TNF-α Secretion Research Articles

The Anorexigenic Peptide, Nesfatin-1, Dampens the B Cell Response to Receptor Mediated Stimulation

Obesity is an inflammatory condition marked by an increase in the accumulation of dysregulated B cells in adipose tissue (AT). These dysregulated B cells secrete pro-inflammatory mediators, cytokines and antibodies, that interfere with the normal function of the AT and contribute to the development of insulin resistance. Furthermore, depletion of the B cell compartment in a murine model has been shown to attenuate the metabolic aberrations associated with obesity. These data emphasize a clear role for B cell contribution to the comorbidities associated with obesity; however, the mechanism(s) regulating their accumulation and function are unknown. Therefore, discovery of targets that normalize the interaction between B cells and adipocytes has the potential to significantly reduce the burden of the inflammatory comorbidities of obesity that lead to metabolic disorders. Nesfatin-1 is a peptide hormone that is cleaved from the nucleobindin 2 (NUCB2) precursor along with two other peptides, nesfatin-2,3. Nesfatin-1 was originally described based on its anorexigenic effects in vivo, however recent reports have documented its anti-inflammatory activity. When NUCB2 is genetically deleted, there is a resultant increase in the presence of pro-inflammatory mediators in subcutaneous white AT (WAT). This is intriguing because humans with obesity have reduced serum nesfatin-1 in addition to low-grade chronic inflammation in WAT. However, the underlying mechanisms of how nesfatin-1 exerts its effects and its sites of action within the immune system have not been fully elucidated. In this study, we sought to determine a mechanism by which this peptide might directly interact with the immune system starting with a human B cell line, Raji. Here, we show that nesfatin-1 directly inhibits lipopolysaccharide (LPS) and B cell receptor (BCR) stimulated B cell proliferation while also protecting from inflammatory cell death. Furthermore, nesfatin-1 prevented stimulation induced secretion of pro-inflammatory mediators IgM and TNFa. Finally, we show that this effect is likely mediated via nesfatin-1’s ability to inhibit inflammatory pathway activation in this B cell line. Taken together, this work highlights a novel action of nesfatin-1 to directly bind to and inhibit B cell responses. These fundamental studies will serve as a springboard for the future development of nesfatin-1-based therapeutics for the treatment of obesity-associated inflammation, thus enabling potential new preventative strategies for forestalling the development of inflammation associated diseases, such as T2D. Saint Louis University. 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.

Abstract 3072: GDF15 Represses The Interferon Response Early In Vascular Senescence

Senescent cells have been identified as major contributors of the progression of vascular diseases including atherosclerosis by increasing the senescence-associated secretory phenotype (SASP) and dysregulating vascular cells. Notably, senescence and the SASP can contribute to plaque rupture and atherosclerotic progression by promoting inflammation and plaque vulnerability. A time course microarray analysis of vascular smooth muscle cell (VSMC) senescence revealed p53-regulated GDF15 mRNA was increased early and remained elevated throughout senescence. Notably, GDF15 has been implicated as a biomarker for cardiovascular disease mortality and is part of the conserved SASP across cell types and tissues. Interestingly, intracellular or secreted GDF15 protein levels were increased in monkeys fed a high-fat diet or in mice fed a high-fat diet and in individuals with high Framingham risk scores, respectively. We sought to identify the role of GDF15 in senescent VSMCs. Using CRISPRi, we silenced GDF15 during early senescence and performed RNA-sequencing. Pathway analysis revealed a enrichment in interferon signaling with the loss of GDF15. Coinciding with the increase in interferon signaling, phosphorylation of STAT1 and CREB were increased by loss of GDF15 in early senescence, aiding in the elevation of interferon expression. Given the role of interferons in communication and the prominence of macrophages in atherosclerosis, we tested the effects of conditioned media, derived from either proliferating or senescent VSMCs, on the activation and the expression of scavenger receptors on human macrophages. Senescent conditioned media increased expression of ABCG1 , CD36 , LOX-1 , SCARF1 , and GDF15 mRNA as well as TNFα and CCL2 secretion in macrophages. We propose that elevated GDF15 levels in early senescence suppress interferon signaling, thus preventing macrophage chemotaxis and in turn leading to the accumulation of senescent cells in the vasculature. Thus, disrupting the senescent VSMC-macrophage communication through secreted proteins may offer promising avenues of therapeutic development to prevent or improve inflammatory vascular diseases.

