Decreased Cytokine Secretion Research Articles

Targeting microglial GLP1R in epilepsy: A novel approach to modulate neuroinflammation and neuronal apoptosis

BackgroundEpilepsy is a prevalent disorder of the central nervous system. Approximately, one-third of patients show resistance to pharmacological interventions. The pathogenesis of epilepsy is complex, and neuronal apoptosis plays a critical role. Aberrantly reactive astrocytes, induced by cytokine release from activated microglia, may lead to neuronal apoptosis. This study investigated the role of glucagon-like peptide 1 receptor (GLP1R) in microglial activation in epilepsy and its impact on astrocyte-mediated neurotoxicity. MethodsWe used human hippocampal tissue from patients with temporal lobe epilepsy and a pilocarpine-induced epileptic mouse model to assess neurobiological changes in epilepsy. BV2 microglial cells and primary astrocytes were used to evaluate cytokine release and astrocyte activation in vitro. The involvement of GLP1R was explored using the GLP1R agonist, Exendin-4 (Ex-4). ResultsOur findings indicated that reduced GLP1R expression in hippocampal microglia in both epileptic mouse models and human patients, correlated with increased cytokine release and astrocyte activation. Ex-4 treatment restored microglial homeostasis, decreased cytokine secretion, and reduced astrocyte activation, particularly of the A1 phenotype. These changes were associated with a reduction in neuronal apoptosis. In addition, Ex-4 treatment significantly decreased the frequency and duration of seizures in epileptic mice. ConclusionsThis study highlights the crucial role of microglial GLP1R in epilepsy pathophysiology. GLP1R downregulation contributes to microglial- and astrocyte-mediated neurotoxicity, exacerbating neuronal death and seizures. Activation of GLP1R with Ex-4 has emerged as a promising therapeutic strategy to reduce neuroinflammation, protect neuronal cells, and control seizures in epilepsy. This study provides a foundation for developing novel antiepileptic therapies targeting microglial GLP1R, with the potential to improve outcomes in patients with epilepsy.

Allyl isothiocyanate mitigates airway inflammation and constriction in a house dust mite-induced allergic asthma model via upregulation of tight junction proteins and the TRPA1 modulation

Asthma is a chronic inflammatory disease that has been associated with insufficient vegetable intake. Allyl Isothiocyanate (AITC) is a natural isothiocyanate found in cruciferous plants with anti-inflammatory and antioxidant abilities. Our study aimed to investigate the potential effect of AITC on tracheal constriction in a house dust mite (HDM)-induced asthma animal model, and explore the underlying mechanisms. To investigate the effects of AITC on HDM-induced allergic asthma model, established by intranasally administering extracts of HDM and AITC or DEX was given orally for four weeks. Flexivent SCIREQ, H&E staining, ELISA were employed to evaluate the lung function and the cytokine secretion. Possible mechanisms were determined by Western blot. Rat tracheae contraction was measured by Labscribe. We utilized lung epithelial cells (BEAS-2B) to assess the adhesion response to the combination of inflammatory factors TNF-α and IL-4. The results of the study showed that AITC significantly reduced tracheal constriction in ex vivo experiments and improved lung function in in vivo experiments compared to HDM-induced mice. Additionally, AITC decreased cytokine secretion, inflammatory cell infiltration in the lung, and constriction-related proteins expression in both lung and tracheae. Moreover, AITC increased tight junction-related protein expression in lung tissues. In vitro experiments showed that AITC had a protective effect through TRPA1 channel without affecting cell viability. Our results demonstrate that AITC has potential anti-asthma effects in HDM-induced asthma models by alleviating airway inflammation and airway constriction through increasing tight junction-related protein expression and suppressing Ca2+ signaling. These findings suggest that AITC may be a beneficial adjuvant therapy in asthma treatment.

Glucose metabolism and its role in the maturation and migration of human CD1c+ dendritic cells following exposure to BCG.

