IL-17A Gene Expression Research Articles

PD-1 regulation of pathogenic IL-17-secreting γδ T cells in experimental autoimmune encephalomyelitis.

The PD-1-PD-L1 immune checkpoint helps to maintain self-tolerance and prevent the development of autoimmune diseases. Immune checkpoint inhibitors are successful immunotherapeutics for several cancers, but responding patients can develop immune-mediated adverse events. It is well established that PD-1 regulates CD4 and CD8 T-cell responses, but its role in controlling the activation of pathogenic γδ T cells is less clear. Here we examined the role of PD-1 in regulating γδ T cells in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We found that PD-1 was highly expressed on CD27- Vγ4 γδ T cells in the lymph node (LN) and CNS of mice with EAE. Treatment of mice with anti-PD-1 significantly augmented IL-17A-producing CD27- Vγ4 γδ T cells in the LN and CNS and enhanced the severity of EAE. The exacerbating effect of anti-PD-1 on EAE was lost in Tcrd-/- mice. Conversely, ligation of PD-1 suppressed Il17a and Rorc gene expression and IL-17A production by purified Vγ4 γδ T cells stimulated via the TCR, but not with IL-1β and IL-23. Our study demonstrates that PD-1 regulates TCR-activated CD27- Vγ4 γδ T cells, but that cytokine-activated IL-17A producing γδ T cells escape the regulatory effects of the PD-1-PD-L1 pathway.

Th17 pathway‐related immune signatures in the pathobiology of myasthenia gravis: Integrating the roles of regulatory/effector cytokines and transcription factors

AbstractObjectivesMyasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptor (AChR), muscle specific kinase (MuSK), and other antigens in the neuromuscular junction. Antibody production is influenced by T lymphocytes. The T helper 17 (Th17) subset, an inflammatory lineage of Th cells, is associated with several autoimmune diseases. Functional interactions between T lymphocytes and pathogenic antibody responses including aberrant Th17 cell responses have been reported in MG. However, the precise mechanism(s) underlying the activation and/or pathogenic transformation of Th17 cells are not clearly known. The current study aimed at simultaneously exploring the roles of the inducers, master regulator transcription factors, and effectors of Th17 cells in patients with MG.MethodsIn this cross‐sectional study, quantification of gene expression of IL6, IL17A, STAT3, and RORC in the peripheral mononuclear cells by quantitative polymerase chain reaction (qPCR) as well as estimation of plasma levels of IL‐1β, IL‐6, and IL‐17A cytokines by multiplex suspension assay were carried out in 59 patients with MG and 61 healthy controls.ResultsGene expression levels of IL17A were significantly upregulated in patients as compared to healthy controls. The plasma levels of IL‐1β, IL‐6, and IL‐17A were significantly elevated in patients compared to healthy controls. IL17A as well as RORC gene expressions correlated with the clinical features. IL17A gene expression levels were higher in AChR‐MG patients.ConclusionThe present study supports the crucial role of the Th17 pathway in the pathobiology of MG, including its potential influence on disease severity.

Study on gene expression of IL-17A in eutopic and ectopic tissue sample of endometriosis patients and comparison with control group

Study on gene expression of IL-17A in eutopic and ectopic tissue sample of endometriosis patients and comparison with control group

Cardiac Left Ventricular miRNA-26a Is Downregulated in Ovariectomized Mice, Upregulated upon 17-Beta Estradiol Replacement, and Inversely Correlated with Collagen Type 1 Gene Expression.

