IL-17A Production Research Articles

803 Epidermal inflammatory activity is an important driver of hidradenitis suppurativa lesions

Hidradenitis suppurativa (HS) is an inflammatory skin disorder of the pilosebaceous unit defined by painful nodules, tunnels, and scarring. Previous studies documented dermal cell infiltrate and the abundance of inflammatory cytokines in HS lesions, including TNFα, IL-17, and IL-1β. However, not much is currently known about the contribution of keratinocytes and immune cell activity within the epidermis in HS. We found that the degree of epidermal inflammation in HS lesions positively correlated with Hurley stage of disease (r =0.746; p=0), indicating the evolution of epidermal responses with disease severity. Keratinocytes were the primary producers of TNFα (p<0.05) and IL-6 (p<0.05) in HS lesions at all Hurley stages. Increased expression of CXCL3 (5-fold; p<0.01) in keratinocytes of stage III lesions positively correlated with increased recruitment of neutrophils to stage III lesional epidermis. Compared to healthy keratinocytes, enhanced production of chemokines from HS keratinocytes, including CCL22 (stage II 4-fold; p=0.09) and CCL3 (stages II and III p<0.05; 30-fold and 10-fold respectively), was associated with CD8 T cell infiltration and corresponding chemokine receptor expression in the epidermis. These recruited immune cells strongly produce or enable the production of IFNγ (>50 fold), IL-17A (500-fold), and IL-1β (5 to 10-fold), as well as IFNβ1, CXCL10, and STAT1 (HS II/III lesions; p<0.05 for all) by multiple cell types locally in the epidermis, likely generating positive feedback for increased inflammation. As epidermal inflammation evolves during disease progression and contributes to immune cell recruitment, our data suggest that topical therapeutics that block cytokine and chemokine production from the epidermis may be efficacious in HS. In all, our results collectively indicate that keratinocytes are not simply bystanders in HS inflammatory events, but rather actively contribute to HS pathogenesis.

LB987 Tapinarof inhibits the formation, cytokine production, and persistence of resident memory T cells in vitro

Tapinarof, a novel, first-in-class small-molecule topical therapy with combined aryl hydrocarbon receptor (AhR) agonist and antioxidant activities, is effective in the treatment of both psoriasis (PsO) (NCT03956355; NCT03983980; NCT04053387) and atopic dermatitis (AD) (NCT02564055). In previous experiments in culture, tapinarof increased keratinocyte expression of skin barrier proteins and reduced T cell production of IL-17A and IL-17F. Psoriasis patients in phase 3 trials who cleared after topical tapinarof remained clear for a median of four months after therapy was discontinued, as defined by a PGA of 0 (clear) or 1 (almost clear) while off therapy after achieving complete disease clearance (PSOARING studies 1, 2, and 3).

Obesity and Fatty Acids Promote Mitochondrial Translocation of STAT3 Through ROS-Dependent Mechanisms.

Obesity promotes the onset and progression of metabolic and inflammatory diseases such as type 2 diabetes. The chronic low-grade inflammation that occurs during obesity triggers multiple signaling mechanisms that negatively affect organismal health. One such mechanism is the persistent activation and mitochondrial translocation of STAT3, which is implicated in inflammatory pathologies and many types of cancers. STAT3 in the mitochondria (mitoSTAT3) alters electron transport chain activity, thereby influencing nutrient metabolism and immune response. PBMCs and CD4+ T cells from obese but normal glucose-tolerant (NGT) middle-aged subjects had higher phosphorylation of STAT3 on residue serine 727 and more mitochondrial accumulation of STAT3 than cells from lean subjects. To evaluate if circulating lipid overabundance in obesity is responsible for age- and sex-matched mitoSTAT3, cells from lean subjects were challenged with physiologically relevant doses of the saturated and monounsaturated fatty acids, palmitate and oleate, respectively. Fatty acid treatment caused robust accumulation of mitoSTAT3 in all cell types, which was independent of palmitate-induced impairments in autophagy. Co-treatment of cells with fatty acid and trehalose prevented STAT3 phosphorylation and mitochondrial accumulation in an autophagy-independent but cellular peroxide–dependent mechanism. Pharmacological blockade of mitoSTAT3 either by a mitochondria-targeted STAT3 inhibitor or ROS scavenging prevented obesity and fatty acid–induced production of proinflammatory cytokines IL-17A and IL-6, thus establishing a mechanistic link between mitoSTAT3 and inflammatory cytokine production.

