Differences In Cytokine Production Research Articles

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Cytokine measurement using a multiplex technique with spectral flow cytometry: application in the study of the immunobiology of ascariasis

To quantify the production of Th1/Th2/Th17 cytokines induced by Ascaris lumbricoides antigens in peripheral blood mononuclear cells (PBMCs) using a multiplex technique. PBMCs were cultured from individuals with mild A. lumbricoides infection (n = 20) and uninfected individuals (n = 21) and stimulated with A. lumbricoides extract (ExtAscaris), a mix of anti-CD2/CD3/CD28 (CDmix) as a positive control, and only medium (negative control). Cytokines in the supernatants were measured using the BD™ Cytometric Bead Array Human Th1/Th2/Th17 kit, to identify IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2, and IL-17A. Readings were taken on a spectral cytometer (Northern Lights, Cytek, USA), and analysis was performed using R software with packages "tidyverse," "beadplexr," "flowCore," and "arsenal." Cytokine concentrations were calculated using a 5-parameter logistic curve. The t-test was used to compare cases and controls, and statistical significance was set at p < 0.05. The study was approved by the Ethics Committee of the University of Cartagena and the participants provided informed consent. This study was financially supported by the Colombian Sistema General de Regalías under the BPIN2020000100405 - BPIN2020000100364. Efficient fluorescence intensity extraction for each cytokine was achieved using detection channel R8 and the "mclust" clustering model (Figure 1). No significant differences were found in the levels of the seven cytokines between cases and controls (Figure 2). Although the IFN-γ response to ExtAscaris was higher in cases than in controls (252.5 ng/mL vs. 173.1 ng/mL), the difference was not significant. IL-17A (detection limit: 18.9 pg/mL) was more detectable in cases than controls (5 cases, 23% vs. 2 controls, 9.5%). IL-4 was only detected in the supernatants from CDmix-stimulated cultures but not with the Ascaris extract (Figure 2). The multiplex technique using spectral flow cytometry combined with open-source software analysis proved applicable for quantifying cytokines induced by A. lumbricoides antigens in PBMCs. However, a more sensitive method is needed to evaluate IL-4 response in the context of ascariasis. The results did not reveal significant differences in cytokine production between cases and controls for the evaluated stimuli.

Identifying Ferroptosis-Related Genes Associated with Weight Loss Outcomes and Regulation of Adipocyte Microenvironment.

The study is about the influence of ferroptosis-related genes combined with the immune microenvironment exerted on weight control outcomes and systematic analysis. Subcutaneous adipose tissue (sWAT) samples from 11 subjects with good outcome and 10 subjects with poor outcome in weight management are obtained from the Gene Expression Omnibus database. The results are validated in vivo in animal models with different weight loss outcomes. The CIBERSORT algorithm is used to evaluate the differences in immune cell infiltration in each sample. Patients with poor outcome have higher levels of ferroptosis in the adipose tissue. Remarkable differences in cytokine production, nuclear factor kappa-B(NF-κB) transcription factor activity, leukocyte migration involved in the inflammatory response, and other biological processes are also observed compared to that in the well-controlled group. Aldo-keto reductase family 1-member C1(AKR1C1), nuclear receptor coactivator 4(NCOA4), and glutamate-cysteine ligase catalytic subunit(GCLC) are identified as core predictive markers and their expression patterns are confirmed in animal models. Ferroptosis and its mediated inflammation play an important role in long-term weight control, and analyses of the role of ferroptosis-related genes(FRGs) in weight control may provide new potential therapeutic targets for long-term weight control. Anti-inflammatory diets that mitigate inflammatory responses and affect ferroptosis may be considered in the future to improve weight control.

Calming the storm: Immunoregulatory receptor NLRX1 and its role in the immune response to SARS-CoV-2

Abstract One of the most characteristic clinical disease presentations of severe COVID-19 is the ‘Cytokine Storm,’ stemming from dysregulation of the immune response. This overproduction of proinflammatory cytokines in response to SARS-CoV-2 is responsible for Acute Respiratory Distress Syndrome (ARDS) in adult patients and the Multisystem Inflammatory Syndrome (MIS) of pediatric patients hospitalized with severe COVID-19. Pattern recognition receptor NLRX1 is a member of the NOD-like receptor family with complex involvement in antiviral immunity. It has been reported in the literature in many contexts to be anti-inflammatory by inhibiting NF-kB and interferon signaling-critical pathways in anti-SARS-CoV-2 immunity. Here we show that loss of NLRX1 impacts immune responses to SARS-CoV-2 both in cell culture and in mice. While Nlrx1 −/−mice showed similar overall morbidity, there were temporary increases in weight loss at multiple time points post infection. SARS-CoV-2 infection of Nlrx1 −/−mice also resulted in an increase in viral titer in the lungs at 2 dpi, despite having higher degrees of airway inflammation. Data in both cell culture and animal challenges report differences in immune activation, showing differences in cytokine production with changes in expression of NLRX1. Together, these experiments uncover another enigmatic function of NLRX1 while simultaneously offering pathways related to NLRX1 as possible therapeutic targets to help mitigate the effects of the ‘cytokine storm’ of severe COVID-19. Finally, these data also add to the growing field regarding SARS-CoV-2 and its pathobiology in an understudied context of immune regulation and continue to explore NLRX1 and its many curious roles in antiviral immunity.

