CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia-reperfusion injury.

Vascular endothelial inflammation is crucial in hepatic ischemia-reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

Inhibiting S-palmitoylation arrests metastasis by relocating Rap2b from plasma membrane in colorectal cancer.

Rap2b, a proto-oncogene upregulated in colorectal cancer (CRC), undergoes protein S-palmitoylation at specific C-terminus sites (C176/C177). These palmitoylation sites are crucial for Rap2b localization on the plasma membrane (PM), as mutation of C176 or C177 results in cytosolic relocation of Rap2b. Our study demonstrates that Rap2b influences cell migration and invasion in CRC cells, independent of proliferation, and this activity relies on its palmitoylation. We identify ABHD17a as the depalmitoylating enzyme for Rap2b, altering PM localization and inhibiting cell migration and invasion. EGFR/PI3K signaling regulates Rap2b palmitoylation, with PI3K phosphorylating ABHD17a to modulate its activity. These findings highlight the potential of targeting Rap2b palmitoylation as an intervention strategy. Blocking the C176/C177 sites using an interacting peptide attenuates Rap2b palmitoylation, disrupting PM localization, and suppressing CRC metastasis. This study offers insights into therapeutic approaches targeting Rap2b palmitoylation for the treatment of metastatic CRC, presenting opportunities to improve patient outcomes.

Loss of Bcl-3 regulates macrophage polarization by promoting macrophage glycolysis.

M1/M2 macrophage polarization plays an important role in regulating the balance of the microenvironment within tissues. Moreover, macrophage polarization involves the reprogramming of metabolism, such as glucose and lipid metabolism. Transcriptional coactivator B-cell lymphoma-3 (Bcl-3) is an atypical member of the IκB family that controls inflammatory factor levels in macrophages by regulating nuclear factor kappa B pathway activation. However, the relationship between Bcl-3 and macrophage polarization and metabolism remains unclear. In this study, we show that the knockdown of Bcl-3 in macrophages can regulate glycolysis-related gene expression by promoting the activation of the nuclear factor kappa B pathway. Furthermore, the loss of Bcl-3 was able to promote the interferon gamma/lipopolysaccharide-induced M1 macrophage polarization by accelerating glycolysis. Taken together, these results suggest that Bcl-3 may be a candidate gene for regulating M1 polarization in macrophages.

DEPDC5 protects CD8+ T cells from ferroptosis by limiting mTORC1-mediated purine catabolism.

Peripheral CD8+ T cell number is tightly controlled but the precise molecular mechanism regulating this process is still not fully understood. In this study, we found that epilepsy patients with loss of function mutation of DEPDC5 had reduced peripheral CD8+ T cells, and DEPDC5 expression positively correlated with tumor-infiltrating CD8+ T cells as well as overall cancer patient survival, indicating that DEPDC5 may control peripheral CD8+ T cell homeostasis. Significantly, mice with T cell-specific Depdc5 deletion also had reduced peripheral CD8+ T cells and impaired anti-tumor immunity. Mechanistically, Depdc5-deficient CD8+ T cells produced high levels of xanthine oxidase and lipid ROS due to hyper-mTORC1-induced expression of ATF4, leading to spontaneous ferroptosis. Together, our study links DEPDC5-mediated mTORC1 signaling with CD8+ T cell protection from ferroptosis, thereby revealing a novel strategy for enhancing anti-tumor immunity via suppression of ferroptosis.

A novel LAG3 neutralizing antibody improves cancer immunotherapy by dual inhibition of MHC-II and FGL1 ligand binding.

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6 nM) and MHC-II binding (IC50 of 6.2 nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.

Polarized Th2 cells attenuate high-fat-diet induced obesity through the suppression of lipogenesis.

Immune cells, such as macrophages, B cells, neutrophils and T cell subsets, have been implicated in the context of obesity. However, the specific role of Th2 cells in adipose tissue function has remained elusive. Eight-week-old male CD3ε─/─ mice were randomly divided into two groups (≥ 5 mice per group): one received intravenous injection of Th2 cells isolated from LATY136F mice, while the other receiving PBS as a control. Both of groups were subjected to a high-fat diet (HFD). The adoptive transfer of polarized Th2 cells led to a significant reduction in obesity following a HFD. This reduction was accompanied by improvements in hepatic steatosis, glucose intolerance, and insulin resistance. Mechanistically, Th2 cell treatment promoted oxidative phosphorylation of adipocytes, thereby contributing to a reduction of lipid droplet accumulation. These findings suggest that Th2 cell therapy represents a novel approach for treating diet-induced obesity and other diseases involving lipid droplet accumulation disorders.

