, which leads to decreased phosphorylation of IRF3 in the antiviral innate response. Our study identifies SMYD2 as a negative regulator of IFN-I production against virus infection. The new way of regulating IRF3 phosphorylation will provide insight into the understanding of IFN-I production in the innate response and possible intervention of the related immune disorders.

Enzymolysis-based RNA pull-down identifies YTHDC2 as an inhibitor of antiviral innate response.

The innate immune response must be terminated in a timely manner at the late stage of infection to prevent unwanted inflammation. The role of m6A-modified RNAs and their binding partners in this process is not well known. Here, we develop an enzymolysis-based RNA pull-down (eRP) method that utilizes the immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) to fish out m6A-modified RNA-associated proteins. We apply eRP to capture the methylated single-stranded RNA (ssRNA) probe-associated proteins and identify YT521-B homology domain-containing 2 (YTHDC2) as the m6A-modified interferon ß (IFN-ß) mRNA-binding protein. YTHDC2, induced in macrophages at the late stage of virus infection, recruits IFN-stimulated exonuclease ISG20 (IFN-stimulated exonuclease gene 20) to degrade IFN-ß mRNA, consequently inhibiting antiviral innate immune response. In vitro and in vivo deficiency of YTHDC2 increases IFN-ß production at the late stage of viral infection. Our findings establish an eRP method to effectively identify RNA-protein interactions and add mechanistic insight to the termination of innate response for maintaining homeostasis.

MFSD2A potentiates gastric cancer response to anti-PD-1 immunotherapy by reprogramming the tumor microenvironment to activate T cell response.

BACKGROUND: The efficacy of anti-programmed cell death protein 1 (PD-1) immunotherapy in various cancers, including gastric cancer (GC), needs to be potentiated by more effective targeting to enhance therapeutic efficacy or identifying accurate biomarkers to predict clinical responses. Here, we attempted to identify molecules predicting or/and promoting anti-PD-1 therapeutic response in advanced GC (AGC). METHODS: The transcriptome of AGC tissues from patients with different clinical responses to anti-PD-1 immunotherapy and GC cells was analyzed by RNA sequencing. The protein and mRNA levels of the major facilitator superfamily domain containing 2A (MFSD2A) in GC cells were assessed via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Additionally, the regulation of anti-PD-1 response by MFSD2A was studied in tumor-bearing mice. Cytometry by Time-of-Flight, multiple immunohistochemistry, and flow cytometry assays were used to explore immunological responses. The effects of MFSD2A on lipid metabolism in mice cancer tissue and GC cells was detected by metabolomics. RESULTS: Higher expression of MFSD2A in tumor tissues of AGC patients was associated with better response to anti-PD-1 immunotherapy. Moreover, MFSD2A expression was lower in GC tissues compared to adjacent normal tissues, and its expression was inversely correlated with GC stage. The overexpression of MFSD2A in GC cells enhanced the efficacy of anti-PD-1 immunotherapy in vivo by reprogramming the tumor microenvironment (TME), characterized by increased CD8+ T cell activation and reduced its exhaustion. MFSD2A inhibited transforming growth factor ß1 (TGFß1) release from GC cells by suppressing cyclooxygenase 2 (COX2)-prostaglandin synthesis, which consequently reprogrammed TME to promote anti-tumor T cell activation. CONCLUSIONS: MFSD2A potentially serves as a predictive biomarker for anti-PD-1 immunotherapy response in AGC patients. MFSD2A may be a promising therapeutic target to potentiate the efficacy of anti-PD-1 immunotherapy by reprogramming the TME to promote T cells activation.

Iron Overload Causes Ferroptosis But Not Apoptosis in MO3.13 Oligodendrocytes.

Oligodendrocytes are the most iron-rich cells in the brain. Studies have shown that oligodendrocytes are very sensitive to oxidative stress, and iron overload is more likely to cause damage to oligodendrocytes. The purpose of this experiment was to investigate the damaging effect and mechanism of ferric ammonium citrate (FAC) on MO3.13 oligodendrocytes. In FAC treatment group, the intracellular iron concentration and intracellular reactive oxygen species were increased. There were no obvious changes in nucleus and chromatin, but increased mitochondrial membrane density, decreased mitochondrial cristae and mitochondrial length were observed. Glutathione peroxidase 4 (GPX4) expression was decreased, but the ratio of Bcl-2/Bax protein levels and cleaved caspase-3 expression did not change. Moreover, the iron chelator deferoxamine (DFO) and the ferroptosis inhibitor ferrostatin-1(Fer-1) could inhibit the upregulation of GPX4, which indicating that DFO and Fer-1 could inhibit ferroptosis in MO3.13 oligodendrocytes induced by iron overload. Furthermore, the phosphorylation level of p53 was not changed, while the ratio of protein expressions of p-Erk1/2/Erk1/2 were markedly increased. Taken together, our data suggest that iron overload induces ferroptosis but not apoptosis in oligodendrocytes. The mechanism may be related to mitogen-activated protein kinase pathway activation rather than p53 pathway activation.

