IL-10 Protein Research Articles

PAD4 silencing inhibits inflammation whilst promoting trophoblast cell invasion and migration by inactivating the NEMO/NF-κB pathway.

Preeclampsia (PE), presenting with onset hypertension and proteinuria, is a pregnancy-specific disorder that can result in maternal and fetal morbidity and mortality. Insufficient trophoblast invasion and migration has been considered to be an important cause of this disease. The present study aimed to investigate the role of peptidyl arginine deiminase 4 (PAD4), whose knockdown has been previously indicated to reduce inflammation and susceptibility to pregnancy loss in mice, in the development of PE in vitro. Lipopolysaccharide (LPS) was used to treat a human trophoblast cell line (HTR8/SVneo). After PAD4 silencing via transfection with short hairpin RNA against PAD4, the concentrations of inflammatory factors IL-6, IL-12 and monocyte chemoattractant protein (MCP)-1 were measured using ELISA. Cell viability was also measured using Cell Counting Kit-8 assay. HTR8/SVneo cell invasion and migration were detected using Transwell and wound healing assays, respectively. Western blotting was used to measure the expression of citrullinated NF-κB essential modulator (NEMO) and nuclear NF-κB p65 protein levels. TNF-α was applied for evaluating the potential regulatory effects of PAD4 on NF-κB in LPS-stimulated HTR8/SVneo cells. LPS increased the levels of IL-6, IL-12 and MCP-1 and reduced the migration and invasion of HTR8/SVneo cells. PAD4-knockdown was found to markedly reduce the levels of IL-6, IL-12 and MCP-1 secretion. HTR8/SVneo cell invasion and migration was also significantly elevated after PAD4 silencing following LPS exposure. In addition, LPS stimulation notably upregulated the protein levels of citrullinated NEMO and nuclear NF-κB p65, which was restored by PAD4 knockdown. Furthermore, TNF-α treatment partially counteracted the effects of PAD4 knockdown on the secretion of IL-6, MCP-1 and IL-12, which are markers of inflammation, and invasion and migration in LPS-induced HTR8/SVneo cells. To conclude, these results suggest that PAD4 silencing can suppress inflammation whilst promoting invasion and migration by trophoblast cells through inhibiting the NEMO/NF-κB pathway. These findings furthered the understanding in the complex molecular mechanism that can trigger PE and provide a promising target for the treatment of this disease.

Dietary 25-Hydroxyvitamin D3 Supplementation Modulates Intestinal Cytokines in Young Broiler Chickens.

Vitamin D signaling is important for intestinal homeostasis. An increase in vitamin D receptors in immune cells can modulate cell phenotype and cytokine secretion. Cytokines regulate both pro- (interleukin 17; IL-17) and anti-inflammatory (IL-10) responses triggered by external stimuli. Inflammation in intestinal tissues can disrupt the structure and the remodeling of epithelial tight junction complexes, thus, compromising the protective barrier. The objective of the study was to determine the impact of dietary supplementation with 25-hydroxycholecalciferol (25OHD3), a hydroxylated metabolite of vitamin D, on intestinal cytokine abundance and epithelial barrier integrity over time in broilers. A randomized complete block design experiment was conducted to evaluate the effect of dietary 25OHD3 inclusion on relative protein expression of the cytokines, IL-17 and IL-10, and tight junction proteins, Zona Occludens 1 (ZO-1), and Claudin-1 (CLD-1), in broiler chicken duodenum and ileum from 3 to 21 days post-hatch. On day 0, male chicks (n = 168) were randomly assigned to raised floor pens. Experimental corn–soybean meal-based treatments were as follows: (1) a common starter diet containing 5,000 IU of D3 per kg of feed (VITD3) and (2) a common starter diet containing 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (25OHD3) fed from days 0 to 21. On days 3, 6, 9, 12, 15, 18, and 21, 12 birds per treatment were euthanized to collect tissue samples for quantitative, multiplex, and fluorescent Western blot analysis. Target proteins were quantified using Image Quant TL 8.1 and expressed relative to total protein. Feeding 25OHD3 post-hatch decreased ileal IL-10 (anti-inflammatory) protein expression in 21-day-old broilers compared with VITD3 only (P = 0.0190). Broilers fed only VITD3 post-hatch had greater IL-17 (pro-inflammatory) protein expression in the ileum at 18 and 21 days-of-age (P = 0.0412) than those that fed 25OHD3. Dietary inclusion of 25OHD3 lowered the abundance of key inflammatory cytokines in the ileum of young broilers.

