Reduced Cytokine Production Research Articles

Matrix stiffening from collagen fibril density and alignment modulates YAP-mediated T-cell immune suppression

T-cells are essential components of the immune system, adapting their behavior in response to the mechanical environments they encounter within the body. In pathological conditions like cancer, the extracellular matrix (ECM) often becomes stiffer due to increased density and alignment of collagen fibrils, which can have a significant impact on T-cell function. In this study, we explored how these ECM properties—density and fibrillar alignment—affect T-cell behavior using three-dimensional (3D) collagen matrices that mimic these conditions. Our results show that increased matrix stiffness, whether due to higher density or alignment, significantly suppresses T-cell activation, reduces cytokine production, and limits proliferation, largely through enhanced YAP signaling. Individually, matrix alignment appears to lower actin levels in activated T-cells and changes migration behavior in both resting and activated T-cells, an effect not observed in matrices with randomly oriented fibrils. Notably, inhibiting YAP signaling was able to restore T-cell activation and improve immune responses, suggesting a potential strategy to boost the effectiveness of immunotherapy in stiff ECM environments. Overall, this study provides new insights into how ECM characteristics influence T-cell function, offering potential avenues for overcoming ECM-induced immunosuppression in diseases such as cancer.

Protective Role of Astaxanthin in Regulating Lipopolysaccharide-Induced Inflammation and Apoptosis in Human Neutrophils.

Astaxanthin, a keto-carotenoid, is known to have potent antioxidant properties. This study aims to investigate the anti-inflammatory effect of astaxanthin and its mechanism in human neutrophils. The effects of astaxanthin on lipopolysaccharide (LPS)-stimulated human neutrophils were investigated in vitro. Neutrophils were isolated from healthy volunteers and stimulated with LPS in the presence and absence of astaxanthin. We assessed cytokine production, signaling pathway activation via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB), and apoptosis. Astaxanthin's impact was evaluated at different concentrations, and both pretreatment and cotreatment protocols were tested. The results demonstrated that astaxanthin significantly reduced the production of pro-inflammatory cytokines TNF-α and IL-1β in LPS-stimulated neutrophils. It effectively inhibited the phosphorylation of ERK1/2 MAPK, without notably affecting p38 MAPK or NF-κB pathways. Furthermore, astaxanthin promoted apoptosis in neutrophils, counteracting the apoptosis-delaying effects of LPS. These effects were more pronounced with pretreatment. In conclusion, astaxanthin has protective effects on inflammatory responses in neutrophils by reducing cytokine production and enhancing apoptosis while selectively modulating intracellular signaling pathways. Astaxanthin demonstrates significant potential as a therapeutic agent in the management of severe inflammatory conditions.

Altered Body Composition and Cytokine Production in Patients with Elevated HOMA-IR after SARS-CoV-2 Infection: A 12-Month Longitudinal Study.

Altered body composition and cytokine production due to SARS-CoV-2 antigens may affect homeostasis model assessment for insulin resistance (HOMA-IR) after SARS-CoV-2 infection. To elucidate this phenomenon, we conducted a longitudinal study involving 47 COVID-19 patients, who were followed up for 12 months. During recruitment, body composition and glucose indices were measured, and heparin blood samples were collected for measuring cytokine production. HOMA-IR was considered an elevated or non-elevated group based on the ratio between HOMA-IR at 12 months and 1 month of convalescence. Those with elevated HOMA-IR had a significantly higher body mass index, body fat percentage, and visceral fat rating and had a lower lean mass and lean/fat mass ratio than their counterparts. During the convalescent period, the elevated HOMA-IR group had lower TNFα, IFNγ, IL-2, IL-10, and granzyme B expression levels but had higher TNFα/IL-10, IFNγ/IL-10, IL-2/IL-10, and granzyme B/IL-10 ratios than the other group. The reduced cytokine production and pro-/anti-inflammatory imbalance in patients with elevated HOMA-IR may suggest immune cell dysfunction toward SARS-CoV-2. Patients with elevated HOMA-IR after SARS-CoV-2 infection may experience an increase in BMI and body fat percentage, leading to increased immune dysfunction and chronic inflammatory condition. A nutritional approach and promotion of physical activity may help reduce HOMA-IR and ameliorate glucose indices in these patients.

