Induction Of Cytokine Response Research Articles

The aryl hydrocarbon receptor pathway is a marker of lung cell activation but does not play a central pathologic role in engineered stone-associated silicosis.

Engineered stone-associated silicosis is characterised by a rapid progression of fibrosis linked to a shorter duration of exposure. To date, there is lack of information about molecular pathways that regulates disease development and the aggressiveness of this form of silicosis. Therefore, we compared transcriptome responses to different engineered stone samples and standard silica. We then identified and further tested a stone dust specific pathway (aryl hydrocarbon receptor [AhR]) in relation to mitigation of adverse lung cell responses. Cells (epithelial cells, A549; macrophages, THP-1) were exposed to two different benchtop stone samples, standard silica and vehicle control, followed by RNA sequencing analysis. Bioinformatics analyses were conducted, and the expression of dysregulated AhR pathway genes resulting from engineered stone exposure was then correlated with cytokine responses. Finally, we inhibited AhR pathway in cells pretreated with AhR antagonist and observed how this impacted cell cytotoxicity and inflammation. Through transcriptome analysis, we identified the AhR pathway genes (CYP1A1, CYP1B1 and TIPARP) that showed differential expression that was unique to engineered stones and common between both cell types. The expression of these genes was positively correlated with interleukin-8 production in A549 and THP-1 cells. However, we only observed a mild effect of AhR pathway inhibition on engineered stone dust induced cytokine responses. Given the dual roles of AhR pathway in physiological and pathological processes, our data showed that expression of AhR target genes could be markers for assessing toxicity of engineered stones; however, AhR pathway might not play a significant pathologic role in engineered stone-associated silicosis.

Cutting Edge: ATP13A2 Is an Endolysosomal Regulator of TLR9/7 Activation in Human Plasmacytoid Dendritic Cells

Abstract ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN–producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.

Mast Cells are Dependent on Glucose Transporter 1 (GLUT1) and GLUT3 for IgE-mediated Activation.

Mast cells (MCs) are known to have a pathological impact in a variety of settings, in particular in allergic conditions. There is also limited evidence implicating MCs in diabetes, raising the possibility that MC function may be influenced by alterations in glucose levels. However, it is not known whether MCs are directly affected by elevated glucose concentrations. Moreover, it is not known which glucose transporters that are expressed by MCs, and whether MCs are dependent on glucose transporters for activation. Here we addressed these issues. We show that MCs express high levels of both glucose transporter 1 (GLUT1/Slc2A1) and GLUT3 (Slc2A3). Further, we show that the inhibition of either GLUT1 or GLUT3 dampens both MC degranulation and cytokine induction in response to IgE receptor crosslinking, and that combined GLUT1 and GLUT3 inhibition causes an even more pronounced inhibition of these parameters. In contrast, the inhibition of GLUT1 or GLUT3, or combined GLUT1 and GLUT3 inhibition, had less impact on the ability of the MCs to respond to activation via compound 48/80. Elevated glucose concentrations did not affect MC viability, and had no stimulatory effect on MC responses to either IgE receptor crosslinking or compound 48/80. Altogether, these findings reveal that MCs are strongly dependent on glucose transport via GLUT1 and/or GLUT3 for optimal responses towards IgE-mediated activation, whereas MC functionality is minimally affected by elevated glucose levels. Based on these findings, antagonists of GLUT1 and GLUT3 may be considered for therapeutic intervention in allergic conditions.

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children

Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges). We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in invitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays. We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA exvivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3+ cells over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3+ cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for TH2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of TH2 cytokines and pathogenic TH2/T follicular helper markers. Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.

Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens.

House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation. We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract. HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response. Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

Crosstalk between TLR8 and RIG-I-like receptors enhances antiviral immune responses.

