Pro-inflammatory Th1 Cytokines Research Articles

Cooperative tumor inhibition by CpG-oligodeoxynucleotide and cyclic dinucleotide in head and neck cancer involves T helper cytokine and macrophage phenotype reprogramming

Head and neck cancer ranks as the sixth most common cancer worldwide, highlighting the critical need for the development of new therapies to enhance treatment efficacy. The activation of innate immune receptors given their potent immune stimulatory properties aid in the eradication of cancer cells. In this study, we investigated the immune mechanism and anti-tumor function of a Toll-like receptor 9 (TLR9) agonist, CpG-oligodeoxynucleotide-2722 (CpG-2722), in combination with cyclic dinucleotides, which are agonists of stimulator of interferon genes (STING). Our results revealed that CpG-2722 stimulation increased the expression of Th1 pro-inflammatory cytokines. Stimulation by STING agonists exhibited lower expression of Th1 cytokines but higher expression of Th2 cytokines compared to CpG-2722. However, the combination of these two agonists significantly enhanced Th1 cytokines while reducing Th2 cytokines. Moreover, in vivo experiment showed that both CpG-2722 and 2'3'-c-di-AMP suppressed head and neck tumor growth, with their combination proving more effective than the use of these agonists alone. The combined treatment cooperatively promoted the production of Th1 cytokines and type I interferons, while suppressing Th2 cytokines in the tumors as observed in vitro. Additionally, it led to the accumulation of M1 macrophages, dendritic cells, and T cells, shaping a favorable tumor microenvironment for T cell-mediated tumor killing. The anti-tumor activity of the CpG-2722 and 2'3'-c-di-AMP combination depends on the macrophage presence but does not directly activate M1 macrophage polarization, instead working through a reprogrammed cytokine profile. Furthermore, this combination shows a cooperative anti-tumor activity with anti-PD-1 in treating head and neck tumors. Overall, these findings highlight a Th response and macrophage phenotype reprograming involved functional mechanism underlying the cooperative activity of the combination of TLR9 and STING agonists in the immunotherapy of head and neck cancer.

Polyfunctional T cells and unique cytokine clusters imprint the anti rAAV2/rAAV9 vector immune response.

Polyfunctional T cells programmed to perform activities such as degranulation of lytic enzymes and simultaneous production of multiple cytokines are associated with more effective control of viral infections. Immune responses to recombinant adeno-associated virus (rAAV) vector delivery systems can critically influence therapeutic efficacy and safety of gene therapy. However, knowledge of polyfunctional T cells in anti-AAV immune responses is scarce. To bridge this knowledge gap, we have investigated the polyfunctionality of primary human CD4 T cells from healthy donors after in-vitro exposure to rAAV2 or rAAV9 vectors. By performing proliferation assays of co-cultured T cells and rAAV pulsed monocyte-derived dendritic cells from healthy donors we demonstrate T cell reactivity of 43% and 50% to rAAV2 and rAAV9 vectors, respectively. We validated this frequency in a second screen using another set of healthy donors measuring CD25 and CD71 T cell activation. Single T cell secretome analysis of reactive donors uncovered a Th1 pro-inflammatory, cytolytic and chemoattractive cytokine release profile after stimulation with rAAV2 or rAAV9 vectors. 12.4% and 9.6% of the stimulated T cells displayed a polyfunctional cytokine response, respectively, including elevated polyfunctional inflammatory indices. These responses were characterized by cytokine clusters such as Granzyme B, MIP1-α and TNF-α released in combination by single T cells. Overall, our results provide insights into adaptive immunity with rAAV vector serotypes which will be important in advancing gene therapy safety, vector selection, immunogenicity assessment and better patient selection for AAV gene therapy.

JNK Pathway-Associated Phosphatase Deficiency Facilitates Atherosclerotic Progression by Inducing T-Helper 1 and 17 Polarization and Inflammation in an ERK- and NF-κB Pathway-Dependent Manner.

