Unveiling novel insights into human IL-6 - IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis.

The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.

Blocking the TRAIL-DR5 Pathway Reduces Cardiac Ischemia-Reperfusion Injury by Decreasing Neutrophil Infiltration and Neutrophil Extracellular Traps Formation.

PURPOSE: Acute myocardial infarction (AMI) is a leading cause of mortality. Neutrophils penetrate injured heart tissue during AMI or ischemia-reperfusion (I/R) injury and produce inflammatory factors, chemokines, and extracellular traps that exacerbate heart injury. Inhibition of the TRAIL-DR5 pathway has been demonstrated to alleviate cardiac ischemia-reperfusion injury in a leukocyte-dependent manner. However, it remains unknown whether TRAIL-DR5 signaling is involved in regulating neutrophil extracellular traps (NETs) release. METHODS: This study used various models to examine the effects of activating the TRAIL-DR5 pathway with soluble mouse TRAIL protein and inhibiting the TRAIL-DR5 signaling pathway using DR5 knockout mice or mDR5-Fc fusion protein on NETs formation and cardiac injury. The models used included a co-culture model involving bone marrow-derived neutrophils and primary cardiomyocytes and a model of myocardial I/R in mice. RESULTS: NETs formation is suppressed by TRAIL-DR5 signaling pathway inhibition, which can lessen cardiac I/R injury. This intervention reduces the release of adhesion molecules and chemokines, resulting in decreased neutrophil infiltration and inhibiting NETs production by downregulating PAD4 in neutrophils. CONCLUSION: This work clarifies how the TRAIL-DR5 signaling pathway regulates the neutrophil response during myocardial I/R damage, thereby providing a scientific basis for therapeutic intervention targeting the TRAIL-DR5 signaling pathway in myocardial infarction.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation.

The impact of Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3) on cisplatin-induced acute kidney injury was investigated in this study. Cisplatin-induced Tim-3 expression in mice kidney tissues and proximal tubule-derived BUMPT cells in a time-dependent manner. Compared with wild-type mice, Tim-3 knockout mice have higher levels of serum creatinine and urea nitrogen, enhanced TUNEL staining signals, more severe 8-OHdG (8-hydroxy-2' -deoxyguanosine) accumulation, and increased cleavage of caspase 3. The purified soluble Tim-3 (sTim-3) protein was used to intervene in cisplatin-stimulated BUMPT cells by competitively binding to the Tim-3 ligand. sTim-3 obviously increased the cisplatin-induced cell apoptosis. Under cisplatin treatment conditions, Tim-3 knockout or sTim-3 promoted the expression of TNF-α (tumor necrosis factor-alpha) and IL-1ß (Interleukin-1 beta) and inhibited the expression of IL-10 (interleukin-10). NF-κB (nuclear factor kappa light chain enhancer of activated B cells) P65 inhibitor PDTC or TPCA1 lowed the increased levels of creatinine and BUN (blood urea nitrogen) in cisplatin-treated Tim-3 knockout mice serum and the increased cleavage of caspase 3 in sTim-3 and cisplatin-treated BUMPT cells. Moreover, sTim-3 enhanced mitochondrial oxidative stress in cisplatin-induced BUMPT cells, which can be mitigated by PDTC. These data indicate that Tim-3 may protect against renal injury by inhibiting NF-κB-mediated inflammation and oxidative stress.

TRAIL inhibition by soluble death receptor 5 protects against acute myocardial infarction in rats.

Acute myocardial infarction (AMI) is associated with high morbidity and mortality. An effective therapeutic strategy is to rescue cardiomyocytes from death. Apoptosis is a key reason of cardiomyocyte death that can be prevented. In this study, we investigated the role of TNF-related apoptosis-inducing ligand (TRAIL) in initiating apoptosis by binding to death receptor 5 (DR5), and this procession is inhibited by soluble DR5 (sDR5) in rats after AMI. First, we found that the level of TRAIL in serum was down-regulated in AMI patients. Then, TRAIL and DR5 expression was analysed in the myocardium of rats after AMI, and their expression was up-regulated. sDR5 treatment reduced the myocardial infarct size and the levels of CK-MB and cTn-I in serum. The expression of caspase 3 and PARP is decreased, but the anti-apoptotic factor Bcl-2 was increased in sDR5 treatment rats after AMI. DR5 expression was also analysed after sDR5 treatment and it was down-regulated, and a low level of DR5 expression seemed to be beneficial for the myocardium. Overall, our findings indicated that sDR5 decreases myocardial damage by inhibiting apoptosis in rat after AMI. We expect to observe the potential therapeutic effects of sDR5 on AMI in the future.

