Secretion Of IL-16 Research Articles

Immunoinhibitory effects of hypoxia-driven reprogramming of EGR1hi and EGR3 positive B cells in the nasopharyngeal carcinoma microenvironment

Regulatory B (Breg) cells is a type of immune cell that exhibit immunosuppressive behavior within the tumor microenvironment. However, the differentiation and regulatory mechanisms of these Breg cells remain unexplored. Single-cell transcriptome sequencing analysis of human nasopharyngeal carcinoma (NPC) revealed a significant enrichment of B cell subset characterized by high expression of EGR1 and EGR3 in the tumor microenvironment. Notably, in the hypoxic microenvironment, these B cells induce MAPK pathway activation, subsequently triggering the activation of transcription factors EGR1 and EGR3, which further modulate the expression of immunosuppressive factors like TGFB1 and IL10. In transplant experiments using primary B cells induced under hypoxia and co-transplanted with cancer cells, a significant increase in tumor growth was observed. Mechanism experiments demonstrated that EGR1hi and EGR3+ B cells further activate the maturation and immunosuppressive function of Treg cells through the secretion of IL16 and TNF-α. Hence, this study identifies the key transcription factors EGR1 and EGR3 as essential regulators and elucidates the differentiation of Breg cells under hypoxic conditions.

Immune Profiling of Secreted Factors from Human Vestibular Schwannoma Cells and Tumor-associated Macrophages.

This study compared the immune-related secretory capacity of human vestibular schwannoma (VS) and tumor-assisted macrophages (TAMs) with their normal counterparts (Schwann cells [SC] and peripheral blood monocyte-derived macrophages [Mo-MFs], respectively), and examined relationships with presurgical hearing and tumor size. VS tumors (n = 16), auditory nerve (n = 1), blood (n = 9), and great auricular nerves (n = 3) were used. SCs (S100B+ ) and TAMs (CD68+ ) were isolated from VS tissue for culture. The secreted levels of 65 immune-related factors were measured and compared using unpaired t-tests with Welch correction (schwannoma vs. SCs) or Mann-Whitney tests (TAMs and Mo-MFs). Associations between factor concentration and word recognition (WR), pure-tone average (PTA), and tumor size were evaluated with Spearman correlation. Secreted factors with significantly higher concentrations in schwannoma versus SC supernatants included IL-2 and BAFF, whereas MMP-1, IL-6, FGF-2, VEGF-A, MIP-3α, and GRO-α concentrations were significantly higher in TAMs versus Mo-MFs (all p < 0.05). Worse WR was significantly associated with higher secretion of fractalkine, eotaxin-3, CD30, and IL-16 by VS cells; IP-10, eotaxin-3, multiple interleukins, GM-CSF, SCF, and CD30 by TAMs; and TNF-α and MIP-1α by Mo-MFs (all p < 0.05). Worse PTA was significantly correlated with higher secretion of IL-16 by VS cells (p < 0.05). Larger tumor size was significantly correlated with higher secretion of eotaxin by VS cells, and of IL-7, IL-21, and LIF by TAMs (all p = 0.017). Differential secretion of immune-related factors was observed in schwannoma versus normal SCs and in TAMs versus Mo-MFs, some of which were correlated with worse hearing and larger VS tumors. N/A Laryngoscope, 134:S1-S14, 2024.

