Cytokine-induced Activation Research Articles

Toxoplasma gondii infection induces the expression of the chemokine CXCL16 in macrophages to promote chemoattraction of CXCR6+ cells.

CXCL16 is a multifaceted chemokine expressed by macrophages and other immune cells in response to viral and bacterial pathogens. However, few studies have investigated its role in parasitic infections. The obligate intracellular parasite Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis, an infection with potentially deleterious consequences in immunocompromised individuals and the developing fetus of acutely infected pregnant women. Chemokines are critical mediators of host defense and, as such, dysregulation of their expression is a subversion strategy often employed by the parasite to ensure its survival. Herein, we report that types I and II T. gondii strains upregulated the expression of both transmembrane and soluble forms of CXCL16 in infected bone marrow-derived macrophages (BMDM). Exposure to soluble T. gondii antigens (STAg) and to excreted-secreted proteins (TgESP) led to the induction of CXCL16. Cxcl16 mRNA abundance and CXCL16 protein levels increased in a time-dependent manner upon T. gondii infection. Importantly, conditioned medium (CM) collected from T. gondii-infected wild-type (WT) macrophage cultures promoted the migration of RAW264.7 cells expressing CXCR6, the cognate receptor of CXCL16, an effect that was significantly reduced by a neutralizing anti-CXCL16 antibody or use of CM from CXCL16 knockout (KO) macrophages. Lastly, T. gondii-driven CXCL16 expression appeared to modulate cytokine-induced (IL-4 + IL-13) alternative macrophage activation and M2 phenotypic marker expression. Further investigation is required to determine whether this chemokine contributes to the pathogenesis of toxoplasmosis and to elucidate the underlying molecular mechanisms.

GsMTx4 ameliorates spinal cord injury by regulating microglial polarization through the Piezo1/NFκB/STAT6 pathway

ObjectiveInflammatory reactions are recognized as pivotal in spinal cord injury (SCI), with the anti-inflammatory role of polarized microglia crucial in mitigating such injury. The present study aimed to determine the protective effects of GsMTx4 on functional recovery in a mouse model of SCI and investigate the role of GsMTx4 in cytokine-induced microglial activation and associated molecular mechanisms. MethodsWe assessed the effects of GsMTx4 on motor function in a mouse model of SCI, including neuronal survival and activated microglia in the vicinity of the injury after SCI. We also investigated the effects of GsMTx4 on expression of relevant inflammatory factors involved in cytokine-induced microglial activation and the associated signaling pathways. ResultsGsMTx4 effectively promoted functional recovery in mice and alleviated nerve damage after SCI. Additionally, GsMTx4 facilitated the transition of microglia from the M1 phenotype to the M2 phenotype, suppressed microglial activation, and reduced the expression of corresponding inflammatory mediators. These effects may involve modulation of neurogenic inflammation through the Piezo1/NFκB/STAT6 pathway, at least in part. ConclusionGsMTx4 safeguards against SCI by regulating microglial polarization, potentially via the Piezo1/NFκB/STAT6 pathway, offering initial evidence supporting the potential therapeutic efficacy of GsMTx4 for treatment of SCI.

IL-13 and IL-17A activate β1 integrin through an NF-kB/Rho kinase/PIP5K1γ pathway to enhance force transmission in airway smooth muscle

Integrin activation resulting in enhanced adhesion to the extracellular matrix plays a key role in fundamental cellular processes. Although integrin activation has been extensively studied in circulating cells such as leukocytes and platelets, much less is known about the regulation and functional impact of integrin activation in adherent cells such as smooth muscle. Here, we show that two different asthmagenic cytokines, IL-13 and IL-17A, activate type I and IL-17 cytokine receptor families, respectively, to enhance adhesion of airway smooth muscle. These cytokines also induce activation of β1 integrins detected by the conformation-specific antibody HUTS-4. Moreover, HUTS-4 binding is increased in the smooth muscle of patients with asthma compared to nonsmokers without lung disease, suggesting a disease-relevant role for integrin activation in smooth muscle. Indeed, integrin activation induced by the β1-activating antibody TS2/16, the divalent cation manganese, or the synthetic peptide β1-CHAMP that forces an extended-open integrin conformation dramatically enhances force transmission in smooth muscle cells and airway rings even in the absence of cytokines. We demonstrate that cytokine-induced activation of β1 integrins is regulated by a common pathway of NF-κB-mediated induction of RhoA and its effector Rho kinase, which in turn stimulates PIP5K1γ-mediated synthesis of PIP2 at focal adhesions, resulting in β1 integrin activation. Taken together, these data identify a pathway by which type I and IL-17 cytokine receptor family stimulation induces functionally relevant β1 integrin activation in adherent smooth muscle and help to explain the exaggerated force transmission that characterizes chronic airway diseases such as asthma.