Differential Recognition of Clinically Relevant Sporothrix Species by Human Mononuclear Cells.

Sporotrichosis is a human and animal fungal infection distributed worldwide that is caused by the thermodimorphic species of the Sporothrix pathogenic clade, which includes Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa. The cell wall composition and the immune response against the Sporothrix species have been studied mainly in S. brasiliensis and S. schenckii, whilst little is known about the S. globosa cell wall and the immune response that its components trigger. Therefore, in this study, we aimed to analyze the cell wall composition of S. globosa in three morphologies (germlings, conidia, and yeast-like cells) and the differences in cytokine production when human peripheral blood mononuclear cells (PBMCs) interact with these morphotypes, using S. schenckii and S. brasiliensis as a comparison. We found that S. globosa conidia and yeast-like cells have a higher cell wall chitin content, while all three morphologies have a higher β-1,3-glucan content, which was found most exposed at the cell surface when compared to S. schenckii and S. brasiliensis. In addition, S. globosa has lower levels of mannose- and rhamnose-based glycoconjugates, as well as of N- and O-linked glycans, indicating that this fungal cell wall has species-specific proportions and organization of its components. When interacting with PBMCs, S. brasiliensis and S. globosa showed a similar cytokine stimulation profile, but with a higher stimulation of IL-10 by S. globosa. Additionally, when the inner cell wall components of S. globosa were exposed at the surface or N- and O-glycans were removed, the cytokine production profile of this species in its three morphotypes did not significantly change, contrasting with the S. schenckii and S. brasiliensis species that showed different cytokine profiles depending on the treatment applied to the walls. In addition, it was found that the anti-inflammatory response stimulated by S. globosa was dependent on the activation of dectin-1, mannose receptor, and TLR2, but not TLR4. All of these results indicate that the cell wall composition and structure of the three Sporothrix species in the three morphologies are different, affecting their interaction with human PBMCs and generating species-specific cytokine profiles.

CT-167 Using the Sodium Iodide Symporter to Visualize CAR-T Cell Trafficking

Chimeric antigen receptor T (CAR-T) cell therapy revolutionized the treatment of refractory B cell malignancies, but relapses occur through a variety of incompletely understood mechanisms. Currently, tracking CAR-T expansion and persistence in patients is limited to peripheral blood sampling, but this approach provides no insights into how and where CAR-T cells traffic beyond the peripheral blood or how cellular localization may impact persistence and efficacy. Furthermore, costimulatory endodomains that are known to influence CAR-T cell phenotype have unclear downstream effects on how CAR-T cells traffic. The sodium iodide symporter (NIS) is a plasma membrane glycoprotein that actively uptakes iodide and its analogs, and it can be transduced into various cell types in preclinical models allowing sensitive and specific PET/SPECT imaging. Coexpression of NIS with CAR on T cells would allow real-time, in vivo imaging of CAR-T cells and aid in understanding CAR-T cell biology and trafficking. Using Golden Gate modular vector construction, multiple anti-CD20 CAR constructs with specificity for both human and non-human primate (NHP) CD20 antigens were generated. Each construct included one of several costimulatory domains (4-1BB, CD28, or CD27), a standard CD3-zeta signaling domain, and either the rhesus-NIS reporter or truncated EGFR following a short ribosomal skip sequence peptide, allowing transduction under control of the same EF1a promoter. CAR was effectively cotransduced with NIS in both human and NHP primary T cells with no effect of NIS on CAR-T cell-mediated cytotoxicity against CD20-expressing Raji cells in vitro compared to control. Cytotoxicity was antigen-specific as no difference was seen against CD20- control cells. Functional iodide and [18F]-TFB, an iodide analog, uptake via NIS was specific to NIS-transduced cells and was appropriately inhibited by coincubation with KClO4. An NSG mouse xenograft model will be used to track the localization, expansion, efficacy, and persistence of CAR-T constructs against Raji lymphoma cells. Different costimulatory domains appear to elicit only slight differences in cytokine production in vitro but will be further studied for differences in efficacy and trafficking in vivo in mice and later in a syngeneic NHP model.