The role of MNK1-mTORC1 pathway in modulating macrophage responses to Vibrio vulnificus infection.

Mitogen-activated protein kinase (MAPK)-interacting kinase (MNK) plays a role in promoting the production of tumor necrosis factor alpha and interleukin-6 in macrophages during Vibrio vulnificus (Vv) infection. Inhibition or knockout of MNK1 in J774A.1 cells resulted in reduced cytokine production without affecting their transcription levels. MNK1 also impairs phagocytosis, bacterial clearance, and phagosome acidification in Vv-infected cells through the MNK1-mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. The findings highlight the importance of the MNK1-mTORC1 pathway in modulating macrophage responses to Vv infection.

IL-17 signaling pathway: A potential therapeutic target for reducing skeletal muscle inflammation

BackgroundThe interleukin-17 (IL-17) signaling pathway is intricately linked with immunity and inflammation; however, the association between the IL-17 signaling pathway and skeletal muscle inflammation remains poorly understood. The study aims to investigate the role of the IL-17 signaling pathway in skeletal muscle inflammation and to evaluate the therapeutic potential of anti-IL-17 antibodies in reducing muscle inflammation. MethodsA skeletal muscle inflammation model was induced by cardiotoxin (CTX) injection in C57BL6/J mice. Following treatment with an anti-IL-17 antibody, we conducted a comprehensive analysis integrating single-cell RNA sequencing (scRNA-seq), bioinformatics, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blot techniques to elucidate underlying mechanisms. ResultsscRNA-seq analysis revealed a significant increase in neutrophil numbers and activity in inflamed skeletal muscle compared to other cell types, including macrophages, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells. The top 30 differentially expressed genes within neutrophils, along with 55 chemokines, were predominantly enriched in the IL-17 signaling pathway. Moreover, the IL-17 signaling pathway exhibited heightened expression in inflamed skeletal muscle, particularly within neutrophils. Treatment with anti-IL-17 antibody resulted in the suppression of IL-17 signaling pathway expression, accompanied by reduced levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as decreased numbers and activity of Ly6g+/Mpo+ neutrophils compared to CTX-induced skeletal muscle inflammation. ConclusionOur findings suggest that the IL-17 signaling pathway plays a crucial role in promoting inflammation within skeletal muscle. Targeting this pathway may hold promise as a therapeutic strategy for ameliorating the inflammatory micro-environment and reducing cytokine production.

Immunomodulatory effects of antiangiogenic tyrosine kinase inhibitors in renal cell carcinoma models: Impact on following anti-PD-1 treatments

The approval of immune checkpoint inhibitors (ICIs) has revolutionized the management of metastatic renal cell carcinoma (RCC), introducing several ICI-based combinations as the new standard of care for affected patients. Nonetheless, monotherapy with antiangiogenic tyrosine kinase inhibitors (TKIs), such as pazopanib or sunitinib, still represents a first-line treatment option for selected patients belonging to the favorable risk group according to the International mRCC Database Consortium (IMDC) model. After TKI monotherapy, the main second-line option is represented by ICI monotherapy with the anti-Programmed Death Receptor 1(PD-1) nivolumab.To date, the expected clinical outcomes are similar with pazopanib or sunitinib and there is no clear indication for selecting one TKI over the other. Moreover, their impact on subsequent ICI treatment outcomes is not well defined, yet.Based on these premises, we investigated the immunomodulatory activity of these drugs in vitro and in vivo.Both TKIs induced Programmed Cell Death Ligand-1 (PD-L1) expression and soluble PD-L1 release in RCC cells, and hampered T cell activation, reducing cytokine production and the proportion of activated T cells. Nevertheless, in a syngeneic co-culture system with peripheral blood mononuclear cells (PBMCs) and tumor cells, incubation with anti-PD-1 antibody following TKIs treatment significantly restored T cell function, potentiating the cytotoxic effects against tumor cells. Pazopanib and sunitinib followed by anti-PD-1 antibody produced a comparable inhibition of tumor growth in a RCC syngeneic mouse model. Our findings suggest that pazopanib and sunitinib, showing similar immunomodulatory effects, may have a comparable impact on the subsequent effectiveness of PD-1/PD-L1 blockade.