JNK pathway-associated phosphatase (JKAP) regulates T cell-mediated immunity and inflammation, which are involved in atherosclerosis pathogenesis. This study investigated the effects of JKAP on T-helper (Th) cell polarization, inflammation, and atherosclerotic progression. Serum JKAP levels were measured in 30 patients with coronary heart disease (CHD) and 30 controls. CHD blood naïve CD4＋ T cells were acquired, followed by JKAP overexpression and knockdown with or without treatment with PD98059 (ERK inhibitor) or BAY-11-7082 (NF-κB inhibitor) in vitro. CD4＋ T-cell conditional JKAP ablation mice were established in vivo, followed by the construction of an atherosclerosis model. JKAP was reduced and negatively correlated with the Gensini score, CRP, Th1 cells, Th17 cells, and proinflammatory cytokines in patients with CHD. In vitro, JKAP overexpression suppressed Th1 and Th17 cell differentiation and proinflammatory cytokines, whereas JKAP knockdown exerted the opposite effect; however, JKAP modification did not affect Th2 cell differentiation. Interestingly, JKAP negatively regulated the ERK and NF-κB pathways; meanwhile, the PD98059 and BAY-11-7082 treatments repressed Th1 and Th17 cell differentiation, and attenuated the effect of JKAP knockdown on these indices. In vivo, conditional CD4＋ T-cell JKAP ablation increased Th1 and Th17 cell polarization in the spleen, lymph node, blood, and/or aortic root. Furthermore, CD4＋ T-cell conditional JKAP ablation exaggerated atherosclerotic lesions in the aorta, elevated CD4+ cell infiltration and proinflammatory cytokines in the aortic root, and activated the ERK and NF-κB pathways in the aortic root. JKAP ablation facilitates atherosclerosis progression by promoting Th1 and 17 polarization and inflammation through regulation of the ERK and NF-κB pathways.

Intranasal vaccination with engineered BCG expressing CCL2 induces a stronger immune barrier against Mycobacterium tuberculosis than BCG

Intradermal Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccination is currently the only licensed strategy for preventing tuberculosis (TB). It provides limited protection against pulmonary TB. To enhance the efficacy of BCG, we developed a recombinant BCG expressing exogenous monocyte chemoattractant CC chemokine ligand 2 (CCL2) called rBCG-CCL2. Co-culturing macrophages with rBCG-CCL2 enhances their abilities in migration, phagocytosis, and effector molecule expression. In the mouse model, intranasal vaccination with rBCG-CCL2 induced greater immune cell infiltration and a more extensive innate immune response in lung compared to vaccination with parental BCG, as determined by multiparameter flow cytometry, transcriptomic analysis, and pathological assessments. Moreover, rBCG-CCL2 induced a high frequency of activated macrophages and antigen-specific T helper 1 (Th1) and Th17 Tcells in lungs. The enhanced immune microenvironment responded more effectively to intravenous challenge with Mycobacterium tuberculosis (Mtb) H37Ra, leading to significant reductions in H37Ra burden and pathological damage to the lungs and spleen. Intranasal rBCG-CCL2-vaccinated mice rapidly initiated pro-inflammatory Th1 cytokine release and reduced pathological damage to the lungs and spleen during the early stage of H37Ra challenge. The finding that co-expression of CCL2 synergistically enhances the immune barrier induced by BCG provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against TB.

Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

The role of vitamin D in amelioration of oral lichen planus and its effect on salivary and tissue IFN-γ level: a randomized clinical trial