Longitudinal T Cell Responses against Ancestral, Delta, and Omicron SARS-CoV-2 Variants Determined by Rapid Cytokine Release Assay in Whole Blood.

T cell immunity to natural SARS-CoV-2 infection may be more robust and longer lived than Ab responses. Accurate assessment of T cell responses is critical for understanding the magnitude and longevity of immunity across patient cohorts, and against emerging variants. By establishing a simple, accurate, and rapid whole blood test, natural and vaccine-induced SARS-CoV-2 immunity was determined. Cytokine release in whole blood stimulated with peptides specific for SARS-CoV-2 was measured in donors with previous PCR-confirmed infection, suspected infection, or with no exposure history (n = 128), as well as in donors before and after vaccination (n = 32). Longitudinal assessment of T cell responses following initial vaccination and booster vaccination was also conducted (n = 50 and n = 62, respectively). Cytokines were measured by ELISA and multiplex array. IL-2 and IFN-γ were highly elevated in PCR-confirmed donors compared with history-negative controls, with median levels ∼33-fold and ∼48-fold higher, respectively. Receiver operating curves showed IL-2 as the superior biomarker (area under the curve = 0.9950). Following vaccination, all donors demonstrated a positive IL-2 response. Median IL-2 levels increased ∼32-fold from prevaccination to postvaccination in uninfected individuals. Longitudinal assessment revealed that T cell responses were stable up to 6 mo postvaccination. No significant differences in cytokine production were observed between stimulations with Wuhan, Delta, or Omicron peptides. This rapid, whole blood-based test can be used to make comparable longitudinal assessments of vaccine-induced T cell immunity across multiple cohorts and against variants of concern, thus aiding decisions on public health policies.

T cells dominate peripheral inflammation in obesity-associated diabetes

Abstract Many studies have examined obese/insulin-resistant mice to conclude that macrophages dominate pathogenic inflammation in metabolic disease, but few studies have ranked chronic sources of peripheral inflammation in obese people across a natural spectrum of metabolic health. We compared the relative contributions of myeloid and T cells to peripheral inflammation in samples from metabolically healthy and unhealthy obese people to address disappointing outcomes from clinical trials of anti-inflammatories in obesity-associated type 2 diabetes (T2D). We stimulated PBMCs from obese subjects with euglycemia, prediabetes, or T2D with T cell-targeting CD3/CD28 or myeloid-targeting LPS, and measured supernatant cytokines over time. Cytokines increased with time following CD3/CD28, but LPS responses were time-independent. A series of surface marker and intracellular staining assays taking into account these temporal differences in cytokine production unexpectedly showed that T cells contribute more TNFα to peripheral inflammation than do myeloid cells from all subject groups. Bioinformatic modeling combining cytokine outcomes from all subjects, stimuli, and time points, indicated IFNγ generated by CD3/CD28 stimulation was most important for differentiating peripheral inflammation in T2D compared to non-T2D samples. This result was consistent with the previously identified Th1/Th17 profile in T2D. We conclude that human T cells dominate peripheral inflammation in obesity-associated T2D, and thus targeting T cells may be an effective approach for prevention/management of obesity-associated metabolic diseases. Supported by NIH Training Grants T32 DK007778 and TL1 UL1TR001998, R01DK108056, the Shared Resource Facility of the University of Kentucky Markey Cancer Center P30 CA177558, University of Kentucky College of Medicine, Barnstable Brown Diabetes and Obesity Center, The Center for Clinical and Translational Research (UL1TR000117), and NIH National Center for Advancing Translational Sciences UL1TR001998.

Phenotypic Changes in Mammary Adenocarcinoma (4T1) cells In Vitro after Treatment with Carcinosinum.

The present study aimed to identify possible phenotypic changes in 4T1 (murine mammary adenocarcinoma) cells in vitro, including viability, HER-2 (human epidermal growth factor receptor-type 2) expression, and metastatic potential, after treatment with Carcinosinum in different homeopathic dilutions (12cH, 30cH, 200cH) shaken mechanically in pure, sterile, water from a commercial stock dilution. Treated cells were cultured in R10 medium, using 24-well plates, 105 cells per well, and treated with vehicle, Carcinosinum 12cH, 30cH or 200cH; untreated cells were used as the baseline control. After 24 hours of treatment, the percentage of apoptotic cells was analyzed by annexin V. Cell morphology was evaluated by microscopy after hematoxylin-eosin and Giemsa staining, whilst HER-2 expression was assessed using immunocytochemistry. The metastatic potential was determined by the expression and activity of the enzyme matrix metalloproteinase 9 (MMP-9) using zymography. The cytokine profile was established using the cytometric bead array method. Treatment of 4T1 cells in vitro with Carcinosinum 30cH produced an increase in the number of annexin V-positive cells (apoptosis) and decreased expression of proactivated MMP-9. Cells treated with Carcinosinum 200cH presented hyper-expression of HER-2 on the plasma membrane, identified by immunocytochemistry. There were no differences in cytokine production among treatments. The data show promising results for Carcinosinum 30cH in vitro, but in vivo studies are also required to evaluate the role of tumor microenvironment in its effects.