Design, synthesis and in vitro biological evaluation of marine phidianidine derivatives as potential anti-inflammatory agents

Phidianidines A and B are novel marine indole alkaloids with various biological activities. Based on their potential anti-inflammatory properties, a series of phidianidine derivatives were designed, synthesized, and tested for their effects on IL-17A production in PMA/ionomycin-stimulated T-cell-lymphoma EL-4 cells. Compounds 9a and 22c exhibited excellent anti-inflammatory activity and low toxicity, with IC50 values of 7.7 μM and 5.3 μM for IL-17A production in PMA/ionomycin-stimulated EL-4 cells, respectively. Further mechanistic study showed that 9a could decrease the STAT3 phosphorylation at Y705 to inhibit IL-17A production in EL-4 cells, indicating its ability of preventing the differentiation of Th17 cells and their possible function. This research may give an insight for the discovery of marine indole alkaloid derived anti-inflammatory drug leads for the treatment of T cell-mediated diseases.

Characterization of a small molecule modulator of inflammatory cytokine production

In the present study, the effect(s) of the immunomodulatory drug GLS-1027 on various cell types involved in inflammation were investigated. GLS-1027 reduced LPS-stimulated secretion of pro-inflammatory cytokines by macrophage or monocytic cells and cell lines. This reduction was likely due in part to decreased activation of NF-κB family transcription factors and inhibition of p38 MAPK signaling in GLS-1027-treated cells. Independent from its effects on macrophages, GLS-1027 inhibited dendritic cell maturation and differentiation of naïve CD4+ T cells into Th17 cells, reducing the production of typical pro-inflammatory cytokines associated with both processes. In vivo administration of GLS-1027 prevented the development of type 1 diabetes in NOD mice which correlated with reduced serum levels of IL17A in GLS-1027 treated animals and reduced ex vivo production of IL17A from both spleen and lymph-node cells. Overall, our data show that GLS-1027 can reduce inflammation through multiple actions, including the reduction of pro-inflammatory cytokine production by innate immune cells, the inhibition of dendritic cells maturation, and the inhibition of Th17 cells polarization.

Chitosan/Calcium-Coated Ginsenoside Rb1 Phosphate Flower-like Microparticles as an Adjuvant to Enhance Immune Responses

Simple SummaryThe field-level control over IBD is primarily via vaccination. The development of high effective IBV vaccine has drawing great attentions worldwide. Herein, the GRb1 was encap-sulated into Calcium phosphate and chitosan core-structure nanoparticles microspheres, which con-stitute a novel system for nanoparticle delivery (GRb1/IL-4@CS/Cap). The new nano-adjuvant de-livery system could induce the activation of chicken dendritic cells ( DCs ), with up-regulate the expression of MHC II and CD80, and increase the production of IL-1β and TNF-α. At the same time, it can trigger higher levels of IBDV-specific IgG and higher IgG2a/IgG1 ratio, and promote the production of IFN-γ, TNF-α, IL-4, IL-6, IL-1α, and IL-1βand other cytokines in chicken serum after vaccination, it provides an effective adjuvant system for the development of chicken IBDV attenu-ated vaccine.Infectious bursal disease (IBD) is a highly contagious immunocompromising disorder that caused great economic losses in the poultry industry. The field-level control over IBD is primarily via vaccination. The development of a highly effective IBV vaccine has drawn great attention worldwide. Chitosan/Calcium Phosphate (CS/CaP) nanoparticle was a newly developed effective biological delivery system for drug and antigen. Ginsenoside Rb1 is one of the main bioactive components of ginseng root extract, which has antioxidant, anti-inflammatory and immunological enhancement effects. Until now, the combined effect of CS/CaP and ginsenoside Rb1 on the chicken immune response had remained unknown. In this study, the GRb1 and IL-4 were encapsulated into Calcium phosphate and chitosan core structure nanoparticles microspheres (GRb1/IL-4@CS/CaP), and the effect of a newly developed delivery system on an infectious bursal disease virus (IBDV) attenuated vaccine was further evaluated. The results demonstrated that GRb1/IL-4@CS/CaP treatment could induce the activation of chicken dendritic cells (DCs), with the upregulated expression of MHCII and CD80, and the increased production of IL-1β and TNF-α. Importantly, GRb1/IL-4@CS/CaP could trigger a higher level of IBDV-specific IgG and a higher ratio of IgG2a/IgG1 than the traditional adjuvant groups, promoting the production of cytokine, including IFN-γ, TNF-α, IL-4, IL-6, IL-1α, and IL-1β, in chicken serum after 28 d and 42 d post-vaccine. Taken in all, GRb1/IL-4@CS/CaP could elicit prolonged vigorous immune responses for IBDV attenuated vaccine in chicken, which might provide an effective adjuvant system for avian vaccine development.