Tuberculosis (TB) still kills over 1 million people annually. The only approved vaccine, BCG, prevents disseminated disease in children but shows low efficacy at preventing pulmonary TB. Myeloid dendritic cells (mDCs) are promising targets for vaccines and immunotherapies to combat infectious diseases due to their essential role in linking innate and adaptive immune responses. DCs undergo metabolic reprogramming following exposure to TLR agonists, which is thought to be a prerequisite for a successful host response to infection. We hypothesized that metabolic rewiring also plays a vital role in the maturation and migration of DCs stimulated with BCG. Consequently, we investigated the role of glycolysis in the activation of primary human myeloid CD1c+ DCs in response to BCG. We show that CD1c+ mDC mature and acquire a more energetic phenotype upon challenge with BCG. Pharmacological inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) decreased cytokine secretion and altered cell surface expression of both CD40 and CCR7 on BCG-challenged, compared to untreated, mDCs. Furthermore, inhibition of glycolysis had differential effects on infected and uninfected bystander mDCs in BCG-challenged cultures. For example, CCR7 expression was increased by 2-DG treatment following challenge with BCG and this increase in expression was seen only in BCG-infected mDCs. Moreover, although 2-DG treatment inhibited CCR7-mediated migration of bystander CD1C+ DCs in a transwell assay, migration of BCG-infected cells proceeded independently of glycolysis. Our results provide the first evidence that glycolysis plays divergent roles in the maturation and migration of human CD1c+ mDC exposed to BCG, segregating with infection status. Further investigation of cellular metabolism in DC subsets will be required to determine whether glycolysis can be targeted to elicit better protective immunity against Mtb.

Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis

BackgroundLymphocyte activation gene-3 (LAG-3) inhibits T cell activation and interferes with the immune response by binding to MHC-II. As antigen presentation is central in rheumatoid arthritis (RA) pathogenesis, we studied aspects of LAG-3 as a serological marker and mediator in the pathogenesis of RA. Since Galectin-3 (Gal-3) is described as an additional binding partner for LAG-3, we also aimed to study the functional importance of this interaction.MethodsPlasma levels of soluble (s) LAG-3 were measured in early RA patients (eRA, n = 99) at baseline and after 12 months on a treat-to-target protocol, in self-reportedly healthy controls (HC, n = 32), and in paired plasma and synovial fluid (SF) from chronic RA patients (cRA, n = 38). Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were examined for LAG-3 expression by flow cytometry. The binding and functional outcomes of LAG-3 and Gal-3 interaction were assessed with surface plasmon resonance (SPR) and in cell cultures using rh-LAG3, an antagonistic LAG-3 antibody and a Gal-3 inhibitor.ResultsBaseline sLAG-3 in the plasma was increased in eRA compared to HC and remained significantly elevated throughout 12 months of treatment. A high level of sLAG-3 at baseline was associated with the presence of IgM-RF and anti-CCP as well as radiographic progression. In cRA, sLAG-3 was significantly increased in SF compared with plasma, and LAG-3 was primarily expressed by activated T cells in SFMCs compared to PBMCs. Adding recombinant human LAG-3 to RA cell cultures resulted in decreased cytokine secretion, whereas blocking LAG-3 with an antagonistic antibody resulted in increased cytokine secretion. By SPR, we found a dose-dependent binding between LAG-3 and Gal-3. However, inhibiting Gal-3 in cultures did not further change cytokine production.ConclusionssLAG-3 in the plasma and synovial fluid is increased in both early and chronic RA patients, particularly in the inflamed joint. High levels of sLAG-3 are associated with autoantibody seropositivity and radiographic progression in eRA, and LAG-3 plays a biologically active role in cRA by decreasing inflammatory cytokine production. This functional outcome is not affected by Gal-3 interference. Our results suggest that LAG-3 is a faceted regulator of inflammation in early and chronic RA.