Antifungal Activity and Biocompatibility of α-AgVO3, α-Ag2WO4, and β-Ag2MoO4 Using a Three-Dimensional Coculture Model of the Oral Mucosa.

Fungal infections have become a major concern in the medical community, especially those caused by Candida spp. Within this species, Candida albicans stands out for being an opportunistic commensal fungus that can cause superficial and invasive infections. Current antifungal therapy involves the local and/or systemic use of drugs such as azoles, polyenes, and echinocandins. These antifungals are based on highly specific target sites, and the development of resistance may occur with changes in the enzymatic pathways that serve as the drug targets. Thus, the development of new antifungal drugs is highly recommended to prevent drug resistance. The present investigation evaluated the antifungal activity of silver-containing microcrystals such as silver vanadate (α-AgVO3), silver tungstate (α-Ag2WO4), and silver molybdate (β-Ag2MoO4). In addition to having antimicrobial activity, such compounds should not cause damage to underlying tissues. Thus, to better assess the biocompatibility of new compounds, a new three-dimensional (3D) coculture model involving three cell lines was developed. The validation of the model was based on fluorescent markers and confocal laser microscopy. The biocompatibility of silver-containing microcrystals was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3D coculture was infected with C. albicans biofilm and challenged with α-AgVO3, α-Ag2WO4, and β-Ag2MoO4. The action of microcrystals on C. albicans biofilm was evaluated by colony-forming units (CFU/ml) and LIVE/DEAD staining. In addition, production of proinflammatory cytokines interleukin 6 (IL-6), IL-8, IL-1β, and tumor necrosis factor α (TNF-α) was measured by cytometric bead array kit using flow cytometry. The 3D coculture model described here proved to be adequate to assess both the biocompatibility of the new materials and the infectious processes. Regarding the biocompatibility of the microcrystals, only α-AgVO3 (15.62 µg/ml) showed a decrease in cell viability. The antibiofilm activity of α-Ag2WO4 was similar to that of the standard drug (fluconazole). Although α-Ag2WO4 was able to induce the production of IL-6, IL-8, and IL-1β, no differences in cytokine production were observed between noninfected and infected models treated with this microcrystal. β-Ag2MoO4 inhibits the production of TNF-α in the infected model; however, it showed no antibiofilm activity. Based on the biocompatibility and antifungal findings, α-Ag2WO4 is a promising material for treating C. albicans infection.

Egg-Phosphatidylcholine Attenuates T-Cell Dysfunction in High-Fat Diet Fed Male Wistar Rats.

Obesity is associated with immune dysfunction including an impaired T-cell function characterized by a lower IL-2 (proliferation marker) production after stimulation. Phosphatidylcholine (PC), a form of choline mostly found in eggs, has been shown to beneficially modulate T-cell responses during the lactation period by increasing the production of IL-2. To determine the impact of egg-PC as part of a high-fat diet on immune function we randomly fed male Wistar rats one of three diets containing the same amount of total choline but differing in the form of choline: 1—Control low fat [CLF, 10% wt/wt fat, 100% free choline (FC)]; 2— Control high-fat (CHF, 25% wt/wt fat, 100% FC); 3— PC high-fat (PCHF, 25% wt/wt, 100% PC). After 9 weeks of feeding, rats were euthanized. Cell phenotypes and ex vivo cytokine production by splenocytes stimulated with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I), lipopolysaccharide (LPS) and pokeweed (PWM) were measured by flow cytometry and ELISA, respectively. Rats fed the PCHF diet had a lower proportion of CD3+ cells when compared to both the CLF and the CHF. Following PMA+I stimulation, splenocytes from the CHF group produced less IL-2 and TNF-α compared to CLF and PCHF groups. No significant differences in cytokine production were found among groups after LPS and PWM stimulation. Our results show that feeding a high-fat diet impairs T-cell responses, as measured by ex vivo cytokine production, which can be attenuated by providing egg-PC.

Metabolic regulation of macrophage proliferation and function in atherosclerosis.