The Hippo-YAP signaling pathway drives CD24-mediated immune evasion in esophageal squamous cell carcinoma via macrophage phagocytosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world with poor prognosis. Despite the promising applications of immunotherapy, the objective response rate is still unsatisfactory. We have previously shown that Hippo/YAP signaling acts as a powerful tumor promoter in ESCC. However, whether Hippo/YAP signaling is involved in tumor immune escape in ESCC remains largely unknown. Here, we show that YAP directly activates transcription of the "don't eat me" signal CD24, and plays a crucial role in driving tumor cells to avoid phagocytosis by macrophages. Mechanistically, YAP regulates CD24 expression by interacting with TEAD and binding the CD24 promoter to initiate transcription, which facilitates tumor cell escape from macrophage-mediated immune attack. Our animal model data and clinical data show that YAP combined with CD24 in tumor microenvironment redefines the impact of TAMs on the prognosis of ESCC patients which will provide a valuable basis for precision medicine. Moreover, treatment with YAP inhibitor altered the distribution of macrophages and suppressed tumorigenesis and progression of ESCC in vivo. Together, our study provides a novel link between Hippo/YAP signaling and macrophage-mediated immune escape, which suggests that the Hippo-YAP-CD24 axis may act as a promising target to improve the prognosis of ESCC patients. A proposed model for the regulatory mechanism of Hippo-YAP-CD24-signaling axis in the tumor-associated macrophages mediated immune escape.

Palmitoylated Sept8-204 modulates learning and anxiety by regulating filopodia arborization and actin dynamics.

Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.

RNF144A promotes antiviral responses by modulating STING ubiquitination.

Stimulator of interferon (IFN) genes (STING, also named MITA, ERIS, MPYS, or TMEM173) plays an essential role in DNA virus- or cytosolic DNA-triggered innate immune responses. Here, we demonstrate that the RING-in-between RING (RBR) E3 ubiquitin ligase family member RING-finger protein (RNF) 144A interacts with STING and promotes its K6-linked ubiquitination at K236, thereby enhancing STING translocation from the ER to the Golgi and downstream signaling pathways. The K236R mutant of STING displays reduced activity in promoting innate immune signal transduction. Overexpression of RNF144A upregulates HSV-1- or cytosolic DNA-induced immune responses, while knockdown of RNF144A expression has the opposite effect. In addition, Rnf144a-deficient cells exhibit impaired DNA virus- or cytosolic DNA-triggered signaling, and RNF144A protects mice from DNA virus infection. In contrast, RNF144A does not affect RNA virus- or cytosolic RNA-triggered innate immune responses. Taken together, our findings identify a new positive regulator of DNA virus- or cytosolic DNA-triggered signaling pathways and a critical ubiquitination site important for fully functional STING during antiviral responses.

Defective LAT signalosome pathology in mice mimics human IgG4-related disease at single-cell level.

Mice with a loss-of-function mutation in the LAT adaptor (LatY136F) develop an autoimmune and type 2 inflammatory disorder called defective LAT signalosome pathology (DLSP). We analyzed via single-cell omics the trajectory leading to LatY136F DLSP and the underlying CD4+ T cell diversification. T follicular helper cells, CD4+ cytotoxic T cells, activated B cells, and plasma cells were found in LatY136F spleen and lung. Such cell constellation entailed all the cell types causative of human IgG4-related disease (IgG4-RD), an autoimmune and inflammatory condition with LatY136F DLSP-like histopathological manifestations. Most previously described T cell-mediated autoimmune manifestations require persistent TCR input. In contrast, following their first engagement by self-antigens, the autoreactive TCR expressed by LatY136F CD4+ T cells hand over their central role in T cell activation to CD28 costimulatory molecules. As a result, all subsequent LatY136F DLSP manifestations, including the production of autoantibodies, solely rely on CD28 engagement. Our findings elucidate the etiology of the LatY136F DLSP and qualify it as a model of IgG4-RD.

Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid.

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein that regulates the activity of FoF1-ATP synthase. Mice lacking ATPIF1 throughout their bodies (Atpif1-/-) exhibit a reduction in the number of neutrophils. However, it remains unclear whether the inactivation of ATPIF1 impairs the antibacterial function of mice, this study aimed to evaluate it using a mouse peritonitis model. Mice were intraperitoneally injected with E. coli to induce peritonitis, and after 24 h, the colonies of E. coli were counted in agarose plates containing mice peritoneal lavage fluids (PLF) or extract from the liver. Neutrophils were analyzed for glucose metabolism in glycolysis following LPS stimulation. Reactive oxygen species (ROS) and lactic acid (LA) levels in neutrophils were measured using flow cytometry and Seahorse analysis, respectively. N-Acetylcysteine (NAC) and 2-Deoxy-d-glucose (2-DG) were employed to assess the role of ROS and LA in neutrophil bactericidal activity. RNA-seq analysis was conducted in neutrophils to investigate potential mechanisms. In ATPIF1-/- neutrophils, bactericidal activity was enhanced, accompanied by increased levels of ROS and LA compared to wildtype neutrophils. The augmented bactericidal activity of ATPIF1-/- neutrophils was reversed by pretreatment with NAC or 2-DG. RNA-seq analysis revealed downregulation of multiple genes involved in glutathione metabolism, pyruvate oxidation, and heme synthesis, along with increased expression of inflammatory and apoptotic genes. This study suggests that the inactivation of the Atpif1 gene enhances glucose metabolism in neutrophils, resulting in increased bactericidal activity mediated by elevated levels of ROS and LA. Inhibiting ATPIF1 may be a potential approach to enhance antibacterial immunity.