Gastric Th17 Cells Specific for H+/K+-ATPase and Serum IL-17 Signature in Gastric Autoimmunity.

Human gastric autoimmunity [autoimmune gastritis (AIG)] is characterized by inflammation of the gastric mucosa and parietal cell loss. The gastric parietal cell proton pump H+/K+-adenosine triphosphatase (H+/K+-ATPase) is the major autoantigen in AIG. Our work aimed to investigate the gastric H+/K+-ATPase-specific T helper 17 (Th17) responses in AIG and serum interleukin (IL)-17 cytokine subfamily in AIG patients, in healthy subjects [healthy controls (HCs)], and in patients with iron deficiency anemia (IDA) without AIG. We analyzed the activation of gastric lamina propria mononuclear cells (LPMCs) by H+/K+-ATPase and the IL-17A and IL-17F cytokine production in eight patients with AIG and four HCs. Furthermore, we compared serum levels of IL-17A, IL-17F, IL-21, IL-17E, IL-22, and IL-23 in 43 AIG patients, in 47 HCs, and in 20 IDA patients without AIG. Gastric LPMCs from all AIG patients, but not those from HCs, were activated by H+/K+-ATPase and were able to proliferate and produce high levels of IL-17A and IL-17F. AIG patients have significantly higher serum IL-17A, IL-17F, IL-21, and IL-17E (393.3 ± 410.02 pg/ml, 394.0 ± 378.03 pg/ml, 300.46 ± 303.45 pg/ml, 34.92 ± 32.56 pg/ml, respectively) than those in HCs (222.99 ± 361.24 pg/ml, 217.49 ± 312.1 pg/ml, 147.43 ± 259.17 pg/ml, 8.69 ± 8.98 pg/ml, respectively) and those in IDA patients without AIG (58.06 ± 107.49 pg/ml, 74.26 ± 178.50 pg/ml, 96.86 ± 177.46 pg/ml, 10.64 ± 17.70 pg/ml, respectively). Altogether, our results indicate that IL-17A and IL-17F are produced in vivo in the stomach of AIG patients following activation with H+/K+-ATPase and that serum IL-17A, IL-17F, IL-21, and IL-17E levels are significantly elevated in AIG patients but not in patients without AIG. These data suggest a Th17 signature in AIG and that IL-17A, IL-17F, IL-21, and IL-17E may represent a relevant tool for AIG management.

Angiotensin AT2 Receptor Stimulation Alleviates Collagen-Induced Arthritis by Upregulation of Regulatory T Cell Numbers.

The angiotensin AT2 receptor (AT2R) is a main receptor of the protective arm of the renin-angiotensin system and exerts for instance anti-inflammatory effects. The impact of AT2R stimulation on autoimmune diseases such as rheumatoid arthritis (RA) is not yet known. We investigated the therapeutic potential of AT2R-stimulation with the selective non-peptide AT2R agonist Compound 21 (C21) in collagen-induced arthritis (CIA), an animal model for inflammatory arthritis. Arthritis was induced by immunization of DBA/1J mice with collagen type II (CII). Prophylactic and therapeutic C21 treatment alleviates arthritis severity and incidence in CIA. Joint histology revealed significantly less infiltrates of IL-1 beta and IL-17A expressing cells and a well-preserved articular cartilage in C21- treated mice. In CIA, the number of CD4+CD25+FoxP3+ regulatory T (Treg) cells significantly increased upon C21 treatment compared to vehicle. T cell differentiation experiments demonstrated increased expression of FoxP3 mRNA, whereas IL-17A, STAT3 and IFN-gamma mRNA expression were reduced upon C21 treatment. In accordance with the mRNA data, C21 upregulated the percentage of CD4+FoxP3+ cells in Treg polarizing cultures compared to medium-treated controls, whereas the percentage of CD4+IL-17A+ and CD4+IFN-gamma+ T cells was suppressed. To conclude, C21 exerts beneficial effects on T cell-mediated experimental arthritis. We found that C21-induced AT2R-stimulation promotes the expansion of CD4+ regulatory T cells and suppresses IL-17A production. Thus, AT2R-stimulation may represent an attractive treatment strategy for arthritis.

The Role of PD-L1 on Langerhans Cells in the Regulation of Psoriasis

Langerhans cells (LCs) are skin-resident cells with potent antigen-presenting cell capabilities, which reportedly play some roles in the development of psoriasis, an inflammatory skin disease mediated by IL-17A‒producing cells, T helper 17 cells, and TCR-γδlow T cells. LCs in psoriatic skin lesions but not in normal skin express PD-L1, which binds to PD-1, an immune checkpoint molecule, to negatively regulate immune reactions. The aim of this study is to elucidate the regulatory role of LCs through the PD-1/PD-L1 axis in a murine model of imiquimod-induced psoriasis-like dermatitis. Imiquimod application on wild-type C57BL/6J mice induced PD-L1 expression on LCs both in the ear skin and skin-draining lymph nodes. To further identify the functional role of PD-L1 expressed on LCs, we generated conditional knockout mice lacking PD-L1 expression on LCs (Pd-l1-cKO mice). Pd-l1-cKO mice presented significantly more severe imiquimod-induced psoriasis-like dermatitis than their control littermates. Flow cytometric analysis showed that the frequency of activated IL-17A‒producing γδlow T cells was increased in the ear skin samples, and IL-17A production by CCR6+ migrating γδlow T cells increased in the skin-draining lymph nodes in imiquimod-applied Pd-l1-cKO mice than in control littermates. Collectively, LCs disrupt the exacerbation of psoriasis through PD-L1.

Istradefylline, an adenosine A2a receptor antagonist, inhibits the CD4+ T-cell hypersecretion of IL-17A and IL-8 in humans

Extracellular adenosine produced from ATP plays a role in energy processes, neurotransmission, and inflammatory responses. Istradefylline is a selective adenosine A2a receptor (A2aR) antagonist used for the treatment of Parkinson's disease. We previously showed using mouse models that adenosine primes hypersecretion of interleukin (IL)-17A via A2aR, which plays a role in neutrophilic inflammation models in mice. This finding suggests that adenosine is an endogenous modulator of neutrophilic inflammation. We, therefore, investigated the in vitro effect of istradefylline in humans. In the present study, using human peripheral blood mononuclear cells (PBMCs), we tested the effect of adenosine, adenosine receptor agonists and istradefylline on cytokine responses using mixed lymphocyte reaction (MLR), PBMCs, CD4+ T cells, and Candida albicans antigen (Ag)-stimulated PBMCs. We showed that adenosine and an A2aR agonist (PSB0777) promoted IL-17A and IL-8 production from human PBMCs, and istradefylline suppressed this response. In addition, istradefylline inhibited not only the IL-17A and IL-8 production induced by adenosine but also that from C. albicans Ag-stimulated PBMCs. These results indicate that adenosine-mediated IL-17A and IL-8 production plays a role in neutrophilic inflammation, against which istradefylline should be effective.

Turning Hot into Cold: Immune Microenvironment Reshaping for Atherosclerosis Attenuation Based on pH-Responsive shSiglec-1 Delivery System.

Current atherosclerosis treatment is based on a combination of cholesterol-lowering medication and low-fat diets; however, the clinical effect is unsatisfactory. It has been shown that the level of immune cell infiltration and pro-inflammatory factors in the atherosclerotic immune microenvironment (AIM) play important roles in the development and progression of atherosclerosis. Therefore, we hypothesized that reshaping "hot AIM" into "cold AIM" could attenuate atherosclerosis. For this purpose, we designed a pH-responsive and charge-reversible nanosystem, referred to as Au-PEI/shSiglec-1/PEI-acetylsalicylic acid (ASPA NPs) to effectively deliver shSiglec-1, which blocked the interactions between macrophages with CD8+ T/NKT cells, thus inhibiting immune cell infiltration. Further, we demonstrated that acetylsalicylic acid (ASA), detached from the pH-responsive PEI-ASA polymer, and inhibited lipid accumulation in macrophage, thereby decreasing the lipid antigen presentation. Additionally, reduced macrophage-produced inflammatory factors by ASA and low CD8+ T/NKT cell infiltration levels synergistically inhibit Th17 cell differentiation, thus further dramatically attenuating inflammation in AIM by decreasing the IL-17A production. Eventually, ASPA NPs efficiently reshaped AIM by inhibiting immune cell infiltration, lipid antigen presentation, and pro-inflammation, which provided a feasible therapeutic strategy for atherosclerosis immunotherapy.

Immunogenicity and clinical efficacy of anti‐SARS‐CoV‐2 vaccination in patients with hematological malignancies: Results of a prospective cohort study of 365 patients

Immunogenicity and clinical efficacy of anti‐SARS‐CoV‐2 vaccination in patients with hematological malignancies: Results of a prospective cohort study of 365 patients

P1425: ACTIVATED AND EXPANDED NATURAL KILLER CELLS TRANSDUCED WITH AN NKG2D CHIMERIC ANTIGEN RECEPTOR AGAINST ACUTE MYELOID LEUKEMIA

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy incurable for almost all patients. Chimeric Antigen Receptor (CAR) therapy is showing promising results in other hematological disorders but remains challenging in AML since no specific antigens have been described yet. Using NKG2D as a CAR, a natural NK receptor with 8 ligands overexpressed in several tumors, could surmount AML targeting limitations. T cells are considered the gold standard immune effector cells for CAR therapy, but they show toxicities that could be overcome using other cells such as activated and expanded natural killer cells (NKAE), that can be used in an allogeneic context with no GvHD, have natural anti-tumor properties and a short lifespan within the organism. In this project we analyze the in vitro efficacy and security of peripheral blood NKAE cells lentivirally transduced with an NKG2D-41BB-CD3z CAR. Aims: The primary objective of this project is to assess the efficacy and security of a cell immunotherapy against AML based on the use of NKAE cells transduced with an NKG2D CAR. Methods: NKAE cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (HD) and/or AML patients after 7 days of coculture with the irradiated cell line k562-mb21-41BBL by magnetic immunodepletion, when they were transduced with lentiviral vectors carrying NKG2D-41BB-CD3z CAR construct. CAR expression was measured up to 15 days post-transduction by flow cytometry. Cytotoxic activity against AML cell lines was performed by Propidium Iodide and Annexin V staining. Toxicity was evaluated by Europium-TDA assays. Results: AML can be targeted with an NKG2D CAR since all patients studied expressed at least one NKG2DL (Fig.1). Primary NK cells can be lentivirally transduced with an NKG2D CAR, showing a modest but stable CAR expression up to 15 days after transduction (23% ±4,7% NKG2D+, 33,35% ±4,25% GFP+). CAR-NKAE cells perform robust cytotoxicity towards MOLM-13 AML cell line after 24h of coculture at an effector:target ratio of 1:1, exerting a quasi-total lysis of AML cells (Fig.2A). This represents a significant increase in anti-leukemia effect compared to untransduced NKAE (92,6% ±0,3% vs 72,2% ±10%, p= 0,0138). Surface expression of relevant molecules for NK activity showed that NKp30, NKp44 (natural cytotoxicity receptors), CD69 (early activation marker), CD25 (IL2R alpha chain), FasL (mediates FasL-mediated cytotoxicity), NKp80 (C-type lectin-like surface-activating receptor) and TRAIL (TNF-Related Apoptosis Inducing Ligand) were more expressed by transduced NKAE (Fig.2B). Cytokine release profile revealed a higher production of IL-6, IL-17A, sFasL and IFNg by CAR-NKAE (Fig.2C). We did not observe relevant toxic effects of none of the cells against healthy PBMCs and very low toxicities were found against NL-20 lung cell line, but there were no differences between NKAE and CAR-NKAE. Image:Summary/Conclusion: Our preliminary results show that primary NK cells can be lentivirally transduced with a second generation NKG2D CAR, exerting a robust anti-leukemia activity towards AML cell lines. Transduced cells show a more cytotoxic immunophenotype as well as a higher release of cytolytic molecules and proinflammatory and proapoptotic proteins, that could be responsible of the increased anti-tumor response. Hence, NKG2D-CAR transduced NKAE cells could be a suitable approach to treat AML. Other costimulatory domains are being studied in order to assess which one triggers a higher cytotoxic activity, thus allowing decreasing the effector:target ratios and therefore, on-target off-tumor effect.

A vitronectin-derived peptide prevents and restores alveolar bone loss by modulating bone re-modelling and expression of RANKL and IL-17A.

This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n= 10/group). VnP-16 (100 μg/10μl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 μg/5μl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.

A New Live Auxotrophic Vaccine Induces Cross-Protection against Klebsiella pneumoniae Infections in Mice.

The development of a whole-cell vaccine from bacteria auxotrophic for D-amino acids present in the bacterial cell wall is considered a promising strategy for providing protection against bacterial infections. Here, we constructed a prototype vaccine, consisting of a glutamate racemase-deficient mutant, for preventing Klebsiella pneumoniae infections. The deletion mutant lacks the murI gene and requires exogenous addition of D-glutamate for growth. The results showed that the K. pneumoniae ΔmurI strain is attenuated and includes a favourable combination of antigens for inducing a robust immune response and conferring an adequate level of cross-protection against systemic infections caused by K. pneumoniae strains, including some hypervirulent serotypes with elevated production of capsule polysaccharide as well as multiresistant K. pneumoniae strains. The auxotroph also induced specific production of IL-17A and IFN-γ. The rapid elimination of the strain from the blood of mice without causing disease suggests a high level of safety for administration as a vaccine.

Abstract 1334: MerTK tumorigenesis and immune evasion mechanisms in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) cells lack estrogen, progesterone, and HER2 receptors. TNBC tends to be more aggressive than most types of breast cancer and is more likely to recur even after surgery and treatment. Recently, a few targeted therapies were approved for TNBC, but additional molecular targeting agents are still needed. Previous studies have found that activated MerTK is associated with many types of human cancers, including breast cancer. In this study, we focused on MerTK expression and its tumorigenesis and immunoregulatory functions in TNBC. We first examined MerTK expression levels in human TNBC tissue samples by analyzing tissue microarrays (TMA) using immunohistochemistry (IHC). IHC revealed that ~40% of TNBC samples expressed high levels of MerTK. We further examined MerTK expression levels in ten TNBC cell lines by immunoblot analysis. Three cell lines (BT549, MDAMB231 and MDAMB436) showed strong MerTK expression levels. Next, to evaluate MerTK’s effect on cell proliferation, we treated MDAMB231 cells with MerTK siRNA, which inhibited cell proliferation by 20% compared to siNon-target (siNT). These data indicate that TNBC cells remain dependent on MerTK-activated signaling pathways for proliferation and survival. To further investigate the impact of MerTK expression in TNBC cells, we stably overexpressed MerTK in the TNBC cell line SUM102, which naturally has very low MerTK levels. The results indicated that overexpression of MerTK in SUM102 cells led to: 1) increased proliferation in vitro 2) robust tumor growth, 3) marked migration potential and 4) increased metastasis in mice. Collectively, these results demonstrate that activation of a MerTK-driven pathway could be involved in tumorigenesis in TNBC. To understand the role of MerTK in cell signaling and pro-tumor immunity, we utilized a cytokine array to examine which molecules were impacted in MerTK-overexpressing SUM102 cells. The results showed that production of CXCL4, CD74, and IL-1α was increased, and production of CXCL10 was decreased in MerTK-overexpressing SUM102 cells as compared to control cells. Interestingly, PD-L1 was upregulated and MHC class I was downregulated in MerTK-overexpressing SUM102 cells as compared to control cells. These preliminary findings suggested that MerTK-overexpressing TNBC could be creating a favorable immune microenvironment for proliferation of tumor cells. To further explore the cytokine, PD-L1, and MHC class I results, we overexpressed MerTK in 4T1 murine TNBC-like cells. Immunoblot analysis showed that PD-L1 was upregulated in MerTK-overexpressing 4T1 cells. We will utilize this cell line to evaluate tumor growth and immunosuppression in the tumor microenvironment in BALB/C mice. Citation Format: Mari Iida, Kourtney Kostecki, Carlene A. Kranjac, Christine Glitchev, David T. Yang, Deric L. Wheeler. MerTK tumorigenesis and immune evasion mechanisms in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1334.

Plasma interleukin-1α and interleukin-8 in female dogs with non-metastatic and metastatic malignant mammary gland tumours.

In this study plasma concentrations of IL-1α and IL-8 in 29 female dogs with malignant mammary gland tumours (19 without metastasis and 10 with metastasis) and in 10 healthy control animals were determined. Concentrations of IL-1α and IL-8 were analysed using a specific canine ELISA assay. Mean plasma concentrations of IL-1α and IL-8 were significantly higher (p⟨0.05) in female dogs with both non-metastatic and metastatic malignant tumours compared to the healthy animals. The concentrations of both tested cytokines were significantly increased (p⟨0.05) in the dogs with metastasis. In female dogs with mammary carcinomas, the plasma concentration of IL-1α was significantly higher (p⟨0.05) in the animals with grade 3 tumours compared to the dogs with grade 1 tumours. The concentration of IL-8 was significantly higher (p⟨0.05) in the dogs with grade 3 tumours compared to that found in the animals with grade 1 and grade 2 tumours. A moderate correlation (r=0.433) was found between IL-1α and IL-8 concentrations in the female dogs. These findings suggest that increased malignancy and invasiveness of canine mammary tumours is associated with an increased production of IL-1α and IL-8 in the tumour microenvironment, which, in turn, leads to an increase in their circulating levels. This may indicate that circulating levels of the cytokines investigated could be considered as diagnostic and prognostic markers in canine malignant mammary tumours. However, further studies in this fields are needed.

Protective Effect against Neosporosis Induced by Intranasal Immunization with Neospora caninum Membrane Antigens Plus Carbomer-Based Adjuvant.

Neospora caninum is an obligate intracellular protozoan responsible for abortion and stillbirths in cattle. We previously developed a mucosal vaccination approach using N. caninum membrane proteins and CpG adjuvant that conferred long-term protection against neosporosis in mice. Here, we have extended this approach by alternatively using the carbomer-based adjuvant Carbigen™ in the immunizing preparation. Immunized mice presented higher proportions and numbers of memory CD4+ and CD8+ T cells. Stimulation of spleen, lungs and liver leukocytes with parasite antigens induced a marked production of IFN-γ and IL-17A and, less markedly, IL-4. This balanced response was also evident in that both parasite-specific IgG1 and IgG2c were raised by immunization, together with specific intestinal IgA. Upon intraperitoneal infection with N. caninum, immunized mice presented lower parasitic burdens than sham-immunized controls. In the infected immunized mice, memory CD4+ T cells predominantly expressed T-bet and RORγt, and CD8+ T cells expressing T-bet were found increased. While spleen, lungs and liver leukocytes of both immunized and sham-immunized infected animals produced high amounts of IFN-γ, only the cells from immunized mice responded with high IL-17A production. Since in cattle both IFN-γ and IL-17A have been associated with protective mechanisms against N. caninum infection, the elicited cytokine profile obtained using CarbigenTM as adjuvant indicates that it could be worth exploring for bovine neosporosis vaccination.

CD69 expression on Treg cells prevents chronic heart damage after myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia e Innovación (MCIN), through the Carlos III Institute of Health (ISCIII)-Fondo de Investigación Sanitaria (PI19/00545) Background Increasing evidences advocate for an important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the molecular mechanisms remain elusive. Result and Methods In this study, a broad analysis of immune markers in 283 patients show a significant CD69 overexpression on Treg cells after MI. Our results in mice demonstrate that CD69 expression on Treg cells increases survival after left-anterior-descending coronary artery (LAD)-ligation. Cd69-/- mice develop strong IL17A+ gdT cell responses after ischemia that increase myocardial inflammation and, consequently, worsen cardiac function. CD69+ Treg cells induce apoptosis and decrease IL-17A production in gdT cells by a CD39-dependent mechanism. Adoptive transfer of CD69+ Treg cells to Cd69-/- mice after LAD-ligation reduces IL17A+ gdT cell recruitment increasing survival. Consistently, clinical data from two independent cohorts of patients indicate that increased CD69 expression in peripheral blood cells after acute MI is associated with a lower risk of re-hospitalization for chronic heart failure (CHF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex and traditional cardiac damage biomarkers (OR 0.929, 95% CI, 0.838-0.980; p&amp;lt;0.0409). Conclusion Our data highlight CD69 expression on T cells as a therapeutic and prognostic target to prevent CHF after MI.

Riboflavin Attenuates Influenza Virus Through Cytokine-Mediated Effects on the Diversity of the Gut Microbiota in MAIT Cell Deficiency Mice.

Influenza is a serious respiratory disease that continues to threaten global health. Mucosa-associated invariant T (MAIT) cells use T-cell receptors (TCRs) that recognize microbial riboflavin derived intermediates presented by the major histocompatibility complex (MHC) class I-like protein MR1. Riboflavin synthesis is broadly conserved, but the roles or mechanisms of riboflavin in MR1–/– mouse influenza infection are not well understood. In our study, immunofluorescence techniques were applied to analyze the number and distribution of viruses in lung tissue. The amount of cytokine expression was assessed by flow cytometry (FCM), ELISA, and qPCR. The changes in the fecal flora of mice were evaluated based on amplicon sequencing of the 16S V3-V4 region. Our study showed that MAIT cell deficiency increased mortality and that riboflavin altered these effects in microbiota-depleted mice. The oral administration of riboflavin inhibited IL-1β, IL-17A, and IL-18 production but significantly increased the expression of IFN-γ, TNF-α, CCL2, CCL3, and CCL4 in a mouse model. The analysis of the mouse flora revealed that riboflavin treatment significantly increased the relative abundance of Akkermansia and Lactobacillus (p < 0.05) and decreased that of Bacteroides. In contrast, MR1–/– mice exhibited a concentrated aggregation of Bacteroides (p < 0.01), which indicated that MAIT cell deficiency reduced the diversity of the bacterial population. Our results define the functions of MAIT cells and riboflavin in resistance to influenza virus and suggest a potential role for riboflavin in enhancing MAIT cell immunity and the intestinal flora diversity. Gut populations can be expanded to enhance host resistance to influenza, and the results indicate novel interactions among viruses, MAIT cells, and the gut microbiota.