Macrophage accumulation within atherosclerotic plaque is a primary driver of disease progression. However, recent advances in both phenotypic and functional heterogeneity of these cells have allowed for improved insight into potential regulation of macrophage function within lesions. In this review, we will discuss recent insights on macrophage heterogeneity, lipid processing, metabolism, and proliferation in atherosclerosis. Furthermore, we will identify outstanding questions in the field that are pertinent to future studies. With the recent development of single-cell RNA sequencing, several studies have highlighted the diverse macrophage populations within plaques, including pro-inflammatory, anti-inflammatory, lipid loaded and tissue resident macrophages. Furthermore, new data has suggested that differential activation of metabolic pathways, including glycolysis and fatty acid oxidation, may play a key role in determining function. Recent works have highlighted that different populations retain varying capacity to undergo proliferation; regulating the proliferation pathway may be highly effective in reducing plaque in advanced lesions. Macrophage populations within atherosclerosis are highly heterogeneous; differences in cytokine production, lipid handling, metabolism, and proliferation are seen between subpopulations. Understanding the basic cellular mechanisms that drive this heterogeneity will allow for the development of highly specific disease modulating agents to combat atherosclerosis.

Comparison of T Cell Subsets and Cytokines between Steady-State and G-CSF-Primed Bone Marrow and Its Association with Graft-Versus-Host-Disease

BackgroundThe widespread use and success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be affected by graft-versus-host-disease (GVHD), an immunological syndrome that involves tissue damage mediated by donor lymphocytes. Both, acute and chronic GVHD, result from an imbalance between the effector and regulatory arms of the immune system. On the other hand, worldwide, the main source of stem cells is peripheral blood (PBSC). However, its use has been associated with higher incidence of GVHD when compared with bone marrow. Although the fast engraftment using PBSC as a source of stem cells compares to that seen with G-CSF-primed bone marrow (G-BM), the latter, also associates with a decreased incidence of GVHD. At our institution, patients transplanted with G-BM, have a lower incidence of GVHD when compared to those transplanted with PBSC and steady state bone marrow (SS-BM). Some microenvironment characteristics of these sources of hematopoietic stem cells (HSCS) remain unknown, including differences between Tregs, Th17 cells, and cytokines, and its association with GVHD.AimThe aim of this study was to analyze lymphocyte subsets and associated cytokines in donor G-BM and SS-BM, comparing patients who developed GVHD and those who did not.Material and MethodsA prospective analysis was performed in 48 donors SS-BM and G-BM collected from March 2002 to April 2017. Twenty two samples collected since March 2002 were thawed; since 2014, samples (20 mL) were immediately processed and analyzed. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay).From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN -γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Comparison between variables was made by means of Student's t test using SPSS v.21.ResultsWe found increased Tregs (1.46% ± 0.69 vs 1.01% ± 0.49, p=0.01) and Th2 (1.55% ± 0.36 vs 1.28% ± 0.39, p=0.02) subsets in G-BM compared to SS-BM. Nonetheless, although differences in cytokine production were observed, we did not find any statistical significance. Acute GVHD was diagnosed in 9% (n=3) and 20% (n=3) of patients receiving G-BM and SS-BM, respectively. Chronic GVHD was seen in 33% of patients in both groups (receiving SS-BM or G-BM). Thirty one patients did not present either GVHD. In all the cohort, indistinctively from the HSCs used, acute GVHD was characterized by a predominant pro-inflammatory profile, with increased TNFα (59.28 ± 56.6 vs 14.21 ± 36.2 pg/mL, p=0.011), and a higher percentage of Th1 (22.55% ± 4.8 vs 20.71% ± 4.8, p=0.04) and Th17 (0.87% ± 0.33 vs 0.57% ± 0.24, p=0.009) cells. Furthermore, a deficient suppressor response, with decreased IL-10 production (1.17 ± 1.26 vs 33.55 ± 49.3 pg/mL, p<0.0001) as well as lower Tregs percentage (0.76% ± 0.12 vs 1.40% ± 0.68, p<0.0001) was found. Likewise, chronic GVHD showed the same profile, but with more striking differences in pro-inflammatory cytokine production. The T cell subset profile in chronic GVHD also showed an imbalance between pro-inflammatory and suppressor subsets percentage (figure 1 ).ConclusionsThis is the first study exploring the differences in T cells and cytokine profile between G-BM and SS-BM. Our results showed that G-BM has an enhanced suppressive phenotype compared to SS-BM. Also, patients who developed GVHD were characterized by an imbalance in pro-inflammatory and suppressor responses, with enhanced Th17 cells, as well as increased TNF-α, IL-6 and IL-17A production in contrast to a dampened Tregs frequency and IL-10 production which can explain the low frequency of GVHD in our transplanted patients using G-BM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Circulating B Cells, Plasma Cells, and Treg Associate with ANCA Levels in ANCA-associated Vasculitis