Toll-like receptor (TLR) agonists have been investigated due to their potential dual effects as latency reverting agents and immune modulatory compounds in people living with HIV (PLWH). Here, we investigated whether co-stimulation of TLR7/8 agonists with RIG-I-like receptor (RLR) agonists enhances antiviral immunity. Peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (DCs) were incubated with TLR and RLR-agonists for 24 h and innate and adaptive immune responses were determined (maturation markers, cytokines in supernatant, ISG expression). Both TLR7 and TLR8 agonists induced pro-inflammatory cytokines in DCs as well as PBMCs. TLR8 agonists were more potent in inducing cytokine responses and had a stronger effect on DC-induced immunity. Notably, while all compounds induced IL-12p70, co-stimulation with TLR8 agonists and RLR agonist polyI: C induced significantly higher levels of IL-12p70 in PBMCs. Moreover, crosstalk between TLR8 and RLR agonists induced a strong type I Interferon (IFN) response as different antiviral IFN-stimulated genes were upregulated by the combination compared to the agonists alone. Our data strongly suggest that TLR crosstalk with RLRs leads to strong antiviral immunity as shown by induction of IL-12 and type I IFN responses in contrast to TLRs alone. Thus, co-stimulation of TLRs and RLRs might be a powerful strategy to induce reactivation of latent reservoir as well as antiviral immunity that eliminates the reactivated cells.

Distinct breast milk microbiota, cytokine, and adipokine profiles are associated with infant growth at 12 months: an in vitro host-microbe interaction mechanistic approach.

Breast milk (BM) is important for adequate infant development, and it contains bioactive compounds, such as bacteria, cytokines and some adipokines which play a role in infant microbial, metabolic, and immunological maturation. However, little is known about its impact on growth and development in early life. The objective of this study was to evaluate the impact of milk microbiota, cytokine, and adipokine profiles on the risk of overweight at 12 months of life to find the possible mechanisms of host-microbe interactions. In this study, BM samples from 100 healthy women collected during 15 d after birth were included. BM microbiota was analysed by 16S rRNA gene sequencing, and cytokine and adipokine levels were measured by the Luminex approach. In addition, infant weight and length were recorded during the first 12 months and z-scores were obtained according to the WHO databases. Infants were classified as risk of overweight (ROW) and no-risk of overweight (NOROW) based on their body mass index z-score (BMIZ) and infant weight-for-length z-score (WLZ) at 12 months. In order to study host-microbe interactions, epithelial intestinal and mammary cell lines were exposed to milk microbes to assess the host response by interleukin (IL)-6 production as a potential inflammatory marker. BM was dominated by Staphylococcus and Streptococcus genera, and the most abundant cytokines were IL-6 and IL-18. Leptin levels were positively correlated with the pregestational body mass index (BMI). Higher relative abundance of the Streptococcus genus was associated with higher IL-10 and higher relative abundance of the Bifidobacterium genus was associated with lower IL-6 concentrations in milk. Infant WLZ at 12 months could be partially predicted by Streptococcus genus proportions and IL-10 and IL-18 levels in BM. BM microbiota significantly induced cytokine responses in mammary epithelial cells. Higher levels of IL-6 production were observed in mammary cells exposed to BM microbiota from mothers with ROW offspring compared to mothers with NOROW offspring. In conclusion, BM microbiota is related to the cytokine profile. IL-10 and IL-18 levels and the abundance of the Streptococcus genus could affect early infant development. Further research is needed to clarify the specific impact of BM microbiota and cytokines on infant growth and the risk of overweight.