Photobiomodulation mitigates Bothrops jararacussu venom-induced damage in myoblast cells by enhancing myogenic factors and reducing cytokine production.

Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. C2C12 myoblast cells were exposed to BjsuV (12.5 μg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кβ, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-β, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-β, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

Targeting glutaminolysis pathway in trained immunity induced by oxidized low-density lipoprotein

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft DFG Atherosclerosis is a chronic inflammatory disease of the arterial wall that causes cardiovascular disease. Monocyte-derived macrophages importantly contribute to atherogenesis. Monocytes can be primed to exhibit increased responsiveness upon a secondary non-related stimulus, a phenomenon called trained immunity. Intracellular metabolic and epigenetic reprogramming lays the basis of trained immunity. One of the stimuli that can induce trained immunity is oxidized low-density lipoprotein (oxLDL). oxLDL-trained monocytes exhibit an increase in metabolic pathways such as glycolysis, OXPHOS, and cholesterol biosynthesis. The glutaminolysis pathway that fuels the tricarboxylic acid (TCA) cycle is essential for trained immunity induction by b-glucan, the cell wall component of Candida albicans. The goal of this project is to investigate the involvement of the glutaminolysis pathway in oxLDL-induced trained immunity in isolated human primary monocytes. We exposed monocytes for 24 hours to oxLDL, allowed subsequent differentiation into macrophages for six days in culture medium, and assessed cytokine production capacity upon restimulation. Co-incubation of oxLDL with the inhibitor that blocks glutaminase, the enzyme that generates glutamate from glutamine, significantly reduced cytokine production capacity in oxLDL-trained cells. We further showed that glutaminolysis is necessary for oxLDL-induced increase in oxygen consumption (OCR) and glycolysis rate (ECAR). Single nucleotide polymorphisms in glutaminolysis genes were shown to correlate with ex vivo oxLDL-induced trained immunity in 243 healthy volunteers. These data suggest an important role of glutaminolysis in the establishment of oxLDL-induced trained immunity. To unravel the molecular mechanisms behind this observation we plan to perform epigenetic as well as metabolic analyses of the monocytes trained in the presence of the inhibitor.

TRPM2 channels are essential for regulation of cytokine production in lung interstitial macrophages.

Interstitial macrophages (IMs) are essential for organ homeostasis, inflammation, and autonomous immune response in lung tissues, which are achieved through polarization to a pro-inflammatory M1 and an M2 state for tissue repair. Their remote parenchymal localization and low counts, however, are limiting factors for their isolation and molecular characterization of their specific role during tissue inflammation. We isolated viable murine IMs in sufficient quantities by coculturing them with stromal cells and analyzed mRNA expression patterns of transient receptor potential (TRP) channels in naïve and M1 polarized IMs after application of lipopolysaccharide (LPS) and interferon γ. M-RNAs for the second member of the melastatin family of TRP channels, TRPM2, were upregulated in the M1 state and functional channels were identified by their characteristic currents induced by ADP-ribose, its specific activator. Most interestingly, cytokine production and secretion of interleukin-1α (IL-1α), IL-6 and tumor necrosis factor-α in M1 polarized but TRPM2-deficient IMs was significantly enhanced compared to WT cells. Activation of TRPM2 channels by ADP-ribose (ADPR) released from mitochondria by ROS-produced H2O2 significantly increases plasma membrane depolarization, which inhibits production of reactive oxygen species by NADPH oxidases and reduces cytokine production and secretion in a negative feedback loop. Therefore, TRPM2 channels are essential for the regulation of cytokine production in M1-polarized murine IMs. Specific activation of these channels may promote an anti-inflammatory phenotype and prevent a harmful cytokine storm often observed in COVID-19 patients.