Modulation of neuroinflammation by interleukin-10 in mouse microglial cells

Abstract Microglia are resident macrophages of the central nervous system that are important for maintaining brain homeostasis. Persistent activation of microglia is associated with neuroinflammation in neurodegenerative disorders. Interest in immortalized microglia cell lines as cell culture models to study their biology and role in neuroinflammation has increased in the past decade, with mouse microglial BV-2 cells emerging as an in vitro model for neuronal inflammation similar to primary mouse microglial cells. We report here IL-10 pretreatment of BV-2 cells compared to primary microglia from CD-1 mice. A 45-plex Luminex immunoassay was used to measure IL-10 pretreatment attenuation of TNF-a, IL-6, CCL5/RANTES, and G-CSF secretion from cells treated with IFN-g and LPS. In addition, we demonstrate the effects of small molecule inhibitors MLN4924, tofacitinib (CP 690550), and Cardamonin on IL-10 pretreatment. MLN4924 inhibits the NFkB pathway and decreased secretion of multiple cytokines and chemokines, including TNF-a, IL-6, CCL2/MCP-1, CCL5, CCL3/MIP-1a, and CCL4/MIP-1b. Tofacitinib inhibits the Jak1/Tyk2-STAT3 pathway, decreasing STAT-3 TYR705 phosphorylation, but had little effect on IL-10 cytokine suppression. Interestingly, IL-10 pretreatment increased secretion of IL-1ra, an M2b microglia phenotype marker, and chemokines CCL2 and CCL3. Multiplex immunoassays are a valuable tool for investigating the mechanisms of neuroinflammation in primary and immortalized cell lines.

Berberine alleviates diabetic retinopathy by regulating the Th17/Treg ratio

BackgroundDiabetic retinopathy (DR) stands as a prominent complication of diabetes. Berberine (BBR) has reported to be effective to ameliorate the retinal damage of DR. Studying the potential immunological mechanisms of BBR on the streptozotocin (STZ) induced DR mouse model will explain the therapeutic mechanisms of BBR and provide theoretical basis for the clinical application of this drug. MethodsC57BL/6 J mice were induced into a diabetic state using a 50 mg/(kg·d) dose of STZ over a 5-day period. Subsequently, they were subjected to a high-fat diet (HFD) for one month. Following a 5-week treatment with 100 mg/(kg·d) BBR, the concentrations of inflammatory factors in the mice's peripheral blood were determined using an enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin staining was employed to scrutinize pathological changes in the mice's retinas, while flow cytometry assessed the proportions of T-lymphocyte subsets and the activation status of dendritic cells (DCs) in the spleen and lymph nodes. CD4+T cells and DC2.4 cell lines were utilized to investigate the direct and indirect effects of BBR on T cells under high glucose conditions in vitro. ResultsFollowing 5 weeks of BBR treatment in the streptozotocin (STZ) mouse model of DR, we observed alleviation of retinal lesions and a down-regulation in the secretion of inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in the serum of these mice. And in the spleen and lymph nodes of these mice, BBR inhibited the proportion of Th17 cells and promoted the proportion of Treg cells, thereby down-regulating the Th17/Treg ratio. Additionally, in vitro experiments, BBR directly inhibited the expression of the transcription factor RORγt and promoted the expression of the transcription factor Foxp3 in T cells, resulting in a down-regulation of the Th17/Treg ratio. Furthermore, BBR indirectly modulated the Th17/Treg ratio by suppressing the secretion of TNF-α, IL-1β, and IL-6 by DCs and enhancing the secretion of indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta (TGF-β) by DCs. This dual action inhibited Th17 cell differentiation while promoting Treg cells. ConclusionOur findings indicate that BBR regulate T cell subpopulation differentiation, reducing the Th17/Treg ratio by directly or indirectly pathway. This represents a potential therapeutic avenue of BBR for improving diabetic retinopathy.