Exploration of the induced cytokine responses in European Lyme neuroborreliosis: A longitudinal cohort study

Lyme neuroborreliosis (LNB) is a prevalent tick-borne disease in Europe caused by Borrelia burgdorferi sensu lato complex. Slightly suppressed induced Th1- and Th17-responses are seen at diagnosis. The induced immune response following antibiotic therapy is unknown. We hypothesized that the immune responses normalize after completing antibiotic treatment.An observational longitudinal cohort study investigating the induced immune response in adult patients with LNB at diagnosis, three and six months after treatment. Whole blood was added to three TruCulture® (Myriad RBM, Austin, USA) tubes each containing one stimulation. An additional TruCulture® tube was without stimulation representing the in vivo activation of blood immune cells. Nine cytokines were measured using Luminex (LX200, R&D Systems, BIO-Teche LTD). Changes in immune response were analyzed with linear mixed model including follow-up as categorical fixed effect.A total of 21 patients with 55 samples were included. All had clinical improvement, but 5/21 patients reported residual symptoms after six months. The non-induced release of IL-17A and IL-1β increased significantly from diagnosis to six month follow-up. Six months after treatment only IFN-α and TNF-α were below the reference range.Minor variations in the induced immune responses were seen during the study period. Th1- and Th17-responses continued to be low with low IFN-γ, IL-12p40, and IL-17A in multiple stimulations.Overall little dynamic was observed. The changes in the cytokine responses are most likely not linked to LNB pathogenesis and our results do not support the implementation of TruCulture® in the diagnostics or follow-up of LNB.

SARS-CoV-2 Induces Cytokine Responses in Human Basophils.

Basophils play a key role in the orientation of immune responses. Though the interaction of SARS-CoV-2 with various immune cells has been relatively well studied, the response of basophils to this pandemic virus is not characterized yet. In this study, we report that SARS-CoV-2 induces cytokine responses and in particular IL-13, in both resting and IL-3 primed basophils. The response was prominent under IL-3 primed condition. However, either SARS-CoV-2 or SARS-CoV-2-infected epithelial cells did not alter the expression of surface markers associated with the activation of basophils, such as CD69, CD13 and/or degranulation marker CD107a. We also validate that human basophils are not permissive to SARS-CoV-2 replication. Though increased expression of immune checkpoint molecule PD-L1 has been reported on the basophils from COVID-19 patients, we observed that SARS-CoV-2 does not induce PD-L1 on the basophils. Our data suggest that basophil cytokine responses to SARS-CoV-2 might help in reducing the inflammation and also to promote antibody responses to the virus.

A functional genomics approach in Tanzanian population identifies distinct genetic regulators of cytokine production compared to European population

Humans exhibit remarkable interindividual and interpopulation immune response variability upon microbial challenges. Cytokines play a vital role in regulating inflammation and immune responses, but dysregulation of cytokine responses has been implicated in different disease states. Host genetic factors were previously shown to significantly impact cytokine response heterogeneity mainly in European-based studies, but it is unclear whether these findings are transferable to non-European individuals. Here, we aimed to identify genetic variants modulating cytokine responses in healthy adults of East African ancestry from Tanzania. We leveraged both cytokine and genetic data and performed genome-wide cytokine quantitative trait loci (cQTLs) mapping. The results were compared with another cohort of healthy adults of Western European ancestry via direct overlap and functional enrichment analyses. We also performed meta-analyses to identify cQTLs with congruent effect direction in both populations. In the Tanzanians, cQTL mapping identified 80 independent suggestive loci and one genome-wide significant locus (TBC1D22A) at chromosome 22; SNP rs12169244 was associated with IL-1b release after Salmonella enteritidis stimulation. Remarkably, the identified cQTLs varied significantly when compared to the European cohort, and there was a very limited percentage of overlap (1.6% to 1.9%). We further observed ancestry-specific pathways regulating induced cytokine responses, and there was significant enrichment of the interferon pathway specifically in the Tanzanians. Furthermore, contrary to the Europeans, genetic variants in the TLR10-TLR1-TLR6 locus showed no effect on cytokine response. Our data reveal both ancestry-specific effects of genetic variants and pathways on cytokine response heterogeneity, hence arguing for the importance of initiatives to include diverse populations into genomics research.

Impact of First-Line Antimicrobials on Chlamydia trachomatis-Induced Changes in Host Metabolism and Cytokine Production.