Interferon Regulatory Factor 4 (IRF4) Promotes Lipopolysaccharide-Induced Colonic Mucosal Epithelial Cell Proliferation by Regulating Macrophage Polarization

Introduction: Ulcerative colitis (UC) is a chronic disease characterized by diffuse inflammation of the mucosa of colon and rectum. Interferon regulatory factor 4 (IRF4) mediates macrophage anti-inflammatory phenotype (alternatively activated macrophages [M2]). This study aimed to investigate the mechanism of IRF4 in lipopolysaccharide (LPS)-induced colonic mucosal epithelial cell proliferation via the regulation of macrophage polarization. Methods: Human bone marrow-derived macrophages were subjected to interleukin 4 (IL-4) induction. M2 macrophages were identified using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). IRF4 expression in M2 macrophages was detected using Western blot and qRT-PCR. IRF4 expression was silenced in M2 macrophages. IL-10 mRNA expression and protein level were detected using qRT-PCR and Western blot. The binding relation between IRF4 and IL-10 was verified using dual-luciferase and chromatin immunoprecipitation assays. Macrophages under different treatments were cocultured with LPS-induced human colonic mucosal epithelial cells. The levels of inflammatory factors (TNF-α, IL-6, and IL-1β) were detected using enzyme-linked immunosorbent assay. The proliferation of inflammatory cells was measured using Cell Counting Kit-8 assay, and the healing of inflammatory cells was detected using wound healing assay. Results: M2 macrophages alleviated LPS-induced inflammatory responses. IRF4 bound to IL-10 and promoted IL-10 expression. Inhibition of IRF4 reduced IL-10 expression and attenuated the alleviating effect of M2 macrophages on inflammatory responses. Inhibition of IRF4 combined with IL-10 overexpression enhanced the promoting effect of M2 macrophages on inflammatory healing. Conclusion: IRF4 promoted colonic mucosal epithelial cell proliferation by increasing IL-10 expression and regulating macrophage polarization to M2 phenotype, which might be related to UC mucosal healing.

Computational study of Cu2+, Fe2+, Mn2+, Mn3+, Fe3+, CrO42-, Si4+, and Hg+ binding sites identification on cytokines to predict dental metal allergy: An in silico study

Context: Metal allergy is a general term to describe allergic diseases due to the release of metal ion reactions in the body which are mediated by T cells and involve inflammatory cytokines that can cause morbidity and mortality. Molecular docking is an analysis that can be used to assess the interaction of ligand bonds with target proteins that are used to predict metal allergies caused by metal ions that stimulate cytokines. Aims: To analyze the binding sites of Cu2+, Fe2+, Mn2+, Mn3+, Fe3+, CrO42-, Si4+, and Hg+ ions on cytokines to predict dental metal allergy through a bioinformatics approach, in silico. Methods: Metal ion particles consisting of Cu2+, Fe2+, Mn2+, Mn3+, Fe3+, CrO42-, Si4+, and Hg+ were predicted to bind tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL) IL-1b, IL-2, IL-4, IL-10, IL-13, IL-17, IL-23, and IL-33 act as target proteins were examined. Results: The blind docking simulation succeeded in identifying the comparison of the binding activity of metal ion particles on cytokines target proteins. The docking simulation results show that the metal ion with the most negative binding affinity value binds to the IL-17 protein. Conclusions: Metal ion particles consisting of Cu2+, Fe2+, Mn2+, Mn3+, Fe3+, CrO42-, Si4+, and Hg+ have the most negative binding affinity values for binding to IL-17 protein, which can cause allergic reactions predicted by molecular docking, in silico.