IL16 PRODUCTION IS A MECHANISM OF RESISTANCE TO BTK INHIBITORS AND TO R‐CHOP

Introduction: Covalent and non-covalent BTK inhibitors (BTKi) are used or being explored for the treatment of patients with B-cell malignancies, such as marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) of the activated B-cell type (ABC). The identification of mechanisms of resistance can lead to improvements in the current therapeutic approaches. We had reported IL16 production in a MZL model of secondary resistance to ibrutinib (Arribas ENA2019). Here, we expand the initial observation to additional lymphoma types and treatments modalities, performing therapeutic interventions to block it and providing clinical validation data on its expression and role in patients. Methods: VL51 (marginal zone lymphoma, MZL) parental (PAR) and Ibrutinib-resistant (RES) cells underwent RNA (mRNA & miRNA by RNA-Seq), DNA (promoter methylation, WES) and secretome (Cytokine array) profiling. RES cells exhibited resistance to covalent and non-covalent BTK inhibitors. No DNA copy number variations or mutations were detected by WES in RES compared to PAR cells, including those affecting BTK or PLCG2 genes. Results: Multi-omics (DNA, mRNA, miRNA, secretome) characterization of RES and PAR cells identified hypomethylation, up-regulation and secretion of IL16 in RES but not in PAR cells. Elevated levels of p-AKT and p-ERK was observed in RES compared to PAR, suggesting a signaling activation in RES cells. Stimulation with recombinant IL16 reduced sensitivity to BTKi not only in VL51 PAR cells, but also in models derived from MCL (REC1) and CLL (MEC1). Conversely, the use of IL16 blocking antibody recovered sensitivity to BTKi in RES cells, along with other lines with high expression of IL16 and low sensitivity to ibrutinib derived from MCL (MAVER1) and MZL (SSK41). IL16 is part of the ABC-DLBCL signature. We observed that high IL16 expression was associated with shorter overall survival in ABC but not in germinal center like DLBCL patients (n = 233, log-rank test p = 0.013, multivariate Cox: HR (95% CI) 0.43 (0.22–0.85), p = 0.016). IL16 stimulation also decreased sensitivity to RCHOP in ABC DBCL cells (OCILy10, HBL1). Finally, secreted levels of IL16 in the serum of CLL patients (n = 17) with resistance to ibrutinib (in absence of BTK or PLCG2 mutations) were significantly higher compared to patients responding to the drug and paired for clinical features. Conclusions: Starting from a MZL model of secondary resistance to ibrutinib, we showed that IL16 can sustain resistance to BTK inhibitors also in CLL and MCL. Furthermore, increased expression of IL16 associated with shorter outcome in ABC DLBCL patients and the interleukin induced in vitro resistance to RCHOP. The demonstration that IL16 blocking therapies recovered sensitivity to therapies can lead to novel therapeutic approaches to overcome resistance in lymphoma patients. The research was funded by: Swiss National Science Foundation (SNSF 31003A_163232/1) Keyword: molecular targeted therapies Conflicts of interests pertinent to the abstract A. Arribas Educational grants: Astra Zeneca

Abstract 5169: Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer

Abstract Introduction: The tumor microenvironment (TME) is composed of different cell types and is involved in tumor progression. Several immunosuppressive signaling pathways can promote tumor growth by inhibiting anti-tumor immunity in TEM. Therefore, targeting the immunosuppressive signaling pathways is a potential cancer treatment. Recent studies reported that inhibition of oncoproteins could lead to reduced anti-tumor immunity in several cancers; therefore, it’s critical to identify the potential oncoproteins which can lead to enhanced anti-tumor immunity. Aurora-A is a serine/threonine kinase that plays an important role in the centrosome, cell mitosis, and chromosome stability. Previous studies indicated that cancer patients with poor prognoses are associated with a high Aurora-A level in tumor cells. Inhibition of Aurora-A is considered as a promising strategy for cancer treatment. However, Aurora-A inhibitors have not had a breakthrough in phase II or phase III clinical trials. The role of Aurora-A in tumor immunity is still unclear. Here, we investigated the role of Aurora-A in tumor immunity. Materials and Methods: shRNA was used to knock down Aurora-A, and Alisertib was added to inhibit Aurora-A. RT-qPCR and western blot were performed to determine the IL-16 expression. The secretion of cytokines was measured by cytokine array and ELISA. BALB/c and NOD/SCID mice were subcutaneously injected with CT26 and then administered daily with Alisertib or treated with anti-IL-16 antibody. The tumor-infiltrating immune populations were analyzed by flow cytometry. IHC and H/E stain were performed to analyze Aurora-A expression and lymphocyte infiltration in human CRC specimens. Results: CRC patients with lower lymphocyte infiltration and higher Aurora-A are associated with poor prognosis and vice versa. Knockdown of Aurora-A in CT26 promotes tumor growth in immunocompetent mice by inhibiting the infiltration and cytotoxic activity of CD8+ T cells. Aurora-A negatively regulates IL-16 expression in a kinase-dependent manner in CRC cells. Blockade of IL-16 by anti-IL-16 antibody can reverse the tumor growth and CD8+ T cell cytotoxic activity in Aurora-A knocked-down CT26 tumors. Alisertib can inhibit CT26 tumor growth and promote IL-16 secretion in TME in mice. A combination of Alisertib and IL-16 antibody can promote the therapeutic effect of Alisertib in CT26 tumors. Conclusion: Tumor-intrinsic Aurora-A plays a diverse role in tumor progression depending on the status of lymphocyte infiltration in TME. Aurora-A can promote anti-tumor immunity via negatively regulating IL-16 in a kinase activity-dependent manner in hot CRC tumors. In tumors with high lymphocyte infiltration, Aurora-A inhibition can suppress cancer cell growth and reduce anti-tumor immunity; the combination of Aurora-A inhibitors and IL-16 antibodies may provide a novel and effective strategy for cancer therapy in hot tumors. Citation Format: Shiang-Jie Yang, Ming-Derg Lai, Liang-Yi Hung. Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5169.