Abstract Or110: Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific peptidase 20

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser 334 augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in Ldlr -/- mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both in vitro and ex vivo models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in Usp20 -/- compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3 Usp20 -/- , P<0.0001; females, N=2 WT; N=3, Usp20 -/- P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser 334 diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.

Abstract We126: Myocarditis Following mRNA Vaccination for COVID-19 is Mediated by CXCL10 and IFN-γ

Introduction: mRNA vaccines have shown remarkable efficacy against symptomatic COVID-19, reshaping the pandemic's trajectory. Despite a robust safety profile, ongoing monitoring is crucial due to the rare but concerning myocarditis cases, predominantly affecting males under 30. Symptoms appear around 2.6 days post-vaccination, with chest pain, shortness of breath, and palpitations. Elevated cTnI and ST-segment changes are common, along with late gadolinium enhancement in cardiac MRI. Understanding these rare adverse effects is essential for the expanding scope of mRNA technology, spanning infectious disease vaccines and anti-cancer immunotherapies. Hypothesis: Myocarditis linked to COVID-19 mRNA vaccines involves cytokine production from myeloid cells, leading to impaired cardiac function. Methodology: a, Reanalysis of proteomics data of healthy and myocarditis individuals following COVID-19 vaccination; b, Macrophages (N = 7 lines) were exposed to BNT162b2 or mRNA-1273, and analyzed by cytokine array and RNA-seq; c, Cardiomyocytes (N = 4 lines) were treated with CXCL10 (1 and 10 ng/mL) and IFN-γ (0.5 and 5 ng/mL), with or without genistein (10 μM), followed by functional assessment, transcriptomic and proteomic profiling; d, Mice (6 per group) were administered with CXCL10 and IFN-γ, with or without genistein pretreatment (7 days). Myeloid cell infiltration in the heart, serum cTnI levels, RNA-seq, and immunoproteasome activity were analyzed. Results: Examination of post-vaccination plasma revealed a notable rise in CXCL10 and IFN-γ levels in patients. Similarly, macrophages exposed to mRNA vaccines displayed heightened production of these cytokines. When cardiomyocytes were exposed to these cytokines, they exhibited compromised contractility, increased arrhythmogenicity, and gene expression patterns reminiscent of myocarditis. Genistein, an anti-inflammatory phytoestrogen, demonstrated significant alleviation of these effects, preventing cytokine-induced proteasomal activation and preserving contractile function. Mouse model of cytokine-induced myocarditis showed that genistein substantially reduced cardiac injury markers and immune cell infiltration. Conclusion: These findings highlight CXCL10 and IFN-γ as pivotal contributors to myocarditis following mRNA vaccination and suggest genistein as a potential therapeutic intervention to mitigate associated cardiovascular risks.

Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4’s neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Preparation of β-1,3-glucan mimics via modification of polymer backbone, and evaluation of cytokine production using the polymer library in immune activation

