PRMT6 Epigenetically Drives Metabolic Switch from Fatty Acid Oxidation toward Glycolysis and Promotes Osteoclast Differentiation During Osteoporosis.

Epigenetic regulation of metabolism profoundly influences cell fate commitment. During osteoclast differentiation, the activation of RANK signaling is accompanied by metabolic reprogramming, but the epigenetic mechanisms by which RANK signaling induces this reprogramming remain elusive. By transcriptional sequence and ATAC analysis, this study identifies that activation of RANK signaling upregulates PRMT6 by epigenetic modification, triggering a metabolic switching from fatty acids oxidation toward glycolysis. Conversely, Prmt6 deficiency reverses this shift, markedly reducing HIF-1α-mediated glycolysis and enhancing fatty acid oxidation. Consequently, PRMT6 deficiency or inhibitor impedes osteoclast differentiation and alleviates bone loss in ovariectomized (OVX) mice. At the molecular level, Prmt6 deficiency reduces asymmetric dimethylation of H3R2 at the promoters of genes including Ppard, Acox3, and Cpt1a, enhancing genomic accessibility for fatty acid oxidation. PRMT6 thus emerges as a metabolic checkpoint, mediating metabolic switch from fatty acid oxidation to glycolysis, thereby supporting osteoclastogenesis. Unveiling PRMT6's critical role in epigenetically orchestrating metabolic shifts in osteoclastogenesis offers a promising target for anti-resorptive therapy.

Hyperglycemia-induced Sirt3 downregulation increases microglial aerobic glycolysis and inflammation in diabetic neuropathic pain pathogenesis.

BACKGROUND: Hyperglycemia-induced neuroinflammation significantly contributes to diabetic neuropathic pain (DNP), but the underlying mechanisms remain unclear. OBJECTIVE: To investigate the role of Sirt3, a mitochondrial deacetylase, in hyperglycemia-induced neuroinflammation and DNP and to explore potential therapeutic interventions. METHOD AND RESULTS: Here, we found that Sirt3 was downregulated in spinal dorsal horn (SDH) of diabetic mice by RNA-sequencing, which was further confirmed at the mRNA and protein level. Sirt3 deficiency exacerbated hyperglycemia-induced neuroinflammation and DNP by enhancing microglial aerobic glycolysis in vivo and in vitro. Overexpression of Sirt3 in microglia alleviated inflammation by reducing aerobic glycolysis. Mechanistically, high-glucose stimulation activated Akt, which phosphorylates and inactivates FoxO1. The inactivation of FoxO1 diminished the transcription of Sirt3. Besides that, we also found that hyperglycemia induced Sirt3 degradation via the mitophagy-lysosomal pathway. Blocking Akt activation by GSK69093 or metformin rescued the degradation of Sirt3 protein and transcription inhibition of Sirt3 mRNA, which substantially diminished hyperglycemia-induced inflammation. Metformin in vivo treatment alleviated neuroinflammation and diabetic neuropathic pain by rescuing hyperglycemia-induced Sirt3 downregulation. CONCLUSION: Hyperglycemia induces metabolic reprogramming and inflammatory activation in microglia through the regulation of Sirt3 transcription and degradation. This novel mechanism identifies Sirt3 as a potential drug target for treating DNP.

ISGylation by HERCs facilitates STING activation.

Optimal activation of stimulator of interferon genes (STING) protein is crucial for host defenses against pathogens and avoiding detrimental effects. Various post-translational modifications control STING activity. However, the function of interferon (IFN)-stimulated gene (ISG) 15 modification (ISGylation) in controlling STING stability and activation is unclear. Here, we show that the E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice) facilitate STING activation by mediating ISGylation of STING at K150, preventing its K48-linked ubiquitination and degradation. Concordantly, Herc6 deficiency suppresses herpes simplex virus 1 infection-induced type I IFN responses and facilitates viral replication both in vitro and in vivo. Notably, severe acute respiratory syndrome coronavirus 2 protein papain-like protease cleaves HERC5-mediated ISGylation of STING, suppressing host antiviral responses. These data identify a mechanism by which HERCs-mediated ISGylation controls STING stability and activation and uncover the correlations and interactions of ISGylation and ubiquitination during STING activation.

Changes in SLITRK1 Level in the Amygdala Mediate Chronic Neuropathic Pain-Induced Anxio-Depressive Behaviors in Mice.

BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.

Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis.

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

PRMT6 deficiency or inhibition alleviates neuropathic pain by decreasing glycolysis and inflammation in microglia.