Heart failure with preserved ejection fraction (HFpEF) is more prevalent in post- compared to pre-menopausal women. The underlying mechanisms are not fully understood. Data in humans is confounded by age and co-morbidities. We investigated the effects of ovariectomy and estrogen replacement on the left ventricular (LV) gene expression of pro-inflammatory and pro-fibrotic factors involved in HFpEF and putative regulating miRNAs. Nine-week-old C57BL/6 female mice were subjected to ovariectomy (OVX) or SHAM operation. OVX and SHAM groups were sacrificed 1-, 6-, and 12-weeks post-surgery (T1/SHAM; T1/OVX; T6/SHAM; T6/OVX, T12/SHAM). 17β-estradiol (E2) or vehicle (VEH) was then administered to the OVX groups for 6 weeks (T12/OVX/E2; T12/OVX/VEH). Another SHAM group was sacrificed 12-weeks post-surgery. RNA and miRNAs were extracted from the LV apex. An early 3-fold increase in the gene expression of IL-1α, IL-6, Mmp9, Mmp12, Col1α1, and Col3α1 was observed one-week post-surgery in T1/OVX vs. T1/SHAM, but not at later time points. miRNA-26a was lower in T1/OVX vs. T1/SHAM and was inversely correlated with Col1α1 and Col3α1 expression 1-week post-surgery (r = -0.79 p < 0.001; r = -0.6 p = 0.007). miRNAs-26a, 29b, and 133a were significantly higher, while Col1α1, Col3α1, IL-1α, IL-6, Tnfα, Mmp12, and FasL gene expression was significantly lower in E2- compared to vehicle-treated OVX mice. miRNA-26a was inversely correlated with Col3α1 in T12/OVX/ E2 (r = -0.56 p = 0.02). OVX triggered an early increase in the gene expression of pro-inflammatory and pro-fibrotic factors, highlighting the importance of the early phase post-cessation of ovarian function. E2 replacement therapy, even if it was not immediately initiated after OVX, reversed these unfavorable changes and upregulated cardiac miRNA-26a, previously unknown to be affected by menopausal status.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant

Malaria infection is a multifactorial disease partly modulated by host immuno-genetic factors. Recent evidence has demonstrated the importance of Interleukin-17 family proinflammatory cytokines and their genetic variants in host immunity. However, limited knowledge exists about their role in parasitic infections such as malaria. We aimed to investigate IL-17A serum levels in patients with severe and uncomplicated malaria and gene polymorphism’s influence on the IL-17A serum levels. In this research, 125 severe (SM) and uncomplicated (UM) malaria patients and 48 free malaria controls were enrolled. IL-17A serum levels were measured with ELISA. PCR and DNA sequencing were used to assess host genetic polymorphisms in IL-17A. We performed a multivariate regression to estimate the impact of human IL-17A variants on IL-17A serum levels and malaria outcomes. Elevated serum IL-17A levels accompanied by increased parasitemia were found in SM patients compared to UM and controls (P < 0.0001). Also, the IL-17A levels were lower in SM patients who were deceased than in those who survived. In addition, the minor allele frequencies (MAF) of two IL-17A polymorphisms (rs3819024 and rs3748067) were more prevalent in SM patients than UM patients, indicating an essential role in SM. Interestingly, the heterozygous rs8193038 AG genotype was significantly associated with higher levels of IL-17A than the homozygous wild type (AA). According to our results, it can be concluded that the IL-17A gene rs8193038 polymorphism significantly affects IL-17A gene expression. Our results fill a gap in the implication of IL-17A gene polymorphisms on the cytokine level in a malaria cohort. IL-17A gene polymorphisms also may influence cytokine production in response to Plasmodium infections and may contribute to the hyperinflammatory responses during severe malaria outcomes.

Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes.

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

Case report: Interleukin-17 targeted biological therapy in netherton syndrome

Netherton syndrome (NS) is rare and multisystemic congenital skin disorder classically distinguied as a triad of congenital ichthyosiform erythroderma, trichorrhexis invaginata (TI), and an atopic diathesis. Recent advances in pathogenesis have explored the role of IL-23/Th17 pathway in NS. Herein, we present a 17 years old girl harbouring homozygous four base pair deletion in exon 26 of the SPINK5 gene, presented with pruritus, scaling, dry skin and generalized eczematous lesions. She was administered anti IL17A (subcutaneous secukinumab) therapy. The treatment was well tolerated and resulted in a favourable clinical response, reduction of the IL17A gene expression and CD4 + Th17 cell population after 6 months which revealed an abrogation of Th17-skewing during therapy.

Pedagogical insights from immune responses: analyzing Covid-19's severity through systemic immune-inflammation indexes and gene expressions

Following the COVID-19 pandemic, it's crucial to understand the relationship between immune responses and clinical results, which also underpins interdisciplinary educational discussions. This study goes beyond standard medical evaluation, linking clinical medicine to sociodemography, genetics, and education, emphasizing the importance of an interconnected view for a comprehensive understanding. Severe COVID-19 cases show imbalances in the immune system, highlighting the need for early and precise prognosis, given its wider sociodemographic consequences. This study aims to investigate potential associations between systemic immune-inflammation indexes, gene expression of IFNA1, IFN receptors, IL17A, and plasmatic IFN-gamma in COVID-19 patients. Our observational study recruited 67 COVID-19 patients, both mild (28) or severe (39). Data was gathered on demographics, clinical symptoms, and laboratory results. Blood tests were used to analyze gene expressions and plasma IFNG levels. Most severe cases were males (61.5%) and of the black ethnic group (87.2%), with a clear age difference compared to mild cases. Elevated levels of systemic immune-inflammation measures were seen in severe cases, with gene expressions of IFNA1 and IL17A increasing, whereas IFNAR1 was more common in mild cases. From a pedagogical standpoint, the study emphasizes the link between clinical findings, sociodemographic and genetics data, underlining its profound implications for medical education. It suggests using specific gene expressions and hematological tests as COVID-19 severity indicators and underscores the need for further research to validate and explore broader clinical and pedagogical implications.

#4201 DYNAMICS OF INFLAMMATION EARLY AND LATE AFTER RENAL TRANSPLANTATION: CHARACTERIZATION OF THE IMMUNOLOGICAL PROCESSES IN A RAT RENAL TRANSPLANT MODEL

Abstract Background and Aims The pathophysiology of rejection after renal transplantation (RTx) has not been unraveled completely. In previous studies, Th17 cells were shown to be involved in several autoimmune diseases affecting the kidney, e.g. lupus nephritis, glomerulonephritis and ANCA vasculitis. The aim of this study was to decipher the dynamics of rejection after RTx with focus on the Th17 cells and IL17 cytokine family. Method For this purpose, the established Fischer to Lewis rat RTx model was used. In this model, renal rejection is elicited and develops over time. The donor renal grafts were harvested from Fischer rats and exposed to static cold storage using HTK or UW buffer. The Lewis recipients were treated for 10 days post-transplant with ciclosporin A and then immunosuppressive therapy stopped. Recipients were harvested early (2 hours, 30 days) and late after RTx (12 weeks, 28 weeks). To analyze the dynamics of inflammation, kidney sections were stained for CD3+ (T-cells) and MPO (neutrophils). RT-PCR was used to detect cytokine gene expression of several target genes within the renal grafts. IL17A Elispot was established to analyse circulating Th17 cells in the recipients. Results Generally, the cold ischemia time of the renal allograft was associated with the lifetime of the recipients. Furthermore, with increasing cold ischemia time of the graft, intra-renal CD3+ T-cell infiltration was enhanced. An increased CD3+ and a diminished number of MPO+ neutrophils were detected in the renal grafts late after RTx compared to early timepoints. Moreover, the intra-graft IL17C gene expression was enhanced late after renal transplantation while CXCL1 and CXCL2 were upregulated early after renal transplantation. IL17A gene expression reached higher levels than IFNg in later timepoints e.g. 30 days, while in early stages IL17A was below IFNg. The impact of cold storage buffer on the immunogenicity of the renal transplant was investigated, too. The use of UW led to a higher intra-renal CD3+ T-cell infiltration at later timepoints compared with HTK. In early stages after transplantation, higher levels of intra-renal MPO+ neutrophils were found upon cold storage using UW compared to cold storage using HTK. Conclusion The inflammatory process during chronic kidney rejection is T-cell driven and may involve the IL17C cascade. The early phase is dominated by neutrophils with enhanced intra-renal expression of CXCL1 and CXCL2. Furthermore, the polarization of the T-cell driven immune response may change depending on the time point after transplantation.

Reduction of Chlamydia pecorum and Koala Retrovirus subtype B expression in wild koalas vaccinated with novel peptide and peptide/recombinant protein formulations

Koalas are an endangered species under threat of extinction from several factors, including infections agents. Chlamydia pecorum infection results in morbidity and mortality from ocular and urogenital diseases while Koala Retrovirus (KoRV) infection has been linked to increased rates of cancer and chlamydiosis. Both C. pecorum and KoRV are endemic in many wild Australian koala populations, with limited treatment options available. Fortunately, vaccines for these pathogens are under development and have generated effective immune responses in multiple trials. The current study aimed to improve vaccine formulations by testing a novel peptide version of the Chlamydia vaccine and a combination Chlamydia – KoRV vaccine. Utilising a monitored wild population in Southeast Queensland, this trial followed koalas given either a ‘Chlamydia only’ vaccine (utilising four peptides from the chlamydial Major Outer Membrane Protein, MOMP), a combination ‘Chlamydia and KoRV’ vaccine (comprised of the chlamydial peptides plus a KoRV recombinant envelope protein (rEnv)) or no treatment. Clinical observations, C. pecorum and KoRV gene expression, serum IgG, and mucosal immune gene expression were assessed over a 17-month period. Overall, both vaccine formulations resulted in a decrease in chlamydiosis mortality, with decreases in C. pecorum, CD4, CD8β and IL-17A gene expression observed. In addition, the combination vaccine group also showed an increase in anti-KoRV IgG production that corresponded to a decrease in detected KoRV-B expression. While these results are favourable, the chlamydial peptide vaccine did not appear to outperform the established recombinant chlamydial vaccine and suggests that a combination vaccine formulated with recombinant MOMP plus KoRV rEnv could capitalize on the demonstrated benefits of both for the betterment of koalas into the future.

Neuropeptide Y potentiates T-Lymphocyte inflammation after psychological trauma

Post-traumatic stress disorder (PTSD) is a psychiatric disorder characterized by severe behavioral alterations, but also demonstrates elevated levels of systemic inflammation, sympathoexcitation, and a potentiated risk for autoimmune and cardiovascular diseases. These physiological changes suggest perturbations to the immune system, however, the signaling mechanisms linking psychological trauma to the immune system are not well understood. Utilizing a pre-clinical mouse model of PTSD known as repeated social defeat stress (RSDS), we previously demonstrated that psychological trauma causes robust T-lymphocyte-driven inflammation in the spleen as evidenced by increased pro-inflammatory cytokine production (primarily interleukin 17A, IL-17A), inflammatory polarization, and enhanced mitochondrial reactive oxygen species. Moreover, targeted denervation of the spleen was able to completely ablate this T-lymphocyte inflammatory response after psychological trauma, suggesting the signal was autonomic and neural in nature. Given that the splenic nerve possesses only sympathetic neurons (no direct parasympathetic innervation), we hypothesized that psychological trauma mediates splenic T-lymphocyte inflammation via enhanced release of sympathetic neurotransmitters. To test this, we first exposed naïve primary T-lymphocytes to various sympathetic catecholamines, including dopamine, norepinephrine, and epinephrine. Contrary to our original hypothesis, we observed no significant changes in IL-17A expression or mitochondria reactive oxygen species. Next, we assessed the physiological response of naïve T-lymphocytes exposed to neuropeptide Y (NPY) given its role in both sympathoexcitation as well as in PTSD. Interestingly, naïve T-lymphocytes treated with NPY also demonstrated no effect, but when NPY was given in combination of norepinephrine, we observed both an increase in IL-17A gene expression (~2 fold) and intracellular redox (~2 fold). To further investigate this effect, we examined NPY levels in our RSDS mouse model over-time. Circulating NPY levels spiked immediately after only one day of RSDS (~4.5 fold, p=0.0317) but steadily declined over the remaining 10 day psychological trauma induction. Intriguingly, NPY levels were strongly correlated with both circulating IL-17A and T-lymphocyte mitochondrial reactive oxygen species levels after RSDS, supporting our in vitro observations. Overall, these data suggest NPY may be playing a critical role in shaping the inflammatory milieu after psychological trauma, which may enhance the likelihood of inflammatory comorbidities. NIH R01HL158521 This is the full abstract presented at the American Physiology Summit 2023 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.

Prohibitin is Upregulated on Donor T cells in Murine Acute Graft-versus-Host Disease and Promotes Th17 Response

Abstract Introduction: Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation recipients. Here, we investigate PHB in Th17-mediated inflammation and as a therapeutic target for acute GVHD. Methods: Irradiated mice received T cell depleted bone marrow cells and allogeneic or syngeneic splenocytes. At day 25 post-transplant, blood, spleen, liver, and colon were harvested and analyzed for surface PHB, IFNγ, and IL-17 on donor T cells by flow cytometry. Splenocytes were cultured in the presence of PHB pharmacological inhibitor rocaglamide (RocA) and analyzed for PHB and IL-17 expression in donor T cells. Naïve murine CD4 T cells were cultured in Th17 polarizing conditions with RocA. CRISPR/Cas9-mediated knockout of PHB was performed followed by evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR. Results: Surface expression of PHB is increased in murine allogeneic donor T cells in liver, spleen, colon, and peripheral blood in acute GVHD. PHB-expressing splenic T cells secrete IL-17, which is reduced with treatment with a PHB inhibitor in ex vivo experiments. PHB inhibition of murine CD4 T cells in vitro impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Knockout of PHB reduces transcription of Il17 and Il22 genes. Conclusions: These findings show relevancy of PHB in Th17-mediated inflammation and suggest a potential role for PHB in acute GVHD. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PHB inhibition to treat this fatal disease. Supported by grants from AAI (KS, PR), ACS (PR; RSG-22-053-01-IBCD), and NIH NCI (PR; R01 CA252469).

The gut microbiota in multiple sclerosis varies with disease activity

BackgroundMultiple sclerosis is a chronic immune-mediated disease of the brain and spinal cord resulting in physical and cognitive impairment in young adults. It is hypothesized that a disrupted bacterial and viral gut microbiota is a part of the pathogenesis mediating disease impact through an altered gut microbiota-brain axis. The aim of this study is to explore the characteristics of gut microbiota in multiple sclerosis and to associate it with disease variables, as the etiology of the disease remains only partially known.MethodsHere, in a case-control setting involving 148 Danish cases with multiple sclerosis and 148 matched healthy control subjects, we performed shotgun sequencing of fecal microbial DNA and associated bacterial and viral microbiota findings with plasma cytokines, blood cell gene expression profiles, and disease activity.ResultsWe found 61 bacterial species that were differentially abundant when comparing all multiple sclerosis cases with healthy controls, among which 31 species were enriched in cases. A cluster of inflammation markers composed of blood leukocytes, CRP, and blood cell gene expression of IL17A and IL6 was positively associated with a cluster of multiple sclerosis-related species. Bacterial species that were more abundant in cases with disease-active treatment-naïve multiple sclerosis were positively linked to a group of plasma cytokines including IL-22, IL-17A, IFN-β, IL-33, and TNF-α. The bacterial species richness of treatment-naïve multiple sclerosis cases was associated with number of relapses over a follow-up period of 2 years. However, in non-disease-active cases, we identified two bacterial species, Faecalibacterium prausnitzii and Gordonibacter urolithinfaciens, whose absolute abundance was enriched. These bacteria are known to produce anti-inflammatory metabolites including butyrate and urolithin. In addition, cases with multiple sclerosis had a higher viral species diversity and a higher abundance of Caudovirales bacteriophages.ConclusionsConsiderable aberrations are present in the gut microbiota of patients with multiple sclerosis that are directly associated with blood biomarkers of inflammation, and in treatment-naïve cases bacterial richness is positively associated with disease activity. Yet, the finding of two symbiotic bacterial species in non-disease-active cases that produce favorable immune-modulating compounds provides a rationale for testing these bacteria as adjunct therapeutics in future clinical trials.

Promoter methylation status of RORC, IL17A, and TNFA in peripheral blood leukocytes in adolescents with obesity-related asthma

A higher Th17-immune response characterises obesity and obesity-related asthma phenotype. Nevertheless, obesity-related asthma has a more significant Th17-immune response than obesity alone. Retinoid-related orphan receptor C (RORC) is the essential transcription factor for Th17 polarisation. Previous studies have found that adolescents with obesity-related asthma presented upregulation of RORC, IL17A, and TNFA. However, the mechanisms that cause these higher mRNA expression levels in this asthmatic phenotype are poorly understood. Methylation directly regulates gene expression by adding a methyl group to carbon 5 of dinucleotide CpG cytosine. Thus, we evaluated the relationship between RORC, IL17A, and TNFA methylation status and mRNA expression levels to investigate a possible epigenetic regulation. A total of 102 adolescents (11–18 years) were studied in the following four groups: 1) healthy participants (HP), 2) allergic asthmatic participants (AAP), 3) obese participants without asthma (OP), and 4) non-allergic obesity-related asthma participants (OAP). Real-time qPCR assessed the methylation status and gene expression levels in peripheral blood leukocytes. Remarkably, the OAP and AAP groups have lower promoter methylation patterns of RORC, IL17A, and TNFA than the HP group. Notably, the OAP group presents lower RORC promoter methylation status than the OP group. Interestingly, RORC promoter methylation status was moderately negatively associated with gene expression of RORC (rs = −0.39, p < 0.001) and IL17A (rs = −0.37, p < 0.01), respectively. Similarly, the promoter methylation pattern of IL17A was moderately negatively correlated with IL17A gene expression (rs = −0.3, p < 0.01). There is also a moderate inverse relationship between TNFA promoter methylation status and TNFA gene expression (rs = −0.3, p < 0.01). The present study suggests an association between lower RORC, IL17A, and TNFA gene promoter methylation status with obesity-related asthma and allergic asthma. RORC, IL17A, and TNFA gene promoter methylation patterns are moderately inversely correlated with their respective mRNA expression levels. Therefore, DNA methylation may regulate RORC, IL17A, and TNF gene expression in both asthmatic phenotypes.

Effect of methionine and trace minerals (zinc, copper and manganese) supplementation on growth performance of broilers subjected to Eimeria challenge.

Coccidiosis is a major intestinal challenge that causes economic loss to the broiler industry. Two battery cage studies were conducted to evaluate the effect of trace minerals, source and dose of methionine on growth performance and gut health of broilers subjected to Eimeria challenge. Experiment #1 consisted of 9 treatments of 2 × 2 × 2 factorial design + 1 arrangement with main factors of methionine (Met) sources (DL-Met vs. 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa)), total sulfur amino acid (TSAA) levels (high vs. low; ±5% of recommended level), and sources of trace minerals (TM) Zn:Cu:Mn in the form Inorganic trace minerals (ITM) in sulfates (80:20:100ppm) vs. mineral methionine hydroxy-analogue bis-chelate (MMHAC, 40:10:50ppm), each with 8 pens of 10 birds. Experiment #2 consisted of 2 treatments--ITM [ZnSO4:tribasic copper chloride (TBCC):MnSO4 110:125:120ppm] and MMHAC (Zn:Cu:Mn, 40:30:40ppm), each with 36 pens of 10 birds. All birds except for treatment 9 in experiment #1 were orally gavaged with 1x, 4x and 16x recommended dose of coccidiosis vaccine on d0, d7 and d14, respectively. Data were subjected to one-way and/or three-way ANOVA, and means were separated by Fisher's protected LSD test with significance at p ≤ 0.05. In experiment #1, factorial analysis revealed the main effects of TSAA level and TM, but not Met source. High TSAA level improved body weight and cumulative feed intake at 14, 20, and 27d. MMHAC improved body weight at 14, and 27d; feed intake at 14, 20, and 27d; and cumulative FCR at 27d vs. sulfates. One-way ANOVA analysis showed that birds fed MMHAC and high levels of TSAA regardless of Met source had similar body weight as unchallenged birds on d27. In experiment #2, MMHAC improved body weight and cumulative FCR, and reduced jejunal IL-17A gene expression on d28. In summary, in broilers subjected to Eimeria challenge, supplementation of the reduced levels of bis-chelated trace minerals MMHAC improved growth performance compared to high levels of ITM (sulfates or TBCC), which might partially result from better immune response, high levels of TSAA improved growth performance, Met source had no effect. Supplementation of both bis-chelated trace minerals MMHAC and high levels of TSAA could overcome the growth performance challenge issue due to coccidiosis.

Abstract C045: CA19-9-mediated remodeling of the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDA) is an intractable common malignancy with a pro-tumorigenic and immunosuppressive microenvironment (ME) that often limits treatment efficacy. The glycan CA19-9 is used to follow treatment response in PDA patients, but its functional role in PDA remained unknown until recently because rodents lack this carbohydrate. CA19-9 elevation in a KRAS-mutant background promotes rapid progression to invasive carcinoma and induces ME remodeling in mice. The tumor ME includes cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) that contribute to desmoplasia, immunosuppression, and therapeutic resistance. All prior work investigating the tumor ME in PDA mouse models was performed in a CA19-9 negative context, potentially missing a key element of PDA biology. Elevation of CA19-9 in mice caused expansion of both CAFs and TAMs, but the mechanisms by which CA19-9 contributes to ME remodeling remains largely unknown. We aim to uncover the direct and indirect mechanisms of CA19-9-mediated ME remodeling. We identified CA19-9 modified ligands by immunoprecipitation and mass spectrometry that may contribute to reprogramming of the PDA ME. In addition to exploring changes in abundance, localization, and protein interactions, we will functionally characterize these candidate effectors via CRISPR ablation and antibody blockade using a novel Macrophage, Organoid, and Fibroblast (MOrF) co-culture platform. MOrF co-cultures can be sustained for at least 10 days in defined media conditions and enable compartment specific delineation of paracrine and juxtacrine signals. While CA19-9 modification may play a direct role in stromal remodeling, we also investigate effectors that are altered in response to CA19-9 elevation. CA19-9 induction in KRAS-mutant organoid mono-cultures increased gene expression of IL1α, CSF1,and TGFβ. In addition, CA19-9 expression increased gene set enrichment of inflammatory programs inorganoid mono-culture and led to increased expression of CAF and TAM differentiation programs in MOrF co-culture. These results are being validated as discussed above as well as by evaluation in vivo in CA19-9 positive and negative syngeneic orthotopic transplantation and autochthonous genetically engineered mouse models using scRNA-seq, pharmacologic and genetic perturbation, as well as other approaches. Using these orthogonal approaches, we will identify the mechanisms by which CA19-9 directly and indirectly contributes to PDA ME remodeling and ultimately, contributes to treatment response in the future. Citation Format: Jasper Hsu, Sejin Chung, Angelica E. Rock, Shira R. Okhovat, Dannielle D. Engle. CA19-9-mediated remodeling of the pancreatic tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C045.

PI3Kγ controls IL-17A expression and attenuates alveolar bone loss in an experimental periodontitis model.

In this study, we investigated the modulatory effects of PI3Kγ on IL-17A expression and the progression of experimental periodontitis in vivo. Ligature-induced periodontitis was developed around the first molar of mice. Animals were treated with anti-mouse IL-17A or IPI-549 (PI3Kγ inhibitor). In addition, PI3Kγ-deficient mice (PI3Kγ-/-) were used in the study. Alveolar bone loss was measured and real-time PCR of Il17a and Rankl genes was performed. A bioinformatics analysis was carried out using the Gene Set Enrichment Analysis computational tool. Nine days after ligature placement, alveolar bone loss scores were significantly increased, with upregulation of Il17a and Rankl genes in the gingival tissues. Treatment with anti-mouse IL-17A (100µg/mice) significantly attenuated alveolar bone loss. Mice with ligature-induced periodontitis treated with IPI-549 (3mg/kg) or PI3Kγ-/- mice showed reduced alveolar bone loss and downregulation of Il17a and Rankl gene expression in the gingival tissues. Consistent with this, the bioinformatics analysis showed upregulation of IL17F, IL17A, IL17D, and STAT3 genes, as well as greater activation of IL-17 and PI3KCI pathways (upregulation of PIK3CG gene) in the gingival tissue of patients with periodontitis. PI3Kγ plays an important role in modulating IL-17A expression and alveolar bone loss in vivo and can be considered a promising pathway for the management of periodontal disease and the development of new therapies.

