Induction Of Inflammatory Cytokines Research Articles

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

Abstract 5294: Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy

Abstract Introduction: Tumor-specific recruitment of co-stimulatory bispecific antibodies (bsAbs) has emerged as a promising therapeutic strategy. Here, we investigated pH-selective CD28xVISTA bsAbs to act selectively within the acidic tumor microenvironment (TME). Our CD28xVISTA bsAbs are designed for tripartite “trans-activation” of CD28 in the TME, aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. Experimental Procedures: We evaluated various CD28xVISTA bsAbs, focusing on a prototype 1+2 format with monovalent CD28 binding, bivalent VISTA binding and Fc receptor interaction null mutations (BS2). BS2 was tested for T-cell trans-activation using Jurkat-IL-2-luciferase reporter cells in the presence of HEK293 cells expressing membrane bound OKT3-scFv and CHO cells expressing human VISTA. BS2’s potency was further evaluated in an xCelligence-based human T-cell killing assay that dynamically monitors growth of LNCaP prostate cancer cells co-cultured with VISTA+ Kasumi-3 cells, alone or in combination with a CD3xPSMA bispecific T-cell engager. T-cell activation and proliferation were also measured using flow cytometry with CD3, CD4, CD8 and CD25 markers. Additionally, we modified BS2 to introduce pH-selective VISTA binding, limiting its activity to the TME, and tested this version in a syngeneic mouse model in combination with anti-murine PD-1 (anti-mPD-1). Finally, cytokine release from human PBMCs, co-cultured with human umbilical vein endothelial cells (HUVECs) and treated with the pH-selective BS2 (or TGN1412 as a positive control), was measured using a bead-based multiplex immune assay. Results: Our data show that the combination of anti-mPD-1 and our prototype CD28xVISTA bsAb (BS2) with pH-selective VISTA binding significantly inhibited tumor growth in vivo. In vitro, CD28xVISTA co-stimulation in trans by BS2 potentiated the activity of a CD3xPSMA bispecific T-cell engager in our human T-cell killing assay. Cytokine release assessments demonstrated negligible induction of inflammatory cytokines, indicating a favorable safety profile for this antibody. Conclusion: Our study demonstrates the feasibility of cis and trans CD28 co-stimulation using a CD28xVISTA bsAb (BS2). This approach, which bypasses the need for tumor-associated antigens (TAAs) required by other CD28xTAA bispecifics in development, suggests a potentially safer alternative for T-cell engagement and stimulation. Moreover, our novel myeloid-directed TME-selective co-stimulation strategy with a CD28xVISTA bsAb may broaden the potential for T-cell engagement approaches in solid tumors by enabling rational combinations with existing CD3xTAA T-cell engagers without competing for the same target. Citation Format: Thomas Thisted, Zhi-Gang Jiang, Zuzana Biesova, Adejumoke Onumajuru, Yuliya Kleschenko, Kanam Malhotra, Vikas Saxena, Arnab Mukherjee, F. Donelson Smith, Edward H. van der Horst. Conditionally active CD28xVISTA bispecific antibodies induce myeloid-driven tumor-specific T-cell co-stimulation for improved cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5294.

SCLEROSTIN-MEDIATED IMPAIRED OSTEOGENESIS BY FIBROBLAST-LIKE SYNOVIOCYTES IN THE PARTICLE-INDUCED OSTEOLYSIS MODEL

Wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of RANL in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particletreated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis.

Mechanistic Studies of Cytokine Release Syndrome (CRS) with Roles of Interferon-Gamma (IFN-g) and Tumor Necrosis Factor Alpha (TNF-α) While Maintaining CAR-T Function in Vitro