Degradation of HDAC10 by autophagy promotes IRF3-mediated antiviral innate immune responses.

Histone deacetylases (HDACs) play important roles in immunity and inflammation. Through functional screening, we identified HDAC10 as an inhibitor of the type I interferon (IFN) response mediated by interferon regulatory factor 3 (IRF3). HDAC10 abundance was decreased in mouse macrophages in response to innate immune stimuli and was reduced in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) compared with that in PBMCs from healthy donors. Deficiency in HDAC10 in mouse embryonic fibroblasts and in mice promoted the expression of genes encoding type I IFNs and of IFN-stimulated genes (ISGs), leading to enhanced antiviral responses in vitro and in vivo. HDAC10 bound in a deacetylase-independent manner to IRF3 in uninfected cells to inhibit the phosphorylation of IRF3 at Ser396 by TANK-binding kinase 1 (TBK1). Upon viral infection, HDAC10 was targeted for autophagy-mediated degradation through its interaction with LC3-II. Consequently, IRF3 phosphorylation was increased, which resulted in enhanced type I IFN production and antiviral responses. Our findings identify a potential target for improving host defense responses against pathogen infection and for treating autoimmune disease.

RNA-binding protein ZCCHC4 promotes human cancer chemoresistance by disrupting DNA-damage-induced apoptosis.

RNA-binding proteins (RBPs) play important roles in cancer development and treatment. However, the tumor-promoting RBPs and their partners, which may potentially serve as the cancer therapeutic targets, need to be further identified. Here, we report that zinc finger CCHC domain-containing protein 4 (ZCCHC4) is of aberrantly high expression in multiple human cancer tissues and is associated with poor prognosis and chemoresistance in patients of hepatocellular carcinoma (HCC), pancreatic cancer and colon cancer. ZCCHC4 promotes chemoresistance of HCC cells to DNA-damage agent (DDA) both in vitro and in vivo. HCC cell deficiency of ZCCHC4 reduces tumor growth in vivo and intratumoral interference of ZCCHC4 expression obviously enhances the DDA-induced antitumor effect. Mechanistically, ZCCHC4 inhibits DNA-damage-induced apoptosis in HCC cells by interacting with a new long noncoding RNA (lncRNA) AL133467.2 to hamper its pro-apoptotic function. Also, ZCCHC4 blocks the interaction between AL133467.2 and γH2AX upon DDA treatment to inhibit apoptotic signaling and promote chemoresistance to DDAs. Knockout of ZCCHC4 promotes AL133467.2 and γH2AX interaction for enhancing chemosensitivity in HCC cells. Together, our study identifies ZCCHC4 as a new predictor of cancer poor prognosis and a potential target for improving chemotherapy effects, providing mechanistic insights to the roles of RBPs and their partners in cancer progression and chemoresistance.

6-Hydroxydopamine Induces Abnormal Iron Sequestration in BV2 Microglia by Activating Iron Regulatory Protein 1 and Inhibiting Hepcidin Release.

Disrupted iron homeostasis in the substantia nigra pars compacta (SNpc) is an important pathological mechanism in Parkinson's disease (PD). It is unclear what role microglia play in iron metabolism and selective iron deposition in the SNpc of PD brain. In this study, we observed that 6-hydroxydopamine (6-OHDA) induced the expression of divalent metal transporter-1 (DMT1) and iron influx in BV2 microglia cells, which might be associated with the upregulation of iron regulatory protein 1 (IRP1) expression. Moreover, we found that 6-OHDA had no significant effect on the expression of ferroportin 1 (FPN1) and iron efflux in BV2 microglial cells, which might be the combined action of IRP1 upregulation and reduced hepcidin levels. Furthermore, 6-OHDA treatment activated BV2 microglia and enhanced the release of pro-inflammatory cytokines. Interestingly, iron overloading suppressed IRP1 expression, thus downregulating DMT1 and upregulating FPN1 levels in these microglial cells. On the contrary, iron deficiency activated IRP1, leading to increased expression of DMT1 and decreased expression of FPN1-which indicates that activated IRP1 induces iron overloading in 6-OHDA-treated microglia, but not iron overloading modulates the expression of IRP1. Taken together, our data suggest that 6-OHDA can regulate the expression of DMT1 and FPN1 by activating IRP1 and inhibiting hepcidin release, thus leading to abnormal iron sequestration in microglia. In addition, 6-OHDA can activate microglia, which leads to increased release of pro-inflammatory factors that can further induce genome damage in dopaminergic neurons.

Regulated cell death: discovery, features and implications for neurodegenerative diseases.

Regulated cell death (RCD) is a ubiquitous process in living organisms that is essential for tissue homeostasis or to restore biological balance under stress. Over the decades, various forms of RCD have been reported and are increasingly being found to involve in human pathologies and clinical outcomes. We focus on five high-profile forms of RCD, including apoptosis, pyroptosis, autophagy-dependent cell death, necroptosis and ferroptosis. Cumulative evidence supports that not only they have different features and various pathways, but also there are extensive cross-talks between modes of cell death. As the understanding of RCD pathway in evolution, development, physiology and disease continues to improve. Here we review an updated classification of RCD on the discovery and features of processes. The prominent focus will be placed on key mechanisms of RCD and its critical role in neurodegenerative disease. Video abstract.