Interleukin-18 in Brazilian Rheumatoid Arthritis Patients: Can Leflunomide Reduce It?

Objectives Rheumatoid arthritis affects about 1% of the world's population. This is a chronic autoimmune disease. It is predominant in females with progressive joint damage. Immune cells are involved, especially Th1/Th17 lymphocytes and their inflammatory cytokines. These proteins have different functions in the immune system, such as IL-16 is a chemotactic factor; IL-18 can activate NFκB transcription producing inflammatory proteins; IL-31 can activate the JAK/STAT pathway which leads to the production of inflammatory factors in chronic diseases; IL-33 promotes IL-16 secretion which causes lymphocyte recruitment, and IL-32 and IL-34 appear to increase TNF secretion by macrophages activation in AR. The aim of this study was to evaluate serum levels of IL-16, IL-18, IL-31, IL-32, IL-33, and IL-34 and compare them with the severity and treatment of RA patients if there are any correlations. Methods A total of 140 RA patients and 40 healthy donors were recruited from the Department of Rheumatology at Hospital das Clínicas from the Federal University of Pernambuco. 60 AR patients were naïve for any treatment. Serum cytokine levels were determined using an ELISA kit. Results Serum IL-16 (p = 0.0491), IL-18 (p < 0.0001), IL-31 (p = 0.0004), and IL-32 (p = 0.0040) levels were significantly increased in RA patients compared with healthy donors. It was observed that patients using leflunomide had the lowest IL-18 levels, close to controls levels (p = 0.0064). Conclusion IL-16, IL-18, IL-31, and IL-32 are increased in the serum of RA patients. IL-18 is at lower levels in those AR who are taking leflunomide as treatment.

Zinc Aspartate Induces IL-16 Secretion and Apoptosis in Human T Cells.

T cell activation mediates immunity to pathogens. On the flipside, T cells are also involved in pathological immune responses during chronic autoimmune diseases. We recently reported that zinc aspartate, a registered drug with high bioavailability, dose-dependently inhibits T cell activation and Th1/Th2/Th17 cytokine production of stimulated human and mouse T cells. To understand the suppressive effect of zinc on T cell function, we here investigated the influence of zinc aspartate on human T cells focusing on the secretion of immunosuppressive cytokines, induction of apoptosis, and caspase 3/7 activity. To this end, we monitored either freshly stimulated or pre-activated human T cells in the presence of zinc aspartate from 40–140 µM over a period of 72 h. Under both experimental conditions, we observed a dose-dependent suppression of human T cell proliferation. While IL-1ra, latent TGF-β1, and IL-10 were dose-dependently reduced, we, unexpectedly, detected elevated levels of IL-16 upon zinc supplementation. In addition, the number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis, steadily increased at zinc aspartate concentrations exceeding 100 µM. Taken together, our findings suggest that zinc aspartate impairs T cell fitness and might be beneficial for the treatment of T cell-mediated autoimmune diseases.

Abstract 243: Myocardial-associated B Cells Modulate the Composition of Cardiac Resident Macrophages