Urogenital infections with Chlamydia trachomatis (C. trachomatis) are the most common bacterial sexually transmitted diseases worldwide. As an obligate intracellular bacterium, chlamydial replication and pathogenesis depends on the host metabolic activity. First-line antimicrobials such as doxycycline (DOX) and azithromycin (AZM) have been recommended for the treatment of C. trachomatis infection. However, accumulating evidence suggests that treatment with AZM causes higher rates of treatment failure than DOX. Here, we show that an inferior efficacy of AZM compared to DOX is associated with the metabolic status of host cells. Chlamydial metabolism and infectious progeny of C. trachomatis were suppressed by therapeutic relevant serum concentrations of DOX or AZM. However, treatment with AZM could not suppress host cell metabolic pathways, such as glycolysis and mitochondrial oxidative phosphorylation, which are manipulated by C. trachomatis. The host cell metabolic activity was associated with a significant reactivation of C. trachomatis after removal of AZM treatment, but not after DOX treatment. Furthermore, AZM insufficiently attenuated interleukin (IL)-8 expression upon C. trachomatis infection and higher concentrations of AZM above therapeutic serum concentration were required for effective suppression of IL-8. Our data highlight that AZM is not as efficient as DOX to revert host metabolism in C. trachomatis infection. Furthermore, insufficient treatment with AZM failed to inhibit chlamydial reactivation as well as C. trachomatis induced cytokine responses. Its functional relevance and the impact on disease progression have to be further elucidated in vivo.

Ex Vivo Evaluation of the Sepsis Triple Therapy High-Dose Vitamin C in Combination with Vitamin B1 and Hydrocortisone in a Human Peripheral Blood Mononuclear Cells (PBMCs) Model.

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.

Application of the TruCulture® whole blood stimulation system for immune response profiling in cattle

Capturing the phenotypic variation in immune responses holds enormous promise for the development of targeted treatments for disease as well as tailored vaccination schedules. However, accurate detection of true biological variation can be obscured by the lack of standardised immune assays. The TruCulture® whole blood stimulation system has now been extensively used to detect basal and induced immune responses to a range of pathogen-associated molecular patterns (PAMPs) in human peripheral blood. This study demonstrates the optimisation of this commercially available assay for systemic immune phenotyping in cattle. The early immune response in Holstein-Friesian bull calves (n = 10) was assessed by haematology, flow cytometry and cytokine expression profiling after 24 h ex-vivo PAMP (LPS, poly (I:C) and zymosan) stimulation in TruCulture® tubes. A comparative analysis was also performed with a traditional whole blood stimulation assay and cell viability using both systems was also evaluated. Results: Supernatant collected from TruCulture® tubes showed a significant increase in IL-1β and IL-8 expression compared to null stimulated tubes in response to both LPS and zymosan. In contrast, a detectable immune response was not apparent at the standard concentration of poly (I:C). Conventional whole blood cultures yielded similar response profiles, although the magnitude of the response was higher to both LPS and zymosan, which may be attributed to prokaryotic strain-specificity or batch of the stimulant used. Despite being a closed system, HIF1A expression – used as a measure of hypoxia was not increased, suggesting the TruCulture® assay did not negatively affect cell viability. This represents the first reported use of this novel standardised assay in cattle, and indicates that the concentration of poly (I:C) immunogenic in humans is insufficient to induce cytokine responses in cattle. We conclude that the low blood volume and minimally invasive TruCulture® assay system offers a practical and informative technique to assess basal and induced systemic immune responses in cattle.

Abstract 4983: Discovery and characterization of next-generation small molecule direct STING agonists

Abstract Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment. Citation Format: Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. Discovery and characterization of next-generation small molecule direct STING agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4983.

Discovery and Mechanistic Study of a Novel Human-Stimulator-of-Interferon-Genes Agonist.

