Cytokine Production In Monocytes Research Articles

Associations between Monocyte and T Cell Cytokine Profiles in Autism Spectrum Disorders: Effects of Dysregulated Innate Immune Responses on Adaptive Responses to Recall Antigens in a Subset of ASD Children.

Changes in monocyte cytokine production with toll like receptor (TLR) agonists in subjects with autism spectrum disorders (ASD) were best reflected by the IL-1β/IL-10 ratios in our previous research. The IL-1β/IL-10 based subgrouping (low, normal, and high) of ASD samples revealed marked differences in microRNA expression, and mitochondrial respiration. However, it is unknown whether the IL-1β/IL-10 ratio based subgrouping is associated with changes in T cell cytokine profiles or monocyte cytokine profiles with non-TLR agonists. In ASD (n = 152) and non-ASD (n = 41) subjects, cytokine production by peripheral blood monocytes (PBMo) with TLR agonists and β-glucan, an inflammasome agonist, and T cell cytokine production by peripheral blood mononuclear cells (PBMCs) with recall antigens (Ags) (food and candida Ags) were concurrently measured. Changes in monocyte cytokine profiles were observed with β-glucan in the IL-1β/IL-10 ratio based ASD subgroups, along with changes in T cell cytokine production and ASD subgroup-specific correlations between T cell and monocyte cytokine production. Non-ASD controls revealed considerably less of such correlations. Altered innate immune responses in a subset of ASD children are not restricted to TLR pathways and correlated with changes in T cell cytokine production. Altered trained immunity may play a role in the above described changes.

Frontline Science: Endotoxin-induced immunotolerance is associated with loss of monocyte metabolic plasticity and reduction of oxidative burst.

Secondary infections are a major complication of sepsis and associated with a compromised immune state, called sepsis‐induced immunoparalysis. Molecular mechanisms causing immunoparalysis remain unclear; however, changes in cellular metabolism of leukocytes have been linked to immunoparalysis. We investigated the relation of metabolic changes to antimicrobial monocyte functions in endotoxin‐induced immunotolerance, as a model for sepsis‐induced immunoparalysis. In this study, immunotolerance was induced in healthy males by intravenous endotoxin (2 ng/kg, derived from Escherichia coli O:113) administration. Before and after induction of immunotolerance, circulating CD14+ monocytes were isolated and assessed for antimicrobial functions, including cytokine production, oxidative burst, and microbial (Candida albicans) killing capacity, as well metabolic responses to ex vivo stimulation. Next, the effects of altered cellular metabolism on monocyte functions were validated in vitro. Ex vivo lipopolysaccharide stimulation induced an extensive rewiring of metabolism in naive monocytes. In contrast, endotoxin‐induced immunotolerant monocytes showed no metabolic plasticity, as they were unable to adapt their metabolism or mount cytokine and oxidative responses. Validation experiments showed that modulation of metabolic pathways, affected by immunotolerance, influenced monocyte cytokine production, oxidative burst, and microbial (C. albicans) killing in naive monocytes. Collectively, these data demonstrate that immunotolerant monocytes are characterized by a loss of metabolic plasticity and these metabolic defects impact antimicrobial monocyte immune functions. Further, these findings support that the changed cellular metabolism of immunotolerant monocytes might reveal novel therapeutic targets to reverse sepsis‐induced immunoparalysis.

Human Toll-like Receptor 8 (TLR8) Is an Important Sensor of Pyogenic Bacteria, and Is Attenuated by Cell Surface TLR Signaling.