Background: The murine myocardium contains distinct subsets of macrophages with disparate roles that can be defined by expression levels of CCR2 and MHC-II. The factors that modulate the composition of the myocardial macrophage pool in the naïve heart are not completely known. B cells have been recently shown to modulate the prevalence of T cells in the heart but the relationship between B cells and myocardial macrophage subsets remains unknown. Objective: To test the hypothesis that myocardial-associated B cells modulate the composition of the myocardial macrophage pool. Methods and Results: Hearts from B cell deficient (μMT) or B cell depleted (treated with Anti-CD20 antibody) mice and age/sex matched controls were analyzed via flow cytometry at post-natal day 14 and soon after weaning. Absence of B cells was consistently associated with a decrease in the prevalence of CCR2 - MHC-II high resident macrophages. Immunofluorescence analysis of the heart showed that the myocardial-associated B cells were frequently in close proximity of CD68+ myocardial macrophages. In vitro co-culture of myocardial-derived CD64+ macrophages with B cells or with B cell-conditioned media showed that B cells fostered an increase in the prevalence of CCR2 - MHC-II high macrophages through a direct paracrine effect. In vivo 10X single cells sequencing and flow cytometric analysis together with in vitro ELISA and antibody mediate blocking suggested that the paracrine effect of B cells on myocardial macrophages is mediated, at least in part, by IL-16 secretion. Conclusion: Myocardial-associated B cells modulate the phenotype of myocardial resident macrophages. This is likely the result of a direct paracrine effect mediated, at least in part, by IL-16. These findings describe for the first time a relationship between circulating naïve B cells and tissue resident macrophages and unveil a previously unappreciated relationship between lymphoid and myeloid myocardial cells.

CaSR Antagonist (Calcilytic) NPS 2143 Hinders the Release of Neuroinflammatory IL-6, Soluble ICAM-1, RANTES, and MCP-2 from Aβ-Exposed Human Cortical Astrocytes.

Available evidence shows that human cortical neurons’ and astrocytes’ calcium-sensing receptors (CaSRs) bind Amyloid-beta (Aβ) oligomers triggering the overproduction/oversecretion of several Alzheimer’s disease (AD) neurotoxins—effects calcilytics suppress. We asked whether Aβ•CaSR signaling might also play a direct pro-neuroinflammatory role in AD. Cortical nontumorigenic adult human astrocytes (NAHAs) in vitro were untreated (controls) or treated with Aβ25–35 ± NPS 2143 (a calcilytic) and any proinflammatory agent in their protein lysates and growth media assayed via antibody arrays, enzyme-linked immunosorbent assays (ELISAs), and immunoblots. Results show Aβ•CaSR signaling upregulated the synthesis and release/shedding of proinflammatory interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1) (holoprotein and soluble [s] fragment), Regulated upon Activation, normal T cell Expressed and presumably Secreted (RANTES), and monocyte chemotactic protein (MCP)-2. Adding NPS 2143 (i) totally suppressed IL-6′s oversecretion while remarkably reducing the other agents’ over-release; and (ii) more effectively than Aβ alone increased over controls the four agents’ distinctive intracellular accumulation. Conversely, NPS 2143 did not alter Aβ-induced surges in IL-1β, IL-3, IL-8, and IL-16 secretion, consequently revealing their Aβ•CaSR signaling-independence. Finally, Aβ25–35 ± NPS 2143 treatments left unchanged MCP-1′s and TIMP-2′s basal expression. Thus, NAHAs Aβ•CaSR signaling drove four proinflammatory agents’ over-release that NPS 2143 curtailed. Therefore, calcilytics would also abate NAHAs’ Aβ•CaSR signaling direct impact on AD’s neuroinflammation.

Abstract A127: Secretion of IL16 is associated with resistance to ibrutinib in pre-clinical models of lymphoma