Serum IL-5, POSTN and IL-33 levels in chronic rhinosinusitis with nasal polyposis correlate with clinical severity

BackgroundChronic rhinosinusitis (CRS) is a group of heterogeneous diseases characterized by epithelial inflammation and tissue eosinophilic infiltration. IL-5, POSTN, and IL-33 are important factors that act as chemoattractants for eosinophils, and a tissue-remodeling protein positively correlated with eosinophils in blood and mediators of eosinophilic infiltration. The aim of the study was to determine the expression of IL-5, POSTN and IL-33, at the gene and protein levels, in eosinophilic CRS with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP), and to correlate this expression with clinical severity.Materials and methodsThe study included 40 CRSwNP patients and 53 CRSsNP patients and 40 control subjects. The expression of IL-5, POSTN and IL-33 mRNA was determined in sinonasal mucosal samples and in nasal polyp tissue by real-time PCR. Protein levels in the serum of CRSwNP patients were measured by ELISA. Computed tomography was evaluated according to Lund–Mackay scores, and visual analog scale scores were assessed.ResultsNP tissue demonstrated significantly higher IL-5 and POSTN mRNA expression than the sinonasal tissue in the CRSsNP and CRSwNP groups. CRS groups demonstrated elevated IL-33 mRNA expression in comparison to controls irrespective of the presence of NP. No correlation was found between IL-5, POSTN and IL-33 mRNA expression and disease severity. CRSwNP group demonstrated significantly higher serum IL-5, POSTN and IL-33 protein levels than controls, and this corresponds to disease severity.ConclusionSerum IL-5, POSTN and IL-33 levels may be important markers for classification of eosinophilic CRSwNP patients, along with disease severity.

Identifying the Hub Genes of Glioma Peritumoral Brain Edema Using Bioinformatical Methods

Glioma peritumoral brain edema (GPTBE) is a frequent complication in patients with glioma. The severity of peritumoral edema endangers patients’ life and prognosis. However, there are still questions concerning the process of GPTBE formation and evolution. In this study, the patients were split into two groups based on edema scoring findings in the cancer imaging archive (TCIA) comprising 186 TCGA-LGG patients. Using mRNA sequencing data, differential gene (DEG) expression analysis was performed, comparing the two groups to find the key genes affecting GPTBE. A functional enrichment analysis of differentially expressed genes was performed. Then, a protein–protein interaction (PPI) network was established, and important genes were screened. Gene set variation analysis (GSVA) scores were calculated for major gene sets and comparatively correlated with immune cell infiltration. Overall survival (OS) was analyzed using the Kaplan–Meier curve. A total of 59 DEGs were found, with 10 of them appearing as important genes. DEGs were shown to be closely linked to inflammatory reactions. According to the network score, IL10 was in the middle of the network. The presence of the IL10 protein in glioma tissues was verified using the human protein atlas (HPA). Furthermore, the gene sets’ GSVA scores were favorably linked with immune infiltration, particularly, with macrophages. The high-edema group had higher GSVA scores than the low-edema group. Finally, Kaplan–Meier analysis revealed no differences in OS between the two groups, and eight genes were found to be related to prognosis, whereas two genes were not. GPTBE is linked to the expression of inflammatory genes.

Abstract 584: Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells

Abstract Recent successes with cell therapies, including CAR-T and CAR-NK cells, for hematopoietic cancers have yet to translate into solid tumors. The major obstacle solid tumors present for cell therapy is the highly immunosuppressive tumor microenvironment (TME), which can impair the function of endogenous as well as therapeutic immune cells. To overcome this challenge, Senti Bio is developing gene circuits to arm CAR-NK cells with multiple cytokines to improve the cytotoxicity and persistence of CAR-NK product candidates. Cytokines, as well as other immune effectors, can act in autocrine or paracrine manner, benefiting the CAR-NK cells as well as potentially engaging the local immune system in the TME. Secreted cytokines can be widely distributed and stimulate other immune cells in the TME, however this is often associated with systemic toxicity as well as reduced impact on the CAR-NK cells. On the other hand, membrane-bound cytokines result in a localized and potent stimulation of the CAR-NK cells but have limited impact on the broader TME. Since IL-15 is known to stimulate NK cell survival and function, Senti Bio has created a novel calibrated release (cr) technology to specifically optimize IL-15 signaling in its CAR-NK platform. crIL-15 is tethered to the NK cells via a cleavable linker that allows for IL-15 release upon cleavage by a ubiquitously expressed protease on the cell surface of NK cells. The rate of linker cleavage can be calibrated by engineering the sensitivity of the cleavable linker sequence to the cell surface protease, thereby enabling the tuning of the ratio of membrane tethered versus fully secreted protein. We compared the function of the wildtype IL-15 protein with different variants of crIL-15 that have a faster or slower release. We demonstrated that crIL-15 has a more potent function, stimulating pSTAT5 signaling and driving expansion and survival of NK cells. CAR-NK cells armed with crIL-15 significantly outperformed CAR-NK cells that expressed the wildtype IL-15 protein, had enhanced function, and demonstrated significantly improved cytotoxicity over three rounds of serial killing (p<0.0001). In parallel, we armed CAR-NK cells with gene circuits that encoded various combinations of two or more cytokines. In particular, the combination of IL-15 and IL-21 resulted in a synergistic effect, leading to longer survival in the absence of cytokines compared to NK cells armed with IL-15 alone. Furthermore, CAR-NK cells armed with IL-15 + IL-21 had significantly increased activity and cytotoxicity compared to CAR-NK cells armed with either cytokine alone (up to 4-times increased cytotoxicity, p<0.0001) when co-cultured with tumor cells. Overall, our results suggest the advantage of multi-arming CAR-NK cells with cytokines, using controlled release systems and cytokine combinations, resulting in improved NK cell function in solid tumor models. Citation Format: Alba Gonzalez, Michelle Hung, Marcela Guzman, Aldo Sotelo, Nicholas Frankel, Yin Yin Chong, Deepika Kaveri, Poornima Ramkumar, Elizabeth Leitner, Priscilla Wong, Ronni Ponek, Kelly Lee, Alyssa L. Mullenix, Russell M. Gordley, Gary Lee. Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 584.