Abstract 1868: Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression

Abstract We previously suggested that contactin 4 (CNTN4) negatively regulates T cell activity through binding with amyloid precursor protein on T cells. In addition, we confirmed that CNTN4 is highly expressed in the various types of tumors such as gallbladder, pancreas, stomach, endometrium, liver, prostate, and bladder cancer and melanoma with positive rates over 70% through immunohistochemistry analysis. However, murine cancer cells expressed CNTN4 at a very low level, differently from human tumors or cancer cells. Therefore, it was limited in assessing the anti-tumor efficacy by targeting CNTN4 in syngeneic mice models. Following this background, we have prepared CT26 murine cancer cells overexpressing CNTN4 (CT26/CNTN4) and evaluated anti-tumor efficacy by treating GENA-104A16, the anti-CNTN4 humanized monoclonal antibody in the CT26/CNTN4 syngeneic mice model. The treatment of GENA-104A16 induced fairly improved in vivo efficacy in the model compared to the CT26 WT model. Moreover, to identify the in vivo immune modulatory function of GENA-104A16, we analyzed the proportion of tumor-infiltrated immune cells in CT26/CNTN4 tumors through flow cytometry and single-cell analysis. As a result, the population of Treg cells decreased in the tumors of GENA-104A16-treated mice. In addition, the total amount of tumor-infiltrated immune cells increased by 2-fold, CD4+ T cells by 4-fold, and cytotoxic CD8+ T cells by 1.5-fold in tumors of mice administrated with GENA-104A16. We also performed transcriptomic analysis on samples collected from IgG-treated CT26 WT tumors and CT26/CNTN4 tumors treated with IgG or GENA-104A16. Comparative analysis of gene expression using the Gene Set Enrichment Analysis Hallmark showed that the gene sets of TNFα signaling, IFNγ response, inflammatory response, and apoptosis were decreased in CT26/CNTN4 tumors than in CT26 WT tumors. And the majority of the two gene sets, TNFα signaling and IFNγ response in CT26/CNTN4 tumors, increased by the administration of GENA-104A16. These results indicate that the overexpression of CNTN4 reduces the activity of tumor-infiltrating lymphocytes, resulting in decreased cytokine secretion, which is restored by administering GENA-104A16. Furthermore, we investigated in vitro T cell-mediated cytotoxicity of GENA-104A16 on U2OS (human osteosarcoma cell) endogenously expressing CNTN4 together with CNTN4-knocked out U2OS. GENA-104A16 increased T cell-mediated cytotoxicity when CNTN4-expressing U2OS cancer cells were co-cultured with T cells. Collectively, the function of CNTN4 in negative immune regulation was confirmed repeatedly through the present studies. And the expression level of CNTN4 on tumors was essential for the anti-tumor activity of GENA-104A16. Therefore, the expression of CNTN4 could be a potential biomarker for monitoring clinical responses to anti-CNTN4 therapy. Citation Format: Mi Young Cha, Hyunkyung Yu, Bu-Nam Jeon, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Joo-Yeon Chung, Gihyeon Kim, Changho Park, Kyung Mi Park, Hansoo Park. Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1868.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Role of m6A modification and novel circ_0066715/ miR-486-5p/ ETS1 axis in rheumatoid arthritis macrophage polarization progression.

Rheumatoid arthritis (RA) is a systemic disease dominated by inflammatory synovitis. RA synovial macrophages tend undergo M1-type macrophage polarization. Then, polarized M1-type macrophages secrete abundant pro-inflammatory cytokines, causing joint and cartilage destruction. N6-methyladenosine (m6A) methylation modification, circular RNA (circRNA), microRNA (miRNA), messenger RNA (mRNA), etc. are involved in the inflammatory response of RA. We found that there is an imbalance of inflammatory polarization in RA, which is manifested by a sharp increase in inflammatory markers and a high inflammatory response. Here, we show that RA was closely associated with low expression of circ_0066715. The overexpression of circ_0066715 significantly increased the ETS1 levels in RA-FLS cells, decreased cytokine secretion by M1-type macrophages, elevated M2-type cytokines, and inhibited FLS proliferation. Interestingly, the overexpression of miR-486-5p significantly suppressed the attenuation of the cell function and the effect on M1 macrophage polarization caused by circ_0066715 positive expression. WTAP may be involved in the methylation process of ETS1 in RA. ETS1 m6A methylation levels were altered upon WTAP intervention. The overexpression or interference of circ_0066715 decreased or increased WTAP expression. Our findings provide a novel circRNA/miRNA/mRNA regulatory axis and m6A regulatory mechanism involved in the process of RA macrophage polarization, thereby providing a powerful diagnostic and therapeutic strategy for RA treatment.