β-1,3-Glucans possess therapeutic potential owing to their ability to exhibit immunostimulating activity. β-1,3-Glucans, isolated from various organisms, differ in their chemical structures, molecular weight, and branching degree, potentially forming particulate, helix, or random coil conformations in water. Therefore, this study used synthesized β-1,3-glucan mimic polymers to investigate the difference in binding affinity for dectin-1 and induced cytokine productions based on polymer structures. The β-1,3-glucan mimic polymers were synthesized using β-1,3-glucan tetrasaccharyl monomer, with subsequent modifications to the polymer backbones through the introduction of hydrogen or a hydroxy group. Polymers with different structures in both ligands and polymer backbones were utilized to comprehensively investigate their binding affinity to dectin-1 and cytokine-inducing in macrophages. Hydroxylated polymers exhibited a high binding affinity for dectin-1, similar to that of schizophyllan, whereas the polymer composed of only saccharyl monomers did not bind to dectin-1. Further, when administered to macrophage RAW264 cells, polymers with branched and hydrophobic polymer backbones exhibited strong cytokine-inducing activities. Moreover, the results revealed that the essential factors for cytokine induction include the branches of β-1,3-glucans, high (tens of thousands) molecular weights, and hydrophobicity. The results suggests that artificial polymers comprising these factors exhibit immunostimulating activity and could be developed as therapeutic agents.

Pulsed low dose rate radiation to mitigate tumor-permissive responses in pancreatic cancer-associated fibroblasts: Introducing the HOST-factor.

682 Background: Pancreatic ductal adenocarcinoma (PDAC) features a distinctive tumor microenvironment comprising cancer-associated fibroblasts (CAFs) and their self-produced extracellular matrix (ECM), forming functional CAF units (CAF u). The current total neoadjuvant therapy (TNT), involving conventional chemoradiotherapy (CRT), yields suboptimal responses while promoting a tumor-permissive CAF u state. To address this, we explored pulsed low dose rate radiation (PLDR)-based TNT, as an alternative to CRT, to treat three-dimensional in vivo-like human PDAC CAF u cultures. We assessed gemcitabine combined with PLDR versus CRT using theHOST Factor, a “harmonized output of stroma traits” factor, comprising several established CAF u functional indicators, which inform on the functional status of the PDAC microenvironment. Methods: The unique CAF u functional values used corresponded to: i) ECM anisotropy; ii) persistent cell-ECM signaling; iii) sustained cytokine-induced activation; iv) cytoskeletal bundling; and v) systemic features of fibroblastic activation. The validity of the resultant HOST factor was assessed using the unit’s ECM ability to elicit fibroblastic activation, indicated by a high αSMA/F-actin ratio value. Results: Ex-vivo CRT led to a HOST factor that was 2-fold greater than chemotherapy alone (p=0.6), which was functionally gauged as a tumor-permissive CAF u. In contrast, PLDR averted CRT-induced tumor-permissive response and established a 6-fold lower HOST factor, suggestive of a “normalized” CAF u, when compared to the HOST factor value established when using conventional TNT (P=0.0016 vs. CRT, and P= 0.0086 vs. gemcitabine alone). The multi-parameter HOST factor validity was confirmed as CRT treated CAF u’s ECM activated human pancreatic fibroblasts (αSMA/F-actin = 70% ± 29) while PLDR-treated CAF u's ECM failed to do so (αSMA/F-actin = 32% ± 26), reaching a 30% mean rank difference (P <0.0001). Conclusions: PLDR mitigates conventional CRT-based TNT-induced tumor-permissive responses, restoring the fibroblastic units' tumor-suppressive function. These results lend support to a phase 1 trial designed to evaluate whether radiation with PLDR-based TNT, constitutes a safe and more effective treatment for PDAC patients. The novel HOST factor is being used to appraise specimens from NCT04452357.

Suppression of NK Cell Activation by JAK3 Inhibition: Implication in the Treatment of Autoimmune Diseases.

Natural killer (NK) cell is an essential cytotoxic lymphocyte in our innate immunity. Activation of NK cells is of paramount importance in defending against pathogens, suppressing autoantibody production and regulating other immune cells. Common gamma chain (γc) cytokines, including IL-2, IL-15, and IL-21, are defined as essential regulators for NK cell homeostasis and development. However, it is inconclusive whether γc cytokine-driven NK cell activation plays a protective or pathogenic role in the development of autoimmunity. In this study, we investigate and correlate the differential effects of γc cytokines in NK cell expansion and activation. IL-2 and IL-15 are mainly responsible for NK cell activation, while IL-21 preferentially stimulates NK cell proliferation. Blockade of Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway by either JAK inhibitors or antibodies targeting γc receptor subunits reverses the γc cytokine-induced NK cell activation, leading to suppression of its autoimmunity-like phenotype in vitro. These results underline the mechanisms of how γc cytokines trigger autoimmune phenotype in NK cells as a potential target to autoimmune diseases.

Antiproliferative and anti-inflammatory effects of fractionated crude lectins from boiled kidney beans (Phaseolus vulgaris).

In this study, we investigated the biological profile of lectins isolated from raw and boiled Japanese red Kintoki beans (red kidney beans [RKB]; Phaseolus vulgaris). Lectins in beans showing agglutination activity were retained after heating. Raw and boiled RKB lectins were fractionated using carboxymethyl- and diethylaminoethyl-Sepharose, respectively. Boiled RKB lectins were evaluated for carbohydrate specificity as well as cytokine-inducing and antiproliferative activities against cancer cells and compared with raw RKB lectins. Raw RKB lectins showed specificity for thyroglobulin and fetuin, whereas boiled lectins showed specificity for N-acetylneuraminic acid. Raw RKB lectins showed low resistance to proteases and tolerated temperatures greater than 80°C for 5min. Notably, lectins from raw and boiled beans showed antiproliferative activity against five types of cancer cells B16, LM8, HeLa, HepG2, and Colo 679. In particular, lectins from raw beans exhibited a significantly stronger activity than those from boiled beans. Anti-inflammatory effects were notably observed in crude extracts from raw and boiled beans. Specifically, lectins fractionated from boiled beans markedly inhibited the expression of tumor necrosis factor-α and interleukin-6. Overall, our results showed that RKB lectins from boiled beans exert anti-inflammatory and anticancer effects and could be developed as potential chemopreventive agents. PRACTICAL APPLICATION: Japanese red kidney beans (RKB) are cultivated in numerous parts of the temperate zone and consumed in many countries. Lectins from boiled beans exhibited anticancer activity, similar to lectins from raw beans. Additionally, crude and fractionated lectins from boiled beans showed anti-inflammatory activity. Thus, boiled RKB lectins have the potential to be used as a bioactive protein for medical research and could be developed as anticancer agents.

Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198-222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1.

A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets invitro and signal-induced puncta invivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N-and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

SAT069 Targeting Human β-Cell G Protein-coupled Receptors To Alter Cytokine-mediated Caspase Activation

Abstract Disclosure: K. Rodrigues dos Santos: None. N. Mukherjee: None. L.F. Barella: None. F. Armoo: None. D.L. Eizirik: None. A.T. Templin: None. M.A. Kalwat: None. In type 1 diabetes (T1D) autoimmune destruction of pancreatic islet β-cells causes a lack of insulin, leading to hyperglycemia. The development of therapies that can prevent β-cell death is critical. β-cell proliferation and survival pathways are affected by signaling through G protein-coupled receptors (GPCRs). Using high-depth RNA-seq, we found altered expression of 123 different GPCRs in human islets exposed to cytokines (IL1β 50U/ml + IFNγ 1000U/ml, 48 h) to model the T1D milieu. We hypothesized these candidates may be exploited for β-cell protection. Among the candidates, adenosine receptors (ADORA1 and ADORA2A), calcium-sensing receptor (CASR), and metabotropic glutamate receptor 4 (GRM4) stood out. Cytokines decreased the expression of ADORA1 and CASR, but increased ADORA2A and GRM4. To gauge the impact of agonizing/antagonizing these receptors, we pre-treated human EndoC-β H1 β-cells or islets with receptor ligands and co-treated with cytokines (IL1β 50U/ml + IFNγ or IL1β + 1000U/ml IFNγ + 1000U/ml TNFɑ, 48 h) and measured glucose-stimulated insulin secretion, cell viability, and gene expression. Each cytokine combination induced target gene expression (e.g. CXCL10), but only the combination of IL1β + IFNγ + TNFɑ reduced glucose-stimulated insulin secretion. Preliminary experiments indicated that adenosine receptor ligands had little impact in our assays. However, ligands for CASR and GRM4 exhibited significant suppression of cytokine-induced caspase 3/7 activation in EndoC-βH1 cells. Interestingly, suppressed caspase activation did not result in observable impacts on live/dead cell ratios or live-cell analysis using Incucyte in EndoC-βH1 cells. Consistently, we also observed cytokine-mediated cell death in the presence of the pan-caspase inhibitor ZVAD-fmk. We conclude that a variety of β-cell GPCR-mediated signaling pathways may converge to prevent cytokine-induced caspase activation, but additional measures are needed to enable protection from eventual cell death. Our results and this line of investigation is significant because understanding mechanisms of GPCR-cytokine crosstalk in β-cells will inform strategies to protect islets in other models of T1D. Funding: JDRF 1-INO-2022-1113-A-N and Diabetes Research Connection #33. Presentation: Saturday, June 17, 2023

Influence of the Composition of Cationic Liposomes on the Performance of Cargo Immunostimulatory RNA.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption.

Low-grade inflammation and barrier disruption are increasingly acknowledged for their association with non-communicable diseases (NCDs). Short chain fatty acids (SCFAs), especially butyrate, could be a potential treatment because of their combined anti-inflammatory and barrier- protective capacities, but more insight into their mechanism of action is needed. In the present study, non-activated, lipopolysaccharide-activated and αCD3/CD28-activated peripheral blood mononuclear cells (PBMCs) with and without intestinal epithelial cells (IEC) Caco-2 were used to study the effect of butyrate on barrier function, cytokine release and immune cell phenotype. A Caco-2 model was used to compare the capacities of butyrate, propionate and acetate and study their mechanism of action, while investigating the contribution of lipoxygenase (LOX), cyclooxygenase (COX) and histone deacetylase (HDAC) inhibition. Butyrate protected against inflammatory-induced barrier disruption while modulating inflammatory cytokine release by activated PBMCs (interleukin-1 beta↑, tumor necrosis factor alpha↓, interleukin-17a↓, interferon gamma↓, interleukin-10↓) and immune cell phenotype (regulatory T-cells↓, T helper 17 cells↓, T helper 1 cells↓) in the PBMC/Caco-2 co-culture model. Similar suppression of immune activation was shown in absence of IEC. Butyrate, propionate and acetate reduced inflammatory cytokine-induced IEC activation and, in particular, butyrate was capable of fully protecting against cytokine-induced epithelial permeability for a prolonged period. Different HDAC inhibitors could mimic this barrier-protective effect, showing HDAC might be involved in the mechanism of action of butyrate, whereas LOX and COX did not show involvement. These results show the importance of sufficient butyrate levels to maintain intestinal homeostasis.

Pre-treatment with IL-6 potentiates β-cell death induced by pro-inflammatory cytokines

BackgroundType I Diabetes mellitus (T1D) is characterized by a specific destruction of β-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for β-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of β-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent β-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on β-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on β-cells exposed to pro-inflammatory cytokines.ResultsPre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in β-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression.ConclusionTreatment with IL-6 alone is not beneficial for β-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve β-cells viability under this condition, suggesting that other mechanisms may be involved.

Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial-Mesenchymal Transition in Endometrial Epithelial Cells.

The endometrium undergoes repeated proliferation and shedding during the menstrual cycle. Significant changes to this environment include fluctuations in the partial pressure of oxygen, exposure to a high-cytokine environment associated with intrauterine infection, and inflammation. Chronic endometritis is a condition wherein mild inflammation persists in the endometrium and is one of the causes of implantation failure and miscarriage in early pregnancy. It is thought that the invasion of embryos into the endometrium requires epithelial-mesenchymal transition (EMT)-associated changes in the endometrial epithelium. However, the effects of inflammation on the endometrium remain poorly understood. In this study, we investigated the effects of the intrauterine oxygen environment, hypoxia-inducible factor (HIF), and inflammation on the differentiation and function of endometrial epithelial cells. We elucidated the ways in which inflammatory cytokines affect HIF activity and EMT in an immortalized cell line (EM-E6/E7/TERT) derived from endometrial epithelium. Pro-inflammatory cytokines caused significant accumulation of HIF-1α protein, increased HIF-1α mRNA levels, and enhanced hypoxia-induced accumulation of HIF-1α protein. The combined effect of inflammatory cytokines and hypoxia increased the expression of EMT-inducing factors and upregulated cell migration. Our findings indicate that pro-inflammatory factors, including cytokines and LPS, work synergistically with hypoxia to activate HIF-1 and promote EMT in endometrial epithelial cells.

Immunomodulatory effects of extracellular glyceraldehyde 3-phosphate dehydrogenase of exopolysaccharide-producing Lactiplantibacillus plantarum JCM 1149.

Probiotic lactic acid bacteria evoke immunomodulatory effects in the host; however, the reasons for the different effects of various species and strains remain to be elucidated. To clarify the critical immunomodulatory components and impact of exopolysaccharide (EPS) in Lactiplantibacillus plantarum, 11 types of L. plantarum strains were compared for the production of EPS, inflammatory cytokines, interleukin-6 and -12, and the anti-inflammatory cytokine, interleukin-10, from THP-1 differentiated dendritic cells. EPS in the fermented medium correlated with cytokine-inducing activities. L. plantarum JCM 1149, with the highest production of EPS, also induced interleukin-6, -10, and -12 among the 11 tested strains. Notably, the cytokine-producing activities overlapped with the protein fraction in gel filtration chromatography but not with EPS, which has been reported to exert immunomodulatory effects. The 41 kDa protein that coexisted with EPS was purified as a major active component and identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a known moonlighting protein. GAPDH secretion was reduced when EPS synthesis inhibitors were added to the culture medium. RNA sequencing of GAPDH-treated THP-1 cells revealed an up-regulation in the expression of genes involved in transcriptional regulation, cell surface receptor signalling, immune response, and matrix components. Here, we report, to our knowledge for the first time, that the cell surface-associated L. plantarum GAPDH plays a crucial role in cytokine production in THP-1 cells, but EPS with less activity may help GAPDH secretion.

NDRG1 Signaling Is Essential for Endothelial Inflammation and Vascular Remodeling.

NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β (interleukin-1β) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.

Effect of different types of immunosuppressive therapy on the parameters of TNF receptor expression in patients with rheumatoid arthritis

Background. The balance of TNF receptor expression on immune cells is a key factor determining cytokine-induced activation of proapoptotic or proliferative signaling pathways. As a result, the changes in cytokine level and in expression of its receptors may be one of the mechanisms that regulate the level of systemic and local inflammation in rheumatoid arthritis (RA) and determine the degree of therapy effectiveness. The aim. To study the effect of rheumatoid arthritis therapy on the change in the patterns of TNF receptors expression in terms of co-expression and the number of receptors on the main subpopulations of immunocompetent cells.Materials and methods. A comparative analysis of the profiles of TNF receptors type 1 and 2 (TNFR1/2) co-expression was carried out in patients with RA (n = 16) before and after having inpatient effective therapy and in comparison with a group of healthy individuals (n = 21). We compared the number of receptors and the proportion of cells expressing the corresponding receptor using flow cytometry and studied the subpopulations of regulatory T cells, T cells, B cells, and monocytes. Results. In patients with RA, there is a significant redistribution of TNFR1 and TNFR2 expression on immunocompetent cells, while the intensity of changes is associated not only with disease severity indicators, but also with the therapy received. The key adaptive mechanism of the TNF system in long-term treatment refractory course of RA is a change in the proportion of double-positive TNFR1+TNFR2+ cells, while the effectiveness of therapy and clinical indicators of the disease severity are associated with individual variability in the parameters of type 2 receptors expression. Conclusions. The data obtained confirm the existence of a relationship between an imbalance in the expression of type 1 and type 2 TNF receptors on immunocompetent cells and the effectiveness of response to therapy. The identified patterns of typical changes in TNFR1/2 co-expression in RA can be used as potential therapeutic targets and predictive factors for the effectiveness of therapy.