Stimulator of interferon genes (STING) is an integral ER-membrane protein that can be activated by 2'3'-cGAMP synthesized by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) upon binding of double-stranded DNA. It activates interferon (IFN) and inflammatory cytokine responses to defend against infection by microorganisms. Pharmacologic activation of STING has been demonstrated to induce an antiviral state and boost antitumor immunity. We previously reported a cell-based high-throughput-screening assay that allowed for identification of small-molecule cGAS-STING-pathway agonists. We report herein a compound, 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC), that induces a proinflammatory cytokine response in a human-STING-dependent manner. Specifically, we showed that BNBC induced type I and III IFN dominant cytokine responses in primary human fibroblasts and peripheral-blood mononuclear cells (PBMCs). BNBC also induced cytokine response in PBMC-derived myeloid dendritic cells and promoted their maturation, suggesting that STING-agonist treatment could potentially regulate the activation of CD4+ and CD8+ T lymphocytes. As anticipated, treatment of primary human fibroblast cells with BNBC induced an antiviral state that inhibited the infection of several kinds of flaviviruses. Taken together, our results indicate that BNBC is a human-STING agonist that not only induces innate antiviral immunity against a broad spectrum of viruses but may also stimulate the activation of adaptive immune responses, which is important for the treatment of chronic viral infections and tumors.

Tricarboxylic Acid Cycle Activity and Remodeling of Glycerophosphocholine Lipids Support Cytokine Induction in Response to Fungal Patterns

Increased glycolysis parallels immune cell activation, but the role of pyruvate remains largely unexplored. We found that stimulation of dendritic cells with the fungal surrogate zymosan causes decreases of pyruvate, citrate, itaconate, and α-ketoglutarate, while increasing oxaloacetate, succinate, lactate, oxygen consumption, and pyruvate dehydrogenase activity. Expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on pyruvate dehydrogenase activity. Mechanistically, pyruvate reinforced histone H3 acetylation, and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because it is a substrate of the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator platelet-activating factor (PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) showed reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and its ensuing autocrine function. Acetyl-CoA therefore intertwines fatty acid remodeling of glycerophospholipids and energetic metabolism during cytokine induction.

No association between local levels of molecular biomarkers and knee cartilage volumes early after anterior cruciate ligament injury

