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Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

Summary Upon viral infection, several proteins in the innate signaling pathway form aggregates, which in turn promote the activation of innate antiviral immune response. In this protocol, we use herpes simplex virus type 1 (HSV-1) to infect mouse peritoneal macrophages, and show how to detect the aggregation of TBK1 upon viral infection. The protocol is adaptable for other proteins and other viruses. For complete details on the use and execution of this profile, please refer to Yan et al. (2021)., Graphical Abstract, Highlights • Detailed protocol for HSV-1 virus amplification and titer detection • Detailed description of preparation and viral infection of mouse macrophages • Protocol for the detection of protein aggregation with Native-PAGE, Upon viral infection, several proteins in the innate signaling pathway form aggregates, which in turn promote the activation of innate antiviral immune response. In this protocol, we use herpes simplex virus type 1 (HSV-1) to infect mouse peritoneal macrophages, and show how to detect the aggregation of TBK1 upon viral infection. The protocol is adaptable for other proteins and other viruses.

Stringent regulation of the transcription factor NF-κB signaling is essential for the activation of host immune responses and maintaining homeostasis, yet the molecular mechanisms involved in its tight regulation are not completely understood. In this study, we report that IKK-interacting protein (IKIP) negatively regulates NF-κB activation. IKIP interacted with IKKα/β to block its association with NEMO, thereby inhibiting the phosphorylation of IKKα/β and the activation of NF-κB. Upon LPS, TNF-α, and IL-1β stimulation, IKIP-deficient macrophages exhibited more and prolonged IKKα/β phosphorylation, IκB, and p65 phosphorylation and production of NF-κB–responsive genes. Moreover, IKIP-deficient mice were more susceptible to LPS-induced septic shock and dextran sodium sulfate–induced colitis. Our study identifies a previously unrecognized role for IKIP in the negative regulation of NF-κB activation by inhibition of IKKα/β phosphorylation through the disruption of IKK complex formation.

The IκB kinase (IKK) complex plays a vital role in regulating the NF-κB activation. Aberrant NF-κB activation is involved in various inflammatory diseases. Thus, targeting IKK activation is an ideal therapeutic strategy to cure and prevent inflammatory diseases related to NF-κB activation. In a previous study, we demonstrated that IKK-interacting protein (IKIP) inhibits the phosphorylation of IKKα/β and the activation of NF-κB through disruption of the formation of IKK complex. In this study, we identified a 15-aa peptide derived from mouse IKIP (46–60 aa of IKIP), which specifically suppressed IKK activation and NF-κB targeted gene expression via disrupting the association of IKKβ and NEMO. Importantly, administration of the peptide reduced LPS-induced acute inflammation and attenuated Zymosan-induced acute arthritis in mice. These findings suggest that this IKIP peptide may be a promising therapeutic reagent in the prevention and treatment of inflammatory diseases.

Summary: TBK1 is an essential kinase for the innate immune response against viral infection. However, the key molecular mechanisms regulating the TBK1 activation remain elusive. Here, we identify PRMT1, a type I protein arginine methyltransferase, as an essential regulator of TBK1 activation. PRMT1 directly interacts with TBK1 and catalyzes asymmetric methylation of R54, R134, and R228 on TBK1. This modification enhances TBK1 oligomerization after viral infection, which subsequently promotes TBK1 phosphorylation and downstream type I interferon production. More important, myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA and RNA viruses than Prmt1fl/fl mice. Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.

Current evidence supports that depression is accompanied by the activation of the inflammatory-response system, and overproduction of pro-inflammatory cytokines may play a role in the pathophysiology of depressive disorders. Resveratrol has anti-inflammatory, antioxidant and anti-depressant-like properties. Using an animal model of depression induced by a single administration of lipopolysaccharide (LPS), the present study investigated the effects of resveratrol on LPS-induced depressive-like behavior and inflammatory-response in adult mice. Our results showed that pretreatment with resveratrol (80mg/kg, i.p.) for 7 consecutive days reversed LPS-increased the immobility time in the forced swimming test and tail suspension test, and LPS-reduced sucrose preference test. Moreover, the antidepressant action of resveratrol was paralleled by significantly reducing the expression levels of pro-inflammatory cytokines, and up-regulating phosphorylated cAMP response-element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF) expression in prefrontal cortex (PFC) and hippocampus. In addition, resveratrol ameliorated LPS-induced NF-κB activation in the PFC and hippocampus. The results demonstrate that resveratrol may be an effective therapeutic agent for LPS-induced depressive-like behavior, partially due to its anti-inflammatory aptitude and by modulating pCREB and BDNF expression in the brain region of mice.