Background: Chimeric Antigen Receptor (CAR) T cell therapy in patients with hematologic malignancies is limited by immune-related toxicities such as Cytokine Release Syndrome (CRS) in over 30% of patients, whereas no CRS was observed in patients receiving NK cell therapy. CRS is characterized by an early phase with fever, hypotension, and elevations of cytokines, including IFN-γ, GM-CSF, TNF-α , IL-10, and IL-6. Methods: To study the mechanisms of CRS, we developed a model of in vitro CRS assay in which different effector cells, tumor target cells, and immature dendritic cells (iDC) were co-cultured, and the induction of inflammatory cytokines was measured by ELISA. To generate immature dendritic cells (iDC) for the assay, monocytes were cultured in the presence of GM-CSF and IL-4 for 4 to 6 days with the addition of fresh cytokines every 2 or 3 days, and the assay was set up on day 5 of iDC culture. This “priming” step was essential for the production of IL-6 by iDC (confirmed by scRNA-seq) after exposure of CART to target cells. Effector cells including CAR-T cells (targeting CD19 or BCMA), CAR- Memory-Like NK cells (CAR-ML-NK), and CAR invariant NKT (CAR-iNKT) cells were evaluated in our model. Results: In our initial in vitro CRS assay, we conducted an experiment by using CART19 (19-28BBζ, 25,000 cells), which was co-cultured with CD19+ tumor (Ramos CBR/GFP, 50,000 cells) and iDC (5000 cells) for 24 to 48hrs. Secreted IL-6, a surrogate marker of CRS, was determined using a human IL-6 ELISA. In vitro killing efficacy of CART19 was determined by bioluminescent imaging (BLI) 24 hrs post co-culture of CART19 with Ramos CBR/GFP targets. A marked induction of CRS-related cytokines (IL-6, IFNγ, TNFα) was observed 24 and 48 hr post-coculture. Similar results were also found when we used CAR-T cell targeting BCMA with BCMA+ MM.1S target and iDC. In contrast, neglibile levels of IL-6, IFNγ, and TNFα secretion were detected when BCMA CAR Memory-Like Natural Killer cells (MLNK) were used compared to BCMA CAR T cells in spite of comparable levels of in vitro killing of BCMA+ MM.1S target cells by both BCMA-CART and BCMA-CAR-MLNK. With the BCMA CAR-iNKT, levels of IL-6, IFN-γ, and TNF-α were intermediate to those of BCMA CAR T cells and BCMA-CAR-MLNK again in spite of identical killing of BCMA+ targets in vitro. To test the effect of kinase inhibitors (Amatya et al, Blood,140:12686) and neutralizing antibodies to block CRS (IL-6 secretion by iDCs) and their impact on CAR-T cell killing, we performed the CRS assay in the presence or absence of small JAK1/2 inhibitors and PI3Kg/d inhibitors (ruxolitinib and duvelisib) and neutralizing antibodies (against IFN-γ, TNF-α, GM-CSF). Both ruxoliltinib and duvelisib and neutralizing antibodies (anti-IFN-γ and anti-TNF-α but not GM-CSF) significantly reduced IL-6 levels (Fig 1). In contrast to dasatinib which completely blocked both CRS and CART killilng (data not shown), no significant attenuation of CAR-T killing efficacy was found with any of these kinase inhibitors or the neutralizing antibodies. The CART CRS assay was adapted to measure both CRS (IL-6 secretion) and target cell killing in response to BiTEs such as blinatumomab. Similar to CART19 blinatumomab induced a dose-dependent increase in IL-6 secretion in the presence of T cells, target Ramos cells, and iDC. CRS (in vitro production of IL-6) was dependent on the presence of GM-CSF/IL-4 “primed” iDC and abolished by duvelisib while maintaining blinatumomab-induced Ramos killing (Fig 2). Finally, we also developed and in vitro mouse CRS assay by coculturing mouse CART19, target (A20 CBR/GFP) cells and mouse bone marrow-derived macrophages. Marked increase in secreted mouse IL-6 was observed which was completely inhibited by duvelisib, ruxolitinib, anti-IFNγ and anti-TNFα. Conclusion: We have developed in vitro CRS co-culture models using cells from both man and mouse and tested the role of multiple effector cell types including CART cells, CARMLNK cells, and CAR-iNK cells and multiple inhibitors on CRS and CART function in vitro. We show that JAK1/2 and PI3Kg/d kinase inhibitors as well as neutralizing antibodies to IFNγ and TNFα all block in vitro CRS without attenuating the anti-tumor efficacy of CAR-T cells. Our result suggests IFNγ, TNFα, JAK1/2 and PI3Kg/d kinases inhibitors are rational targets for CRS mitigation approaches without compromising CART-mediated anti-tumor efficacy in the clinic.