Background and objectivesOral lichen planus (OLP) is a common, prevalent, immune-mediated, inflammatory disease affecting both the skin and oral mucosa and is considered one of the potentially malignant diseases. Since OLP is regarded as an immunologically mediated disease, some studies suggest the use of vitamin D (VD) for its management as it exhibits immune-modulatory, anti-inflammatory, and antimicrobial properties, as well as anti-proliferative, pro-differentiative, and anti-angiogenic effects. VD has demonstrated a suppressive effect on TH1 pro-inflammatory cytokines, including IFN-γ while augmenting the secretion of anti-inflammatory cytokines. At the same time, VD deficiency is a prevalent public issue. Therefore, the present study aimed to investigate the role of VD as an adjunct to steroids in the management of VD-deficient OLP patients as well as its inhibitory effect on IFN-γ through measurement of salivary and tissue IFN-γ levels in OLP patients.MethodsA total of 40 patients with ulcerative or erythematous OLP, diagnosed according to the World Health Organization’s (WHO) modified criteria for OLP, were randomly allocated into one of the two study groups to receive either systemic steroids in addition to VD supplements (Group A) or systemic steroids only (Group B). Blood samples were collected for the measurement of serum VD level (SVDL) using the enzyme-linked immunosorbent assay (ELISA) to involve only patients with VD deficiency or insufficiency (≤ 30 ng/ml). Clinical evaluation of the lesion involved objective signs and subjective symptoms. Also, changes in salivary and tissue INF-γ levels (in pg/mL and pg/mg, respectively) were determined using the ELISA technique. All parameters were measured at baseline and after 4 weeks of treatment. The clinical pharmacy team devised a checklist to record all team interventions. The interventions were categorized into six domains, including drug interactions and/or adverse reactions, medication dose issues, drug selection issues, support with medication history, patient-related concerns, and suggestions for dental medication.ResultsAfter one month of treatment, a significantly greater number of patients in group A showed complete pain relief and resolution of clinical lesions, as well as a greater number of patients showing a reduction in the clinical severity of lesions than in group B (P = 0.005). Also, there was a statistically significant reduction in average VAS pain scores and clinical scores in group A compared to group B after 1 month of treatment (P = 0.001 and 0.002, respectively). Furthermore, there was a statistically significant greater reduction in salivary and tissue IFN-γ levels in group A than in group B (P ≤ 0.001 and 0.029, respectively) after 1 month of treatment.ConclusionCurrent evidence suggests a significant preventive and therapeutic role for VD as an adjunct to standard therapies indicated for OLP lesions. These protective and therapeutic functions are achieved through the suppressive effect of VD on pro-inflammatory cytokines, particularly IFN-γ. Also, salivary IFN-γ appears to be a valuable prognostic marker for monitoring the progression of OLP. In addition, the inter-professional collaboration between dentists and clinical pharmacists helped to deliver complete, patient-centered primary care and ensured the quality of the medications included in patient kits, thus improving patient treatment and management. Nevertheless, further studies with larger sample sizes, longer follow-ups, and standardized designs may still be needed.

Lung microbiota participated in fibrous microplastics (MPs) aggravating OVA-induced asthma disease in mice

Environmental pollution is one of the risk factors for asthma. Currently, whether micro-plastics could aggravate asthma, is still unclear. In the air, fibrous MPs are the predominant shape. Since fibrous micro-plastics are reported to be detected in the lower respiratory tract and other body parts, the relationship of fibrous MP and asthma, as well as the potential mechanism is not well investigated. In this study, we produced fibrous MPs, whose lengths and widths were in accordance with the natural environment, and further, investigated the potential adverse effect of which on the asthma in a OVA (ovalbumin)-induced mice model, aiming at exploring the true life hazard of MP to the respiratory system. Following nasal exposure to fibrous MPs, the airway inflammation, mucus hypersecretion and fibrosis were aggravated in asthmatic mice. Fibrous MPs exposure also significantly increased the levels of total IgE, and, cardinal Th2 and Th1 pro-inflammatory cytokines participated in the etiopathogenesis of allergic airway inflammation. In addition, MP fibers exposure induced lung epithelial cells apoptosis, disruption of epithelial barrier integrity and activation of NLRP3 related signaling pathways. Moreover, fibrous MPs significantly altered the bacterial composition at the genus level. Compared to the control group, the relative abundance of Escherichia-Shigella and Uncultured were decreased to 4.47% and 0.15% in OVA group, while Blautia and Prevotella were elevated to 4.96% and 2.94%. For the OVA + MPs group, the relative abundance of Blautia and Uncultured were decreased to 2.27% and 0.006%, while Prevotella was increased to 3.05%. Our study highlights the detrimental effect of fibrous MPs on asthmatic population and facilitates an indication of the latent mechanisms of fibrous MPs induced airway pathology.

Tapinarof cream 1% once daily: Significant efficacy in the treatment of moderate to severe atopic dermatitis in adults and children down to 2 years of age in the pivotal phase 3 ADORING trials

Tapinarof cream 1% once daily (QD), a topical aryl hydrocarbon receptor agonist, downregulates pro-inflammatory Th2 cytokines, upregulates skin-barrier components, and reduces oxidative stress. To assess tapinarof efficacy and safety in adults and children down to 2 years of age with atopic dermatitis (AD). Eight hundred and thirteen patients were randomized to tapinarof or vehicle QD in two 8-week phase 3 trials. The primary efficacy endpoint, Validated Investigator Global Assessment for Atopic Dermatitis score of 0 or 1 and ≥2-grade improvement from baseline at Week 8, was met with statistical significance in both trials: 45.4% versus 13.9% and 46.4% versus 18.0% (tapinarof vs vehicle; both P<.0001). Significantly superior Eczema Area and Severity Index 75 (EASI75) responses were also observed with tapinarof versus vehicle at Week 8: 55.8% versus 22.9% and 59.1% versus 21.2% (both P<.0001). Rapid improvements in patient-reported pruritus were also significant with tapinarof versus vehicle. Common adverse events (≥5%) of folliculitis, headache, and nasopharyngitis were mostly mild or moderate, with lower discontinuations due to adverse events in the tapinarof groups than with vehicle. Long-term efficacy was not assessed. Tapinarof demonstrated highly significant efficacy and favorable safety and tolerability in a diverse population of patients with AD down to 2years of age.

Effect of Short Peptides of Pregnancy-Specific β1-Glycoprotein on Differentiation of Human Regulatory T Cells In Vitro and on Their Cytokine Profile.

Pregnancy-specific β1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor β (TGFβ) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1β, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1β, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.

Melatonin pretreatment improves endometrial regenerative cell-mediated therapeutic effects in experimental colitis

BackgroundEndometrial regenerative cells (ERCs) have been proven to be an effective strategy for attenuating experimental colitis, but the complex in vivo microenvironment such as oxidative stress may largely limit and weaken ERC efficacy. Melatonin (MT) works as an anti-oxidative agent in a variety of preclinical diseases, and has been identified to promote mesenchymal stem cell-mediated therapeutic effects in different diseases. However, the ability of MT to enhance ERC-mediated effects in colitis is currently poorly understood. MethodsMenstrual blood was collected from healthy female volunteers to obtain ERCs and identified. In vitro, H2O2-induced oxidative stress was introduced to test if MT could prevent ERCs from damage through detection of intracellular reactive oxidative species (ROS) and apoptosis assay. In vivo, dextran sodium sulfate (DSS)-induced acute colitis was treated by ERCs and MT-primed ERCs, therapeutic effects were assayed by the disease activity index (DAI), histological features, and macrophage and CD4+ T cell in the spleen and colon, and cytokine profiles in the sera and colon were also measured. ResultsIn vitro, ERCs that underwent MT-precondition were found to possess more anti-oxidative potency in comparison to naïve ERCs, which were characterized by decreased apoptosis rate and intracellular ROS under H2O2 stimulation. In vivo, MT pretreatment can significantly enhance the therapeutic effects of ERCs in the attenuation of experimental colitis, including decreased DAI index and damage score. In addition, MT pretreatment was found to promote ERC-mediated inhibition of Th1, Th17, and M1 macrophage and pro-inflammatory cytokines, increase of Treg, and immunomodulation of cytokines in the spleen and colon. ConclusionsMT pretreatment facilitates the promotion of cell viability under oxidative stress in vitro, while also enhancing ERC-mediated therapeutic effects in experimental colitis.

Endotoxin Inflammatory Action on Cells by Dysregulated-Immunological-Barrier-Linked ROS-Apoptosis Mechanisms in Gut-Liver Axis.

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut-liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.

Molecular and Cellular Evaluation of Horses With Summer Pasture Associated Asthma Syndrome

Equine asthma is an airway disease that affects a large number of horses annually leading to considerable economic losses in the horse industry. Despite advances in research in this area, there is still a lack of information on its etiology and molecular characterization in pasture associated asthma. The objective of the current study was to characterize the inflammatory disease of lower airways in horses maintained on pasture through cytologic and immunologic profile during the summer in a tropical environment by analysis of the gene expression of Th1 cytokines (IFN- λ, IL-8), Th2 cytokines (IL-4 and IL-5), and pro-inflammatory cytokines (IL-1, TNF-α) in the bronchoalveolar lavage (BAL) fluid in healthy and asthma horses on pasture. A group 39 of clinically healthy horses maintained on native pasture and supplemented with concentrate was evaluated by BAL analyzed for differential cellular count and assigned into a control and an asthma group. The gene expression of pro-inflammatory cytokines was analyzed in the BAL by reverse time PCR (RT-PCR) (IL-1α (alpha), IL-4, IL-5, IL-8, TNF-α alpha and IFN-λ), using β-actin as housekeeping gene. Higher gene expression of IL-1, IL-4, IL-5, IL-8, IFN-λ in the BAL of asthma horses was found. Current results indicate an increase in Th2, characterizing an allergic inflammatory reaction due to the significant increase in IL-5 in asthmatic horses (10.3 ± 1.13), when compared to the values ​​obtained in normal horses (3.27 ± 0.46). The only down regulated cytokine in the asthma group was TNF-α, suggesting a chronic antigenic reaction.

Hypothyroidism-associated immunosuppression involves induction of galectin-1-producing regulatory T cells.

Hypothyroidism exerts deleterious effects on immunity, but the precise role of the hypothalamic-pituitary-thyroid (HPT) axis in immunoregulatory and tolerogenic programs is barely understood. Here, we investigated the mechanisms underlying hypothyroid-related immunosuppression by examining the regulatory role of components of the HPT axis. We first analyzed lymphocyte activity in mice overexpressing the TRH gene (Tg-Trh). T cells from Tg-Trh showed increased proliferation than wild-type (WT) euthyroid mice in response to polyclonal activation. The release of Th1 pro-inflammatory cytokines was also increased in Tg-Trh and TSH levels correlated with T-cell proliferation. To gain further mechanistic insights into hypothyroidism-related immunosuppression, we evaluated T-cell subpopulations in lymphoid tissues of hypothyroid and control mice. No differences were observed in CD3/CD19 or CD4/CD8 ratios between these strains. However, the frequency of regulatory T cells (Tregs) was significantly increased in hypothyroid mice, and not in Tg-Trh mice. Accordingly, in vitro Tregs differentiation was more pronounced in naïve T cells isolated from hypothyroid mice. Since Tregs overexpress galectin-1 (Gal-1) and mice lacking this lectin (Lgals1-/- ) show reduced Treg function, we investigated the involvement of this immunoregulatory lectin in the control of Tregs in settings of hypothyroidism. Increased T lymphocyte reactivity and reduced frequency of Tregs were found in hypothyroid Lgals1-/- mice when compared to hypothyroid WT animals. This effect was rescued by the addition of recombinant Gal-1. Finally, increased expression of Gal-1 was found in Tregs purified from hypothyroid WT mice compared with their euthyroid counterpart. Thus, a substantial increase in the frequency and activity of Gal-1-expressing Tregs underlies immunosuppression associated with hypothyroid conditions, with critical implications in immunopathology, metabolic disorders, and cancer.

Expression kinetics of cytokines and the humoral antibody response concerning short-term protection induced by radiation-attenuated Trypanosoma evansi in bovine calves.

'Surra', an economically important disease of livestock, is caused by the parasitic blood protozoon Trypanosoma evansi. Both innate and adaptive immunity contribute to the protection against this infection. T-helper cells play a crucial role in the antibody-mediated clearance of T. evansi. We present here the data on the kinetics of expression of important Th1, Th2 and Th17 cytokines, vis-a-vis the dynamics of humoral response in bovine calves following immunization with γ-radiation-attenuated live T. evansi and later challenged with homologous virulent T. evansi. Significant upregulation of the pro-inflammatory Th1 and Th17 cytokines was correlated with the IgG2-mediated protection in the immunized bovine calves post-challenge. The calves were immunized with 5×106 500Gy γ-radiation-attenuated live T. evansi (horse isolate) thrice at 15days intervals through the subcutaneous route and subsequently, challenged with 1×103 virulent T. evansi on day 50. Significantly high serum IgG (1:1600) and IgM (1:800) titres were recorded on week 2 PC, whereas the peak serum IgG2 titre (1:800) was recorded on week 6 PC. Significant upregulation of IFN-γ, TNF, IL-1β, and IL-2 was recorded between days 1 to 3 PC, while the same for IL-17 was recorded on day 14 PC. The immunized calves were free from parasitemia post-challenge and were clinically healthy till the end of the experiment. Significant upregulation of IL-10 and IL-4 transcripts and a corresponding increase of serum IgG1 titre in the placebo group helped patency of the parasite in an anti-inflammatory environment and clinical exacerbation of the disease. The expression of the important Th1 cytokines was crucial for antibody-mediated short-term protection against a lethal challenge of T. evansi in cattle.

Coal waste-derived synthesis of yellow oxidized graphene quantum dots with highly specific superoxide dismutase activity: characterization, kinetics, and biological studies.

The disintegration of coal-based precursors for the scalable production of nanozymes relies on the fate of solvothermal pyrolysis. Herein, we report a novel economic and scalable strategy to fabricate yellow luminescent graphene quantum dots (YGQDs) by remediating unburnt coal waste (CW). The YGQDs (size: 7-8 nm; M.W: 3157.9 Da) were produced using in situ "anion-radical" assisted bond cleavage in water (within 8 h; at 121 °C) with yields of ∼87%. The presence of exposed surface and edge groups, such as COOH, C-O-C, and O-H, as structural defects accounted for its high fluorescence with εmax ∼530 nm at pH 7. Besides, these defects also acted as radical stabilizers, demonstrating prominent anti-oxidative activity of ∼4.5-fold higher than standard ascorbic acid (AA). In addition, the YGQDs showed high biocompatibility towards mammalian cells, with 500 μM of treatment dose showing <15% cell death. The YGQDs demonstrated specific superoxide dismutase (SOD) activity wherein 15 μM YGQDs equalled the activity of 1-unit biological SOD (bSOD), measured using the pyrogallol assay. The Km for YGQDs was ∼10-fold higher than that for bSOD. However, the YGQDs retained their SOD activity in harsh conditions like high temperatures or denaturing reactions, where the activity of bSOD is completely lost. The binding affinity of YGQDs for superoxide ions, measured from isothermal calorimetry (ITC) studies, was only 10-fold lower than that of bSOD (Kd of 586 nM vs. 57.3 nM). Further, the pre-treatment of YGQDs (∼10-25 μM) increased the cell survivability to >75-90% in three cell lines during ROS-mediated cell death, with the highest survivability being shown for C6-cells. Next, the ROS-induced apoptosis in C6-cells (model for neurodegenerative diseases study), wherein YGQDs uptake was confirmed by confocal microscopy, showed ∼5-fold apoptosis alleviation with only 5 μM pretreatment. The YGQDs also restored the expression of pro-inflammatory Th1 cytokines (TNF-α, IFN-γ, IL-6) and anti-inflammatory Th2 cytokines (IL-10) to their basal levels, with a net >3-fold change observed. This further explains the molecular mechanism for the antioxidant property of YGQDs. The high specific SOD activity associated with YGQDs may provide the cheapest alternative source for producing large-scale SOD-based nanozymes that can treat various oxidative stress-linked disorders/diseases.

An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients.

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.

Trace elements and Immunity: A brief overview

Trace elements or micronutrients play a vital role in the human body. They are required as cofactors and function with enzymes to catalyze various biochemical reactions. Around 14 trace elements are found in the human body which are crucial to various physiological and biological functions in the cells. Some of these elements have therapeutic potential. Impaired immunity and increased susceptibility to infections are a few responses that develop under nutritional deficiency of these trace elements. A comprehensive literature search was accomplished on PubMed, SCOPUS and Google Scholar to identify articles on trace elements and their pivotal role in immunity. Antibody responses, cell-mediated immunity and natural killer (NK) cell activity are few of the immune processes likely to be influenced by trace element levels. Elements such as zinc, selenium and other trace elements activate the cytokine-mediated immune response by triggering the synthesis of TH1 cells and proinflammatory cytokines. In addition, some trace elements possess antiviral properties, interfering with the entry and replication of the viruses. Based on the above perspectives and the significance of trace elements in immunity, the current review was undertaken to shed light on the role of trace elements important in immunity.

S230: HUMAN ΒETA-DEFENSIN 2 TREATMENT MODULATES THE INTESTINAL MICROBIOME AND THE ALLOGENEIC T CELL RESPONSE TO LIMIT ACUTE GRAFT-VERSUS-HOST DISEASE

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy option for patients with hematological malignancies. Acute graft-versus-host disease (GVHD) is primarily driven by allogeneic donor T cells and remains a serious cause of morbidity and mortality post allo-HCT. In particular, acute GVHD of the gastrointestinal tract (GI GVHD) is associated with a high mortality rate and treatment options are limited. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide, which is induced by inflammatory stimuli and possesses both antimicrobial and immunomodulatory functions. Aims: In this study, we aimed to investigate the expression of beta defensins in human and murine GI GVHD and the therapeutic effect of recombinant hBD-2 on acute GVHD development in mouse models. Methods: To investigate the functional role of hBD-2 post allo-HCT, we employed established murine GVHD and graft-versus-leukemia (GVL) models and analyzed the allogeneic T cell response using transcriptome and kinome profiling. Shotgun metagenomic sequencing was used to examine the effect of hBD-2 on the intestinal microbiome. Furthermore, we studied beta-defensin expression in two independent acute GVHD patient cohorts and in mice with acute GVHD. Results: We found that expression of murine beta-defensin 4 (mBD-4), the murine orthologue for hBD-2, was reduced in the colon and ileum of mice developing acute GVHD. Oral treatment of mice with recombinant hBD-2 post allo-HCT reduced weight loss and acute GVHD severity and mortality. Furthermore, hBD-2 treatment affected the intestinal microbial composition, including a shift towards higher abundance of Bacteroides species in hBD-2 treated compared to vehicle treated mice. The changes in the microbiome resulted in reduced neutrophil infiltration of the ileum during acute GVHD induction in hBD-2 treated mice. Additionally, hBD-2 dampened pro-inflammatory Th1 cytokine production (TNF, IFN-gamma) by allogeneic T cells in vivo, while preserving the beneficial GVL effect in two different leukemia models. Mechanistically, oral hBD-2 treatment decreased alloreactive T cell infiltration and the expression of genes involved in T cell receptor (TCR) signaling in the ileum of mice with acute GVHD. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation and metabolism by reducing proximal TCR signaling. In patients with acute GI GVHD, intestinal hBD-2 expression was inadequately induced in response to inflammation at the mRNA and protein level when compared to healthy subjects and patients with ulcerative colitis. Summary/Conclusion: In conclusion, our study demonstrates that hBD-2 reduces acute GVHD severity, likely through its effects in shaping the intestinal microbiota, reducing neutrophil infiltration in the ileum and dampening allogeneic T cell responses. Both human and murine acute GVHD are characterized by a lack of intestinal beta-defensin induction and recombinant hBD-2 represents a potential novel therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

METABOLIC INSULTS ELICIT EPITOPE-DEPENDENT IMMUNE AUTOREACTIVITY AGGRAVATING HEPATIC DAMAGE IN TYPE 2 DIABETES

Abstract A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs including the liver, ultimately driving non-alcoholic steatohepatitis. The environment-driven lipotoxicity and glucotoxicity induces liver damage, which promotes dendritic cell adjuvanticity and generates an MHC-II immunopeptidome enriched of peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and stress responses. Here, we demonstrate that metabolic insults promote the presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3, which is involved in immunogenic cell death) on MHC Class II, ultimately supporting pathogenic autoreactivity. Additionally, PDIA3-driven T cell responses infiltrating the hepatic parenchyma, favored the release of TH1 and TH17 pro-inflammatory cytokines and, PDIA3-driven B cell responses were associated with antibodies that aggravated hepatic toxicity in mice subjected to high-fat and high-sucrose diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions including autoimmune hepatitis, primary biliary cholangitis and type 2 diabetes. Altogether our data indicate that metabolic insult caused by high-sucrose diet, cause tissue damage, which, by supplying PDIA3 T cell and B cell epitopes further contributes to epitope-dependent pathogenic immune autoreactivity Supported by NIHAI146180 and NIH AI137198