Soluble Flt-1 in AMI Patients Serum Inhibits Angiogenesis of Endothelial Progenitor Cells by Suppressing Akt and Erk's Activity.

In acute myocardial infarction (AMI), endothelial progenitor cells (EPCs) are essential for the recovery of collateral circulation via angiogenesis. Clinical research has shown that the poor prognosis of the patients with AMI is closely associated with the cell quantity and function of EPCs. Whether there are differences in the biological features of EPCs from AMI patients and healthy subjects is worth exploring. In this study, EPCs were isolated from human peripheral blood and identified as late-stage EPCs by flow cytometry, immunofluorescence, and blood vessel formation assay. Compared to healthy subjects, AMI patients had more EPCs in the peripheral blood compared to healthy subjects. In addition, EPCs from AMI patients exhibited higher migration ability in the transwell assay compared to EPCs from healthy subjects. However, no difference in the angiogenesis of EPCs was observed between AMI patients and healthy subjects. Further studies revealed that soluble vascular endothelial growth factor receptor 1 (sFlt-1) in the serum of AMI patients was involved in the inhibition of EPCs angiogenesis by suppressing the Akt and Erk pathways. In conclusion, this study demonstrated that elevated serum sFlt-1 inhibits angiogenesis of EPC in AMI patients. Our findings uncover a pathogenic role of sFlt-1 in AMI.

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

1,25(OH)2 D3 alleviates DSS-induced ulcerative colitis via inhibiting NLRP3 inflammasome activation.

1,25-dihydroxyvitamin D3 (1,25(OH)2 D3, VitD3) is the major active ingredient of vitamin D and has anti-inflammatory activity; however, the mechanism for this remains poorly understood. In this study, we found that VitD3 was able to abolish NOD-like receptor protein 3 (NLRP3) inflammasome activation and subsequently inhibit caspase-1 activation and IL-1ß secretion via the vitamin D receptor (VDR). Furthermore, VitD3 specifically prevented NLRP3-mediated apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) oligomerization. In additional to this, NLRP3 binding to NIMA-related kinase 7 (NEK7) was also inhibited. Notably, VitD3 inhibited autophagy, leading to the inhibition of the NLRP3 inflammasome. Uncoupling protein 2-reactive oxygen species signaling may be involved in inflammasome suppression by VitD3. Importantly, VitD3 had both preventive and therapeutic effects on mouse model of ulcerative colitis, via inhibition of NLRP3 inflammasome activation. Our results reveal a mechanism through which VitD3 represses inflammation and prevents the relevant diseases, and suggest a potential clinical use of VitD3 in autoimmune syndromes or other NLRP3 inflammasome-driven inflammatory diseases.

The E3 ubiquitin ligase Itch is required for B-cell development.

The E3 ubiquitin ligase Itch interacts with Foxo1 and targets it for ubiquitination and degradation during follicular helper T-cell differentiation, whereas the transcription factor Foxo1 plays a critical role in B-cell development. Thus, we proposed that Itch mediates B-cell differentiation. Unexpectedly, we found that Itch deficiency downregulated Foxo1 expression in B cells. Itch cKO (conditional knock out in B cells) mice had fewer pro-B cells in the bone marrow, more small resting IgM-IgD-B cells in the periphery, and lower B-cell numbers in the lymph nodes through decreased Foxo1-mediated IL-7Rα, RAG, and CD62L expression, respectively. Importantly, Itch deficiency reduced Foxo1 mRNA expression by up-regulating JunB-mediated miR-182. Finally, Foxo1 negatively regulated JunB expression by up-regulating Itch. Thus, we have identified a novel regulatory axis between Itch and Foxo1 in B cells, suggesting that Itch is essential for B-cell development.

Post-transcriptional regulator Rbm47 elevates IL-10 production and promotes the immunosuppression of B cells.

