Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease marked by joint destruction and functional impairment. Tumor necrosis factor (TNF) plays a critical role in RA pathogenesis. Although TNF-targeting drugs are clinically effective, their need for frequent and long-term administration often results in poor patient adherence and suboptimal outcomes. This study developed a gene therapy approach using engineered adeno-associated virus (AAV) vectors to deliver an anti-TNF agent directly into the joint cavity of RA animal models. Animals receiving this therapy demonstrated sustained improvement in clinical scores, inflammatory markers, and joint tissue health. Immunofluorescence staining revealed that AAV vectors could transduce various cell types, including T cells, type A synoviocytes, and dendritic cells. Our results indicate that a single administration of this gene therapy provided long-term efficacy. This suggests that AAV-mediated anti-TNF gene therapy can offer prolonged relief from clinical symptoms and reduce inflammatory damage in a mouse model of RA. This innovative approach presents a promising new therapy with significant clinical prospects to treat patients with RA.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn\'s disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.

UNASSIGNED: Lung adenocarcinoma (LUAD) has become one of the most lethal cancers, for which the recurrence and survival rates remain unfavorable. The tumor necrosis factor (TNF) family is involved in tumorigenesis and tumor progression. Various long non-coding RNAs (lncRNAs) play important roles by mediating the TNF family in cancer. Therefore, this study aimed to construct a TNF-related lncRNA signature to predict prognosis and immunotherapy response in LUAD.
UNASSIGNED: The expression of TNF family members and their related lncRNAs in a total of 500 enrolled LUAD patients was collected from The Cancer Genome Atlas (TCGA). Univariate Cox and the least absolute shrinkage and selection operator (LASSO)-Cox analysis was used to construct a TNF family-related lncRNA prognostic signature. Kaplan-Meier (KM) survival analysis was used to evaluate survival status. The time-dependent area under the receiver operating characteristic (ROC) curve (AUC) values were used to assess the predictive value of the signature to 1-, 2-, and 3-year overall survival (OS). Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied to identify the signature-related biological pathways. Furthermore, tumor immune dysfunction and exclusion (TIDE) analysis was employed to evaluate immunotherapy response.
UNASSIGNED: A total of 8 TNF-related lncRNAs significantly associated with OS of LUAD patients were used to construct a TNF family-related lncRNA prognostic signature. According to risk score, these patients were divided into high- and low-risk subgroups. The KM survival analysis indicated that patients in the high-risk group showed significantly less favorable OS than that of low-risk group. The AUC values in predicting 1-, 2-, and 3-year OS were 0.740, 0.738, and 0.758, respectively. Moreover, the GO and KEGG pathway analyses demonstrated that these lncRNAs were closely involved in immune-related signaling pathways. The further TIDE analysis indicated that high-risk patients had a lower TIDE score than that of low-risk patients, indicating that high-risk patients may be appropriate candidates for immunotherapy.
UNASSIGNED: For the first time, this study constructed and validated a prognostic predictive signature of LUAD patients based on TNF-related lncRNAs, and the signature showed good performance to predict immunotherapy response. Therefore, this signature may provide new strategies for individualized treatment of LUAD patients.

Objective: Tumor necrosis factor (TNF) receptor type II (TNFR2) is expressed by a wide spectrum of tumor cells including colon cancer, non-Hodgkin lymphoma, myeloma, renal carcinoma and ovarian cancer, and its exact role remains to be fully understood. In this study, we examined the effect of genetic ablation of TNFR2 on in vitro and in vivo growth of mouse MC38 and CT26 colon cancer cells. Methods: CRISPR/Cas9 technology was used to knockout TNFR2 on mouse MC38 and CT26 colon cancer cells. In vitro growth and colony formation of wild-type (W.T.) and TNFR2 deficiency of MC38 and CT26 cells, as well as the potential mechanism, was studied. The growth of W.T. and TNFR2 deficient MC38 and CT26 tumors in mice and intratumoral CD8 CTLs were also examined. Results: TNFR2 deficiency impaired in vitro proliferation and colony formation of cancer cells. This was associated with the inhibition of protein kinase B (AKT) phosphorylation and enhanced autophagy-induced cell death. Moreover, deficiency of TNFR2 also markedly impaired in vivo growth of MC38 or CT26 in the syngeneic C57BL/6 mice or BALB/c mice, respectively, accompanied by the decrease in soluble TNFR2 levels in the circulation and the increase in the number of tumor-infiltrating IFNγ+ CD8 cells. Conclusion: TNFR2 plays a role in the growth of mouse colon cancers. Our study provides further experimental evidence to support the development of TNFR2 antagonistic agents in the treatment of cancer.