Abstract 4169: A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy

Abstract Background: Interleukin-10 (IL-10) has demonstrated utility in aiding CD8 T cell-mediated tumor control in mouse models but phase 2 clinical trials with pegylated IL-10 have so far not been entirely successful. More recent studies revealed that another half-life-extended form of IL-10 (IL10-Fc) can rejuvenate terminally-exhausted CD8+ T cells to boost anti-tumor activity. We investigated whether combination of a PDL-1-blocking antibody with a C-terminal IL-10 fusion may enhance anti-tumor immunity, and possibly achieve synergistic effects. Methods: Recombinant proteins were produced using an expiCHO system (Thermo Fisher). PDL-1 blocking activity was evaluated by ELISA, PD-1/PDL-1 signaling (Eurofins) and mixed lymphocyte reaction assays. IL-10 activity was evaluated by MC/9 cell proliferation and CD8 T cell activation and effector assays. Mouse CT26 colon cancer and EMT6 breast cancer studies employed the services of Oneness Biotech and Crown Bioscience, respectively. Results: To develop a bifunctional anti-PDL-1 antibody/IL-10 fusion protein, anti-human PDL-1 antibodies were first developed by screening an in-house human variable domain phage display library. Three different antibodies were then C-terminally-fused with human IL-10 polypeptide. All three showed high binding affinity to PDL-1 and blocked PD-1 signaling through PD-1/PDL-1 interaction in a dose-dependent manner. In CD4 T cell/dendritic cell co-cultures, the lead anti-PDL1/IL10 protein enhanced IL-2 and IFNγ production, and it enhanced IFNγ and granzyme B production from activated CD8 T cells as well as proliferation of MC/9 cells in an IL-10-dependent manner. Also, a mouse ‘surrogate’ version (BF11, using avelumab anti-PDL-1) showed superior efficacy compared to either IL10-Fc or anti-PDL1/TGFbR in a mouse CT26 colon tumor model, and correlated with increased serum levels of CXCL9 and IL-18. BF11 also allowed greater EMT6 tumor control than anti-PDL-1 alone or anti-PDL1/TGFbR fusion protein. Such BF11-treated mice also showed long-lasting immunity against a secondary EMT6 tumor challenge. This ability of BF11 to enhance tumor control was in part dependent on increased activity of tumor-resident cells as co-treatment with FTY720, which inhibits lymphocyte egress from secondary lymphoid organs, did not appear to reduce anti-tumor activity of BF11 in some, albeit not all, EMT6-tumor-bearing mice. Conclusions: Anti-PDL1/IL10 fusion protein not only targets PDL-1 for checkpoint blockade but also activates the IL-10 pathway to boost T cell response, presumably including revitalization of exhausted T cells as recently reported. Effective anti-tumor activities of a mouse surrogate fusion protein, BF11, are demonstrated in CT26 and EMT6 tumor models. Taken together, our findings support this dual mechanism-strategy to potentiate anti-tumor immunity. Citation Format: Huey-Wen Hsiao, Chih-Lun Hsiao, Pandelakis Koni, Hung-Kai Chen. A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4169.