Cardioprotective anti-inflammatory activities of Artemisia lactiflora Wall. ex DC. extract and fractions in a rat cardiomyoblast (H9c2) model of inflammatory sepsis

Context: Sepsis is a progressive inflammatory response commonly caused by gram-negative bacteria via activation of the NF-κB-dependent pathway. Severe sepsis can cause cardiac dysfunction and increases the risk of death. Artemisia lactiflora is a Chinese medicinal plant that contains phenolics and flavonoids with probable anti-inflammatory activities. Aims: To investigate the anti-inflammatory activities of A. lactiflora leaves extract in a cardiac sepsis model. Methods: A. lactiflora leaves were extracted and fractioned using solvents of different polarities. The total phenolic and flavonoid content was quantified. The antioxidants were quantified in vitro using DPPH and ABTS radicals. The cytotoxicity of the extract and fractions was evaluated and calculated using the neutral red assay and curve-fitting analysis in cardiomyoblasts (H9c2). The anti-inflammatory activities of A. lactiflora were observed by treating H9c2 with extract and fractions in the presence of Escherichia coli. Expression of pro-inflammatory genes (TNF and IL6) and secretion of pro-inflammatory cytokines (TNF-α and IL-6) were measured by RT-qPCR and ELISA. Results: Extract and fractions of A. lactiflora contained noticeably high quantities of phenolics and flavonoids with considerably high anti-DPPH and anti-ABTS activities. Co-treatment with A. lactiflora extract and all fractions significantly downregulated the expression of TNF and IL6 as well as decreased the secretion of TNF-α and IL-6 (p<0.0001) compared with E. coli-stimulated cardiomyoblasts. The butanol fraction had the highest potency in reducing pro-inflammatory cytokine secretions in cardiomyoblasts under in vitro sepsis conditions. Conclusions: A. lactiflora leaf extract demonstrated therapeutic potential in a cardiac sepsis model by alleviating the inflammatory responses. This plant can potentially be developed as an alternative medicine for inflammation and sepsis.

Structural Characterization of Polygonatum Cyrtonema Polysaccharide and Its Immunomodulatory Effects on Macrophages.

A neutral Polygonatum cyrtonema polysaccharide (NPCP) was isolated and purified from Polygonatum cyrtonema by various chromatographic techniques, including DEAE-52 and Sephadex-G100 chromatography. The structure of NPCP was characterized by HPLC, HPGPC, GC-MS, FT-IR, NMR, and SEM. Results showed that NPCP is composed of glucose (55.4%) and galactose (44.6%) with a molecular weight of 3.2 kDa, and the sugar chain of NPCP was →1)-α-D-Glc-(4→1)-β-D-Gal-(3→. In vitro bioactivity experiments demonstrated that NPCP significantly enhanced macrophages proliferation and phagocytosis while inhibiting the M1 polarization induced by LPS as well as the M2 polarization induced by IL-4 and IL-13 in macrophages. Additionally, NPCP suppressed the secretion of IL-6 and TNF-α in both M1 and M2 cells but promoted the secretion of IL-10. These results suggest that NPCP could serve as an immunomodulatory agent with potential applications in anti-inflammatory therapy.

PLK1-activating IFI16-STING-TBK1 pathway induces apoptosis of intestinal epithelial cells in patients with intestinal Behçet's syndrome.

Inflammatory signals from immunological cells may cause damage to intestinal epithelial cells (IECs), resulting in intestinal inflammation and tissue impairment. Interferon-γ-inducible protein 16 (IFI16) was reported to be involved in the pathogenesis of Behçet's syndrome (BS). This study aimed to investigate how inflammatory cytokines released by immunological cells and IFI16 participate in the pathogenesis of intestinal BS. RNA sequencing and real-time quantitative PCR (qPCR) showed that the positive regulation of tumor necrosis factor-α (TNF-α) production in peripheral blood mononuclear cells (PBMCs) of intestinal BS patients may be related to the upregulation of polo like kinase 1 (PLK1) in PBMCs (P = 0.012). The plasma TNF-α protein level in intestinal BS was significantly higher than in healthy controls (HCs; P = 0.009). PBMCs of intestinal BS patients and HCs were co-cultured with human normal IECs (NCM460) to explore the interaction between immunological cells and IECs. Using IFI16 knockdown, PBMC-NCM460 co-culture, TNF-α neutralizing monoclonal antibody (mAb), stimulator of interferon genes (STING) agonist 2'3'-cGAMP, and the PLK1 inhibitor SBE 13 HCL, we found that PLK1 promotes the secretion of TNF-α from PBMCs of intestinal BS patients, which causes overexpression of IFI16 and induces apoptosis of IECs via the STING-TBK1 pathway. The expressions of IFI16, TNF-α, cleaved caspase 3, phosphorylated STING (pSTING) and phosphorylated tank binding kinase 1 (pTBK1) in the intestinal ulcer tissue of BS patients were significantly higher than that of HCs (all P < 0.05). PLK1 in PBMCs of intestinal BS patients increased TNF-α secretion, inducing IEC apoptosis via activation of the IFI16-STING-TBK1 pathway. PLK1 and the IFI16-STING-TBK1 pathway may be new therapeutic targets for intestinal BS.