RUFY3 regulates endolysosomes perinuclear positioning, antigen presentation and migration in activated phagocytes.

Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 (RUFY3) exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for phosphatidylinositol 3-phosphate on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in primary immune cells and up-regulated upon activation by microbes and Interferons. iRUFY3 is necessary for ARL8b + /LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ, while being important for intracellular Salmonella replication. Specific inactivation of rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights the role of iRUFY3 in controlling endo-lysosomal dynamics, which contributes to phagocyte activation and immune response regulation.

Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis.

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

Immunotherapy of Epstein-Barr virus (EBV) infection and EBV-associated hematological diseases with gp350/CD89-targeted bispecific antibody.

Acute and persistent infection of Epstein-Barr virus (EBV) is associated with several life-threatening hematological disorders, including lymphoproliferative disorders (LPD), hemophagocytic lymphohistiocytosis (HLH), and chronic active Epstein-Barr virus infection (CAEBV). Currently, there are no efficacious virus-targeted therapies for EBV-driven hematological diseases. To explore the potential of phagocytosis-based immunotherapy, we created a bispecific antibody by targeting the viral envelope protein gp350 with a novel EBV-neutralizing antibody (named R1) that was paired with a monoclonal antibody against CD89 for redirecting macrophages and neutrophils. In vitro study showed that the bispecific antibody enabled efficient phagocytosis of EBV and killing of gp350 + lymphoma cells in the presence of PBMC. In vivo studies in NSG mice inoculated with EBV showed that bispecific antibody dramatically reduced the viral load in blood, solid organs and tissues. Treatment of mice implanted with EBV-harboring Raji lymphoma cells efficiently prevented tumor formation and massive metastasis to solid organs. Treatment of mice implanted with whole blood from EBV-HLH patients was effective in reducing viral levels in blood and solid organ. The gp350/CD89 bispecific antibody was highly effective in clearing EBV and immunotherapy of EBV-driven hematological diseases such as LPD and EBV-HLH.

Myeloid-restricted CD68 deficiency attenuates atherosclerosis via inhibition of ROS-MAPK-apoptosis axis.

In atherosclerosis, macrophages derived from blood monocytes contribute to non-resolving inflammation, which subsequently primes necrotic core formation, and ultimately triggers acute thrombotic vascular disease. Nevertheless, little is known about how inflammatory cells, especially the macrophages fuel atherosclerosis. CD68, a unique class D scavenger receptor (SRD) family member, is specifically expressed in monocytes/macrophages and remarkably up-regulated upon oxidized low-density lipoprotein (ox-LDL) stimulation. Nonetheless, whether and how myeloid-specific CD68 affects atherosclerosis remains to be defined. To determine the essential in vivo role and mechanism linking CD68 to atherosclerosis, we engineered global and myeloid-specific CD68-deficient mice on an ApoE-null background. On Western diet, both the mice with global and the myeloid-restricted deletion of CD68 on ApoE-null background attenuated atherosclerosis, accompanied by diminished immune/inflammatory cell burden and necrotic core content, but increased smooth muscle cell content in atherosclerotic plaques. In vitro experiments revealed that CD68 deficiency in macrophages resulted in attenuated ox-LDL-induced macrophage apoptosis. Additionally, CD68 deficiency suppressed ROS production, while removal of ROS can markedly reversed this effect. We further showed that CD68 deficiency affected apoptosis through inactivation of the mitogen-activated protein kinase (MAPK) pathway. Our findings establish CD68 as a macrophage lineage-specific regulator of "ROS-MAPK-apoptosis" axis, thus providing a previously unknown mechanism for the prominence of CD68 as a risk factor for coronary artery disease. Its therapeutic inhibition may provide a potent lever to alleviate the cardiovascular disease.

p38-mediated FOXN3 phosphorylation modulates lung inflammation and injury through the NF-κB signaling pathway.

NF-κB activates the primary inflammatory response pathway responsible for methicillin-resistant Staphylococcus aureus (MRSA)-induced lung inflammation and injury. Here, we report that the Forkhead box transcription factor FOXN3 ameliorates MRSA-induced pulmonary inflammatory injury by inactivating NF-κB signaling. FOXN3 competes with IκBα for binding to heterogeneous ribonucleoprotein-U (hnRNPU), thereby blocking ß-TrCP-mediated IκBα degradation and leading to NF-κB inactivation. FOXN3 is directly phosphorylated by p38 at S83 and S85 residues, which induces its dissociation from hnRNPU, thus promoting NF-κB activation. After dissociation, the phosphorylated FOXN3 becomes unstable and undergoes proteasomal degradation. Additionally, hnRNPU is essential for p38-mediated FOXN3 phosphorylation and subsequent phosphorylation-dependent degradation. Functionally, genetic ablation of FOXN3 phosphorylation results in strong resistance to MRSA-induced pulmonary inflammatory injury. Importantly, FOXN3 phosphorylation is clinically positively correlated with pulmonary inflammatory disorders. This study uncovers a previously unknown regulatory mechanism underpinning the indispensable role of FOXN3 phosphorylation in the inflammatory response to pulmonary infection.