Abstract Background.The first-in-class BTK inhibitor ibrutinib has been approved by the U.S. Food and Drug Administration (FDA) for different indications including the treatment of patients with marginal zone lymphoma, who are in need of systemic therapy and have received at least one prior anti-CD20-based therapy. We have generated and characterized a model resistant to ibrutinib and derived from splenic marginal zone lymphoma. Materials and Methods.The splenic MZL VL51 cell line was kept under ibrutinib (IC90) until acquisition of resistance or with no drug (parental, PAR). Cell identity was confirmed by STR DNA fingerprinting. Resistance was determined by MTT assay as stable if present after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome profiling (RNA-Seq), whole exome sequencing, lipidomic profiling, pharmacological screening (348 compounds), and immunophenotypic analysis (FACS). Secreted cytokines and growth factors were analyzed by ELISA. Results. We developed a stable ibrutinib resistance model derived from VL51 cell line (VL51-ibR) with 8-fold times higher IC50 than parental cells. Specific mutations associated with resistance were not detected, including those in BTKor PLCG2 genes. Conditioned media from VL51-ibR conferred resistance to ibrutinib in the parental cells, indicating the involvement of secreted factors in the mechanism of resistance. At transcriptome level, VL51-ibR exhibited overexpression of genes coding for secreted molecules (IL16, CXCL10), integrins (ITGAM, ITGA1), members of the NFKB (TNF, LTA) and RAS-RAF (RASGRP4, RASGRP2) signaling pathways. The pharmacologic screening identified acquired sensitivity to a RAS- inhibitor. Lipidomic profiling showed high levels of specific triacylglycerols, glycerophosphocholines and cardiolipins with a down-regulation of sphingomyelins. Also, in agreement with transcriptomic data, VL51-ibR had increased levels of p-PLCG2 and p-ERK, paired with the presence of IL6 and CXCL10 in the medium and double positive surface expression of CXCR5 and CD49d. We extended our findings to other models and to clinical specimens. Lastly, we investigated whether these results might be extrapolated into different in vitro models and splenic marginal zone lymphoma clinical cases. First, IL16 and CXCL10 expression levels were inversely correlated with sensitivity to ibrutinib in a panel of 13 B-cell lymphoma cell lines (Tarantelli et al, CCR 2018) (P&amp;lt;0.05). Second, we determined the top 200 genes positively correlated genes with IL16 in a series of splenic marginal zone lymphoma clinical cases (Arribas et al, Mod Pathol 2013), and we observed that these genes were also more enriched in the VL51-ibR when compared to the parental VL51. Conclusions. We have developed and characterized a preclinical model, driven by secreted factors, of secondary resistance to the BTK-inhibitor ibrutinib in splenic marginal zone lymphoma. The current work provides new insights into the mechanisms of resistance to ibrutinib and can lead to novel therapeutic approaches to overcome the resistance. Citation Format: Alberto J. Arribas, Sara Napoli, Eugenio Gaudio, Luciano Cascione, Alessandra Di Veroli, Chiara Tarantelli, Filipppo Spriano, Antonella Zucchetto, Francesca Rossi, Giulio Sartori, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Valter Gattei, Gabriele Cruciani, Emanuele Zucca, Davide Rossi, Francesco Bertoni. Secretion of IL16 is associated with resistance to ibrutinib in pre-clinical models of lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A127. doi:10.1158/1535-7163.TARG-19-A127

Cellular and Molecular Effects of High-Molecular-Weight Heparin on Matrix Metalloproteinase 9 Expression.

Blood sampling with different anticoagulants alters matrix metalloproteinase (MMP-) 9 expression, thus influencing its concentration and diagnostic validity. Here, we aimed to evaluate the effects of different anticoagulants on MMP-9 regulation. MMP-9 expression was assessed in response to ethylenediaminetetraacetic acid, citrate, and high-/low-molecular-weight heparin (HMWH, LMWH) in co-culture experiments using THP-1, Jurkat, and HT cells (representing monocytes, T, and B cells). Triple and double cell line co-culture experiments revealed that HMWH treatment of THP-1 and Jurkat led to a significant MMP-9 induction, whereas other anticoagulants and cell type combinations had no effect. Supernatant of HMWH-treated Jurkat cells also induced MMP-9 in THP-1 suggesting monocytes as MMP-9 producers. HMWH-induced cytokine/chemokine secretion was assessed in co-culture supernatant, and the influence of cytokines/chemokines on MMP-9 production was analyzed. These experiments revealed that Jurkat-derived IL-16 and soluble intercellular adhesion molecule (sICAM-) 1 are able to induce MMP-9 and IL-8 production by THP-1. As a consequence, the increased MMP-9 expression found in HMWH blood samples may be influenced by HMWH-dependent secretion of IL-16 and sICAM-1 by T cells resulting in an increased production of MMP-9 and IL-8 by monocytes. IL-8, in turn, may support MMP-9 and its own expression in a positive autocrine feedback loop.

Neutrophil-Derived Interleukin 16 in Premetastatic Lungs Promotes Breast Tumor Cell Seeding.

The premetastatic niche in distant organs prior to metastatic cell arrival emerged as an important step in the metastatic cascade. However, molecular mechanisms underlying this process are still poorly understood. In particular, whether neutrophil recruitment at a premetastatic stage promotes or inhibits metastatic cell seeding has to be clarified. We aimed at unraveling how neutrophil infiltration in lung parenchyma induced by the distant primary tumor influences the establishment of lung metastasis. Elevated neutrophil counts and IL-16 levels were found in premetastatic lungs in a syngenic mouse model using 4T1 tumor cells. 4T1 cell–derived soluble factors stimulated IL-16 secretion by neutrophils. The functional contribution of IL-16 is supported by metastasis burden reduction in lungs observed on instillation of an IL-16 neutralizing antibody. Moreover, IL-16 promotes in vitro 4T1 cell adhesiveness, invasiveness, and migration. In conclusion, at a premetastatic stage, neutrophil-derived IL-16 favors tumor cell engraftment in lung parenchyma.

The expression of IGF-1R in Helicobacter pylori-infected intestinal metaplasia and gastric cancer.

Overexpression of IGF-1R has been demonstrated in gastrointestinal cancers, and its expression is reported as the result of the loss of tumor suppressors. IL-16 is involved in the pathophysiological process of chronic inflammatory diseases. The aim of this study is to determine the changes in the expression of IGF-1R in intestinal metaplasia (IM) and gastric cancer (GC) as well as the effect of Helicobacter pylori (H. pylori) and IL-16 on cell proliferation and IGF-1R expression in gastric cells. AGS cells were incubated with combinations of IL-16 and H. pylori. Gastric cell proliferation was studied by BrdU uptake. In H. pylori infected mucosa, IGF-1R was significantly higher in IM than chronic gastritis (CG), and also higher in GC than CG and IM. H. pylori significantly decreased BrdU uptake. IL-16 increased BrdU uptake and IGF-1R on AGS cells which had been decreased by H. pylori. Co-incubation with IL-16 increased the expression of IGF-1R mRNA in H. pylori infected cells. We conclude that the expression of IGF-1R in H. pylori infected gastric mucosa may indicate an early stage of carcinogenesis. The IL-16 secretion by H. pylori can be a trigger for the expression of IGF-1R, and it may also be a factor for gastric carcinogenesis.

Cationic antimicrobial peptide, magainin down-regulates secretion of pro-inflammatory cytokines by early placental cytotrophoblasts

BackgroundHuman placental villous cytotrophoblasts exhibit relative externalization of negatively charged moieties to the outer leaflet of the plasma membrane during the time of syncytialization rendering their reactivity to positively charged cationic antimicrobial peptides (CAMPs) during the window of implantation and early placentation. Vaginal administration of a synthetic CAMP, Ala8,13,18-magainin II amide (AMA) inhibited blastocyst implantation and early placentation in monkeys. Furthermore, the administration of AMA resulted in significant inhibition of cell differentiation, enhancement in apoptosis and loss of viability in first trimester placental villous cytotrophoblasts in primary culture. The present study examines the effect of in vitro application of different doses (0, 1, 10, 100, 1000 ng/ml) of AMA on the secreted cytokine profiles of cytotrophoblasts obtained from placental villi samples (n = 13) collected during 8–9 weeks of gestation and grown on three-dimensional collagen matrix in vitro.MethodsA panel of forty-eight (48) cytokines in conditioned medium was analysed using multiplex immunoassays technique. Further, the steady state transcript levels of four cytokines (CCL4, CCL5, IL1B, IL6), the concentrations of which were affected by AMA in the isolated cytotrophoblasts, as well as, two cytokines (IL1A and TNF) which were not affected by AMA were estimated. Input list of cytokines secreted by cytotrophoblasts and showing differential secretion in response to AMA were used in enrichment analysis for the generation of biological networks.ResultsPlacental cytotrophoblasts secreted 27 cytokines, 13 of which are affected by AMA in vitro with significantly decreased secretion of CCLs-2, 3, 4, 5, CXCLs-1 and 8, FGF2 and MCSF and that of IL1B, IL6 and MIF, and increased secretion of IL16 and IL-2RA. Of the above cytokines showing differential secretion, only IL-2RA, IL16 and MIF showed significant correspondence in the steady state expression of their respective transcript levels. Post-hoc Enrichment analysis revealed Toll-like receptor (TLR) mediated pathways were the top-scored target pathways that were affected by AMA.ConclusionsAdministration of a CAMP causes shift in the balance of immune-inflammatory responses involving downstream pathways of TLRs in cytotrophoblast function. Further verification of functions of placental trophoblasts on administration of CAMP with pregnancy outcome is necessary.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0119-8) contains supplementary material, which is available to authorized users.

Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that IL-16, a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram negative pathogens or LPS. IL-16 production involved multiple signals including ligation of TNFR family members or EGFR, both receptors for SpA and generation of Ca2+ fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4+ cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S.aureus to activate TNFR1 and Ca2+/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

Inhibitory CD8 + T cells in autoimmune disease

Rheumatologists have long been focused on developing novel immunotherapeutic agents to manage such prototypic autoimmune diseases as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The ultimate challenge in providing immunosuppressive treatment for patients with RA and SLE has derived from the dilemma that both protective and harmful immune responses result from adaptive immune responses, mediated by highly diverse, antigen-specific T and B cells endowed with powerful effector functions and the ability for long-lasting memory. As regulatory/suppressor T cells can suppress immunity against any antigen, including self-antigens, they emerge as an ideal therapeutic target. Several distinct subtypes of CD8 + suppressor cells (Ts) have been described that could find application in treating RA or SLE. In a xenograft model of human synovium, CD8 +CD28 −CD56 + T cells effectively suppressed rheumatoid inflammation. Underlying mechanisms involve conditioning of antigen presenting cells (APC). Adoptively transferred CD8 + T cells characterized by IL-16 secretion have also exhibited disease-inhibitory effects. In mice with polyarthritis, CD8 + Ts suppressed inflammation by IFNγ–mediated modulation of the tryptophan metabolism in APC. In SLE animal models, CD8 + Ts induced by a synthetic peptide exerted suppressive activity mainly via the TGFβ-Foxp3-PD1 pathway. CD8 + Ts induced by histone peptides were found to downregulate disease activity by secreting TGFβ. In essence, disease-specific approaches may be necessary to identify CD8 + Ts optimally suited to treat immune dysfunctions in different autoimmune syndromes.

Serotonin Activates Human Monocytes and Prevents Apoptosis

Monocytes play a critical role in chronic atopic dermatitis (AD) and are the primary leukocytes that interact with activated platelets. Although activated platelets release a variety of mediators, the role of platelets in cutaneous allergic inflammation remains unclear. Serotonin (5-hydroxytryptamine, 5-HT) is one of the prototypic mediators produced by activated platelets. We examined the effect of 5-HT on the function and lifespan of human monocytes. Normal human monocytes treated with 5-HT exhibited upregulated expression of costimulatory molecules, enhanced capacity to produce cytokines following lipopolysaccharide treatment, and to stimulate allogeneic CD4+ T cells. 5-HT also attenuated the apoptosis in normal human monocytes in a dose-dependent manner. The plasma levels of 5-HT were increased in patients with AD compared with controls and correlated with the SCORAD index. 5-HT also inhibited monocyte apoptosis in these patients. 5-HT upregulated Bcl-2 and Mcl-1, and inhibited the activation of caspase-3. The effects of 5-HT on monocyte apoptosis were mediated by the 5-HT1 and/or 5-HT7 receptors. 5-HT and a 5-HT(1/6/7)-receptor agonist induced phosphorylation of extracellular signal-regulated kinase1/2 and activation of nuclear transcription factor-kappaB. These findings support that 5-HT activates monocytes and inhibits apoptosis, allowing them to remain in the tissue and contribute to chronic inflammation.

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes.

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases. This antiserum, termed PAP-1 (phospho antibody for proteomics-1), has proven useful for probing the serine phosphoproteome of antigen receptor-activated T lymphocytes. The present study shows that PAP-1 can also be used to explore serine kinases activated by cytokines and chemokines in T cells. Using PAP-1, together with proteomic analysis, the precursor form of the cytokine IL-16 (ProIL-16) was shown to be phosphorylated on Ser144 in antigen receptor-, SDF1alpha- and IL-2-activated T cells. Genetic and pharmacological-inhibitor experiments showed that the phosphorylation of ProIL-16 is dependent on activation of the kinases Erk1/2. IL-16 is secreted by mitogen-activated T cells, and the biochemical link between ProIL-16 and Erk1/2, revealed by studies with PAP-1, prompted analysis of the role of MAP kinases in this response. We show that TCR-mediated secretion of IL-16 is dependent on MAP kinases. The present study thus reveals how phosphoproteomic analysis opens previously unrecognized avenues for research, and yields novel insights about targets for MAP kinases in T lymphocytes.

Tumor necrosis factor-alpha-induced synthesis of interleukin-16 in airway epithelial cells: priming for serotonin stimulation.