RSK1 Is an Exploitable Dependency in Myeloid Malignancies

Myeloid malignancies harbor distinct molecular drivers but share convergence of oncogenic signaling pathways and propagation by ripe pro-inflammatory niches. To delineate these hallmarks across the spectrum of myeloid disease states, we established the largest comprehensive atlas of myeloproliferative neoplasms (MPN) and secondary acute myeloid leukemia (sAML) through RNA-sequencing and mass cytometry (CyTOF) of 370 primary samples encompassing CD34+ hematopoietic stem/progenitor cells and CD14+ monocytes, revealing aberrant of PI3K/AKT/mTOR signalling and NFκB-mediated hyper-inflammation. As a central mediator of these hyperactive signaling pathways, ribosomal S6 kinases (RSKs) represent potentially attractive novel therapeutic targets warranting further evaluation. Using both genetic approaches and a first-in-class oral RSK inhibitor, PMD-026, that is currently being evaluated in phase 1/1b clinical trials (NCT04115306) in metastatic and triple negative breast cancer, we found that RSK1 ( RPS6KA1) inhibition potently induced apoptosis and G2/M arrest in leukemia models in conjunction with suppression of PI3K/AKT/mTOR and NFκB signaling. Further NFκB cascade profiling following RSK1 perturbation revealed reduced phosphorylation of IKKα/β and p65/RELA and prevention of p65/RELA nuclear translocation. Using RNA-seq, ATAC-seq and H3K27ac CUT&Tag, we found that PMD-026 treatment altered chromatin accessibility and inhibited NFKB1, TNF, and cell cycle regulators CCNA1/2 and CDK1/2. In cytokine CyTOF, PMD-026 nearly abrogated all induction of inflammatory cytokines including TNF, IL-6, IL-8, CCL3, and CCL4 in primary MPN monocytes to levels substantially below basal conditions. We further extended our findings to de novo AML harboring FLT3 internal tandem duplication ( FLT3-ITD), where FLT3-ITD patients exhibited elevated RPS6KA1 expression and those who developed resistance to gilteritinib demonstrated progressively increased RPS6KA1 expression. FLT3-ITD cells exhibited preferentially sensitivity to RSK1 inhibition, where we uncovered bi-directional regulation. Through cycloheximide, MG-132, and ubiquitination assays, we found that RSK1 regulates FLT3-ITD activity and protein stability through deubiqutinase USP1, and where either RPS6KA1 or USP1 inhibition resulted in concomitant downregulation of FLT3 protein and cell lethality. Multivariate analysis of the TCGA and BeatAML cohorts (n= 568 patients) revealed high median USP1 or RPS6KA1 expression to be significantly associated with poor survival, thus defining a RSK1-USP1-FLT3 axis with prognostic relevance. Across in vivo models, PMD-026 ameliorated MPN disease features driven by MPL W515L including leukocytosis, splenomegaly, and bone marrow fibrosis, while prolonging survival, in conjunction with suppression of inflammatory cytokines including TNF, IL-6 and IL-1b. We then evaluated PMD-026 across five patient-derived xenograft (PDX) models spanning the spectrum of myeloid malignancies including myelofibrosis (MF), sAML, and chronic myelomonocytic leukemia (CMML) encompassing various driver and multiple high-risk mutations. Treatment in MF PDX mice decelerated disease progression, extended survival, and reduced splenomegaly, while in CMML/sAML PDXs, PMD-026 led to a 67 to 96 percent reduction in human CD45+ cells engraftment. Finally, PMD-026 treatment in both mice xenografted with MV4-11 cells and syngeneic Flt3ITDTet2KO mice significantly reduced leukemic cell engraftment and prolonged survival. These data indicate potent efficacy of RSK inhibition across diverse models of chronic and acute myeloid malignancies. Lastly, we investigated the impact of PMD-026 on hematological parameters from 41 breast cancer patients enrolled in the phase 1/1b clinical trial. Overall, there was no sustained suppressive effect on hematological parameters and there were no hematological PMD-026-related toxicities meeting criteria for dose modification during the trial period. These clinical data demonstrate that PMD-026 did not cause myelosuppression. Our findings uncover a novel therapeutic avenue for a conserved RSK1 dependency across chronic and acute myeloid neoplasms. The potent and consistent disease-ameliorating effects across numerous leukemia mouse models demonstrates promise for repurposing PMD-026 for the treatment of myeloid malignancies.