Microglia induced chronic inflammation is the critical pathology of Neuropathic pain (NP). Metabolic reprogramming of macrophage has been intensively reported in various chronic inflammation diseases. However, the metabolic reprogramming of microglia in chronic pain remains to be elusive. Here, we reported that immuno-metabolic markers (HIF-1α, PKM2, GLUT1 and lactate) were related with increased expression of PRMT6 in the ipsilateral spinal cord dorsal horn of the chronic construction injury (CCI) mice. PRMT6 deficiency or prophylactic and therapeutic intrathecal administration of PRMT6 inhibitor (EPZ020411) ameliorated CCI-induced NP, inflammation and glycolysis in the ipsilateral spinal cord dorsal horn. PRMT6 knockout or knockdown inhibited LPS-induced inflammation, proliferation and glycolysis in microglia cells. While PRMT6 overexpression exacerbated LPS-induced inflammation, proliferation and glycolysis in BV2 cells. Recent research revealed that PRMT6 could interact with and methylate HIF-1α, which increased HIF-1α protein stability. In sum, increased expression of PRMT6 exacerbates NP progress by increasing glycolysis and neuroinflammation through interacting with and stabilizing HIF-1α in a methyltransferase manner, which outlines novel pathological mechanism and drug target for NP.

HSV-1 reactivation results in post-herpetic neuralgia by upregulating Prmt6 and inhibiting cGAS-STING.

Chronic varicella zoster virus (VZV) infection induced neuroinflammatory condition is the critical pathology of post-herpetic neuralgia (PHN). The immune escape mechanism of VZV remains elusive. As to mice have no VZV infection receptor, herpes simplex virus type 1 (HSV-1) infection is a well established PHN mice model. Transcriptional expression analysis identified that the protein arginine methyltransferases 6 (Prmt6) was upregulated upon HSV-1 infection, which was further confirmed by immunofluorescence staining in spinal dorsal horn. Prmt6 deficiency decreased HSV-1-induced neuroinflammation and PHN by enhancing antiviral innate immunity and decreasing HSV-1 load in vivo and in vitro. Overexpression of Prmt6 in microglia dampened antiviral innate immunity and increased HSV-1 load. Mechanistically, Prmt6 methylated and inactivated STING, resulting in reduced phosphorylation of TANK binding kinase-1 (TBK1) and interferon regulatory factor 3 (IRF3), diminished production of type I interferon (IFN-I) and antiviral innate immunity. Furthermore, intrathecal or intraperitoneal administration of the Prmt6 inhibitor EPZ020411 decreased HSV-1-induced neuroinflammation and PHN by enhancing antiviral innate immunity and decreasing HSV-1 load. Our findings revealed that HSV-1 escapes antiviral innate immunity and results in PHN by upregulating Prmt6 expression and inhibiting the cGAS-STING pathway, providing novel insights and a potential therapeutic target for PHN.

Tofacitinib Promotes Functional Recovery after Spinal Cord Injury by Regulating Microglial Polarization via JAK/STAT Signaling Pathway.

Background: The JAK/STAT signaling pathway is the main inflammatory signal transduction pathway, whether JAK/STAT contributes the pathology of SCI and targeting the pathway will alleviate SCI needs to be addressed. Here, we explored the therapeutic effect of pan-JAK inhibitor tofacitinib (TOF) on secondary injury after SCI and explained the underlying mechanisms. Methods: SCI model in rat was established to evaluate the therapeutic effects of TOF treatment in vivo. Histological and behavioral analyses were performed at different time points after SCI. In vitro, the effects of TOF on pro-inflammatory activation of primary microglia and BV2 cells were analyzed by western blot analysis, fluorescent staining, qPCR and flow cytometry. The neuroprotection of TOF was detected using a co-culture system with primary neurons and microglia. Results: TOF can effectively improve motor dysfunction caused by spinal cord injury in rats. TOF administration in the early stage of inflammation can effectively inhibit neuronal apoptosis and scar tissue formation, and promote the repair of axons and nerve fibers. Further studies have demonstrated that TOF suppresses inflammation caused by spinal cord injury by inhibiting the activation of microglia to pro-inflammatory phenotype in vivo and in vitro. Additionally, an interesting phenomenon is revealed in our results that TOF exhibits superior neuronal protection during inflammation in vitro. Conclusions: Our study showed that TOF could regulate microglial activation via JAK / STAT pathway and promote the recovery of motor function after SCI, which is of great significance for the immunotherapy of SCI.

CCL2-mediated inflammatory pathogenesis underlies high myopia-related anxiety.

High myopia is a leading cause of blindness worldwide. It may lead to emotional defects that rely closely on the link between visual sensation and the central nervous system. However, the extent of the defects and its underlying mechanism remain unknown. Here, we report that highly myopic patients exhibit greater anxiety, accompanied by higher CC chemokine ligand 2 (CCL2) and monocyte levels in the blood. Similar findings are found in the mouse model of high myopia. Mechanistic evaluations using GFP-positive bone marrow chimeric mice, parabiotic mouse model, enhanced magnetic resonance imaging, etc., show that highly myopic visual stimulation increases CCL2 expression in eyes, aggravates monocyte/macrophage infiltration into eyes and brains, and disrupts blood-ocular barrier and blood-brain barrier of mice. Conversely, Ccl2-deficient highly myopic mice exhibit attenuated ocular and brain infiltration of monocytes/macrophages, reduced disruption of the blood-ocular barrier and blood-brain barrier, and less anxiety. Substantial alleviation of high myopia-related anxiety can also be achieved with the administration of CCL2-neutralizing antibodies. Our results establish the association between high myopia and anxiety, and implicate the CCL2-mediated inflammatory pathogenesis as an underlying mechanism.

i-CRISPR: a personalized cancer therapy strategy through cutting cancer-specific mutations.