Increased CD8+ T cells in patients with Henoch–Schonlein purpura nephritis exhibit suppressive immune activity

BackgroundAbout 40% to 50% of children with Henoch-Schonlein purpura often suffer from nephritis, which can cause irreversible renal damage. Significantly increased peripheral T lymphocytes and reduced B lymphocytes have been widely reported as hallmarks of Henoch-Schonlein purpura nephritis (HSPN) differing from Henoch-Schonlein purpura without nephritis (HSP). While the role of peripheral immune cells, especially CD8+ T cells, in the development of nephritis of Henoch-Schonlein purpura is not clear. ObjectivesTo explore the changes of peripheral CD8+ T cells and the association of CD8+ T cell markers with indicators of renal function in HSP and HSPN patients. Patients and MethodsA total of 27 HSP and 16 HSPN patients were included in this study. The serum urea, serum creatinine, 24-hour urinary protein and peripheral white blood cell counts were collected from hospital registry systems. The T cell surface markers (CD28, CD107a and CD69) and cytokine (TNFα and IFNγ) secretion capacity were measured by flow cytometry. ResultsCompared with HSP patients, The number of CD8+ T cells in HSPN patients increased significantly (p=0.0003) and demonstrated with decreased CD69 expression (p<0.0001) and decreased cytokine secretion. The expression level of CD69 in CD3+, CD4+ and CD8+ T cells all significantly correlated negatively with serum creatinine and 24-hour urinary protein in HSP and HSPN children. ConclusionsThe inhibition of CD8+ T cell activity was significantly related to the decline of renal function in HSP and HSPN patients. It is possible to monitor renal function by detecting the expression of CD69 on CD8+ T cells in HSP and HSPN patients.

Shufeng Jiedu capsule inhibits inflammation and apoptosis by activating A2AAR and inhibiting NF-κB to alleviate LPS-induced ALI

Ethnopharmacological relevanceShufeng Jiedu capsule (SFJDC) is a pure form of traditional Chinese medicine (TCM) that contains eight medicinal plants. Known for its anti-inflammatory and antipyretic effects, it is mostly used to treat upper respiratory tract infections and other infectious diseases, such as colds, pharyngitis, laryngitis, and tonsillitis. Both acute lung injury (ALI) and COVID-19 are closely related to lung damage, primarily manifesting as lung inflammation and epithelial cell damage. However, whether SFJDC can improve ALI and by what mechanism remain unclear. The purpose of this study was to explore whether SFJDC could be used as a prophylactic treatment for COVID-19 by improving acute lung injury. Aim of the studyThe purpose of this study was to determine whether SFJDC could protect against ALI caused by lipopolysaccharide (LPS), and we wanted to determine how SFJDC reduces inflammation and apoptosis pharmacologically and molecularly. Materials and methodsPreadministering SFJDC at 0.1 g/kg, 0.3 g/kg, or 0.5 g/kg for one week was followed by 5 mg/kg LPS to induce ALI in mice. Observations included the study of lung histomorphology, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) secretion, as well as the ratio of lung wet/dry weights. In addition, RAW264.7 cells were treated for 24 h with 1 μg/mL LPS after being pretreated for 1 h with 0.5 mg/mL SFJDC. In the samples, we detected TNF-α, IL-1β, and IL-6. Cell apoptosis was detected by stimulating A549 cells for 24 h with RAW264.7 supernatant. Both in vitro and in vivo, the levels of A2A adenosine receptor (A2AAR), PKA, IκB, p-IκB, NF-κB P65 (P65), p–NF–κB P65 (p-P65), cleaved caspases-3 (Cc3), Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) proteins were determined using Western blot analysis. ResultsLung tissue morphology was improved as SFJDC decreased cytokine secretion, the ratio of lung wet/dry weights, and lung tissue secretion of proinflammatory cytokines. The expression of A2AAR was increased by SFJDC, and the phosphorylation of NF-κB was inhibited. TUNEL staining and flow cytometry showed that SFJDC inhibited apoptosis by reducing the expression of Cc3 and the ratio of Bax/Bcl-2. ConclusionsAccording to the results of this study, SFJDC can reduce inflammation and inhibit apoptosis. A2AAR activation and regulation of NF-κB expression are thought to make SFJDC anti-inflammatory and anti-apoptotic. A wide range of active ingredients may result in an anti-inflammatory and antipyretic effect with SFJDC.