Nerve Growth Factor (NGF) as Partaker in the Modulation of UV-Response in Cultured Human Conjunctival Fibroblasts.

Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m2), either alone or supplemented with NGF (1–10–100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific “inflammatory to remodeling” pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling (DNMT3a, HDAC1, NRF2 and KEAP1)) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkANGFR/p75NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 transcripts, while it significantly upregulated NRF2 at lowest NGF doses and did not change KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkANGFR/p75NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

Immune-related genes are differentially associated with negative symptoms subdomains in patients with schizophrenia

IntroductionNegative symptoms (NS) are a core feature of schizophrenia. NS show heterogeneity proven by factor analysis, which revealed two distinct negative symptoms subdomains: diminished expression (DE) and avolition/apathy (AA) (Marder et al. 2017, Fleischhacker et al. 2019). Some studies showed the different effects of these subdomains on clinical features of schizophrenia that suggest different pathophysiological mechanisms for their development (Stanculete 2021). It has been also shown that the levels of peripheral interleukins (IL) specifically correlate with NS (Enache et al. 2021), in particular, increased IL levels were determined in patients with deficit syndrome compared to non-deficit schizophrenia (Goldsmith et al. 2018).ObjectivesTo search for the association of genes for IL-4, IL-6, IL-10 and C-reactive protein (CRP) with NS subdomains.MethodsThe total sample included 551 patients (women 51,4%, aged 18-72 years) with ICD-10 diagnosis of schizophrenia. NS were assessed by PANSS. PANSS-derived factors include AA (items N2, N4, G16) and DE (N1, N3, N6, G5, G7, G13). Genotyping was performed for the following polymorphisms: C-589T IL-4, C-174G IL-6, C-592A IL-10, G-1082A IL-10, CRP (rs2794521).ResultsThere are effects of C-592A IL-10 (p=0.017) and G-1082A IL-10 (p=0.012) on AA subdomain. Post-hoc Bonferroni corrected comparisons show that carriers of the haplotype AA (C-592A)- AA (G-1082A) have the highest AA score. A significant effect of CRP (rs2794521) on AA is identified (p=0.007). There is a trend towards the association of C-589T IL-4 and C-174G IL-6 with AA. No association of these polymorphisms with DE was found.ConclusionsAA and DE may have different genetic background.DisclosureNo significant relationships.

Single-cell profiling of immune cells associated with response to neoadjuvant chemoimmunotherapy in IIIA non-small cell lung cancer (NSCLC).

e20584 Background: The combination of immune checkpoint inhibitors (ICIs) with chemotherapy (chemoimmunotherapy) in the neoadjuvant setting have achieved favorable clinical benefits in non-small cell lung cancer (NSCLC), but the underlying molecular mechanism has not been fully elucidated. Methods: To identify factors associated with the clinical outcome, single-cell RNA/TCR sequencing was performed using 10x Genomics on CD45+ immune cells isolated from tumor and multiple immune-relevant tissues and peripheral blood of four treatment-naïve and eight neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients (responders versus non-responders). Bioinformatics analysis was used to screen immune cell factors associated with therapy response in single-cell sequencing profiles, and results were verified at protein level by flow cytometry, multiplex fluorescent immunohistochemistry (mIHC) and ELISA. Results: A total of 186,477 immune cells from 48 samples were acquired after quality-control filtering. The synergistic increase of B cells and CD4+ T cells are associated with positive therapeutic response of neoadjuvant chemoimmunotherapy. B cell class switching to IgG1 and IgG3 plays a critical role in anti-tumor immune response in tumor lesion, and this process is driven by increased IL-21 protein secreted by infiltrated T follicular helper (Tfh) cells after neoadjuvant chemoimmunotherapy. Several critical events lead to the positive clinical outcome during neoadjuvant chemoimmunotherapy, including the diminished activated TNFRSF4+ regulatory T cells (Tregs), increased LAMP3+ dendritic cells (DCs), high pretreatment peripheral blood T-cell diversity, and the expansion of intratumoral CD4+ T clones and peripheral CD8+ T clones. A validation cohort of 26 treatment-naïve and 30 neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients verified these findings. Conclusions: Single-cell profiling of the immune response to neoadjuvant chemoimmunotherapy uncovered several critical factors of anti-tumor immune response and provided insight of potential novel predictive factors and therapeutic targets for improving the efficacy of treatment in NSCLC.

