Pin Ling

Background: Dysregulation of the oxytocin (OXT) system is implicated in the pathophysiology of several neuropsychiatric and neurological disorders, particularly autism spectrum disorder (ASD). Our previous study using the Coiled-coil and C2 domain containing 1a ( Cc2d1a ) conditional knockout (cKO) mouse model of ASD showed that restoring OXT levels effectively ameliorates irritability-like behavior; however, the mechanisms by which loss of Cc2d1a in forebrain excitatory neurons leads to decreased OXT expression in the paraventricular nucleus of the hypothalamus (PVN) remain elusive. Methods: We used the bottle-brush test (BBT) to assess irritability-like behavior in wild-type (WT) and Cc2d1a cKO mice. Retrograde and anterograde trans-synaptic viral tracing were used to map associated neural circuits, and chemogenetic methods were employed to modify neuronal activity. Fiber photometry monitored OXT dynamics in situ, and whole-cell voltage-clamp recordings in ex vivo brain slices assessed synaptic transmission onto PVN OXT neurons. Results: We found that decreased OXT expression in both magnocellular and parvocellular PVN neurons is evident in adult male Cc2d1a cKO mice, without corresponding changes in mRNA levels. Chronic silencing of PVN OXT neurons during adolescence leads to fewer OXT-immunoreactive neurons and heightened irritability-like behavior in adult male WT mice. Using both retrograde and anterograde trans-synaptic viral tracing techniques, we identified that the prelimbic cortex (PrL) indirectly regulates PVN OXT neuronal activity. In male Cc2d1a cKO mice, there was a preferential decrease in excitatory synaptic transmission onto PVN OXT neurons. In vivo real-time measurements of OXT dynamics in the posteroventral medial amygdala revealed reduced OXT release during the BBT in male Cc2d1a cKO mice. Chronic chemogenetic silencing of the PrL-PVN pathway during adolescence reduces irritability-like behavior in adult male Cc2d1a cKO mice. Conclusions: Our study unveils how forebrain Cc2d1a loss reduces OXT expression in the PVN at both molecular and circuit levels, underscoring the critical role of the OXT system as a potential therapeutic target for managing irritability in individuals with ASD.

CC2D1A is a multidomain scaffold protein implicated in transcriptional regulation and autosomal recessive non-syndromic intellectual disability (NSID), yet its molecular mechanism is still poorly understood due to a lack of structural information. Here, we present the crystal structure of the human CC2D1A491–810 fragment, encompassing the fourth DM14 domain, a coiled-coil region, and a C-terminal C2 domain. These elements form a compact, integrated architecture, with the C2 domain mediating symmetric dimerization through conserved electrostatic interactions. In addition, a unique antiparallel β1–β10 sheet connects the coiled-coil and C2 domains, stabilizing the tertiary structure. Fluorescence polarization assays reveal micromolar DNA-binding affinity, likely mediated by the basic surface of the DM14 domain. Comparison with the Drosophila homolog Lgd highlights conserved topology with added structural features, offering insights into CC2D1A’s vertebrate-specific functions and NSID-related mutations.

Embryonic development and tumor genesis share numerous similarities, with OCT4 standing out as a pivotal transcription factor in embryonic development. Expression of OCT4 is associated with poor prognosis of lung adenocarcinoma. VEGF-correlated chemokine-1 (VCC-1), also known as C-X-C motif chemokine ligand 17 (CXCL17), has been suggested to play a role in promoting tumor angiogenesis and metastasis. In the present study, we show a positive correlation between OCT4 expression levels and tumor metastatic potential, where an increase in OCT4 expression parallels an upregulation of VCC-1 in lung cancer. This relationship was substantiated through DNA microarray analysis and further confirmed by tissue staining of clinical lung cancer samples, demonstrating a positive correlation between OCT4 and VCC-1 expression. In A549 and H1299 human lung cancer cells, modulations in OCT4 expression directly influenced VCC-1 levels, as evidenced by the reporter assay of the VCC-1 promoter, indicating the regulatory role of OCT4 in transactivating VCC-1 expression. Furthermore, enhanced VCC-1 expression in H1299 cells promoted transforming growth factor-β (TGF-β) secretion, contributing to lung cancer cell aggressiveness. Additionally, VCC-1 secretion by H1299 cells could attract THP-1 macrophages, further implicating its role in tumor progression. NOD/SCID mice inoculated with VCC-1-knockdown A549 lung cancer cells exhibited significantly smaller tumors than those inoculated with control cells. On the basis of these findings, we highlight the importance of the OCT4-VCC-1 axis in lung cancer progression. Our findings also provide therapeutic targets for lung cancer.