Regulatory B cells (Bregs) are a functionally defined B cell subset, and IL-10 is crucial for the suppressive functions of Bregs. However, little is known regarding how IL-10 production is regulated in B cells. To explore the mechanisms by which IL-10 is regulated in B cells, we used mRNA microarrays to screen for molecules that are upregulated in IL-10-producing B cells and identified RNA-binding motif protein 47 (Rbm47) as a post-transcriptional regulator. Rbm47 was found to promote IL-10 production in B cells. We found that Rbm47 promotes the stability of IL-10 mRNA by binding to AU-rich elements in the 3' untranslated region of Il10 mRNA. In addition, we demonstrated that the overexpression of Rbm47 enabled B cells to facilitate Foxp3+ regulator T-cell induction and reduce the severity of DSS-induced ulcerative colitis. Taken together, these results suggest that Rbm47 plays an important role in regulating IL-10 at the post-transcriptional level, thus promoting the regulatory functions of B cells. The findings presented in this study not only increase our understanding of the post-translational regulation of IL-10 in B cells but also identify a novel strategy for the potential application of Bregs.

Gypenosides improve diabetic cardiomyopathy by inhibiting ROS-mediated NLRP3 inflammasome activation.

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti-hyperglycaemia and anti-inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL-1ß and IL-18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down-regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C-reactive proteins (CRPs), IL-1ß and IL-18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS-mediated NLRP3 inflammasome activation.

A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice.

Interleukin-12 family cytokines have emerged as critical regulators of immunity with some members (IL-12, IL-23) associated with disease pathogenesis while others (IL-27, IL-35) mitigate autoimmune diseases. Each IL-12 family member is comprised of an α and a ß chain, and chain-sharing is a key feature. Although four bona fide members have thus far been described, promiscuous chain-pairing between alpha (IL-23p19, IL-27p28, IL-12/IL-35p35) and beta (IL-12/IL-23p40, IL-27/IL-35Ebi3) subunits, predicts six possible heterodimeric IL-12 family cytokines. Here, we describe a new IL-12 member composed of IL-23p19 and Ebi3 heterodimer (IL-39) that is secreted by LPS-stimulated B cells and GL7(+) activated B cells of lupus-like mice. We further show that IL-39 mediates inflammatory responses through activation of STAT1/STAT3 in lupus-like mice. Taken together, our results show that IL-39 might contribute to immunopathogenic mechanisms of systemic lupus erythematosus, and could be used as a possible target for its treatment.

Pre-existing CD19-independent GL7(-) Breg cells are expanded during inflammation and in mice with lupus-like disease.

Interleukin 10 (IL-10)-producing regulatory B-cells (Bregs) suppress inflammatory responses that mediate autoimmune diseases. However, it is unknown whether Bregs derive from a pre-existing dedicated B-cell lineage or if any B-cell can differentiate into Bregs in response to BCR or TLR activation. GL7(+) B-cells are antigen-experienced differentiated B-cells while GL7(-/lo) are at an early stage of B-cell differentiation. While both GL7(-/lo) and GL7(+) B cells can produce IL-10, differentiation of GL7(-) B-cells into Bregs does not require CD19- or Bcl6-induced signals, suggesting that BCR-induced proliferation or Ig class-switching is not necessary for generation of Breg cells. Of particular importance, we show that GL7(-) Breg cells are dramatically expanded in lupus-like mice and GL7(-) Bregs suppressed inflammatory responses in lupus-like mice by inducing expansion of Foxp3(+)Treg cells. Taken together, these results suggest that pre-existing GL7(-)IL-10(+) cells are expanded during inflammation, differentiate into GL7(+) Bregs and contribute to immune-regulation in lupus-like mice.

Blockade of B-cell activating factor with TACI-IgG effectively reduced Th1 and Th17 cells but not memory T cells in experimental allergic encephalomyelitis mice.

B-cell activating factor (BAFF) is regarded as a new therapeutic target in autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Along with other researchers, we have demonstrated that BAFF inhibitor atacicept (TACI-IgG) suppresses lupus and experimental allergic encephalomyelitis (EAE) by reducing the mature B-cell number but not memory B cells. It is however unclear whether TACI-Ig affects pathogenic T cells and memory T cells. In the present study, we found that blocking BAFF with TACI-IgG effectively reduces the pathogenic Th1 and Th17 cells in EAE mice. However, TACI-IgG did not reduce memory CD62L(+)CD44(hi)CD4(+) and CD62L(+)CD44(hi)CD8(+) T cells in EAE mice. When interleukin (IL)-15 was neutralized, memory CD62L(+)CD44(hi) T cells were significantly reduced in TACI-IgG-treated EAE mice. These results suggest that TACI-IgG is effective in effective controlling Th1 and Th17 cells, but it also increases IL-15 to upregulate memory T cells in EAE mice. The study provides hints for the clinical application of the combination of BAFF- and IL-15-specific therapeutic agents.