Therapeutic effect of ginkgetin on smoke-induced airway inflammation by down-regulating the c/EBPβ signaling pathway and CCL2 expression

Ethnopharmacological relevanceGinkgo biloba leaf and seed have been traditionally used in ancient China for the treatment of cough and asthma. However, there is limited literature available on the anti-COPD effects and mechanisms of Ginkgo biloba. Aims of the studyThe aim of this study was to comprehensively investigate the therapeutic potential of ginkgo extracts in COPD through a combination of in vivo and in vitro functional experiments. Transcriptomic analyses were also employed to uncover novel molecular mechanisms underlying the therapeutic effects of ginkgetin in COPD. Materials and methodsThe therapeutic efficacy of ginkgo extracts was assessed in a COPD model. The anti-inflammatory effects of ginkgetin and its underlying molecular mechanisms were examined in A549 cells treated with cigarette smoke extract (CSE). Additionally, transcriptomic analyses were conducted to identify novel molecular pathways influenced by ginkgetin. These findings were further validated using quantitative real-time polymerase chain reaction (qPCR) and Western blot techniques. ResultsThe ethyl acetate extract of Ginkgo biloba L. seeds and ginkgetin treatment significantly reduced cytokine production in COPD mice. Following drug administration, lung function improved in different groups. The transcriptome data strongly supports the inhibitory effect of ginkgetin on CSE-induced inflammation through the downregulation of the c/EBPβ signaling pathway and subsequent inhibition of CCL2 expression. ConclusionOur results demonstrate that ginkgetin, one of the biflavones found in Ginkgo biloba, exhibits inhibitory effects on smoke-induced airway inflammation. This effect is achieved through the downregulation of the c/EBPβ signaling pathway and the reduction of CCL2 expression.

Impaired NK cell-mediated antiviral responses to HIV following BCG vaccination: implications for neonatal vaccination

Abstract Bacillus Calmette-Guérin (BCG) vaccine is the only approved vaccine against tuberculosis (TB). BCG has been shown to elicit nonspecific protection as well as detrimental effects during diseases unrelated to TB. We investigate the impact of BCG on modulating NK cell responses and its effect on altering susceptibility to HIV acquisition. The C57BL/6 mouse model was used to compare splenic NK cell responses of control (n=4) and BCG vaccinated mice (n=4) following subsequent exposure to Mycobacterium bovis (Mtb) and HIV Gag peptide antigens. Statistical comparisons were made using Wilcoxon ranked sum test. The data acquired though flowcytometry suggested that mature, BCG trained memory-like (KLRG1+ Ly49H+) NK cells showed increased proinflammatory cytokine responses and polyfunctionality upon re-exposure to Mtb antigens. However, when these trained cells were exposed to HIV Gag antigen, we observed a significant reduction in cytokine production and polyfunctionality. Specifically, these BCG trained NK cells showed marked decrease in IFNγ, TNFα, IL1β and Granzyme B production alone or in combination (p<0.05). Furthermore, negative correlations between NK polyfunctionality and mature BCG trained NK cells were observed, following HIV Gag peptide exposure. This suggests that NK cells are impaired in their antiviral effects during HIV exposure, soon after BCG vaccination. This may suggest higher risk of HIV acquisition in BCG vaccinated neonates that are frequently exposed to HIV.