Epithelial cells from individuals with asthma or from allergen-sensitized mice contain intracellular interleukin (IL)-16 protein, not present in epithelial cells from individuals without asthma or unsensitized mice. IL-16 is only present in the bronchoalveolar lavage (BAL) fluid following airway challenge with either allergen or vasoactive amine. This suggests that the initial response to allergen (sensitization) results in synthesis but not secretion of IL-16. In this study, we investigated what factors produced during the sensitization phase are responsible for epithelial cell priming for IL-16 production. We determined that ovalbumin (OVA)-sensitized mice have an increase in systemic tumor necrosis factor-alpha levels, and that serum or BAL fluid stimulation of bronchial epithelial cells results in production of IL-16 that is subsequently secreted only following serotonin stimulation. The mechanism for IL-16 production was shown to be caspase-3-dependent, and serotonin-induced secretion of IL-16 required binding of the serotonin type 2 receptor. The relevance of the priming effect associated with sensitization for IL-16 production and storage was confirmed in vivo by serotonin airway challenge of OVA-sensitized mice, resulting in rapid secretion of IL-16 into BAL fluid. As IL-16 has been shown to regulate CD4+ cell recruitment and activation, and is detected early following airway challenge of individuals with asthma, this two-step process for IL-16 production by epithelial cells may represent a rapid response mechanism in the orchestration of allergic airway inflammation.

Evidence for a role of Langerhans cell-derived IL-16 in atopic dermatitis

Background: The factors controlling infiltration of inflammatory cells into atopic dermatitis (AD) lesions remain to be fully explored. Recently, epidermal cells in lesional AD were reported to contain increased messenger (m)RNA levels of IL-16, a cytokine that induces chemotactic responses in CD4+T cells, monocytes, and eosinophils. Objectives: We sought to determine the expression of IL-16 in epidermal cells in normal skin and skin from AD lesions and to investigate whether Langerhans cell (LC)-derived IL-16 may contribute to the initiation of atopic eczema. Methods: The cutaneous expression of IL-16 was investigated by in situ hybridization and immunohistochemistry. Expression of IL-16 was also investigated in freshly isolated LCs and in keratinocytes by intracellular cytokine staining, quantitative real-time RT-PCR, and ELISA. Results: Low levels of IL-16 mRNA, but no stored IL-16 protein, were detected in keratinocytes and LCs isolated from normal skin. Synthesis, storage, and secretion of IL-16 could be induced in LCs, but not keratinocytes, by activation with phorbol ester and ionomycin. In normal skin (n = 10) neither keratinocytes nor LCs expressed IL-16. In contrast, IL-16 was contained in approximately 40% of CD1a+LCs in patients with active AD (n = 16). IL-16 expression in LCs in patients with AD correlated with the number of infiltrating CD4+cells (r = .72, P = .0017) and was completely downregulated parallel to the clinical response of AD lesions to topical treatment with FK506. Conclusion: LC-derived IL-16 may participate in the recruitment and activation of inflammatory cells in AD. (J Allergy Clin Immunol 2002;109:681-7.)

Processing, Secretion, and Anti-HIV-1 Activity of IL-16 With or Without a Signal Peptide in CD4+ T Cells

CD4+ T cells transfected with the C-terminal 130 aa of human IL-16 are rendered resistant to HIV infection. Whether the constitutively expressed IL-16 acts intracellularly, extracellularly, or both is not clear. To address this question and to further study the processing of IL-16, new constructs containing either the C-terminal 130 aa or the C-terminal 100 aa (PDZ-like motif) were constructed with and without a signal peptide. Pulse-chase experiments and treatment of cells with brefeldin A and/or tunicamycin showed that IL-16 is secreted despite the absence of a signal peptide, but with a signal peptide IL-16 is processed through the endoplasmic reticulum-golgi pathway and is glycosylated. Cells expressing IL-16 linked to a signal peptide secrete considerably more IL-16 into the supernatant than cells expressing IL-16 without a signal peptide and are considerably more resistant to HIV replication. Resistance extends to almost 25 days for cells expressing IL-16 with signal peptide as compared with only 15 days for cells without signal peptide. Cells expressing the C-terminal 100 aa not linked to a signal peptide are poor secretors of IL-16 and show little if any resistance to HIV. In contrast, cells expressing the C-terminal 100 aa linked to a signal peptide secrete IL-16 and are resistant to HIV replication. It is concluded that the secretion of IL-16 is required for HIV inhibition.