A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

M351-0056 is a novel low MW compound modulating the actions of the immune-checkpoint protein VISTA.

Background and PurposeThe protein V‐domain immunoglobulin suppressor of T‐cell activation (VISTA) is a novel immune‐checkpoint molecule that belongs to the B7 family and regulates a broad spectrum of immune responses. So far, low MW compounds targeting VISTA for the treatment of autoimmune diseases or inflammation, have not been identified.Experimental ApproachWe developed a homology modelling for VISTA 3D structure and subsequent virtual screening for low MW ligands binding to VISTA. Visualization of the binding postures of docked ligands with protein VISTA indicated that compound M351‐0056 targeted VISTA. The biological activities of compound M351‐0056 targeting VISTA were investigated in vitro using monocytes and T cells and in vivo, using mice with imiquimod‐induced dermatitis.Key ResultsThe K D value of M351‐0056 for human VISTA‐extracellular domain was 12.60 ± 3.84 μM as assessed by microscale thermophoresis. M351‐0056 decreased cytokine secretion from PBMCs or human CD4+ T cells, suppressed proliferation of PBMCs and enhanced expression of Foxp3+ T cells. These effects of M351‐0056 modulating VISTA involved the JAK2–STAT2 pathway. Daily administration of M351‐0056 ameliorated imiquimod‐induced psoriasis‐like dermatitis. Expression of mRNA and protein of inflammatory cytokines in psoriatic lesions was decreased after M351‐0056 treatment.Conclusion and ImplicationsThe compound M351‐0056 showed high affinity for VISTA and may modulate its immune function in vitro and in vivo. Our finding provides a lead compound for therapeutically enhancing VISTA‐mediated pathways to benefit the treatment of autoimmune diseases or inflammation.

Commensal fungi and their cell-wall β-glucans direct differential responses in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that β-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to β-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and β-glucans activated Syk and ERK in IEC lines. However, only β-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with β-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with β-glucans alone. Finally, exvivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than β-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.

Evaluation of periodontal status and cytokine/chemokine profile of GCF in patients with severe congenital neutropenia.

Severe congenital neutropenia (SCN) is a primary immunodeficiency characterized by defect in neutrophil count. Increased risk of infections in addition to periodontal problems, such as ulcerations of oral mucosa, gingival inflammation, and rapid loss of attachment are common in the course of the disease. The aim of the present study is to define the causal relationship between the severity of periodontal inflammation and severe congenital neutropenia through identification of cytokine profile in gingival crevicular fluid (GCF). A case-control study was performed in patients diagnosed with SCN and healthy controls. Demographic data, the molecular defect, laboratory work-up were gathered from the hospital registry. Periodontal indices were recorded and GCF samples were analyzed using multiplex analysis for the simultaneous measurements of the particular cytokines and chemokines. The present study included 14 patients and 22 control subjects. Both groups were comparable in terms of age and sex. Severity of gingival inflammation measured by the criteria of Löe was higher in the SCN cases (p < 0.05). Moreover, GCF levels of IFN-α, TNF-α, IL-10, IL-13, IL-15, IL-17, IL-2, IL-7, IL-33, IP-10, MIG, MIP-1β were significantly higher in the controls. Decreased cytokine secretion seems to correlate with the decrease in neutrophil counts. The severity of gingival inflammation in SCN patients may be due to the bacterial overgrowth and the change in the content of the oral flora due to the decreased neutrophil counts. Therefore, regular periodontal examinations, the motivation of oral hygiene as well as the compliance with therapy in SCN patients contribute to the periodontal health.

Transient Receptor Potential Vanilloid 4 (TRPV4) Protects the Lung from Bacterial Pneumonia via MAPK Molecular Switching

Abstract Mechanical cell-matrix interactions can drive the innate immune responses to infection, however the molecular underpinnings of these responses remain elusive. We have discovered that the biophysical properties of the matrix in the range of injured fibrotic lung (≥ 25 kPa) conditions the macrophage response to LPS and infection respectively through the mechanosensitive cation channel, TRPV4 in vitro and in vivo. Studies suggest that LPS-induced macrophage activation is controlled in part by the MAPK pathway (i.e. p38, ERK, JNK). Thus, we investigated if TRPV4 plays a role in the macrophage activation response after LPS through alteration of the MAPK pathway. TRPV4 KO mice exhibited reduced lung bacterial clearance by macrophages (6-fold, p=0.012) after intratracheal P. aeruginosa administration and increased lung injury as measured by inflammatory cell infiltration (≥80±3%, p&amp;lt;0.05), vascular permeability (BAL total protein ≥63±6%, p&amp;lt;0.05), and pro-inflammatory cytokine secretion in BALF (IL-6, CCL2, CXCL1 ≥71±4%, p&amp;lt;0.05). LPS-induced p38 activation was decreased (69%, p&amp;lt;0.05) while JNK activation was increased (2-fold, p&amp;lt;0.05) in a stiffness-dependent manner with no change in ERK activation in TRPV4 KO BMDMs. Inhibition of p38 (SB203580, BIRB796) decreased phagocytosis whereas inhibition of JNK (SP600125) decreased cytokine secretion (IL-6, CCL2, and CXCL1) after LPS (2 fold, p&amp;lt;0.05). DUSP1/MKP1 (MAPK phosphatase) protein was reduced by 3-fold in TRPV4 KO and inhibition of DUSP1 decreased phagocytosis and selectively increased activation of JNK. These data are the first to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner, through MAPK activation switching.

Autophagy is critical for group 2 innate lymphoid cell metabolic homeostasis and effector function

Abstract Allergic asthma is a chronic inflammatory disorder that is characterized with airway hyperreactivity (AHR) and driven by Th2 cytokine production. Group 2 innate lymphoid cells (ILC2s) secrete high amount of Th2 cytokines and contribute to the development of AHR. Autophagy is a cellular degradation pathway that recycles cytoplasmic content. However, the role of autophagy in ILC2s remains to be fully elucidated. We characterized the effects of autophagy deficiency on ILC2 effector functions and metabolic balance. ILC2s from autophagy deficient mice were isolated to evaluate homeostasis, cytokine secretion, gene expression and cell metabolism. Also, autophagy deficient ILC2s were adoptively transferred into Rag−/−GC−/− mice, which were them challenged with IL-33 and assessed for AHR and lung inflammation. We demonstrate that autophagy is extensively used by activated ILC2s to maintain their homeostasis and effector functions. Deletion of the critical autophagy gene Atg5 resulted in decreased cytokine secretion and increased apoptosis. Moreover, lack of autophagy among ILC2s impaired their ability to utilize fatty acid oxidation and promoted glycolysis as evidenced by our transcriptomic and metabolite analyses. This shift of fuel dependency led to impaired homeostasis and Th2 cytokine production, thus inhibiting the development of ILC2-mediated AHR. Notably, this metabolic reprogramming was also associated with an accumulation of dysfunctional mitochondria producing excessive reactive oxygen species. These findings provide new insights into the metabolic profile of ILC2s and suggest that modulation of fuel dependency by autophagy is a potentially new therapeutic approach to target ILC2 dependent inflammation.