Inhibition of SGL2 Prevents Albumin Induced Proximal Tubule Inflammation

Albuminuria is an independent risk factor for cardiovascular disease and renal damage in hypertension. It is associated with a higher risk of cardiovascular morbidity and mortality, and with declining kidney function. The Na+-glucose cotransporter-2 (SGLT2) plays a crucial role in glucose and sodium reabsorption in the proximal tubule. Clinical trials have shown that SGLT2 inhibitors can reduce cardiovascular events, lower blood pressure, and decrease the risk of end-stage kidney disease in individuals with type 2 diabetes mellitus. These trials have also demonstrated the potential of SGLT2 inhibitors to provide additional benefits beyond their glucose control, including decreased albuminuria. In this study, we hypothesized that exposure to elevated albumin causes proximal tubule injury and the release of cytokines from the basolateral membrane. We propose that by reducing transport in the proximal tubule, SGLT2 inhibitors may alleviate kidney damage by reducing cytokine production and subsequently renal inflammation. To test our hypothesis, we conducted experiments using Dahl salt-sensitive (SS) rats and immortalized human proximal tubule cells (RPTEC-TERT1). To determine how early albuminuria and proximal injury occur in the development of salt-sensitive hypertension, 24-hour urine was collected from SS rats fed either a control salt (CS, 0.4% NaCl, n=10) or a high salt (HS, 4.0% NaCl, n=11) diet for 7 days. Significant increases in albuminuria were observed after 1 day of HS (CS: 25±7 vs. HS: 59±13 mg/day p=0.016). This was followed by significant increases in urinary kidney injury molecule-1 (Kim-1) after 2 days of HS (CS: 13±2 vs. HS: 23±3 pg/day p=0.021). Preliminary analysis found substantial increases in urinary CCL2 after 4 days of HS (CS: 58±7 vs. HS: 135±25 pg/mg, n=5). To investigate whether the renoprotective effects of SGLT2 inhibitors are in part due to their ability to reduce inflammation in the proximal tubule, we exposed the apical membrane of RPTEC-TERT1 cells to human serum albumin (HSA, 10mg/ml, n=6) with and without Dapagliflozin (Dapa, 15ng/ml, n=5) for 24-hours and measured the levels of CCL2 in the basolateral media. HSA caused a significant increase in CCL2 levels relative to control cells (Control: 42.5±5.6 vs. HSA: 84.1±8.7 pg/mg, p=0.00). This increase was prevented by co-treatment with Dapa and CCL2 levels were equivalent to those in the control cells (HSA+Dapa: 44.0±4.5 pg/mg, p=0.988). In conclusion, our findings support the hypothesis that albuminuria precedes proximal tubule injury and may contribute to the development of renal inflammation in salt-sensitive hypertension. The ability of Dapa to reduce prevent albumin induced CCL2 production in RPTEC-TERT1 cells suggests that SGLT2 inhibitors may reduce renal inflammation in salt-sensitive hypertension and is an area of ongoing studies. Studies funded by R01HL152166 to LE. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes invivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

Metabolic heterogeneity is a determinant of immune cell function. The normal physiological metabolic reprogramming of pregnancy that ensures the fuel requirements of mother and baby are met, might also underpin changes in immunity that occur with pregnancy and manifest as altered responses to pathogens and changes to autoimmune disease symptoms. Using peripheral blood from pregnant women at term, we reveal that monocytes lose M2-like and gain M1-like properties accompanied by reductions in mitochondrial mass, maximal respiration, and cardiolipin content in pregnancy; glycolysis is unperturbed. We establish that muramyl dipeptide (MDP)-stimulated cytokine production relies on oxidative metabolism, then show in pregnancy reduced cytokine production in response to MDP but not LPS. Overall, mitochondrially centered metabolic capabilities of late gestation monocytes are down-regulated revealing natural plasticity in monocyte phenotype and function that could reveal targets for improving pregnancy outcomes but also yield alternative therapeutic approaches to diverse metabolic and/or immune-mediated diseases beyond pregnancy.