Developing a strategy to specifically kill cancer cells without inducing obvious damage to normal cells may be of great clinical significance for cancer treatment. In the present study, we developed a new precise personalized strategy named "i-CRISPR" for cancer treatment through adding DNA damage repair inhibitors(i) and inducing cancer cell-specific DNA double strand breaks by CRISPR. Through in vitro and in vivo experiments, we confirmed the efficacy of this strategy in multiple cancer models and revealed the mechanism of cell death. Our strategy might provide a novel concept for precise cancer therapy.

Demalonylation of DDX3 by Sirtuin 5 promotes antiviral innate immune responses.

Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnß transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnß transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.

Author Correction: Bromodomain protein Brd3 promotes Ifnb1 transcription via enhancing IRF3/p300 complex formation and recruitment to Ifnb1 promoter in macrophages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Integrin CD11b attenuates colitis by strengthening Src-Akt pathway to polarize anti-inflammatory IL-10 expression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction to: The methyltransferase PRMT6 attenuates antiviral innate immunity by blocking TBK1-IRF3 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Siglec-G Deficiency Ameliorates Hyper-Inflammation and Immune Collapse in Sepsis via Regulating Src Activation.

Hyper-inflammation during acute phase and sequential hypo-inflammation during immunosuppressive phase in macrophages/monocytes lead to multiorgan failure syndrome and immune collapse of sepsis, in which toll-like receptor (TLR)-triggered inflammatory responses play a major role. Here, we reported that Siglecg deficiency attenuated TLR4-triggered pro-inflammatory cytokine production and increased anti-inflammatory cytokine [interleukin-10 [IL-10]] production in vivo and in vitro at both acute and immunosuppressive phases. Siglecg deficiency also protected mice from lipopolysaccharide (LPS)-induced sepsis with less inflammation in the lung and less tissue destruction in the spleen. Siglec-G inhibited proto-oncogene tyrosine-protein kinase Src (Src) activation via recruiting and activating tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP1) through immunoreceptor tyrosine-based inhibitory motif (ITIM) domain. Src could inhibit TLR4-induced inflammatory cytokines and promote anti-inflammatory cytokine IL-10. Mechanical investigation showed that Src could interact with and phosphorylate STAT3. Src could also promote HIF1α degradation through activating GSK3ß. Our study reveals that Siglec-G orchestrates TLR-induced inflammation, which outlines that blocking Siglec-G or activating Src may be a promising strategy for both acute and chronic inflammatory diseases.

An endosomal LAPF is required for macrophage endocytosis and elimination of bacteria.

Macrophages can internalize the invading pathogens by raft/caveolae and/or clathrin-dependent endocytosis and elicit an immune response against infection. However, the molecular mechanism for macrophage endocytosis remains elusive. Here we report that LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains) is required for caveolae-mediated endocytosis. Lapf-deficient macrophages have impaired capacity to endocytose and eliminate bacteria. Macrophage-specific Lapf-deficient mice are more susceptible to Escherichia coli (E. coli) infection with higher bacterial loads. Moreover, Lapf deficiency impairs TLR4 endocytosis, resulting in attenuated production of TLR-triggered proinflammatory cytokines. LAPF is localized to early endosomes and interacts with caveolin-1. Phosphorylation of LAPF by the tyrosine kinase Src is required for LAPF-Src-Caveolin complex formation and endocytosis and elimination of bacteria. Collectively, our work demonstrates that LAPF is critical for endocytosis of bacteria and induction of inflammatory responses, suggesting that LAPF and Src could be potential targets for the control of infectious diseases.

The methyltransferase PRMT6 attenuates antiviral innate immunity by blocking TBK1-IRF3 signaling.

Protein arginine methyltransferases (PRMTs) play diverse biological roles and are specifically involved in immune cell development and inflammation. However, their role in antiviral innate immunity has not been elucidated. Viral infection triggers the TBK1-IRF3 signaling pathway to stimulate the production of type-I interferon, which mediates antiviral immunity. We performed a functional screen of the nine mammalian PRMTs for regulators of IFN-ß expression and found that PRMT6 inhibits the antiviral innate immune response. Viral infection also upregulated PRMT6 protein levels. We generated PRMT6-deficient mice and found that they exhibited enhanced antiviral innate immunity. PRMT6 deficiency promoted the TBK1-IRF3 interaction and subsequently enhanced IRF3 activation and type-I interferon production. Mechanistically, viral infection enhanced the binding of PRMT6 to IRF3 and inhibited the interaction between IRF3 and TBK1; this mechanism was independent of PRMT6 methyltransferase activity. Thus, PRMT6 inhibits antiviral innate immunity by sequestering IRF3, thereby blocking TBK1-IRF3 signaling. Our work demonstrates a methyltransferase-independent role for PRMTs. It also identifies a negative regulator of the antiviral immune response, which may protect the host from the damaging effects of an overactive immune system and/or be exploited by viruses to escape immune detection.