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus, GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus, GBS, Streptococcus pneumonia, Pseudomonas aeruginosa, and E. coli. The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus, GBS, and S. pneumonia, the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

Rab7b participation on the TLR4 (Toll-like receptor) endocytic pathway in Shiga toxin-associated Hemolytic Uremic Syndrome (HUS)

BackgroundThe inflammatory response of the host to Shiga toxin and/or lipopolysaccharide (LPS) of Escherichia coli (E. coli) is included in (HUS). The TLR4-LPS complex is internalized and TLR4 induced inflammatory signaling is stopped by targeting the complex for degradation. Rab7b, a small guanosine triphosphatase (GTPase) expressed in monocytes, regulates the later stages of the endocytic pathway. Objective: we studied the Rab7b participation on the TLR4 endocytic pathway and its effect on monocyte cytokine production along the acute course of pediatric Shiga toxin-associated HUS. Methods and resultsMonocytes were identified according to their positivity in CD14 expression. Surface TLR4 expression in monocytes from 18 HUS patients significantly increased by day 1 to 6, showing the highest increase on day 4 compared to monocytes of 10 healthy children. Significant higher surface TLR4 expression was accompanied by increased proinflammatory intracellular cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In contrast, after these time points, surface TLR4 expression and intracellular TNF-α levels, returned to near control levels after 10 days. Furthermore, confocal immunofluorescence microscopy proved colocalization of increased intracellular TLR4/Rab7b determined by Pearson's coefficient in monocytes from HUS patients from day 1 on the highest colocalization of both proteins by day 4. Decreased TLR4/Rab7b colocalization was shown 10 days after HUS onset. ConclusionThe colocalization of TLR4 and Rab7b allows us to suggest Rab7b participation in the control of the TLR4 endocytic pathway in HUS patient monocytes. A consequential fall in cytokine production throughout the early follow up of HUS is demonstrated.

Interleukin-21 enhances Toll-like receptor 2/4-mediated cytokine production via phosphorylation in the STAT3, Akt and p38 MAPK signalling pathways in human monocytic THP-1 cells.

Interleukin-21 (IL-21) is a type I cytokine produced by activated T cells that promotes cytokine production in monocytes. Monocytes are activated by Toll-like receptors (TLRs) to produce pro-inflammatory mediators. However, little is known about the regulatory effect of IL-21 on TLR-mediated inflammation in human monocytes. This study investigated the potential association between IL-21 and TLR2/4-mediated inflammation in human monocytic THP-1 cells. First, the expression of the IL-21 receptor (IL-21R) in human monocytic THP-1 cells was examined by flow cytometry. Then, THP-1 cells were treated with either the TLR2 ligand peptidoglycan (PGN) or the TLR4 ligand lipopolysaccharide (LPS) with or without IL-21. Then, the production of several cytokines (IL-6, IL-8, TNF-α, IFN-γ and IL-10), expression of TLR2/4, and activation of the downstream signaling pathways of mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and nuclear factor-kappa B (NF-κB) were determined. We found that IL-21R was highly expressed in human monocytic THP-1 cells and that IL-21 induced TLR2 and TLR4 expression and further enhanced PGN/LPS-mediated TLR2/4 expression. In addition, IL-21 also upregulated the expression of IL-6, IL-8, TNF-α, IFN-γ and IL-10 and enhanced TLR2/4-mediated cytokine production in THP-1 cells via phosphorylation of the STAT3, Akt and p38 MAPK signalling pathways. Our study suggests, for the first time that IL-21 enhances TLR2/4-mediated cytokine production in human monocytic THP-1 cells by activating the STAT3, PI3K/Akt and p38 MAPK signalling pathways.

Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis.

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16+ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14+ CD16+ monocytes expressed higher levels of these cytokines than CD14+ CD16- cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14hi CD16+ monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.