Circulating B Cells, Plasma Cells, and Treg Associate with ANCA Levels in ANCA-associated Vasculitis

Differences in Cytokine Gene Expression after a Stimulation with Escherichia Coli and Porphyromonas Gingivalis or Lipopolysaccharides Derived from these Bacteria

Monocytes are important cells in innate immunity. The early stage of the innate immunity is regulated by various cytokines produced by monocytes. We conducted a preliminary study to investigate TNFα expression by stimulating THP-1 cells with several bacterial species. The TNFα mRNA levels significantly varied, with the most potent stimulatory effects observed with P. gingivalis. In the present study, we focused on P. gingivalis and compared differences in cytokine expression profiles after the stimulation of THP-1 with E. coli. Bacterial antigen stimulation increased various cytokine gene expressions in THP-1. P. gingivalis had significantly more potent effects on the mRNA expressions of TNFα, IL-1β, and IL-10, but not of IL-12p40, than E. coli. This result suggests the potent ability of P. gingivalis to induce inflammation. THP-1 stimulated with LPS derived from both bacterial species showed that E. coli had significantly more potent effects on the expressions of TNFα, IL-1β, and IL-12p40 than P. gingivalis. The differences in the bacterial antigens and the LPS stimulation effects suggest involvements of different receptors, such as TLR-2 and -4, which recognize bacterial components. The present results suggest that the P. gingivalis somatic cell antigen stimulates a number of pattern recognition receptors at the same time as the synthesis of bacterial components, except LPS. The potent virulence of P. gingivalis and persistence of infection might be affected by differences in cytokine production. Pro-inflammatory responses are dependent not only on the bacterial type, but also bacterial components.

An Exaggerated Monocyte-Derived Cytokine Response to Candida Hyphae in Patients With Recurrent Vulvovaginal Candidiasis.

BackgroundRecurrent vulvovaginal candidiasis (RVVC) affects up to 8% of women. The immunopathogenesis is poorly understood but it has been suggested that RVVC might be due to dysregulated innate immune response. The aim of this study was to compare cytokine profiles in stimulated primary mononuclear cells (PBMCs) from RVVC and healthy individuals.MethodsPBMCs isolated from RVVC patients (n = 24) and healthy volunteers (n = 30) were stimulated with unspecific and pathogen-specific antigens. Cytokine production was assessed after 24 hours, 48 hours, and 7 days using ELISA.ResultsNo significant differences in cytokine production were found in T helper 1 (Th1), Th2, and Th17 immunity in response to both unspecific and pathogen-specific stimulations. Tumor necrosis factor-α (TNF-α) production in response to C. albicans hyphae was significantly higher in patients than controls and within the patient group, a significant positive correlation was found between interleukin-1β (IL-1β) and both TNF-α and IL-6. Both IL-1β/IL-1Ra and TNF-α/IL-10 ratios in Candida hyphae-stimulated PBMCs were significantly higher in patients than controls.ConclusionsWomen affected by RVVC showed increased monocytes-derived cytokine production, which might contribute to an exaggerated vaginal immune response to Candida hyphae. RVVC patients show no defective Th-dependent adaptive immune response upon Candida stimulation.

Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection

Abstract Alveolar macrophages (AMs) are the first cells encountered by TB pathogen and serve as the primary defense against Mycobacterium tuberculosis (Mtb) in the lungs. Studies have shown that liver macrophages (Kupffer cells; KCs) can control Mtb growth but, it is not clear how KCs completely eliminate mycobacterial infections. Thus, identification of these mechanisms will facilitate the development of immunomodulatory strategies to boost AM-mediated immunity to Mtb. In the current study, we compared Mtb growth in murine AMs, peritoneal (PMs), liver macrophages (Kupffer cells; KCs) and bone marrow-derived monocytes (BDMs). KCs restricted Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections. Differences in Mtb growth restriction were not due to differences in cytokine production, expression of Toll-like receptors 2 and 4, M1/M2 paradigm or apoptosis. We provide evidence that the enhanced autophagy efficiently restricts Mtb growth in KCs using flow cytometry, western blot, RT-PCR and confocal microscopy. A metabolomic comparison of Mtb-infected macrophages by liquid chromatography mass spectrometry indicated that ornithine (VIP=1.8) and imidazole (VIP=1.6) were two top-scoring metabolites found in Mtb-infected KCs and that acetylcholine was top-scoring in Mtb-infected AMs. Ornithine and imidazole inhibited Mtb growth in AMs by enhancing AMPK mediated autophagy whereas imidazole directly killed Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or together restricted Mtb growth in Mtb-infected mouse lungs. Our study demonstrates that the metabolic differences in Mtb-infected AMs and KCs leads to differences in the restriction of Mtb growth.