Intratumoral immunotherapy relies on B and T cell collaboration.

Antitumor T cell responses are the primary mediators of cancer immunotherapy. However, many other components of the immune system are needed for efficient T cell responses to be generated. Here, we developed a combinatorial approach where a Toll-like receptor 9 agonist (CpG) and Fc-fused IL-12 protein were injected together into just one of several tumor sites in a mouse. This combination led to body-wide (abscopal) therapeutic responses in multiple cancer models. These systemic responses were dependent not only on T cells but also on B cells. B cells were activated by the treatment and were required for optimal T cell activation. This cross-talk was dependent on MHC and was tumor antigen specific. The addition of an agonistic antibody against OX40 further enhanced T cell activation and therapeutic responses. Our data suggest that the combination of CpG, anti-OX40, and IL-12Fc may have success in patients with cancer and that B and T cell collaboration is crucial for the efficacy of this combination immunotherapy.

Differential Effects of Alarmins on Human and Mouse Basophils.

Epithelial-derived alarmins (IL-33, TSLP, and IL-25) play an upstream role in the pathogenesis of asthma. Basophil-derived cytokines are a pivotal component of allergic inflammation. We evaluated the in vitro effects of IL-33, TSLP, and IL-25, alone and in combination with IL-3 on purified peripheral blood human basophils (hBaso) and bone marrow-derived mouse basophils (mBaso) in modulating the production of IL-4, IL-13, CXCL8 or the mouse CXCL8 equivalents CXCL1 and CXCL2. IL-3 and IL-33, but not TSLP and IL-25, concentration-dependently induced IL-4, IL-13, and CXCL8 release from hBaso. IL-3 synergistically potentiated the release of cytokines induced by IL-33 from hBaso. In mBaso, IL-3 and IL-33 rapidly induced IL-4 and IL-13 mRNA expression and protein release. IL-33, but not IL-3, induced CXCL2 and CXCL1 from mBaso. Differently from hBaso, TSLP induced IL-4, IL-13, CXCL1 and CXCL2 mRNA expression and protein release from mBaso. IL-25 had no effect on IL-4, IL-13, and CXCL1/CXCL2 mRNA expression and protein release even in the presence of IL-3. No synergism was observed between IL-3 and either IL-25 or TSLP. IL-3 inhibited both TSLP- and IL-33-induced CXCL1 and CXCL2 release from mBaso. Our results highlight some similarities and marked differences between the effects of IL-3 and alarmins on the release of cytokines from human and mouse basophils.

The Effect of Low-Dose Dexmedetomidine on Pain and Inflammation in Patients Undergoing Laparoscopic Hysterectomy.

Dexmedetomidine has sedative, sympatholytic, analgesic, and anti-inflammatory effects. We investigated the effects of intraoperative dexmedetomidine infusion without a loading dose in the prevention of pain and inflammation after laparoscopic hysterectomy. In this study, 100 patients undergoing laparoscopic hysterectomy under desflurane anesthesia were randomized to receive either 0.9% saline or dexmedetomidine (0.4 μg/kg/h) after induction to trocar removal. The primary endpoints were postoperative pain and inflammatory response presented by the level of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-10, and C-reactive protein (CRP). The secondary endpoints were hemodynamics during the anesthesia and surgery and postoperative nausea and vomiting. Postoperative pain was decreased in the dexmedetomidine group for every time point, and post-anesthesia care unit (PACU) rescue fentanyl doses were decreased in the dexmedetomidine group. The inflammatory response representing TNF-α, IL-6, IL-10, and CRP were similar across the two groups. Postoperative nausea and vomiting from PACU discharge to 24 h post-surgery were reduced in the dexmedetomidine group. During anesthesia and surgery, the patient’s heart rate was maintained lower in the dexmedetomidine-receiving group. Dexmedetomidine of 0.4 μg/kg/h given as an intraoperative infusion significantly reduced postoperative pain but did not reduce the inflammatory responses in patients undergoing laparoscopic hysterectomy.