Decreased IL-1 β Secretion as a Potential Predictor of Tuberculosis Recurrence in Individuals Diagnosed with HIV.

Background: The mechanisms of the formation of immunological competence against tuberculosis (TB), and especially those associated with HIV co-infection, remain poorly understood. However, there is an urgent need for risk recurrence predictive biomarkers, as well as for predictors of successful treatment outcomes. The goal of the study was to identify possible immunological markers of TB recurrence in individuals with HIV/TB co-infection. Methods: The plasma levels of IFN-γ, TNF-α, IL-10, and IL-1β (cytokines which play important roles in the immune activation and protection against Mycobacterium tuberculosis) were measured using ELISA EIA-BEST kits. The cytokine concentrations were determined using a standard curve obtained with the standards provided by the manufacturer of each kit. Results: A total of 211 individuals were enrolled in the study as follows: 62 patients with HIV/TB co-infection, 52 with HIV monoinfection, 52 with TB monoinfection, and 45 healthy donors. Out of the 62 patients with HIV/TB, 75.8% (47) of patients were newly diagnosed with HIV and TB, and 24.2% (15) displayed recurrent TB and were newly diagnosed with HIV. Decreased levels of IFN-γ, TNF-α, and IL-10 were observed in patients with HIV/TB when compared with HIV and TB patients. However, there was no difference in IFN-γ, TNF-α, or IL-10 secretion between both HIV/TB groups. At the same time, an almost 4-fold decrease in Il-1β levels was detected in the HIV/TB group with TB recurrence when compared with the HIV/TB group (p = 0.0001); a 2.8-fold decrease when compared with HIV patients (p = 0.001); and a 2.2-fold decrease with newly diagnosed TB patients (p = 0.001). Conclusions: Significantly decreased Il-1β levels in HIV/TB patients' cohort with secondary TB indicate that this cytokine can be a potential biomarker of TB recurrence.

P162 Dimethyl fumarate modulates T cell metabolism and function in systemic lupus erythematosus patient samples

Abstract Background/Aims Activated CD4+T cells make a significant contribution to inflammation in systemic lupus erythematosus (SLE) through secretion of proinflammatory cytokines such as IFN-γ and TNF-α. CD4+T cells can also express activating NK receptors such as NKG2D that can engage with stress-induced ligands such as MICA, as noted in lupus nephritis, and mediate nonspecific tissue injury. We know that cellular metabolism regulates the activation of T cells. Evidence from patients with multiple sclerosis indicates that dimethyl fumarate (DMF), an electrophile, targets cellular metabolism to modulate CD4+T cell activation and function. Here, we investigated whether DMF modulates T cell metabolism and function in samples from patients with SLE in a series of in vitro experiments. Methods All experiments were performed using isolated T cells from freshly drawn whole blood samples from patients with SLE. T cells were isolated using negative selection using stem cell or Miltenyei magnetic bead separation kit. Isolated T cells were activated with anti-CD3 and IL-2 and incubated with either DMF at 25μM concentration or DMSO alone for three or seven days at 37 °C and 5% CO2 before harvesting. Results In Seahorse experiments, after three days of incubation, dimethyl fumarate (DMF) inhibited Figure A-i) the oxygen consumption rate (OCR) and A-ii) extra cellular acidification rate (ECAR) in isolated T cells when compared with samples incubated with vehicle, dimethyl sulfoxide (DMSO). Our results revealed that DMF significantly inhibited: 1) aerobic glycolysis and oxidative phosphorylation in activated T cells from patients with SLE (n = 4) (Figure A- i and ii) in vitro; 2) T cell activation and proliferation as assessed by a reduction in the frequency of CD69 (n = 4) and D) Ki67 (n = 2) positivity, respectively (Figure B-E); 3) Collectively, these results suggest that DMF inhibits T cell activation and proliferation in samples from patients with SLE. After 7 days of incubation, DMF significantly inhibited the expression of activating NK receptors CD158a and NKG2D on T cells whereas there was a trend toward enhancing the expression of inhibitory NK receptors NKG2A and CD158b (Figure F-I). After 7 days of incubation, DMF significantly reduced intracellular expression of IFN-γ, TNF-α, IL-17 and secretion of pro-inflammatory cytokines IFN-γ and TNF-α in supernatants (Figure J - N). Conclusion Our preliminary data indicated that, in SLE patient samples, DMF modulates metabolic programming of CD4+ T cells and inhibits glycolysis and oxidative phosphorylation of activated CD4+ T cells. In vitro, DMF inhibited: T cell activation; proliferation; the expression of activating NK receptors; and secretion of proinflammatory cytokines from T cells patients with SLE. Taken together with the existing safety data in humans, these data provide mechanistic rationale for investigating the potential of dimethyl fumarate, with a distinct mechanism of action, as a novel therapy for SLE. Disclosure L. Kell: None. K. Shah: None. S. Taylor: None. R. De Maeyer: None. D.A. Isenberg: None. D. Sen: None. M. Castelino: None. M. Leandro: None. A. Akbar: None. V. Reddy: Grants/research support; UCLH Biomedical research centre funded this work, Dr.Reddy's work was supported through MR/T024968/1 MRC-CARP Fellowship.