PAH-induced metabolic changes related to inflammation in childhood asthma.

Epidemiological studies have shown that PAHs may exert adverse effects on childhood asthma. However, the underlying molecular mechanism remains to be fully elucidated. This study aimed to investigate this process in view of metabolic pathways, especially one-carbon metabolism and tryptophan metabolism. Fifty asthmatic children and 50 control subjects were recruited for this study. Serum IgE and IL-17A levels were detected by ELISA. Serum PAH concentrations were measured by GC-MS. One-carbon-related metabolites and tryptophan metabolites were determined by UPLC-Orbitrap-MS. DNA methylation was analyzed by bisulfite sequencing PCR. ChIP assays were used to examine H3K4me3 enrichment on IL-17A gene. Multivariable linear regression was performed to evaluate the association between PAHs and childhood asthma mediated by intermediators. HE staining in lung tissue, IgE and IL-17A in BALF, metabolic profiles in urine, and Ahr, Il-17a, and Cyp1a1 gene expression were determined in PAH-exposed mice. Serum Fla level was associated with childhood asthma (OR = 1.380, 95% CI: 1.063-1.792), and had a great effect on one-carbon metabolites, especially SAH, SAM, and Ser, which exerted significant mediation effects on the relationship between the Fla concentration and asthma. Moreover, we did find significant mediation effects between serum Fla and asthma by LINE-1 DNA methylation and H3K4me3 levels in the IL-17A promoter region. The differential Trp metabolites, such as Trp, tryptamine, IA, IAA, indole, IAld, and IAAld, indicated that asthmatic children had increased indole-AhR pathway. Mediation analysis failed to show a mediator effect of Trp metabolites in the association between PAHs and childhood asthma. An animal study confirmed that PAH exposure increased methylation levels, and altered Trp metabolite-AhR-IL-17A axis, which may be influenced by gender. PAHs disturbed one-carbon metabolism to influence the methyl group refilling DNA methylation and histone methylation, and disturbed tryptophan metabolism to regulate Th17-cell differentiation, which may elevate serum IL-17A concentration in asthmatic children.

Endogenous Interleukin-17a Contributes to Normal Spatial Memory Retention but Does Not Affect Early Behavioral or Neuropathological Outcomes after Experimental Traumatic Brain Injury.

Interleukin-17 (IL-17) is a proinflammatory cytokine primarily secreted in the brain by inflammatory T lymphocytes and glial cells. IL-17+ T-helper (Th17) cells are increased in the ipsilateral hemisphere after experimental traumatic brain injury (TBI), and IL-17 levels are increased in serum and brain tissue. We hypothesized that il17a and related gene expression would be increased in brain tissue after TBI in mice and il17a–/– mice would demonstrate neuroprotection versus wild type. The controlled cortical impact (CCI) model of TBI in adult male C57BL6/J mice was used for all experiments. Data were analyzed by analysis of variance (ANOVA) or repeated-measures two-way ANOVA with the Bonferroni correction. A value of p < 0.05 determined significance. Expression of il17a was significantly reduced in the ipsilateral cortex and hippocampus by day 3 after TBI, and expression remained low at 28 days. There were no differences between il17a–/– and il17a+/+ mice in beam balance, Morris water maze performance, or lesion volume after CCI. Surprisingly, naïve il17a–/– mice performed significantly (p = 0.02) worse than naïve il17a+/+ mice on the probe trial. In conclusion, sustained depression of il17a gene expression was observed in brains after TBI in adult mice. Genetic knockout of IL-17 was not neuroprotective after TBI. IL-17a may be important for memory retention in naïve mice.