RIG-I–like receptors (RLRs) are cytosolic RNA sensors critical for antiviral immunity. RLR activation is regulated by polyubiquitination and oligomerization following RNA binding. Yet, little is known about how RLRs exploit subcellular organelles to facilitate their posttranslational modifications and activation. Endosomal adaptor TAPE regulates the endosomal TLR and cytosolic RLR pathways. The potential interplay between RIG-I signaling and endosomes has been explored. Here, we report that endosomes act as platforms for facilitating RIG-I polyubiquitination and complex formation. RIG-I was translocated onto endosomes to form signaling complexes upon activation. Ablation of endosomes impaired RIG-I signaling to type I IFN activation. TAPE mediates the interaction and polyubiquitination of RIG-I and TRIM25. TAPE-deficient myeloid cells were defective in type I IFN activation upon RNA ligand and virus challenges. Myeloid TAPE deficiency increased the susceptibility to RNA virus infection in vivo. Our work reveals endosomes as signaling platforms for RIG-I activation and antiviral immunity.

The imbalance of mucosal immunity in the lower gastrointestinal tract can lead to chronic inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis. IBD is a chronic inflammatory disorder that causes small and/or large intestines ulceration. According to previous studies, recombinant interleukin (IL)-10 protein and genetically modified bacteria secreting IL-10 ameliorate dextran sulfate sodium (DSS)-induced colitis in mice. IL-19 is a transcriptional activator of IL-10 and can alter the balance of T helper 1 (Th)1/Th2 cells in favor of Th2. In this study, we aimed to investigate whether the expression of the murine IL-19 gene carried by Salmonella choleraesuis (S. choleraesuis) could ameliorate murine IBD. Our results showed that the attenuated S. choleraesuis could carry and express the IL-19 gene-containing plasmid for IBD gene therapy by reducing the mortality and clinical signs in DSS-induced acute colitis mice as compared to the untreated ones. We also found that IL-10 expression was induced in IL-19-treated colitis mice and prevented inflammatory infiltrates and proinflammatory cytokine expression in these mice. We suggest that S. choleraesuis encoding IL-19 provides a new strategy for treating IBD in the future.

Enterovirus 71 (EV71) is an etiological agent of hand foot and mouth disease and can also cause neurological complications in young children. However, there are no approved drugs as of yet to treat EV71 infections. In this study, we conducted antiviral drug screening by using a Food and Drug Administration (FDA)-approved drug library. We identified five drugs that showed dose-dependent inhibition of viral replication. Sertraline was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index among the five hits. The antiviral activity of sertraline was noted for other EV serotypes. The drug’s antiviral effect is not likely associated with its approved indications as an antidepressant and its mode-of-action as a selective serotonin reuptake inhibitor. The time-of-addition assay revealed that sertraline inhibited an EV71 infection at the entry stage. We also showed that sertraline partitioned into acidic compartments, such as endolysosomes, to neutralize the low pH levels. In agreement with the findings, the antiviral effect of sertraline could be greatly relieved by exposing virus-infected cells to extracellular low-pH culture media. Ultimately, we have identified a use for an FDA-approved antidepressant in broad-spectrum EV inhibition by blocking viral entry through the alkalization of the endolysosomal route.

Enterovirus 71 (EV71) is an etiological agent of hand foot and mouth disease and can also cause neurological complications in young children. However, there are no approved drugs to treat EV71 infections. In this study, we conducted an antiviral drug screening by using a Food and Drug Administration (FDA)-approved drug library. We identified five drugs that showed dose-dependent inhibition of viral replication. Sertraline was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index among the five hits. The antiviral activity of sertraline was noted for other EV serotypes. The drug’s antiviral effect is not likely associated with its approved indications as an antidepressant and its mode-of-action as a selective serotonin reuptake inhibitor. The time-of-addition assay revealed that sertraline inhibited an EV71 infection at the entry stage. We also showed that sertraline partitioned into acidic compartments, such as endolysosomes, to neutralize the low pH levels. In agreement with the findings, the antiviral effect of sertraline could be relieved greatly by exposing virus-infected cells to extracellular low-pH culture media. Together, we have identified an FDA-approved antidepressant with the new indication for the broad-spectrum EV inhibition by blocking viral entry through the alkalization of the endolysosomal route.

Background: Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host. Methods: Luciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-β and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity. Results: Here we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity. Conclusions: Together our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

Enterovirus A71 (EV-A71) is a growing threat to public health, particularly in the Asia-Pacific region. EV-A71 infection is most prevalent in infants and children and causes a wide spectrum of clinical complications, including hand-foot-and-mouth disease (HFMD), pulmonary and neurological disorders. The pathogenesis of EV-A71 infection is poorly understood at present. It is likely that viral factors and host immunity, and their interplay, affect the pathogenesis and outcome of EV-A71 infection. The mammalian innate immune system forms the first layer of defense against viral infections and triggers activation of adaptive immunity leading to full protection. In this review, we discuss recent advances in our understanding of the interaction between EV-A71 and the innate immune system. We discuss the role of pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and inflammasomes, in the detection of EV-A71 infection and induction of antiviral immunity. As a counteraction, EV-A71 viral proteins target multiple innate immune pathways to facilitate viral replication in host cells. These novel insights at the virus-host interphase may support the future development of vaccines and therapeutics against EV-A71 infection.

Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-β upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.