Chromatin remodeler ARID1A binds IRF3 to selectively induce antiviral interferon production in macrophages.

Transcription factor IRF3 is critical for the induction of antiviral type I interferon (IFN-I). The epigenetic regulation of IFN-I production in antiviral innate immunity needs to be further identified. Here, we reported that epigenetic remodeler ARID1A, a critical component of the mSWI/SNF complex, could bind IRF3 and then was recruited to the Ifn-I promoter by IRF3, thus selectively promoting IFN-I but not TNF-α, IL-6 production in macrophages upon viral infection. Myeloid cell-specific deficiency of Arid1a rendered mice more susceptible to viral infection, accompanied with less IFN-I production. Mechanistically, ARID1A facilitates chromatin accessibility of IRF3 at the Ifn-I promoters by interacting with histone methyltransferase NSD2, which methylates H3K4 and H3K36 of the promoter regions. Our findings demonstrated the new roles of ARID1A and NSD2 in innate immunity, providing insight into the crosstalks of chromatin remodeling, histone modification, and transcription factors in the epigenetic regulation of antiviral innate immunity.

Nuclear carbonic anhydrase 6B associates with PRMT5 to epigenetically promote IL-12 expression in innate response.

Interleukin-12 (IL-12) is critical for induction of protective immunity against intracellular bacterial infection. However, the mechanisms for efficient induction of IL-12 in innate response remain poorly understood. Here we report that the B type of carbonic anhydrase 6 (Car6-b, which encoded CA-VI B) is essential for host defense against Listeria monocytogenes (LM) infection by epigenetically promoting IL-12 expression independent of its carbonic anhydrase activity. Deficiency of Car6-b attenuated IL-12 production upon LM infection both in vitro and in vivo. Car6-/- mice were more susceptible to LM infection with less production of IL-12. Mechanistically, the nuclear localized CA-VI B selectively promotes IL-12 expression by interaction with protein arginine N-methyltransferase 5 (PRMT5), which reduces symmetric dimethylation of histone H3 arginine 8 modification (H3R8me2s) at Il12 promoters to facilitate chromatin accessibility, selectively enhancing c-Rel binding to the Il12b promoter. Our findings add insights to the epigenetic regulation of IL-12 induction in innate immunity.

Silencing Snail suppresses tumor cell proliferation and invasion by reversing epithelial-to-mesenchymal transition and arresting G2/M phase in non-small cell lung cancer.

Epithelial-to-mesenchymal transition (EMT) is essential for tumor invasion and metastasis. Snail has been proven to be a key regulator of EMT. Several studies have shown compelling evidence that Snail is also an important regulator of tumor growth and aggression; however, the role of Snail in the cell cycle has not been clarified. We decreased Snail expression by siRNA transfection and lentiviral­mediated RNAi, to explore the effect of silencing Snail on the tumorigenicity and migration of lung carcinoma (lung cancer) cells. The results showed that silencing Snail conferred significant anti-proliferative activity and inhibited cell migration, tumor growth and metastasis both in vitro and in vivo. To understand the mechanism of these effects, we further investigated correlations among Snail expression, EMT and cell cycle. Significantly, Snail knockdown reversed EMT processes in lung cancer cells. Furthermore, the cyclin-dependent kinase inhibitor P21 was upregulated after silencing Snail. P21 upregulation manifested its tumor suppressor effects and arrested cells in the G2/M phase, not the G1/S phase following Snail depletion in lung cancer cells. These data suggest that silencing Snail decreases the malignant behaviors of lung cancer cells by reversing EMT processes and causing cell cycle defects.

Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping.

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression.

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

Conversion of cadherin isoforms in cultured human gastric carcinoma cells.

AIM: To explore the expression of cadherin isoforms in cultured human gastric carcinoma cells and its regulation. METHODS: The expressions of cell adhesion molecules (including E-cadherin, N-cadherin, alpha-catenin, beta-catenin) and cadherin transcription factors including snail, slug and twist were determined by reverse transcriptase-polymerase chain reaction(RT-PCR), immunoblotting and immunofluorescence in SV40-immortalized human gastric cell line Ges-1 and human gastric cancer cell lines MGC-803, BGC-823 and SGC-7901. RESULTS: All cell lines expressed N-cadherin, but not E-cadherin. N-cadherin immunofluorescence was detected at cell membranous adherents junctions where co-localization with immunofluorescent staining of inner surface adhesion proteins alpha- and beta-catenins was observed. The transformed Ges-1 and gastric cancer cell lines all expressed transcription factors (snail, slug and twist) which inhibited the expression of E-cadherin and triggered epithelial-mesenchymal transformation. CONCLUSION: Cadherin isoforms can change from E-cadherin to N-cadherin in transformed human gastric cancer cells, which is associated with intracellular events of stomach carcinogenesis and high expression of corresponding transcription factors.