Bone Marrow-Derived Mesenchymal Stromal Cells (MSCs) Modulate the Inflammatory Character of Alveolar Macrophages from Sarcoidosis Patients.

Sarcoidosis is a devastating inflammatory disease affecting many organs, especially the lungs and lymph nodes. Bone marrow-derived mesenchymal stromal cells (MSCs) can “reprogram” various types of macrophages towards an anti-inflammatory phenotype. We wanted to determine whether alveolar macrophages from sarcoidosis subjects behave similarly by mounting an anti-inflammatory response when co-cultured with MSCs. Fifteen sarcoidosis and eight control subjects underwent bronchoscopy and bronchoalveolar lavage (BAL). Unselected BAL cells (70–94% macrophages) were isolated and cultured with and without MSCs from healthy adults. Following stimulation of the cultured cells with lipopolysaccharide, the medium was removed to measure interleukin 10 and tumor necrosis factor alpha (IL-10 and TNF-α). In two additional sarcoidosis subjects, flow cytometry was used to study intracellular cytokines and surface markers associated with alveolar macrophages to confirm the results. Unselected BAL cells from sarcoidosis subjects co-cultured with MSCs showed a reduction in TNF-α (pro-inflammatory M1) and an increase in IL-10 (anti-inflammatory M2) in 9 of 11 samples studied. Control subject samples showed few, if any, differences in cytokine production. Unselected BAL cells from two additional patients analyzed by flow cytometry confirmed a switch towards an anti-inflammatory state (i.e., M1 to M2) after co-culture with MSCs. These results suggest that, similarly to other macrophages, alveolar macrophages also respond to MSC contacts by changing towards an anti-inflammatory phenotype. Based on our results, we hypothesize that mesenchymal stromal cells applied to the airways might alleviate lung inflammation and decrease steroid need in patients with sarcoidosis.

Abnormal Cytokine Production By Mast Cell Cultures from Sickle Cell Anemia Patients in Response to Inflammatory Stimuli and to Co-Culture with Eosinophils