Mechanism of electro-acupuncture in alleviating intestinal injury in septic mice via polyamine-related M2-macrophage polarization.

The objective of this study was to investigate the impact of electro-acupuncture (EA) on sepsis-related intestinal injury and its relationship with macrophage polarization. A sepsis model was established using cecal ligation and puncture (CLP) to assess the effectiveness of EA. The extent of pathological injury was evaluated using Chiu's score, the expression of ZO-1 and Ocludin, and the impact on macrophage polarization was examined through flow cytometry and immunofluorescence staining. The expression of spermidine, one type of polyamine, and ornithine decarboxylase (ODC) was measured using ELISA and PCR. Once the efficacy was determined, a polyamine depletion model was created, and the role of polyamines was reassessed by evaluating efficacy and observing macrophage polarization. EA treatment reduced the Chiu's score and increased the expression of ZO-1 and Ocludin in the intestinal tissue of septic mice. It inhibited the secretion of IL-1β and TNF-α, promoted the polarization of M2-type macrophages, increased the secretion of IL-10, and upregulated the expression of Arg-1, spermidine, and ODC. However, after depleting polyamines, the beneficial effects of EA on alleviating intestinal tissue damage and modulating macrophage polarization disappeared. The mechanism underlying the alleviation of intestinal injury associated with CLP-induced sepsis by EA involves with the promotion of M2-type macrophage polarization mediated by spermidine expression.

Extraction, Identification, and In Vitro Anti-Inflammatory Activity of Feruloylated Oligosaccharides from Baijiu Distillers' Grains.

The structure and function of phenoyl oligosaccharides in baijiu distillers' grains (BDGs) have not been identified and investigated yet. This study aimed to elucidate the major phenolic oligosaccharides present in BDGs, optimize their extraction process via a central composite design, and assess their anti-inflammatory properties utilizing the LPS-induced RAW264.7 inflammation model. The main results are as follows: feruloylated oligosaccharides (FOs) were identified as the main phenoyl oligosaccharides in BDGs with a structure of ferulic acid esterified on arabinooligosaccharide xylose. Then, the preparation process of FOs was optimized using the following conditions: pH 5, temperature 55 °C, time 12 h, xylanase addition amount 7 g/L, BDG concentration 120 g/L. Furthermore, the acquired FOs demonstrated notable scavenging activity against DPPH and ABTS free radicals, with Trolox equivalent values of 366.8 ± 10.38 and 0.35 ± 0.01 mM Trolox/mg sample, respectively. However, their efficacy was comparatively lower than that of ferulic acid. Finally, the obtained FOs could effectively inhibit the LPS-induced secretion of TNF-α, IL-6, and IL-1β and promote the secretion of IL-10 in RAW264.7 cells. Based on the above results, FOs from BDGs were determined to have certain antioxidant and anti-inflammatory activities.