α-MANGOSTIN/CLOVE OIL-INCORPORATED LAURIC ACID-BASED SOLVENT REMOVAL-INDUCED IN SITU FORMING MATRICES

Typically, the bacteria in periodontal pocket of periodontitis disease are capable of producing a number of virulence factors, which are directly toxic to host tissues or immune cells, or indirectly damage host tissues via induction of inflammatory cytokines. The eradication of these microbes from periodontal pocket is one of crucial processes of periodontitis treatment. The α-mangostin from Garcinia mangostana Linn has the most potent antibacterial activity with less tendency to acquire resistance; therefore, it is interesting for using as active compound for periodontitis treatment. This study focuses on the development of in situ forming matrices (ISM) involving 50% w/w lauric acid, 5% w/w clove oil and different α-mangostin amount in NMP for periodontal drug delivery. Physicochemical properties of ISM were investigated including pH, density, viscosity, surface tension, in vitro matrix formation, and injectability. Antimicrobial activities of formulated ISM against three standard microbes were evaluated using agar cup diffusion methods. The pH and density of all α-mangostin and clove oil-loaded 50% w/w lauric acid matrices was 4.40 ± 0.04 to 4.78 ± 0.20 and 0.960 ± 0.000 to 0.963 ± 0.001 g/cm3, respectively. The increasing α-mangostin amounts significantly increased the viscosity of prepared formulation and injection force. All formulations had low viscosity that it was easy to injection and spreadable in the periodontal pockets. After contact PBS pH 6.8, they transformed as matrix and the hydrophobic manner of clove oil retarded a solvent exchange. These prepared formulations showed the antimicrobial activities against Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Candida albicans ATCC 17011. Hence, they are the potential systems for periodontal drug delivery.

Oestrogen receptor-independent actions of oestrogen in cancer.

Oestrogen, the primary female sex hormone, plays a significant role in tumourigenesis. The major pathway for oestrogen is via binding to its receptor [oestrogen receptor (ERα or β)], followed by nuclear translocation and transcriptional regulation of target genes. Almost 70% of breast tumours are ER + , and endocrine therapies with selective ER modulators (tamoxifen) have been successfully applied. As many as 25% of tamoxifen-treated patients experience disease relapse within 5years upon completion of chemotherapy. In such cases, the ER-independent oestrogen actions provide a plausible explanation for the resistance, as well as expands the existing horizon of available drug targets. ER-independent oestrogen signalling occurs via one of the following pathways: signalling through membrane receptors, oxidative catabolism giving rise to genotoxic metabolites, effects on mitochondria and redox balance, and induction of inflammatory cytokines. The current review focuses on the non-classical oestrogen signalling, its role in cancer, and its clinical significance.

Functional characterization of porcine nucleophosmin (NPM1) gene in promoting the replication of Japanese encephalitis virus and induction of inflammatory cytokines

Nucleophosmin (NPM1) is a multifunctional nucleolar protein that plays a role in cell cycle control, tumorigenesis, induction of the inflammatory cytokine, virus replication, as well as the cellular responses to a variety of stress stimuli. However, its physiological functions in pigs have not been well understood. Here, we cloned the porcine NPM1 (porNPM1) gene and analyzed the functions of the porNPM1 protein in pigs. The full-length porNPM1 gene encoded a 294-amino acid protein with 94.5%–99.3% sequence identity to its orthologues in mammals and was extensively expressed in various pig tissues at the mRNA level. The porNPM1 primarily localizes in the nucleus of ST cells, while it translocates from the nucleus to nucleoplasm upon UV irradiation or H2O2 treatment. Notably, JEV infection blocked the translocation of porNPM1 from the nucleolus to the nucleoplasm. Furthermore, porNPM1 interacted with the JEV C protein and facilitated JEV replication in ST cells. The overexpression and knockdown of porNPM1 respectively enhanced or impaired JEV replication, suggesting the important role of porNPM1 in JEV replication. Additionally, the purified ectodomain of porNPM1 induced the production of inflammatory cytokines (TNF-α, IL-6, and IL-8). Together, these data demonstrated that porNPM1 is involved in cellular stress stimuli, JEV replication, and induction of inflammatory cytokines.

Gelsevirine is a novel STING-specific inhibitor and mitigates STING-related inflammation in sepsis.

Stimulation of IFN genes (STING) is central to the production of interferon and proinflammatory cytokines in response to microbial DNA or self-DNA in the cytosol. The detrimental role of the activation of STING during sepsis has been well documented. Here, we found that gelsevirine (GS) potently inhibit interferon and inflammatory cytokine induction in macrophages exposed to STING agonists (2'3'-cGAMP, IFN stimulatory DNA (ISD), and poly(dA:dT)). I n silico docking analysis and surface plasmon resonance binding study showed that GS bonds with high affinity to the cyclic dinucleotide (CDN)-binding pocket of STING. Biotin pull-down assay also confirmed that GS competitively bonded to STING protein. Furthermore, GS inhibited 2'3'-cGAMP-induced STING dimerization and subsequent activation. In addition, GS induced K48-linked STING ubiquitination and degradation, which was likely through upregulating and recruiting TRIM21. In mice exposed to cecal ligation and puncture (CLP)-induced sepsis, post-operative administration of GS significantly extended the survival period and mitigated acute organ damage. Overall, GS inhibited STING signaling by competitively binding to the CDN-binding pocket to lock STING in an inactive open conformation, while also promoting K48-linked STING ubiquitination and degradation. Our findings identify a novel STING-specific inhibitor that could be applied in the treatment of sepsis.

The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling

BackgroundThe inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, ‘cytokine storm’, harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo.MethodsShort B7 and CD28 receptor dimer interface mimetic peptides were synthesized and tested for their ability to attenuate the inflammatory cytokine response of human peripheral blood mononuclear cells, as well as for their ability to attenuate B7/CD28 intercellular receptor engagement. Mice were used to test the ability of such peptides to protect from lethal superantigen toxin challenge when administered in molar doses far below the toxin dose.ResultsB7 and CD28 homodimer interfaces are remote from the coligand binding sites, yet our finding is that by binding back into the receptor dimer interfaces, short dimer interface mimetic peptides inhibit intercellular B7-2/CD28 as well as the tighter B7-1/CD28 engagement, attenuating thereby pro-inflammatory signaling. B7 mimetic peptides exhibit tight selectivity for the cognate receptor in inhibiting intercellular receptor engagement with CD28, yet each diminishes signaling through CD28. In a prominent example of inflammatory cytokine storm, by attenuating formation of the B7/CD28 costimulatory axis, B7-1 and CD28 dimer interface mimetic peptides protect mice from lethal toxic shock induced by a bacterial superantigen even when administered in doses far submolar to the superantigen.ConclusionsOur results reveal that the B7 and CD28 homodimer interfaces each control B7/CD28 costimulatory receptor engagement and highlight the protective potential against cytokine storm of attenuating, yet not ablating, pro-inflammatory signaling via these receptor domains.

Novel nanoadjuvants balance immune activation with modest inflammation: implications for older adult vaccines

BackgroundAge-associated impairments of immune response and inflammaging likely contribute to poor vaccine efficacy. An appropriate balance between activation of immune memory and inflammatory response may be more effective in vaccines for older adults; attempts to overcome reduced efficacy have included the addition of adjuvants or increased antigenic dose. Next generation vaccine formulations may also use biomaterials to both deliver and adjuvant vaccine antigens. In the context of aging, it is important to determine the degree to which new biomaterials may enhance antigen-presenting cell (APC) functions without inducing potent inflammatory responses of APCs or other immune cell types (e.g., T cells). However, the effect of newer biomaterials on these cell types from young and older adults remains unknown.ResultsIn this pilot study, cells from young and older adults were used to evaluate the effect of novel biomaterials such as polyanhydride nanoparticles (NP) and pentablock copolymer micelles (Mi) and cyclic dinucleotides (CDN; a STING agonist) on cytokine and chemokine secretion in comparison to standard immune activators such as lipopolysaccharide (LPS) and PMA/ionomycin. The NP treatment showed adjuvant-like activity with induction of inflammatory cytokines, growth factors, and select chemokines in peripheral blood mononuclear cells (PBMCs) of both young (n = 6) and older adults (n = 4), yet the degree of activation was generally less than LPS. Treatment with Mi or CDN resulted in minimal induction of cytokines and chemokine secretion with the exception of increased IFN-α and IL-12p70 by CDN. Age-related decreases were observed across multiple cytokines and chemokines, yet IFN-α, IL-12, and IL-7 production by NP or CDN stimulation was equal to or greater than in cells from younger adults. Consistent with these results in aged humans, a combination nanovaccine composed of NP, Mi, and CDN administered to aged mice resulted in a greater percentage of antigen-specific CD4+ T cells and greater effector memory cells in draining lymph nodes compared to an imiquimod-adjuvanted vaccine.ConclusionsOverall, our novel biomaterials demonstrated a modest induction of cytokine secretion with a minimal inflammatory profile. These findings suggest a unique role for biomaterial nanoadjuvants in the development of next generation vaccines for older adults.

Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases.

Changes in the organization and structure of the fibronectin matrix are believed to contribute to dysregulated wound healing and subsequent tissue inflammation and tissue fibrosis. These changes include an increase in the EDA isoform of fibronectin as well as the mechanical unfolding of fibronectin type III domains. In previous studies using embryonic foreskin fibroblasts, we have shown that fibronectin's EDA domain (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for Toll-Like Receptor-4 (TLR4). However, the role of signaling molecules downstream of TLR-4 such as TGF-β Activated Kinase 1 (TAK1) and Mitogen activated protein kinases (MAPK) in regulating the expression of fibronectin DAMP induced inflammatory genes in specific cell types is not known. In the current study, we evaluate the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines: embryonic foreskin, adult dermal, and adult kidney. The fibronectin derived DAMPs each induce the phosphorylation and activation of TAK1 which results in the activation of two downstream signaling arms, IKK/NF-κB and MAPK. Using the specific inhibitor 5Z-(7)-Oxozeanol as well as siRNA, we show TAK1 to be a crucial signaling mediator in the release of cytokines in response to fibronectin DAMPs in all three cell types. Finally, we show that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines.

SARS-CoV-2 induces cardiomyocyte apoptosis and inflammation but can be ameliorated by ACE inhibitor Captopril

Although the clinical manifestation of COVID-19 is mainly respiratory symptoms, approximately 20% of patients suffer from cardiac complications. COVID-19 patients with cardiovascular disease have higher severity of myocardial injury and poor outcomes. The underlying mechanism of myocardial injury caused by SARS-CoV-2 infection remains unclear. Using a non-transgenic mouse model infected with Beta variant (B.1.351), we found that the viral RNA could be detected in lungs and hearts of infected mice. Pathological analysis showed thinner ventricular wall, disorganized and ruptured myocardial fiber, mild inflammatory infiltration, and mild epicardia or interstitial fibrosis in hearts of infected mice. We also found that SARS-CoV-2 could infect cardiomyocytes and produce infectious progeny viruses in human pluripotent stem cell-derived cardiomyocyte-like cells (hPSC-CMs). SARS-CoV-2 infection caused apoptosis, reduction of mitochondrial integrity and quantity, and cessation of beating in hPSC-CMs. In order to dissect the mechanism of myocardial injury caused by SARS-CoV-2 infection, we employed transcriptome sequencing of hPSC-CMs at different time points after viral infection. Transcriptome analysis showed robust induction of inflammatory cytokines and chemokines, up-regulation of MHC class I molecules, activation of apoptosis signaling and cell cycle arresting. These may cause aggravate inflammation, immune cell infiltration, and cell death. Furthermore, we found that Captopril (hypotensive drugs targeting ACE) treatment could alleviate SARS-CoV-2 induced inflammatory response and apoptosis in cardiomyocytes via inactivating TNF signaling pathways, suggesting Captopril may be beneficial for reducing COVID-19 associated cardiomyopathy. These findings preliminarily explain the molecular mechanism of pathological cardiac injury caused by SARS-CoV-2 infection, providing new perspectives for the discovery of antiviral therapeutics.

Identification of Salmonella Pullorum Factors Affecting Immune Reaction in Macrophages from the Avian Host.

The host-specific Salmonella serovar S. Pullorum (SP) modulates the chicken immune response to a Th2-biased response associated with persistent infection. This is different from the Th1-biased immune response induced by the genetically close serovar, S. Enteritidis (SE). Based on core genome differences between SP and SE, we used three complementary bioinformatics approaches to identify SP genes, which may be important for stimulation of the immune response. Defined mutants were constructed in selected genes, and the infection potential and ability of mutants to stimulate cytokine production in avian derived HD11 macrophages were determined. Deletion of large genomic regions unique to SP did not change infection potential nor immune stimulation significantly. Mutants in genes with conserved single nucleotide polymorphisms (SNPs) between the two serovars in the region 100 bp upstream of the start codon (conserved upstream SNPs [CuSNPs]) such as sseE, osmB, tolQ, a putative immune antigen, and a putative persistent infection factor, exhibited differences in induction of inflammatory cytokines compared to wild-type SP, suggesting a possible role of these CuSNPs in immune regulation. Single nucleotide SP mutants correcting for the CuSNP difference were constructed in the upstream region of sifA and pipA. The SNP corrected pipA mutant expressed pipA at a higher level than the wild-type SP strain, and the mutant differentially caused upregulation of proinflammatory cytokines. It suggests that this CuSNP is important for the suppression of proinflammatory responses. In conclusion, this study has identified putative immune stimulating factors of relevance to the difference in infection dynamics between SP and SE in avian macrophages. IMPORTANCE Salmonella Pullorum is host specific to avian species, where it causes life-threatening infection in young birds. It is unknown why it is host restricted and causes systemic disease, rather than gastroenteritis normally seen with Salmonella. In the present study, we identified genes and single nucleotide polymorphisms (SNPs; relative to the broad-host-range type Salmonella Enteritidis), which affected survival and immune induction in macrophages from hens suggesting a role in development of the host specific infection. Further studies of such genes may enable understanding of which genetic factors determine the development of host specific infection by S. Pullorum. In this study, we developed an in silico approach to predict candidate genes and SNPs for development of the host-specific infection and the specific induction of immunity associated with this infection. This study flow can be used in similar studies in other clades of bacteria.

Therapeutic Effects of an Anti-Sialyl Lewis x Antibody in a Murine Model of Acute Lung Injury.

Acute respiratory distress syndrome is a life-threatening acute lung injury (ALI) characterized by the destruction of alveoli leading to pulmonary edema. The infiltration and activation of inflammatory cells and production of inflammatory cytokines are both involved in the pathogenesis of ALI. Here, we show that the infiltration of neutrophils, major inflammatory cells causing ALI, into the lung is mediated by sialyl Lewis x (sLex) glycans, which can be efficiently suppressed by a monoclonal antibody (mAb) against these glycans. In fucosyltransferase-IV and -VII double-deficient mice lacking sLex expression, neutrophil infiltration into the lung was significantly suppressed compared with that observed in wild-type mice in a lipopolysaccharide (LPS)-induced ALI model. Administration of a highly specific anti-sLex mAb F2 3 hours after LPS administration significantly suppressed pulmonary neutrophil infiltration, accompanied by the reduced induction of inflammatory cytokines. It was consistently indicated from ex vivo cell rolling assay that mAb F2 blocked the rolling of mouse neutrophils on P-selectin-expressing cells. Overall, these results indicate that the sLex glycan could serve as a therapeutic target against ALI, and also that mAb F2 would be useful for specific targeting of this glycan.

Dectin-1 deficiency alleviates diabetic cardiomyopathy by attenuating macrophage-mediated inflammatory response

Cardiovascular diseases are the primary cause of mortality in patients with diabetes and obesity. Hyperglycemia and hyperlipidemia in diabetes alters cardiac function, which is associated with broader cellular processes such as aberrant inflammatory signaling. Recent studies have shown that a pattern recognition receptor called Dectin-1, expressed on macrophages, mediates pro-inflammatory responses in innate immunity. In the present study, we examined the role of Dectin-1 in the pathogenesis of diabetic cardiomyopathy. We observed increased Dectin-1 expression in heart tissues of diabetic mice and localized the source to macrophages. We then investigated the cardiac function in Dectin-1-deficient mice with STZ-induced type 1 diabetes and high-fat-diet-induced type 2 diabetes. Our results show that Dectin-1 deficient mice are protected against diabetes-induced cardiac dysfunction, cardiomyocyte hypertrophy, tissue fibrosis, and inflammation. Mechanistically, our studies show that Dectin-1 is important for cell activation and induction of inflammatory cytokines in high-concentration glucose and palmitate acid (HG + PA)-challenged macrophages. Deficiency of Dectin-1 generate fewer paracrine inflammatory factors capable of causing cardiomyocyte hypertrophy and fibrotic responses in cardiac fibroblasts. In conclusion, this study provides evidence that Dectin-1 mediates diabetes-induced cardiomyopathy through regulating inflammation. Dectin-1 may be a potential target to combat diabetic cardiomyopathy.

Interactions of Carboxylated Nanodiamonds With Mouse Macrophages Cell Line and Primary Cells

Nanodiamonds (ND) have attracted significant interest for their use in several biomedical applications. These applications can be very useful if the safety and compatibility of ND are proven. We assessed the effects of ND (100 nm, Carboxylated) on primary macrophages and a macrophage-like cell line and found that these particles are not toxic to these cells at lower concentrations but may interfere with cell functions and differentiation. Internalization of ND by these cells in a time- and dose-dependent manner was mostly via phagocytosis and clathrin-dependent endocytosis and localized to the cytoplasm but not into the nucleus. No significant induction of inflammatory cytokines or reduction in the ability of these cells to respond to lipopolysaccharides (LPS) was noted. However, the endocytic activity of these cells is significantly reduced. In addition, ND exposure reduced the ability of differentiating bone marrow cells to express macrophage surface markers. Measurement of the fluorescence and absorbance of ND-treated cells clearly showed the ability of these particles to produce a signal at different wavelengths. Therefore, it is important to consider interference of ND in different colorimetric and fluorometric assays when testing interactions or effects of ND on cells. Our findings suggest that ND are not cytotoxic to macrophages at the tested concentrations, but it can interfere with macrophage functions and differentiation and may interfere with assays’ result through the production of a signal at different wavelengths.

Discovery of Novel STING Inhibitors Based on the Structure of the Mouse STING Agonist DMXAA.

The stimulator-of-interferon-gene (STING) protein is involved in innate immunity. The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) proved to be a potent murine-STING (mSTING) agonist but had little effect on human-STING (hSTING). In this paper, we draw upon the comparison of different crystal structures and protein-ligand interaction relationships analysis to venture the hypothesis that the drug design of DMXAA variants has the potential to convert STING agonists to inhibitors. Based on our previous discovery of two DMXAA analogs, 3 and 4 (both could bind to STING), we structurally optimized them and synthesized new derivatives, respectively. In binding assays, we found compounds 11 and 27 to represent STING binders that were superior to the original structures and discussed the structure-activity relationships. All target compounds were inactive in cellular assays for the screening of STING agonistic activity. Gratifyingly, we identified 11 and 27 as STING inhibitors with micromolar activity in both hSTING and mSTING pathways. In addition, 11 and 27 inhibited the induction of interferon and inflammatory cytokines activated by 2'3'-cGAMP without apparent cytotoxicity. These findings break the rigid thinking that DMXAA provides the structural basis specifically for STING agonists and open up more possibilities for developing novel STING agonists or inhibitors.

Targeting RIPK1 kinase for modulating inflammation in human diseases.

Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) is a master regulator of TNFR1 signaling in controlling cell death and survival. While the scaffold of RIPK1 participates in the canonical NF-κB pathway, the activation of RIPK1 kinase promotes not only necroptosis and apoptosis, but also inflammation by mediating the transcriptional induction of inflammatory cytokines. The nuclear translocation of activated RIPK1 has been shown to interact BAF-complex to promote chromatin remodeling and transcription. This review will highlight the proinflammatory role of RIPK1 kinase with focus on human neurodegenerative diseases. We will discuss the possibility of targeting RIPK1 kinase for the treatment of inflammatory pathology in human diseases.

Impact of endothelial nitric oxide synthase activation on accelerated liver regeneration in a rat ALPPS model

Although the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) induces more rapid liver regeneration than portal vein embolization, the mechanism remains unclear. To assess the influence of inflammatory cytokines and endothelial nitric oxide synthase (eNOS) activation on liver regeneration in ALPPS. The future liver remnant/body weight (FLR/BW) ratio, hepatocyte proliferation, inflammatory cytokine expression, and activation of the Akt-eNOS pathway were evaluated in rat ALPPS and portal vein ligation (PVL) models. Hepatocyte proliferation was assessed based on Ki-67 expression, which was confirmed using immunohistochemistry. The serum concentrations of inflammatory cytokines were measured using enzyme linked immune-solvent assays. The Akt-eNOS pathway was assessed using western blotting. To explore the role of inflammatory cytokines and NO, Kupffer cell inhibitor gadolinium chloride (GdCl3), NOS inhibitor N-nitro-arginine methyl ester (L-NAME), and NO enhancer molsidomine were administered intraperitoneally. The ALPPS group showed significant FLR regeneration (FLR/BW: 1.60% ± 0.08%, P < 0.05) compared with that observed in the PVL group (1.33% ± 0.11%) 48 h after surgery. In the ALPPS group, serum interleukin-6 expression was suppressed using GdCl3 to the same extent as that in the PVL group. However, the FLR/BW ratio and Ki-67 labeling index were significantly higher in the ALPPS group administered GdCl3 (1.72% ± 0.19%, P < 0.05; 22.25% ± 1.30%, P < 0.05) than in the PVL group (1.33% ± 0.11% and 12.78% ± 1.55%, respectively). Phospho-Akt Ser473 and phospho-eNOS Ser1177 levels were enhanced in the ALPPS group compared with those in the PVL group. There was no difference between the ALPPS group treated with L-NAME and the PVL group in the FLR/BW ratio and Ki-67 labeling index. In the PVL group treated with molsidomine, the FLR/BW ratio and Ki-67 labeling index increased to the same level as in the ALPPS group. Early induction of inflammatory cytokines may not be pivotal for accelerated FLR regeneration after ALPPS, whereas Akt-eNOS pathway activation may contribute to accelerated regeneration of the FLR.