Autophagy is critical for group 2 innate lymphoid cell metabolic homeostasis and effector function

Allergic asthma is a chronic inflammatory disorder characterized by airway hyperreactivity (AHR) and driven by TH2 cytokine production. Group 2 innate lymphoid cells (ILC2s) secrete high amounts of TH2 cytokines and contribute to the development of AHR. Autophagy is a cellular degradation pathway that recycles cytoplasmic content. However, the role of autophagy in ILC2s remains to be fully elucidated. We characterized the effects of autophagy deficiency on ILC2 effector functions and metabolic balance. ILC2s from autophagy-deficient mice were isolated to evaluate proliferation, apoptosis, cytokine secretion, gene expression and cell metabolism. Also, autophagy-deficient ILC2s were adoptively transferred into Rag-/-GC-/- mice, which were then challenged with IL-33 and assessed for AHR and lung inflammation. We demonstrate that autophagy is extensively used by activated ILC2s to maintain their homeostasis and effector functions. Deletion of the critical autophagy gene autophagy-related 5 (Atg5) resulted in decreased cytokine secretion and increased apoptosis. Moreover, lack of autophagy among ILC2s impaired their ability to use fatty acid oxidation and strikingly promoted glycolysis, as evidenced by our transcriptomic and metabolite analyses. This shift of fuel dependency led to impaired homeostasis and TH2 cytokine production, thus inhibiting the development of ILC2-mediated AHR. Notably, this metabolic reprogramming was also associated with an accumulation of dysfunctional mitochondria, producing excessive reactive oxygen species. These findings provide new insights into the metabolic profile of ILC2s and suggest that modulation of fuel dependency by autophagy is a potentially new therapeutic approach to target ILC2-dependent inflammation.

Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.

Twist1 and Twist2 Induce Human Macrophage Memory upon Chronic Innate Receptor Treatment by HDAC-Mediated Deacetylation of Cytokine Promoters.

Intestinal tissues are continuously exposed to microbial products that stimulate pattern-recognition receptors (PRRs). Ongoing PRR stimulation can confer epigenetic changes in macrophages, which can then regulate subsequent immune outcomes and adaptation to the local environment. Mechanisms leading to these changes are incompletely understood. We found that short-term stimulation of the PRR NOD2 in primary human monocyte-derived macrophages resulted in increased H3 and H4 acetylation of cytokine promoters, consistent with the increased cytokine secretion observed. However, with prolonged NOD2 stimulation, both the acetylation and cytokine secretion were dramatically decreased. Chronic NOD2 stimulation upregulated the transcription factors Twist1 and Twist2, which bound to the promoters of the histone deacetylases HDAC1 and HDAC3 and induced HDAC1 and HDAC3 expression. HDAC1 and HDAC3 then mediated histone deacetylation at cytokine promoters and, in turn, cytokine downregulation under these conditions. Similar regulation was observed upon chronic stimulation of multiple PRRs. Consistent with the chronic microbial exposure in the intestinal environment, TWIST1, TWIST2, HDAC1, and HDAC3 were upregulated in human intestinal relative to peripheral macrophages. Importantly, complementing HDAC1 and HDAC3 in Twist1/Twist2-deficient monocyte-derived macrophages restored the reduced histone acetylation on cytokine promoters and the decreased cytokine secretion with chronic NOD2 stimulation. Taken together, we identify mechanisms wherein Twist1 and Twist2 promote chromatin modifications, resulting in macrophage instruction and adaptation to conditions in the intestinal microenvironment.

A focused structure-activity relationship study of psoralen-based immunoproteasome inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. The development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. Here, we describe a focused series of psoralen-based inhibitors of the β5i subunit of the immunoproteasome with different substituents placed at position 4'. The most promising compound was further evaluated through changes at position 3 of the psoralen ring. Despite a small decrease in the inhibitory potency in comparison with the parent compound, we were able to improve the selectivity against other subunits of both the immunoproteasome and the constitutive proteasome. The most potent compounds discriminated between both proteasome types in cell lysates and also showed a decrease in cytokine secretion in peripheral blood mononuclear cells.

Notch signaling mediated by Delta-like ligands 1 and 4 controls the pathogenesis of chronic GVHD in mice

AbstractChronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2→BALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)–driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6→B10.BR major histocompatibility complex–mismatched model with multi–organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.