Effect of Humanin G (HNG) on inflammation in age-related macular degeneration (AMD).

Inflammation plays a crucial role in the etiology and pathogenesis of AMD (Age-related Macular Degeneration). Humanin G (HNG) is a Mitochondrial Derived Peptide (MDP) that is cytoprotective in AMD and can protect against mitochondrial and cellular stress induced by damaged AMD mitochondria. The goal of this study was to test our hypothesis that inflammation-associated marker protein levels are increased in AMD and treatment with HNG leads to reduction in their protein levels. Humanin protein levels were measured in the plasma of AMD patients and normal subjects using ELISA assay. Humanin G was added to AMD and normal (control) cybrids which had identical nuclei from mitochondria-deficient ARPE-19 cells but differed in mitochondrial DNA (mtDNA) content derived from clinically characterized AMD patients and normal (control) subjects. Cell lysates were extracted from untreated and HNG-treated AMD and normal cybrids, and the Luminex XMAP multiplex assay was used to measure the levels of inflammatory proteins. AMD plasma showed reduced Humanin protein levels, but higher protein levels of inflammation markers compared to control plasma samples. In AMD RPE cybrid cells, Humanin G reduced the CD62E/ E-Selectin, CD62P/ P-Selectin, ICAM-1, TNF-α, MIP-1α, IFN–γ, IL-1β, IL-13, and IL-17A protein levels, thereby suggesting that Humanin G may rescue from mtDNA-mediated inflammation in AMD cybrids. In conclusion, we present novel findings that: A) show reduced Humanin protein levels in AMD plasma vs. normal plasma; B) suggest the role of inflammatory markers in AMD pathogenesis, and C) highlight the positive effects of Humanin G in reducing inflammation in AMD.

Interleukin-35 suppresses pyroptosis and protects against neuronal death in retinal ischaemia/reperfusion injury

Retinal ischaemia-reperfusion (I/R) is a pathological process in many eye disorders. Neuroinflammation and cell pyroptosis have been recognized as important in the pathogenesis of tissue damage in retinal I/R. Interleukin (IL)-35 is a novel heterodimeric cytokine that exhibits anti-inflammatory activity in various autoimmune diseases, but its role in retinal I/R and the underlying molecular mechanisms remain unexplored. This research investigated the effect of IL-35 on retinal I/R and the inhibition of pyroptosis and neuronal death. In our study, a murine retinal I/R model was used to explore the neuroprotective effect of recombinant IL-35 protein in vivo. Primary murine microglial cells in pyroptosis and retinal ganglion cells (RGCs) in oxygen and glucose deprivation/reoxygenation (OGD/R) models were employed to assess the antipyroptotic and antiapoptotic effects of IL-35 in vitro. The data showed that IL-35 decreases retinal damage, RGC death, and inner plexiform layer (IPL) thinning in mice with retinal I/R injury, with significant attenuation of pyroptosis in the retina. The study also demonstrated the anti-pyroptotic action of IL-35 in primary microglia stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Furthermore, primary RGC apoptosis induced by OGD/R was directly suppressed by IL-35, and IL-35-mediated neuroprotection was abrogated when miR-21 was blocked. In conclusion, our results suggest the mechanisms of RGC apoptosis and a new therapeutic target, IL-35, that exerts a robust neuroprotective effect against retinal I/R.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-β in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-β. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Elevated Serum Purine Levels in Schizophrenia: A Reverse Translational Study to Identify Novel Inflammatory Biomarkers.

BackgroundImmunological markers and related signaling molecules in the blood are altered in schizophrenia mouse models, in acutely relapsed patients with schizophrenia, and in persons at a clinically high risk for subsequently developing psychosis, highlighting their potential as prognostic and theranostic biomarkers. Therefore, we herein aimed to identify novel potential biomarkers in the serum that are associated with purinergic signaling.MethodsTo our knowledge, this is the first study to assess the correlations among the levels of human serum adenine nucleotides (ATP, ADP), adenosine, P2X7 receptor, and disease activity in patients hospitalized due to an acute relapse of schizophrenia (n = 53) and healthy controls (n = 47). In addition, to validate these findings using a reverse translational approach, we examined the same parameters in an acute phencyclidine-induced schizophrenia mouse model.ResultsWe found consistently elevated levels of ATP, ADP, interleukin (IL)-6, and IL-10 in both schizophrenia groups compared with the controls. The levels of adenosine, IL-1β, IL-12, and C-reactive protein were also increased in the human patient samples. Moreover, ATP and ADP were significantly positively correlated with the Positive and Negative Symptom Scale item “lack of judgment and insight”; IL-1β, IL-12, and tumour necrosis factor alpha were significantly positively correlated with “tension” and “depression”; and “disorientation” and “poor attention” were correlated significantly with IL-6 and IL-8.ConclusionsOur study suggests the promising potential of blood purines and inflammatory markers as future prognostic tools.

