Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor

Drosophila peptidoglycan recognition protein LC (PGRP-LC), a transmembrane protein required for the response to bacterial infection, acts at the top of a cytoplasmic signaling cascade that requires the death-domain protein Imd and an IkappaB kinase to activate Relish, an NF-kappaB family member. It is not clear how binding of peptidoglycan to the extracellular domain of PGRP-LC activates intracellular signaling because its cytoplasmic domain has no homology to characterized proteins. Here, we demonstrate that PGRP-LC binds Imd and that its cytoplasmic domain is critical for its activity, suggesting that PGRP-LC acts as a signal-transducing receptor. The PGRP-LC cytoplasmic domain is also essential for the formation of dimers, and results suggest that dimerization may be required for receptor activation. The PGRP-LC cytoplasmic domain can mediate formation of heterodimers between different PGRP-LC isoforms, thereby potentially expanding the diversity of ligands that can be recognized by the receptor.     

Olfactomedin 4 down-regulates innate immunity against Helicobacter pylori infection

Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 KO mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with WT littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1α was increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF--κB pathway and has a negative feedback effect on NF-κB activation induced by H. pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization.     

Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression

Smac mimetics target cancer cells in a TNFα-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFα-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFα via NF-κB and modulation of the NF-κB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome.     

Interleukin-23 production in dendritic cells is negatively regulated by protein phosphatase 2A

IL-12 and IL-23 are produced by activated antigen-presenting cells but the two induce distinct immune responses by promoting Th1 and Th17 cell differentiation, respectively. IL-23 is a heterodimeric cytokine consisting of two subunits: p40 that is shared with IL-12 and p19 unique to IL-23. In this study, we showed that the production of IL-23 but not IL-12 was negatively regulated by protein phosphatase 2A (PP2A) in dendritic cells (DC). PP2A inhibits IL-23 production by suppressing the expression of the IL-23p19 gene. Treating DC with okadaic acid that inhibits the PP2A activity or knocking down the catalytic subunit of PP2A with siRNA enhanced IL-23 but not IL-12 production. Unlike PP2A, MAP kinase phosphatase-1 or CYLD did not show an effect on IL-23 production supporting the specificity of PP2A. PP2A-mediated inhibition requires a newly made protein that is likely responsible for bringing PP2A and IKKβ together upon LPS stimulation, which then results in the termination of IKK phosphorylation. Thus, our results uncovered an important role of the protein phosphatase in the regulation of IL-23 production and identified PP2A as a previously uncharacterized inhibitor of IL-23p19 expression in DC.     

Elevated Fibroblast Growth Factor 23 Impairs Endothelial Function through the NF-κB Signaling Pathway

Aim: Roles of fibroblast growth factor 23 (FGF23) in endothelial dysfunction remain controversial, and evidence from population-based studies is lacking. The present study aimed to explore the effects of FGF23 on endothelial dysfunction on the basis of both clinical data of patients with coronary artery disease (CAD) and thein vitro research in human umbilical vein endothelial cells (HUVECs). Methods: A total of 321 CAD patients were enrolled after coronary angiography, brachial artery flow-mediated dilation (FMD) was assessed using ultrasound equipment. Serum FGF23, nitric oxide (NO), and endothelin-1 (ET-1) were detected via enzyme-linked immunosorbent assay. Apoptosis was determined using the annexin V-fluorescein isothiocyanate/propidium lodide apoptosis detection kit. Cell migration was evaluated by wound healing and transwell migration assays. Reactive oxide species levels were determined using fluorescent probes, and NF-κB p65 nuclear translocation was assessed via immunofluorescence. Results: Serum FGF23 was significantly increased in CAD patients combined with severe endothelial dysfunction (FMD ＜2%) compared to those with FMD ≥ 2% (P＜0.001). Furthermore, the levels of FGF23 were negatively correlated with NO, whereas positively correlated with ET-1 both in unadjusted analysis and multivariate-adjusted analysis. In HUVECs, FGF23 interfered with the bioavailability of NO via increased oxidative stress. Moreover, FGF23 directly impaired the endothelium by promoting HUVECs apoptosis and attenuating the migration of HUVECs. Additional experiments showed that FGF23 induced endothelial injury through activation of the NF-κB signaling pathway. Conclusions: Elevated FGF23 is clinically associated with endothelial dysfunction in CAD patients, and FGF23 impairs endothelial function through activation of the NF-κB signaling pathway.     

Pharmacologic modulation of intracellular Na+ concentration with ranolazine impacts inflammatory response in humans and mice

Changes in Ca2⁺ influx during proinflammatory stimulation modulates cellular responses, including the subsequent activation of inflammation. Whereas the involvement of Ca²⁺ has been widely acknowledged, little is known about the role of Na⁺. Ranolazine, a piperazine derivative and established antianginal drug, is known to reduce intracellular Na⁺ as well as Ca2⁺ levels. In stable coronary artery disease patients (n = 51) we observed reduced levels of high-sensitive C-reactive protein (CRP) 3 mo after the start of ranolazine treatment (n = 25) as compared to the control group. Furthermore, we found that in 3,808 acute coronary syndrome patients of the MERLIN‐TIMI 36 trial, individuals treated with ranolazine (1,934 patients) showed reduced CRP values compared to placebo-treated patients. The antiinflammatory effects of sodium modulation were further confirmed in an atherosclerotic mouse model. LDL^−/− mice on a high-fat diet were treated with ranolazine, resulting in a reduced atherosclerotic plaque burden, increased plaque stability, and reduced activation of the immune system. Pharmacological Na⁺ inhibition by ranolazine led to reduced express of adhesion molecules and proinflammatory cytokines and reduced adhesion of leukocytes to activated endothelium both in vitro and in vivo. We demonstrate that functional Na⁺ shuttling is required for a full cellular response to inflammation and that inhibition of Na⁺ influx results in an attenuated inflammatory reaction. In conclusion, we demonstrate that inhibition of Na⁺–Ca²⁺ exchange during inflammation reduces the inflammatory response in human endothelial cells in vitro, in a mouse atherosclerotic disease model, and in human patients.

Cardiovascular diseases(CVD) remain the number one cause of mortality in the Western world. According to the European Society of Cardiology, ischemic heart disease caused 45% of all deaths in females and 39% in males during the year 2019 (1). Similarly, the leading cause of death within the United States was heart disease (2). Patients with an underlying atherosclerotic process represent a substantial number among those individuals. The chance of recurrent vascular events was found to be of moderate to high risk of 59% in a large-scale model analysis (3, 4). The fact that atherosclerosis is not just a mere storage disease but rather a chronic inflammatory process has been known since the end of the last century (5), and numerous studies have shown that inflammatory markers like high-sensitive C-reactive protein (hsCRP) predict cardiovascular events (6). The CANTOS trial was able to show that antiinflammatory therapy targeting the interleukin‐1β (IL-1β) innate immunity pathway can lower the rate of recurrent cardiovascular events as compared to treatment with placebo in patients with a history of myocardial infarction and hsCRP ≥2mg/L. Importantly, these findings were independent of lipid levels and substantiate the importance of inflammation in the atherosclerotic process (7). However, the CANTOS trial also demonstrated the usability of hsCRP as an indirect marker as the significant reduction of vascular event rates was in proportion to the magnitude of hsCRP reduction achieved (8). The mode of action of canakinumab, the monoclonal antibody used in the CANTOS trial, is to prevent the downstream activation of IL‐1β targets and the nuclear factor “kappa-light-chain-enhancer” of activated B cells (nuclear factor κB [NFκB]) pathway. A similar antiinflammatory strategy in CVD was presented in the Colchicine Cardiovascular Outcomes Trial (COLCOT) in patients within 30 d after myocardial infarction and in the Low-Dose Colchicine 2 (LoDoCo2) Trial involving patients with chronic coronary disease which indicated a positive effect of the antiinflammatory drug colchicine on the development of ischemic cardiovascular events (9). In a following substudy hsCRP levels were found lower in patients treated with colchicine compared to placebo (10).The NFκB pathway plays a major role in the cellular inflammatory response and can be activated by numerous triggers, including IL‐1β (11). Intracellular electrolyte shifts, especially Ca²⁺ influx, and the Ca²⁺ concentration in itself are thought to be among those factors propagating the inflammatory signal. The association of intracellular Ca²⁺ levels with the activation of the NFκB pathway was shown in various cell lines (12–14). Intracellular calcium signaling might require several proteins, including stromal interaction molecule (STIM) and Orai (15). Interestingly, Orai signaling is also associated with Na⁺ influx (16). To our knowledge, there are no clear data on the influence of intracellular Na⁺ concentration on NFκB activation in vascular cells or on the effects of modulation of Na⁺ to control inflammation in vivo or in vitro.Ranolazine, a piperazine derivative and inhibitor of Na⁺ channel currents, is known to reduce intracellular Na⁺ and Ca²⁺ levels in cardiomyocytes (17). Even though ranolazine is indicated in patients with stable angina [class IIa recommendation for symptom relief in the American College of Cardiology/American Heart Association guidelines (18)], no information of ranolazine treatment regarding inflammation or atherosclerotic disease progression is available and a potential finding of inflammation‐modulating effects might be highly relevant in such patients. The purpose of this study was to investigate possible pleiotropic effects of an established antianginal drug in vivo and to evaluate a potential Na⁺- and Ca²⁺‐dependent inflammatory response via the NFκB pathway in endothelial cells in vitro.     

Mitochondria-dependent synthetic small-molecule vaccine adjuvants for influenza virus infection

Vaccine adjuvants enhance and prolong pathogen-specific protective immune responses. Recent reports indicate that host factors—such as aging, pregnancy, and genetic polymorphisms—influence efficacies of vaccines adjuvanted with Toll-like receptor (TLR) or known pattern-recognition receptor (PRR) agonists. Although PRR independent adjuvants (e.g., oil-in-water emulsion and saponin) are emerging, these adjuvants induce some local and systemic reactogenicity. Hence, new TLR and PRR-independent adjuvants that provide greater potency alone or in combination without compromising safety are highly desired. Previous cell-based high-throughput screenings yielded a small molecule 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide], which enhanced lipopolysaccharide-induced NF-κB and type I interferon signaling in reporter assays. Here compound 81 activated innate immunity in primary human peripheral blood mononuclear cells and murine bone marrow-derived dendritic cells (BMDCs). The innate immune activation by 81 was independent of TLRs and other PRRs and was significantly reduced in mitochondrial antiviral-signaling protein (MAVS)-deficient BMDCs. Compound 81 activities were mediated by mitochondrial dysfunction as mitophagy inducers and a mitochondria specific antioxidant significantly inhibited cytokine induction by 81. Both compound 81 and a derivative obtained via structure–activity relationship studies, 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] modestly increased mitochondrial reactive oxygen species and induced the aggregation of MAVS. Neither 81 nor 2F52 injected as adjuvants caused local or systemic toxicity in mice at effective concentrations for vaccination. Furthermore, vaccination with inactivated influenza virus adjuvanted with 2F52 demonstrated protective effects in a murine lethal virus challenge study. As an unconventional and safe adjuvant that does not require known PRRs, compound 2F52 could be a useful addition to vaccines.

Vaccine adjuvants enhance the immunogenic properties of antigens to enable enhanced and prolonged protective immune responses. Defined ligands for the pattern recognition receptors (PRRs)—including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I like receptors (RLR), and stimulator of interferon genes (STING)—have been developed as adjuvants (1, 2). Although PRR-targeting adjuvants are well-tolerated and show promising protective efficacy, vaccination effectiveness is influenced by host factors causing variance in PRR signaling (e.g., aging, pregnancy, or genetic polymorphisms) (3). In the elderly population, activation of TLR signaling pathways is dysregulated, causing impaired activation or constant low-level activation, thereby reducing immune responses (4). Pregnant women are another vulnerable population where immunologic mechanisms that prevent fetal rejection can also lower the maternal response to vaccination. In addition to physiologic factors, genetic polymorphisms, particularly in TLRs and their adaptor proteins, have been reported to reduce responses to measles, meningococcal, and cytomegalovirus glycoprotein B vaccines (3, 5–7). To overcome variations in vaccine responses by these and other host considerations, adjuvants independent of known PRR should be reevaluated for vaccine development.Saponin and oil emulsions are TLR-independent vaccine adjuvants that have been recently approved by the Food and Drug Administration (FDA) (8, 9). Saponin adjuvants, including QS-21, induce strong humoral and T cell responses, and are FDA-approved for malaria and shingles vaccines (8–11). Saponin can disrupt the cell membrane, causing cytotoxicity, which can be reduced by proper formulation within liposomes or emulsions (12). The vaccines adjuvanted with QS-21 sometimes cause systemic adverse effects, including flu-like symptoms, fever, and malaise (13, 14). Oil-in-water adjuvants, including MF59 and AS03, are efficacious, but are also inflammatory in some recipients, inducing local swelling, pain, and fever (14–16). Therefore, new TLR-independent adjuvants with minimal if any local reactions are highly desirable.To seek additional adjuvants that would act independently or as a coadjuvant to enhance the response to monophosphoryl lipid A (MPLA) (9, 17), an FDA-approved TLR4 agonist, we previously performed a high-throughput screening (HTS) using interferon (IFN)-stimulated response element (ISRE) reporter cells. Compound 81 [N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] that belonged to bis-aryl sulfonamide scaffold was identified as a low molecular weight compound that prolonged activation of ISRE induced by type I IFN (18) and was also found to enhance lipopolysaccharide (LPS)-induced NF-κB responses (19). Further structure–activity relationship (SAR) studies identified the sites critical for immune-stimulatory properties of derivatives of 81. Unlike most PRR ligands, these compounds can be easily synthesized in one to two synthetic steps from readily available commercial reagents. Additionally, compound 81, when used as a coadjuvant with MPLA, showed significant enhancement in antigen-specific immunoglobulin responses compared to MPLA alone (19).This study examines the mechanisms that lead to the downstream signaling pathways and efficacy of the compound 81 scaffold (bis-aryl sulfonamide) as a vaccine adjuvant. We demonstrate that compound 81 and its derivative 2F52 [N-benzyl-N-(4-chloro-2,5-dimethoxyphenyl)-4-ethoxybenzenesulfonamide] increase mitochondrial stress associated with mitochnodrial reactive oxygen species (mtROS) release, enhancing mitochondrial antiviral-signaling protein (MAVS)-dependent cytokine production, independent of known PRRs and inflammasomes. When used as an adjuvant with inactivated influenza A virus (IIAV) in a murine lethal influenza virus challenge model, the lead compound 2F52 protects mice from a lethal infection.     

Highly potent,synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4⁺ T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4⁺ T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4⁺ T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.