Mast cell function (MC) in pathologic states can be studied through their ability to secrete mediators in vitro depending on MC phenotype and the nature of the stimuli. Reports on MC mediators in sickle cell anemia (SCA) patients are scarce, but clinical signs of MC activation syndrome, such as increased plasma histamine in vaso‐occlusive crisis (VOC), and normal or slightly elevated serum tryptase have been reported. However, assessing the biological relevance of MC as a cytokine source is more challenging because it is unclear under which circumstances they secrete those products in vivo, or if the cytokines measured systemically stem from a different cell type. We aimed to investigate the profile of mediators involved in the inflammatory process produced by MC in SCA. Methods:The supernatant of 5-week old MC cultures (17 SCA, 8 HV) obtained from peripheral blood CD34+ cells from 29 SCA patients and 13 healthy volunteers (HV) was analyzed using a multiplex platform and colorimetric assays for endothelin-1 (ET-1) and substance P (SP) (10 SCA, 6 HV). A correlation matrix (Pearson correlations, R software, v. 3.6.1) was generated using laboratory and clinical data chosen based on their value as inflammatory or prognosis markers (hydroxyurea [HU] treatment, fetal hemoglobin [HbF], hemoglobin [Hb], vaso-occlusive crisis [VOC], percentage of peripheral blood neutrophils, eosinophils (Eos), basophils, erythroblasts, and reticulocytes), MC surface expression of CD117, CD48 and CD63, and the supernatant content of 11 cytokines. To investigate MC cytokine release, we tested the supernatants from Eos-MC co-cultures (3:1 ratio), and after stimulation with ET-1 (20 nM), SP (10 µM) and imatinib (20 µg/ml)(n=3 per treatment). Results: Out of 26 cytokines, we found elevated levels of the following in the supernatants of SCA-MC cultures (data represented as mean in pg/ml±SE): TNFα: SCA=88.7±18.4, HV=32.6±3.8; IFNγ: SCA=55.3±11.2, HV=15.7±1.8; MCP1: SCA=555.0±147.2, HV=145.3±35.2; RANTES: SCA=24.7±3.9, HV=10.7±1.8 (p&amp;lt;0.05). However, SCA-MC from patients treated with HU (n = 11) showed higher values of IL-1b, IL-4, IL-5, IL-9, IL-15, and FGF than HV (n=8) and HU-free patients (n=6) (p&amp;lt;0.05). Supernatants from SCA-MC had higher ET-1 production compared to HV-MC (SCA=16.3±1.2, HV=11.93±1.3, pg/ml, p=0.02) but SP production was similar (SCA=27.9±1.3; HV=31.49±0.7 pg/ml). ET-1 stimulation of MC cultures caused 2-fold increase in IL-1AR production on HV-MC, but failed to produce any effect on SCA-MC. Similarly, imatinib reduced FGF only in HV-MC samples (HV: 15.1±3.5, HV-HU: 4.0±1.6, pg/ml). No effect on cytokine production was observed with SP. Conversely, Eos-MC cocultures showed a 10- and 4-fold increase of IL-5 and IL-9, respectively, regardless of the origin of Eos (HV or SCA). SCA-MC/SCA-Eos co-cultures had elevated proinflammatory (IL-1b, IL-12, TNF-α) and angiogenic (FGF, VEGF) cytokines, RANTES, IL-7, IL-4, and IL1-RA compared with SCA-MC/HV-Eos and HV-MC/HV-Eos (p&amp;lt;0.05). Preliminary multiparametric analysis on data from SCA patients showed a strong negative correlation between HU therapy and VGEF production, and between HbF levels and CD63 expression (MC activation marker).We also found a positive correlation between history of VOC and eotaxin-1 produced by SCA-MC. Conclusions: We found that MC responses depend both on the origin of the cultured cell and the stimuli utilized. Despite differences between in vitro and in vivo MC populations, our data show that cultured SCA-MC have a sustained activated state and produce a repertoire of mediators that could contribute to a perivascular microenvironment in favor of leukocyte and endothelium activation. In terms of cytokine production, cultured SCA-MC were more sensitive to stimulation by SCA-Eos than by HV-Eos, which may be relevant to the pathophysiology of airway inflammation in SCA patients with asthma. Differences in cytokine production between SCA-MC cultures from patients treated or not with HU may reflect the variability in adherence to treatment, individual response to each compound, or epigenetic modifications during the MC differentiation process that affect the phenotype of the mature MC. These results support that mediators produced by MC can contribute to the chronic inflammatory state and may be implicated in exacerbated responses to eosinophil activation in SCA. Disclosures Fertrin: Agios Pharmaceuticals, Inc.: Research Funding.

1620P - Cytokine genetic variations and worse quality of life among family caregivers of head and neck cancer patients in palliative care

BackgroundHigh inflammatory cytokine levels can lead to severe fatigue and alter the quality of life (QoL) of family caregivers (FCGs) of patients with head and neck cancer in palliative care (HNCPC). In addition, single nucleotide variants (SNVs) in cytokine genes, such as IL1B c-598T>C (rs16944), IL1R2 c.-2009G>A (rs4141134), IL6 c.-237G>C (rs1800795), and TREML2 c.430A>G (rs3747742), can influence individual differences in cytokine production. However, the roles of the referred SNVs on the QoL of FCGs are unknown. Thus, the aim of our study was to evaluate the influence of the referred SNVs in the QoL of FCGs of patients with HNCPC. MethodsGenomic DNA of 100 FCGs of patients with HNCPC was analyzed by real-time PCR to identify the genotypes. QoL was measured with the abbreviated World Health Organization QoL questionnaire (WHOQOL-bref). The cut-off of the score was lower than 60 for worse QoL. Differences between groups were assessed by Fisher’s exact test, chi-square (bivariate analysis), and multiple linear regression. ResultsFCGs who carried IL6 GG (62.8% vs. 49.2%, odds ratio [OR]: 2.78, 95% confidence interval [CI]: 1.03-7.44, p=0.04) and TREML2 AA (60.0% vs. 44.6%, OR: 2.68, 95% CI: 1.03-7.01, p=0.04) had worse QoL for the overall QoL domain than non-carriers. The frequency of the genotype TREML2 AA was higher in FCGs with lower score for physical domain (61.0% vs. 42.4%, OR: 2.69, 95% CI: 1.05-6.87, p=0.03). FCGs who carried IL1B TT genotype had worse QoL for the social domain than others (48.9% vs. 32.1%, OR: 3.49, 95% CI: 1.03-7.01, p=0.01). ConclusionsGenotypes of the IL1B, IL6, and TREML genes, associated with higher inflammatory levels, seem to influence the QoL of FCGs. We believe that our results can contribute to identify FCGs with worse QoL for physical, psychological, and social aspects and provide better and more specialized care. Legal entity responsible for the studyThe authors. FundingFAPESP. DisclosureAll authors have declared no conflicts of interest.