Inhibition of PI3K p110δ rebalanced Th17/Treg and reduced macrophages pyroptosis in LPS-induced sepsis

Sepsis is a systemic inflammatory response syndrome caused by trauma or infection, which can lead to multiple organ dysfunction. In severe cases, sepsis can also progress to septic shock and even death. Effective treatments for sepsis are still under development. This study aimed to determine if targeting the PI3K/Akt signaling with CAL-101, a PI3K p110δ inhibitor, could alleviate lipopolysaccharide (LPS)-induced sepsis and contribute to immune tolerance. Our findings indicated that CAL-101 treatment improved survival rates and alleviated the progression of LPS-induced sepsis. Compared to antibiotics, CAL-101 not only restored the Th17/regulatory T cells (Treg) balance but also enhanced Treg cell function. Additionally, CAL-101 promoted type 2 macrophage (M2) polarization, inhibited TNF-α secretion, and increased IL-10 secretion. Moreover, CAL-101 treatment reduced pyroptosis in peritoneal macrophages by inhibiting caspase-1/gasdermin D (GSDMD) activation. This study provides a mechanistic basis for future clinical exploration of targeted therapeutics and immunomodulatory strategies in the treatment of sepsis.

In vivo micro-computed tomography evaluation of radiopaque, polymeric device degradation in normal and inflammatory environments

Polymeric biomedical implants are an important clinical tool, but degradation remains difficult to determine post-implantation. Computed tomography (CT) could be a powerful tool for device monitoring, but polymers require incorporation of radiopaque contrast agents to be distinguishable from tissue. In addition, immune response to radiopaque devices must be characterized as it modulates device function. Radiopaque devices and films were produced by incorporating 0–20 wt% TaOx nanoparticles into polymers: polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA). In vitro inflammatory responses of mouse bone marrow-derived macrophages to polymer matrix incorporating TaOx nanoparticles was determined by monitoring cytokine secretion. Nanoparticle addition stimulated a slight inflammatory reaction, increasing TNFα secretion, mediated by changes in polymer matrix properties. Subsequently, devices (PLGA 50:50 + 20 wt% TaOx) were implanted subcutaneously in a mouse model of chronic inflammation, that featured a sustained increase in inflammatory response local to the implant site over 12 weeks. No changes to device degradation rates or foreign body response were noted between a normal and chronically stimulated inflammatory environment. Serial CT device monitoring post-implantation provided a detailed timeline of device collapse, with no rapid, spontaneous release of nanoparticles that occluded matrix visualization. Importantly, repeat CT sessions did not ablate the immune system or alter degradation kinetics. Thus, polymer devices incorporating radiopaque nanoparticles can be used for in situ monitoring and be readily combined with other medical imaging techniques, for a dynamic view biomaterial and tissue interactions. Statement of significanceA growing number of implantable devices are in use in the clinic, exposing patients to inherent risks of implant movement, collapse, and infection. The ability to monitor implanted devices would enable faster diagnosis of failure and open the door for personalized rehabilitation therapies – both of which could vastly improve patient outcomes. Unfortunately, polymeric materials which make up most biomedical devices are not radiologically distinguishable from tissue post-implantation. The introduction of radiopaque nanoparticles into polymers allows for serial monitoring via computed tomography, without affecting device degradation. Here we demonstrate for the first time that nanoparticles do not undergo burst release from devices post-implantation and that inflammatory responses – a key determinant of device function in vivo – are also unaffected by nanoparticle addition.

Novel Fermentates Can Enhance Key Immune Responses Associated with Viral Immunity.

Fermented foods have long been known to have immunomodulatory capabilities, and fermentates derived from the lactic acid bacteria of dairy products can modulate the immune system. We have used skimmed milk powder to generate novel fermentates using Lb. helveticus strains SC234 and SC232 and we demonstrate here that these fermentates can enhance key immune mechanisms that are critical to the immune response to viruses. We show that our novel fermentates, SC234 and SC232, can positively impact on cytokine and chemokine secretion, nitric oxide (NO) production, cell surface marker expression, and phagocytosis in macrophage models. We demonstrate that the fermentates SC234 and SC232 increase the secretion of cytokines IL-1β, IL-6, TNF-α, IL-27, and IL-10; promote an M1 pro-inflammatory phenotype for viral immunity via NO induction; decrease chemokine expression of Monocyte Chemoattractant Protein (MCP); increase cell surface marker expression; and enhance phagocytosis in comparison to their starting material. These data suggest that these novel fermentates have potential as novel functional food ingredients for the treatment, management, and control of viral infection.