Dendritic cell activation and screening for key molecular signatures required for the induction of allergic responses.

The chain of events that leads to the sensitization of the immune system to environmental antigens, resulting in the onset of allergic disease, has been studied in great detail over the past 30 years. However, during this time, the rate of allergic diseases has increased exponentially, indicating the need to concentrate our studies on host-environmental factors that contribute to the onset of disease. Monocyte-derived dendritic cells (DCs) play a key role in driving localized and systemic immune responses. In this study, we developed a platform for screening the molecular signature and phenotypic profile of DCs activated by allergenic stimuli, including TSLP, IL-25, IL-33, IL-1a, Vit-D3 (1α,25-Dihydroxyvitamin D3), PAR1-AP Peptide, Papain, and recombinant human DerP1 protein to induce a type II associated inflammatory signature. Following activation with allergenic stimuli, modulated DCs are subjected to deep phenotyping via flow cytometry for surface and intracellular markers to detect and/or validate immunomodulatory properties. RNA sequencing is further used to compare the gene expression profiles of DCs responding to either allergenic or microbial stimuli, including the TLR3 agonist dsRNA Poly I:C and TLR4 agonist LPS. In our study, we aimed to identify key molecular signatures of DCs involved in the development of asthma and allergy based on their comparative activation with this broad panel of allergens. We expect to determine central control modules of transcription factors in DCs associated with Th2 induction.

Ketone body \u03b2-hydroxybutyrate ameliorates colitis by promoting M2 macrophage polarization through the STAT6-dependent signaling pathway

BackgroundKetone body β-hydroxybutyrate (BHB) has received more and more attentions, because it possesses a lot of beneficial, life-preserving effects in the fields of clinical science and medicine. However, the role of BHB in intestinal inflammation has not yet been investigated.MethodsColonic mucosa of inflammatory bowel disease (IBD) patients and healthy controls were collected for evaluation of BHB level. Besides, the therapeutic effect of exogenous BHB in a murine model of acute dextran sulfate sodium (DSS)-induced colitis were assessed by body weight change, colon length, disease activity index, and histopathological sections. The regulatory effectors of BHB were analyzed by RT-qPCR, immunofluorescence, and microbe analysis in vivo. Moreover, the molecular mechanism of BHB was further verified in bone marrow-derived macrophages (BMDMs).ResultsIn this study, significantly reduced BHB levels were found in the colonic mucosa from IBD patients and correlated with IBD activity index. In addition, we demonstrated that the administration of exogenous BHB alleviated the severity of acute experimental colitis, which was characterized by less weight loss, disease activity index, colon shortening, and histology scores, as well as decreased crypt loss and epithelium damage. Furthermore, BHB resulted in significantly increased colonic expression of M2 macrophage-associated genes, including IL-4Ra, IL-10, arginase 1 (Arg-1), and chitinase-like protein 3, following DSS exposure, suggesting an increased M2 macrophage skewing in vivo. Moreover, an in vitro experiment revealed that the addition of BHB directly promoted STAT6 phosphorylation and M2 macrophage-specific gene expression in IL-4-stimulated macrophages. Besides, we found that BHB obviously increased M2 macrophage-induced mucosal repair through promoting intestinal epithelial proliferation. However, the enhancement effect of BHB on M2 macrophage-induced mucosal repair and anti-inflammation was completely inhibited by the STAT6 inhibitor AS1517499.ConclusionsIn summary, we show that BHB promotes M2 macrophage polarization through the STAT6-dependent signaling pathway, which contributes to the resolution of intestinal inflammation and the repair of damaged intestinal tissues. Our finding suggests that exogenous BHB supplement may be a useful therapeutic approach for IBD treatment.