Ligation of metabotropic glutamate receptor 3 (Grm3) ameliorates lupus-like disease by reducing B cells.

Recently B-cell activating factor (BAFF) was identified by our group and others as a novel therapeutic target for the treatment of autoimmune diseases. To expand upon this, we utilized microarrays to screen for molecules upregulated in B cells from BAFF-inhibited mice with lupus-like disease and identified metabotropic glutamate receptor 3 (Grm3). In addition to confirming the expression of this receptor in B cells, a synthetic agonist of Grm3 was found to downregulate B cells and ameliorate autoimmune symptoms in mice. Conversely, a Grm3 antagonist increased B-cell numbers and further aggravated disease. Thus, these results suggest that activation of Grm3 ameliorates lupus-like disease in mice by reducing B cell numbers. Not only do the findings presented in this study increase our understanding of the inhibitory signals initiated on the surface of B cells, but they also identify a novel potential target for the treatment of autoimmune diseases.

IL-15-secreting γδT cells induce memory T cells in experimental allergic encephalomyelitis (EAE) mice.

With the most recent data suggesting γδT cells as primary producers of the pro-inflammatory autoimmune-associated cytokine, the relationship between γδT cells and Th17 in experimental allergic encephalitis (EAE) mice requires more extensive investigation. By flow cytometry and qPCR, we identified a new subset of IL-15-secreting γδT (γδT15) cells that increased in EAE mice. The capacity of IL-15-secreting γδT cells inducing memory T cells and memory T cells inducing IL-17(+)Th17 was examined by transferring into EAE mice and 7-week-old female nude mice, respectively. We found that γδT15 induced CD44(hi) memory T cells by secreting IL-15. γδT15-induced memory T cells induced EAE by transforming into pathogenic Th17 cells. The data suggest that a new subset of IL-15-secreting γδT cells mediated the production of memory T cells which transformed into pathogenic Th17 cells in EAE mice.

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling.

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8(+) T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.

Exosomes with membrane-associated TGF-ß1 from gene-modified dendritic cells inhibit murine EAE independently of MHC restriction.

We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF-ß1 (sTGF-ß1-EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane-associated TGF-ß1 (mTGF-ß1-EXOs) and found mTGF-ß1-EXOs had more potent immunosuppressive activity than sTGF-ß1-EXOs in vitro. Treatment of mice with mTGF-ß1-EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE even after disease onset. Treatment of mice with mTGF-ß1-EXOs also impaired Ag-specific Th1 and IL-17 responses, but promoted IL-10 responses ex vivo. Treatment with mTGF-ß1-EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down-regulation of the p38, ERK, Stat3, and NF-κB activation and IL-6 expression in DCs. Treatment with mTGF-ß1-EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4(+)Foxp3(+)Treg cells from mTGF-ß1-EXO-treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF-ß1-EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide-induced EAE in BALB/c mice. These results indicate that mTGF-ß1-EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.

Pharmacophore-based drug design and biological evaluation of novel ABCB1 inhibitors.

Overexpression of ABCB1 is one of major barriers for multidrug resistance in chemotherapy and limits drug oral bioavailability. Inhibition of ABCB1 would sensitize multidrug resistance in clinical cancer chemotherapy. With this aim, a 3D pharmacophore model was created based on known ABCB1 inhibitors with correlation coefficient of 0.94, comprising three hydrophobic features and one hydrogen bond acceptor. It was further validated and used to search our in-house 3D database for potential ABCB1 inhibitors. The inhibitory activities of the best hits were evaluated by several biological assays, such as rhodamine 123 accumulation assay, chemosensitization assay, multidrug resistance 1-Madin-Darby canine kidney cells/Madin-Darby canine kidney cells permeability assay. Finally, compounds YZ-3 and YZ-16 were identified as potential leads to be developed in the designing of novel potent ABCB1 inhibitors.