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The resultsindicate that the D2andD3 domainsof FliCEcNhave more complex interactionscomparedtothe D1-D2 domainpair., and thesedomains alsoplay a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 hasmore missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulatehigherlevelsofIL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. Epithelial activation and/or barrier effects upon exposure to 2-50 μg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 μg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 μg/mL of LPS, with or without 2 μM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 μg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 μg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

CD8 T cell-derived perforin regulates macrophage-mediated inflammation in a murine model of gout.

Gout is characterized by hyperuricemia and recurrent inflammatory episodes caused by intra-articular crystal deposition of monosodium urate (MSU). There is a clear relationship between gout and metabolic syndrome. Recent evidence indicates that perforin plays a role in regulating glucose homeostasis and provides protection in diet-induced non-alcoholic steatohepatitis models. However, the impact of perforin on immune inflammation in gout remains unclear. We induced acute gout models in both wild-type (WT) mice and Prf1null mice by administering intra-articular injections of MSU crystals. We compared the ankle joint swelling and the histological score between the two groups. Furthermore, we investigated underlying mechanisms through in vitro co-culture experiments involving CD8 T cells and macrophages. In this study, Prf1null mice showed significantly more pronounced ankle swelling with increased inflammatory cell infiltrations compared with WT mice 24h after local MSU injection. Moreover, MSU-induced Prf1null mice exhibited increased accumulation of CD8 T cells but not NK cells. Perforin-deficient CD8 T cells displayed reduced cytotoxicity towards bone marrow-derived M0 and M1 macrophages and promoted TNF-α secretion from macrophage. Perforin from CD8 T cells limits joint inflammation in mice with acute gout by downregulating macrophage-mediated inflammation. Key Points • Perforin deficiency increased swelling in the ankle joints of mice upon MSU injection. • Perforin deficiency is associated with increased immune cell recruitment and severe joint damage in gout. • Perforin regulated CD8 T cell accumulation in gout and promoted CD8 T cell cytotoxicity towards M0 and M1 macrophages. • CD8 T cell-derived perforin regulated pro-inflammatory cytokine secretion of macrophage.

Abstract P65: Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs)

Abstract Allogeneic Epstein-Barr Virus-specific T cells (EBVSTs) have thus far been a safe immunotherapy product in the clinic, with little graft-versus-host-disease reported in over 300 recipients to date. Consequently, EBVSTs armoured with chimeric antigen receptors (CARs) have emerged as a promising platform for allogeneic T cell therapies. Believing that a deep characterisation of CAR EBVST cell products can enhance insights into their clinical safety, we performed single cell T cell receptor (TCR) sequencing on CAR EBVSTs, taking CD30.CAR EBVSTs as a prototype. Non-transduced (NT) EBVSTs and CD30.CAR EBVSTs generated from three healthy donors exhibited a narrower, more focused TCR repertoire by diversity indices, compared to resting T cells and CD30.CAR-transduced activated T cells (ATCs) from the same donors. EBVSTs harboured a lower frequency of unique TCR clonotypes and a higher proportion of expanded clones, indicating a tendency toward clonal dominance. Narrower TCR repertoires corresponded to greater IFNγ and TNFα secretion in response to stimulation with EBV peptides, with no cross-reactivity toward other common viruses, and were associated with lower allogeneic reactivity in mixed lymphocyte reactions. To investigate whether the EBVST nature of the product might contribute to better CAR activity, we concurrently performed single cell transcriptomic sequencing on NT EBVSTs, CD30.CAR EBVSTs, and CD30.CAR ATCs. Transcriptomic signatures revealed that each sample type contained two major, distinct populations of CD4+ T cells: proliferative and activated. While the proliferative subsets appeared to be closely related, the activated populations had distinct transcriptional profiles. In particular, CD30.CAR EBVSTs appeared to be more polyfunctional and metabolically poised than CD30.CAR ATCs. The restricted TCR repertoire and apparent functional superiority of CD30.CAR EBVSTs may underlie their safety and efficacy observed in early-stage clinical trials and preclinical models, with the larger implication that these qualities extend to CAR EBVSTs targeting antigens beyond CD30. Citation Format: Lindsay Kua, Ting Wei Lim, Fiona Wong, Pei Yun Teo, Lionel Low, Ivan Horak, Jinmiao Chen, Kar Wai Tan. Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs) [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P65.