UNASSIGNED: Breast cancer (BRCA) is the most common malignant tumor in the world. Because of its substantial heterogeneity, its clinical treatment is faced with various problems. Only a small number of patients can benefit from the treatment of immune checkpoint inhibitor (ICI). Costimulatory molecule signature (CMS) plays an essential role in T cell activation and antitumor immune response. Previous studies found that CMS is associated with prognosis-related immune response markers, suggesting that CMS may be a potential therapeutic target. However, the research on their function in BRCA subtype is still inadequate. Our study aims to analyze CMS in BRCA and establish an effective prognostic model.
UNASSIGNED: We extracted 1,222 messenger RNA (mRNA) samples of 1,110 patients registered in the BRCA cohort of The Cancer Genome Atlas (TCGA), including 1,109 tumor tissue mRNA samples and 113 standard tissue samples for model construction and verification. The prognostic significance was determined by least absolute shrinkage and selection operator (LASSO)-Cox proportional hazard regression, which showed that the overall survival (OS) of the high-risk group was shorter than that of the low group (P<0.01).
UNASSIGNED: Although the CMS prognostic model can predict the prognosis well, the receiver operating characteristic (ROC) prediction results were unsatisfactory. The reason for this may be the heteromorphism of BRCA, so we divided the cases into four subtypes according to the PAM50 (PAM50Call_RNAseq) in clinical information. The same method was used to construct the model in the four subtypes and verify the effect of each subtype prognostic model.
UNASSIGNED: The results showed that the submodels constructed in this study can be used to evaluate the prognosis of each subtype.

Rheumatoid arthritis (RA) is a complex autoimmune disease with multiple etiological factors, among which aberrant memory CD4 T cells activation plays a key role in the initiation and perpetuation of the disease. SIGIRR (single immunoglobulin IL-1R-related receptor), a member of the IL-1 receptor (ILR) family, acts as a negative regulator of ILR and Toll-like receptor (TLR) downstream signaling pathways and inflammation. The aim of this study was to investigate the potential roles of SIGIRR on memory CD4 T cells in RA and the underlying cellular and molecular mechanisms.
Single-cell transcriptomics and bulk RNA sequencing data were integrated to predict SIGIRR gene distribution on different immune cell types of human PBMCs. Flow cytometry was employed to determine the differential expression of SIGIRR on memory CD4 T cells between the healthy and RA cohorts. A Spearman correlation study was used to determine the relationship between the percentage of SIGIRR+ memory CD4 T cells and RA disease activity. An AIA mouse model (antigen-induced arthritis) and CD4 T cells transfer experiments were performed to investigate the effect of SIGIRR deficiency on the development of arthritis in vivo. Overexpression of SIGIRR in memory CD4 T cells derived from human PBMCs or mouse spleens was utilized to confirm the roles of SIGIRR in the intracellular cytokine production of memory CD4 T cells. Immunoblots and RNA interference were employed to understand the molecular mechanism by which SIGIRR regulates TNF-α production in CD4 T cells.
SIGIRR was preferentially distributed by human memory CD4 T cells, as revealed by single-cell RNA sequencing. SIGIRR expression was substantially reduced in RA patient-derived memory CD4 T cells, which was inversely associated with RA disease activity and related to enhanced TNF-α production. SIGIRR-deficient mice were more susceptible to antigen-induced arthritis (AIA), which was attributed to unleashed TNF-α production in memory CD4 T cells, confirmed by decreased TNF-α production resulting from ectopic expression of SIGIRR. Mechanistically, SIGIRR regulates the IL-1/C/EBPβ/TNF-α signaling axis, as established by experimental evidence and cis-acting factor bioinformatics analysis.
Taken together, SIGIRR deficiency in memory CD4 T cells in RA raises the possibility that receptor induction can target key abnormalities in T cells and represents a potentially novel strategy for immunomodulatory therapy.

Atherosclerosis, a chronic inflammatory condition that leads to a variety of life-threatening cardiovascular diseases, is a worldwide public health concern. Endothelial cells (ECs), which line the inside of blood vessels, play an important role in atherogenic initiation. Endothelial activation and inflammation are indispensable for the early stage of atherosclerosis. Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme that regulates the stability and activity of target proteins, has been identified as a potential therapeutic target for many inflammatory diseases. However, the role of USP14 on ECs is undefined. In this study, we found that USP14 is downregulated in either atherosclerosis patient specimens or oxidized low-density lipoprotein (ox-LDL)-stimulated ECs as compared to the control group. Overexpression of USP14 in ECs restrains ox-LDL-stimulated nuclear transcription factor kappa B (NF-κB) activation and subsequent adhesion molecule production. USP14 inhibits endothelium proinflammatory activation by suppressing the degradation of the negative regulator of NF-κB signaling, nod-like receptor family caspase recruitment domain family domain containing 5 (NLRC5). Finally, our in vivo experiments confirmed that USP14 adenovirus injection in apolipoprotein E deficient (ApoE-/-) mice fed with a western diet reduced the atherosclerotic lesion size, inhibited macrophage accumulation in the intima, and restricted the progression of atherosclerosis. Our results reveal that USP14 may represent a new therapeutic target for atherosclerosis.

The venomous species Deinagkistrodon acutus has been used as anti-inflammatory medicine in China for a long time. It has been proven to have anti-inflammatory activity, but its specific anti-inflammatory components have not yet been fully elucidated. Tumor necrosis factor receptor-1 (TNFR1), which participates in important intracellular signaling pathways, mediates apoptosis, and functions as a regulator of inflammation, is often used as the target to develop anti-inflammatory drugs. The small peptides of snake venom have the advantages of weak immunogenicity and strong activity. To obtain the specific TNFR1 binding peptides, we constructed a T7 phage library of D. acutus venom glands, and then performed biopanning against TNFR1 on the constructed library. After biopanning three times, several sequences with potential binding capacity were obtained and one 41-amino acid peptide was selected through a series of biological analyses including sequence length, solubility, and simulated affinity, named DAvp-1. After synthesis, the binding capacity of DAvp-1 and TNFR1 was verified using surface plasmon resonance technology (SPR). Conclusively, by applying phage display technology, this work depicts the successful screening of a promising peptide DAvp-1 from D. acutus venom that binds to TNFR1. Additionally, our study emphasizes the usefulness of phage display technology for studies on screening natural product components.

OBJECTIVE: Liver sinusoidal endothelial cells (LSECs) serve as sentinel cells to detect microbial infection and actively contribute to regulating immune responses for surveillance against intrahepatic pathogens. We recently reported that hepatitis B e antigen (HBeAg) stimulation could induce LSEC maturation and abrogate LSEC-mediated T cell suppression in a TNF-α and IL27 dependent manner. However, it remains unclear how HBeAg deficiency during HBV infection influences LSEC immunoregulation function and intrahepatic HBV-specific CD8 T cell responses.
METHODS: The function of LSECs in regulating effector T cell response, intrahepatic HBV-specific CD8 T cell responses and HBV viremia were characterized in both HBeAg-deficient and -competent HBV hydrodynamic injection (HDI) mouse models.
RESULTS: LSECs isolated from HBeAg-deficient HBV HDI mice showed a reduced capacity to promote T cell immunity in vitro compared with those isolated from wild-type HBV HDI mice. HBeAg expression replenishment in HBeAg-deficient HBV HDI mice restored the HBV-induced LSEC maturation, and resulted in potent intrahepatic anti-HBV CD8 T cell responses and efficient control of HBV replication. Moreover, in vivo TNF-α, but not IL27 blockade in HBV HDI mice impaired HBV-specific CD8 T cell immunity and delayed HBV clearance.
CONCLUSIONS: Our study underlines that HBeAg is indispensable for HBV-induced LSEC maturation to trigger intrahepatic HBV-specific T cell activation, and provides a new mechanism to elucidate the intrahepatic immune microenvironment regulation upon HBV exposure.