Inhibition of HDAC1 and 3 in the Presence of Systemic Inflammation Reduces Retinal Degeneration in a Model of Dry Age-Related Macular Degeneration.

Purpose: Previously, we identified increased retinal degeneration and cytokine response in a mouse model of dry age-related macular degeneration (AMD) in the presence of systemic inflammation from rheumatoid arthritis (RA). Histone deacetylases (HDACs) regulate cytokine production by reducing acetylation and are found to be dysregulated in inflammatory diseases, including RA and AMD. Therefore, this current study investigates the effect of HDAC inhibition on AMD progression in the presence of systemic inflammation. Methods: Collagen induced arthritis (CIA) was induced in C57BL6J mice, followed by sodium iodate (NaIO3)-induced retinal degeneration. Mice were treated with a selective HDAC class I inhibitor, MS-275, and retinal structure [optical coherence tomography (OCT)], function (electroretinography), and molecular changes quantitative real-time polymerase chain reaction (RT-qPCR, Western Blot) were assessed. Results: NaIO3 retinal damage was diminished in CIA mice treated with MS-275 (P ≤ 0.05). While no significant difference was observed in retinal pigment epithelium (RPE) function, a trend in increased c-wave amplitude was detected in CIA + NaIO3 mice treated with MS-275. Finally, we identified decreased Hdac1, Hdac3, and Cxcl9 expression in CIA + NaIO3 mouse RPE/choroid when treated with MS-275 (P ≤ 0.05). Conclusions: Our data demonstrate that HDAC inhibition can reduce the additive effect of NaIO3-induced retinal degeneration in the presence of systemic inflammation by CIA as measured by OCT analysis. In addition, HDAC inhibition in CIA + NaIO3 treated mice resulted in reduced cytokine production. These findings are highly innovative and provide additional support to the therapeutic potential of HDAC inhibitors for dry AMD treatment.

RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

137 Light-mediated Vagus Nerve Stimulation Inhibits Pro-inflammatory Cytokine Production in Acute Endotoxemia

INTRODUCTION: Vagus nerve stimulation (VNS) is a form of neuromodulation being explored to treat chronic inflammatory conditions by activating the inflammatory reflex, an arc reducing proinflammatory cytokine release through efferent cholinergic signaling. Current VNS approaches are primarily invasive and use electricity, but alternatives may improve vagal fiber specificity and eliminate surgical risks. Red-light optogenetics, a biological tool utilizing red light-activatable channelrhodopsins (ReaChR), can selectively activate genetically-modified neurons transdermally with light. Here, we show that ReaChR activation of cholinergic vagal fibers reduces cytokine production in a model of acute inflammation. METHODS: Vagus nerve cholinergic fibers were selectively activated using ReaChR expressed on choline acetyltransferase-expressing (ChAT) fibers in transgenic ChAT-ReaChR mice. Sensory afferent vagal fibers were activated in a separate cohort of transient receptor potential vanilloid subfamily member 1 (TRPV1)-ReaChR mice. Lipopolysaccharide (LPS) was injected intraperitoneally under anesthesia to induce acute endotoxemia, as measured by elevated tumor necrosis factor-alpha (TNFa). A 635nm fiber optic was targeted onto an isolated, cervical vagus for optogenetic VNS post-LPS administration. RESULTS: Selective ReaChR activation of cholinergic fibers in the cervical vagus nerve stimulates the inflammatory reflex to inhibit splenic TNFa production to bacterial LPS (ChAT-ReaChR: 1.27 ± 1.44 pg/ug; control: 4.26 ± 0.97 pg/ug, p < 0.001), but no significant differences were observed in serum (ChAT-ReaChR: 538 ± 216 pg/ml; littermate: 447 ± 206 pg/ml, p = 0.81). In contrast to efferent activation, ReaChR stimulation of afferent subsets (TRPV1+) failed to show cytokine regulation in splenic nor in serum .Approximate 20% baseline heart rate drop was seen during both ChAT+ and TRPV1+ fiber excitation, but only cholinergic activation sustained bradycardia throughout the stimulation period. CONCLUSIONS: Red-light optogenetic activation of cholinergic vagal fibers reduces splenic inflammatory cytokine production in murine endotoxemia and holds potential as a non-invasive, light-mediated VNS approach.