Purpose: Trauma induced cytokine response in knee injuries may play a role in the development of osteoarthritis. We hypothesize that the cartilage volume affects the release of cartilage proteins and/or the presence of cytokines in the acute stage after knee injuries. Primary aim of our study was to compare knee cartilage volumes, derived from magnetic resonance images (MRI), with concentrations of biomarkers in biofluids at baseline (early after injury) in patients with anterior cruciate ligament (ACL) injuries. Secondary aim was to examine the correlation of confounders on biomarker levels or cartilage volumes. Methods: The KANON cohort consists of patients with an acute ACL tear to a previously uninjured knee (n = 121; age 26 years at injury, 74% males). In biofluids, collected at a median of 17 days (range 1-44 days) after the ACL injury, biomarkers were assessed using immunoassays: cartilage biomarkers aggrecan ARGS neoepitope (ARGS; in-house assay), cartilage oligomeric matrix protein (COMP; BioVendor) and cross-linked C-telopeptide of type II collagen (CTX-II; IDS); bone biomarker cross-linked N-telopeptide of type I collagen (NTX-I; Osteomark); cytokines (multiplex assay, MSD) interleukin (IL) 6, 8, 10, tumor necrosis factor α (TNF) and interferon gamma (INF-γ). Cartilage volumes were obtained from sagittal 3D SPGR MRI (1.5 T Philips Gyroscan; resolution: 0.29x0.29x3.0mm) based on quality-controlled, manual segmentations in three different cartilage compartments: tibiofemoral joint (TFJ), patellofemoral joint (PFJ) and total knee joint (TFJ + PFJ). MR images were acquired at the same day as the serum and urine sampling, but on average 11 days (range 1-24 days) after collection of synovial fluid. In linear regression models, we estimated cartilage volume as explanatory for molecular biomarker concentrations adjusted for age and time between injury and sampling. Models including both men and women were additionally adjusted for sex. Since both sex and BMI were associated with cartilage volume (see below) and BMI associated with sex (data not shown), we decided to adjust only for sex. 46 synovial fluid, 117 serum and 115 urine samples from individuals with corresponding MRI assessed cartilage volumes were used in linear regression analyses. Biomarkers with values below lower limit of quantification (LLOQ) were imputed with LLOQ divided by 2, (N% with levels above LLOQ): i.e. synovial fluid INF-γ (87%), serum IL-10 (46%) and urine CTX-II (99%). Unadjusted correlation (Pearson), Mann-Whitney U-test and student’s t-test were used for estimation of confounders. P values less than 0.05 were considered significant. Results: None of the synovial fluid biomarkers associated with cartilage volumes: Total knee joint (Table 1), TFJ or PFJ (data not shown); additional adjustment for differences in time between MRI assessment and synovial fluid sampling gave similar results (data not shown). In analyses stratified by sex, serum ARGS and urine CTX-II associated positively with total knee joint and TFJ cartilage volume in men, while in women, serum TNF associated negatively with total knee joint cartilage volume; none of the other biomarkers showed any association to any of the cartilage compartment volumes. Effects of confounders (Table 2): age associated negatively with serum ARGS, urine CTX-II and urine NTX-I concentrations, and positively with serum COMP levels; BMI associated positively with cartilage volumes, serum COMP levels and negatively with urine CTX-II; time between injury and sampling associated negatively with synovial fluid cytokine concentrations and positively with serum ARGS; compared to women, men had 1.3-fold higher serum COMP levels (Mann-Whitney: p = 0.002; men n = 89, women n = 32) and 1.3- and 1.4-fold more cartilage volume (t-test: p < 0.001, men n = 89, women n = 32). No other associations were found between confounders and molecular biomarker levels or cartilage volumes (Table 2). Conclusions: We found no evidence in support of local cytokines or cartilage biomarkers in the synovial fluid to be cross-sectionally associated with cartilage volume in the early phase after ACL injury. An association between cartilage volume and serum ARGS-aggrecan or urine CTX-II concentrations was only found in men. Several confounders affect biomarker levels and the cartilage volume creating a complex pattern of causalities that must be considered in analysis models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

TP1-4 In vitro induced cytokine response of astrocytes modelling conditions in human traumatic brain injury

ObjectivesThe objective of this study was to investigate how in vitro astrocyte cultures respond to cytokine pro- and anti-inflammatory cytokine concentrations, corresponding to those seen in the aftermath of human TBI, by analysing downstream cytokine generation.DesignIn vitro study.SubjectsHuman induced pluripotent stem cells (iPSC)-derived astrocytes.MethodsThe astrocytes were exposed to levels of TNF (1–10,000 pg/ml), IL-6 (100–1,000,000 pg/ml), Interleukin-1β (IL-1β, 1–10,000 pg/ml), Interleukin-4 (IL-4, 1–10,000 pg/ml) and Interleukin-10 (IL-10, 1–10,000 pg/ml). Following 24, 48 and 72 hours, culture supernatant was extracted and analysed using a human cytokine/chemokine 39-plex luminex assay (ThermoFisher).ResultsThe astrocyte secretome revealed concentration-, time- or concentration*time-dependent production of downstream cytokines (12, 8 and 2 cytokines, respectively p&lt;0.05). IL-1β and TNF exposure generated the most downstream cytokine production, while IL-6, IL-4 and IL-10 did not generally induce a robust response.ConclusionsiPSC-derived astrocytes exposed to cytokine concentrations reflecting those in TBI generate an increased downstream cytokine production, especially when exposed to IL-1β and TNF. This is in contrast to our previous work on neuronal cultures where IL-1β only produced a few down-stream cytokines.1 More work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI alone and in combination.

Francisella induced microparticulate caspase-1/gasdermin-D activation is regulated by NLRP3 independent of Pyrin.

Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin.