Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection

Elevated CO₂ levels (hypercapnia) frequently occur in patients with obstructive pulmonary diseases and are associated with increased mortality. However, the effects of hypercapnia on non-neuronal tissues and the mechanisms that mediate these effects are largely unknown. Here, we develop Drosophila as a genetically tractable model for defining non-neuronal CO₂ responses and response pathways. We show that hypercapnia significantly impairs embryonic morphogenesis, egg laying, and egg hatching even in mutants lacking the Gr63a neuronal CO₂ sensor. Consistent with previous reports that hypercapnic acidosis can suppress mammalian NF-κB-regulated innate immune genes, we find that in adult flies and the phagocytic immune-responsive S2* cell line, hypercapnia suppresses induction of specific antimicrobial peptides that are regulated by Relish, a conserved Rel/NF-κB family member. Correspondingly, modest hypercapnia (7–13%) increases mortality of flies inoculated with E. faecalis, A. tumefaciens, or S. aureus. During E. faecalis and A. tumefaciens infection, increased bacterial loads were observed, indicating that hypercapnia can decrease host resistance. Hypercapnic immune suppression is not mediated by acidosis, the olfactory CO₂ receptor Gr63a, or by nitric oxide signaling. Further, hypercapnia does not induce responses characteristic of hypoxia, oxidative stress, or heat shock. Finally, proteolysis of the Relish IκB-like domain is unaffected by hypercapnia, indicating that immunosuppression acts downstream of, or in parallel to, Relish proteolytic activation. Our results suggest that hypercapnic immune suppression is mediated by a conserved response pathway, and illustrate a mechanism by which hypercapnia could contribute to worse outcomes of patients with advanced lung disease, who frequently suffer from both hypercapnia and respiratory infections.     

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.     

From the Cover: Vinpocetine inhibits NF-κB–dependent inflammation via an IKK-dependent but PDE-independent mechanism

Inflammation is a hallmark of many diseases, such as atherosclerosis, chronic obstructive pulmonary disease, arthritis, infectious diseases, and cancer. Although steroids and cyclooxygenase inhibitors are effective antiinflammatory therapeutical agents, they may cause serious side effects. Therefore, developing unique antiinflammatory agents without significant adverse effects is urgently needed. Vinpocetine, a derivative of the alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment. Its role in inhibiting inflammation, however, remains unexplored. Here, we show that vinpocetine acts as an antiinflammatory agent in vitro and in vivo. In particular, vinpocetine inhibits TNF-α–induced NF-κB activation and the subsequent induction of proinflammatory mediators in multiple cell types, including vascular smooth muscle cells, endothelial cells, macrophages, and epithelial cells. We also show that vinpocetine inhibits monocyte adhesion and chemotaxis, which are critical processes during inflammation. Moreover, vinpocetine potently inhibits TNF-α- or LPS-induced up-regulation of proinflammatory mediators, including TNF-α, IL-1β, and macrophage inflammatory protein-2, and decreases interstitial infiltration of polymorphonuclear leukocytes in a mouse model of TNF-α- or LPS-induced lung inflammation. Interestingly, vinpocetine inhibits NF-κB–dependent inflammatory responses by directly targeting IKK, independent of its well-known inhibitory effects on phosphodiesterase and Ca²⁺ regulation. These studies thus identify vinpocetine as a unique antiinflammatory agent that may be repositioned for the treatment of many inflammatory diseases.     

Transient Receptor Potential Melastatin 2 (TRPM2) ion channel is required for innate immunity against Listeria monocytogenes

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2(-/-) mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2(-/-) mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2(-/-) mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2(-/-) mice. The severity of listeriosis we observed in TRPM2(-/-) mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.     

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.     

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.     

幽门螺杆菌CagA与YWHAE互作区段的确定

目的 探讨幽门螺杆菌CagA蛋白与宿主细胞YWHAE蛋白相互作用的区段。方法 先在酵母细胞水平构建CagA区段蛋白[-N、-M、-(N+M)、-C]的表达载体,经“滤纸法”检测、一对一交配试验及“ONPG液相法”分析,筛选互作区段;进而在哺乳动物细胞水平,选用Cos7细胞经共转染、Co-IP方法确定互作区段,选用AGS细胞经上调/下调YWHAE表达、共转染、双荧光素酶报告基因检测等分析细胞NF-κB活性检测CagA候选区段与YWHAE互作的功能。结果 成功构建CagA蛋白的N、M、(N+M)和C区段的酵母表达载体,CagA各区段蛋白“滤纸法”检测反式激活特性均为阴性,“一对一”交配后“液相法”筛选到CagA区段[-N、-(N+M)、-C]分别可与YWHAE在酵母中相互作用;Cos7细胞实验确定CagA区段[-N、-C]可分别沉淀外源性YWHAE,AGS细胞中CagA区段[-N、-C]对细胞NF-κB的激活,上调YWHAE能增强、下调YWHAE则抑制。结论 幽门螺杆菌CagA蛋白N-末端与C-末端均分别与宿主细胞YWHAE蛋白相互作用,并可激活细胞NF-κB。     

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.