Poly(malic acid)-budesonide nanoconjugates embedded in microparticles for lung administration.

To improve the therapeutic activity of inhaled glucocorticoids and reduce potential side effects, we designed a formulation combining the advantages of nanoparticles, which have an enhanced uptake by alveolar cells, allow targeted delivery and sustained drug release, as well as limited drug systemic passage, with those of microparticles, which display good alveolar deposition. Herein, a polymer-drug conjugate, poly(malic acid)-budesonide (PMAB), was first synthesized with either 11, 20, 33, or 43mol% budesonide (drug:polymer from 1:8 to 3:4), the drug creating hydrophobic domains. The obtained conjugates self-assemble into nanoconjugates in water, yielding excellent drug loading of up to 73 wt%, with 80-100nm diameters. In vitro assays showed that budesonide could be steadily released from the nanoconjugates, and the anti-inflammatory activity was preserved, as evidenced by reduced cytokine production in LPS-activated RAW 264.7 macrophages. Nanoconjugates were then embedded into microparticles through spray-drying with L-leucine, forming nano-embedded microparticles (NEMs). NEMs were produced with an aerodynamic diameter close to 1µm and a density below 0.1 g.cm-3, indicative of a high alveolar deposition. NEMs spray-dried with the less hydrophobic nanoconjugates, PMAB 1:4, were readily dissolved in simulated lung fluid and were chosen for in vivo experiments to study pharmacokinetics in healthy rats. As it was released in vivo from NEMs, sustained distribution of budesonide was obtained for 48 h in lung tissue, cells, and lining fluid. With high loading rates, modulable release kinetics, and low cytotoxicity, these nanoconjugates delivered by NEMs are promising for the more efficient treatment of pulmonary inflammatory diseases.

O226: OTD is cytotoxic to A-375 melanoma cells without compromising fibroblast migratory wound healing function – a potential role in perioperative melanoma surgery

Abstract Introduction Melanoma incidence is increasing with 325,000 new cases reported in 2020 worldwide and 57,000 deaths. High recurrence rates are associated with prolonged inflammation and subsequent immunosuppression post-operatively. Surgical adjuvants may play a role in mitigating these adverse immune responses while increasing the cytotoxicity of cancer cells. Our previous research demonstrated anti-inflammatory effects of 1,4,5-oxathiazinane-4,4-dioxide (OTD) on Human Dermal Fibroblasts (HDFs) by the reduction of cytokine production. This study evaluates its cytotoxicity on melanoma cells. Methods OTD treatments of 0.25mM, 0.5mM, 0.75mM, 1mM and 1.25mM were applied to A-375 primary melanoma cells in triplicate for 24h and an MTT assay assessed cell viability. The absorbance was measured at 570nm wavelength. Cell viability was expressed as a percentage of the control. Results were compared using one-way analysis of variance (ANOVA) with GraphPad Prism software. Results Application of OTD treatments demonstrated a cytotoxic effect on melanoma cells at all concentrations. Relative optical density (OD) value was compared for each concentration against the control. A significant difference in cytotoxicity, relative to untreated control, was observed for each concentration; 56.8% at 0.25mM OTD (p*** = 0.001) and 68 to 71.6% at 0.5mM to 1.25mM OTD (p****<0.001). Conclusion OTD induces cytotoxicity in primary melanoma cells at all concentrations. Whereas our previous research demonstrated at 0.75mM it reduced inflammatory cytokine production in HDFs without adversely affecting migration. Therefore, OTD as a surgical adjuvant has the potential to be cytotoxic towards primary melanoma cells, reduce HDF inflammation and preserve migratory wound healing function.