Structural variants of Salmonella Typhimurium lipopolysaccharide induce less dimerization of TLR4/MD-2 and reduced pro-inflammatory cytokine production in human monocytes

Salmonella enterica serovar Typhimurium (S. Typhimurium) changes the structure of its lipopolysaccharide (LPS) in response to the environment. The two main LPS variants found in S. Typhimurium correspond to LPS with a hepta-acylated lipid A (LPS 430) and LPS with modified phosphate groups on its lipid A (LPS 435). We have previously shown that these modified LPS have a lower capacity than wild type (WT) LPS to induce the production of pro-inflammatory cytokines in mice. Nevertheless, it is not know if LPS 430 and LPS 435 could also subvert the innate immune responses in human cells. In this study, we found that LPS 430 and LPS 435 were less efficient than WT LPS to induce the production of pro-inflammatory cytokines by human monocytes, in addition we found a decreased dimerization of the TLR4/MD-2 complex in response to LPS 430, suggesting that structurally modified LPS are sensed differently than WT LPS by this receptor; however, LPS 430 and 435 induced similar activation of the transcription factors NF-κB p65, IRF3, p38 and ERK1/2 than WT LPS. Microarray analysis of LPS 430- and LPS 435-activated monocytes revealed a gene transcription profile with differences only in the expression levels of microRNA genes compared to the profile induced by WT LPS, suggesting that the lipid A modifications present in LPS 430 and LPS 435 have a moderate effect on the activation of the human TLR4/MD-2 complex. Our results are relevant to understand LPS modulation of immune responses and this knowledge could be useful for the development of novel adjuvants and immunomodulators.

Calcineurin inhibitors reduce NFAT-dependent expression of antifungal pentraxin-3 by human monocytes.

Calcineurin (CN) inhibitors are effective clinical immunosuppressants but leave patients vulnerable to potentially fatal fungal infections. This study tested the hypothesis that CN inhibition interferes with antifungal immune defenses mediated by monocytes. We showed that NFAT is expressed by human monocytes, and is activated by exposure to fungal ligands. We confirmed that NFAT translocation potently activated target gene transcription using a human monocytic reporter cell line. Inhibition of CN‐NFAT by cyclosporine A significantly reduced monocyte production of TNF‐α, IL‐10, and MCP‐1 proteins in response to pattern recognition receptor ligands as well as to Aspergillus fumigatus conidia. Moreover, we revealed that human monocytes express the antifungal protein pentraxin‐3 under control of NFAT. In conclusion, clinical CN inhibitors have the potential to interfere with the novel NFAT‐dependent pentraxin‐3 pathway as well as antifungal cytokine production in human monocytes, thereby impeding monocyte‐mediated defenses against fungal infection in immune‐suppressed patients.

Nicotinic acetylcholine receptor silent agonists modulate inflammation

Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune regulatory properties. nAChRs are best known to function as ligand‐gated ion channels in neurons, however, recent evidence suggests that nicotine modulates inflammation by desensitizing non‐neuronal nAChRs, rather than by inducing channel opening. The objective of this study was to determine whether nAChR‐dependent anti‐inflammatory properties are due to channel desensitization. To this end, we made use of various nAChR ligands, including: 1) nicotine, a nAChR non‐selective agonist that also desensitizes the receptors; 2) AR‐R17779, an α7 nAChR subtype‐selective agonist; 3) α‐bungarotoxin, an α7 and α9 nAChR‐selective competitive antagonist, 4) mecamylamine, a non‐selective and non‐competitive antagonist; 5) N,N‐diethyl‐N’‐phenylpiperazine (DEPP)‐based silent agonists that selectively induce the desensitized state of α7 nAChR while producing little or no channel opening. We first evaluated the effects of these various nAChR ligands on the modulation of mouse and human monocyte/macrophage numbers, phenotype and cytokine production. Monocyte phenotype, cellular viability, proliferation and cytokine production were assessed by flow cytometry. In bone marrow‐derived monocyte/macrophage (BMDM) cell cultures, nicotine, mecamylamine and silent agonists reduced pro‐inflammatory monocyte numbers by affecting their viability and proliferation. The silent agonists also significantly reduced both LPS‐ and ATP‐induced cytokine production by mouse BMDMs. In human peripheral blood mononuclear cells (PBMCs), the silent agonists tested had modest effects on monocyte numbers and phenotype. However, LPS‐ and ATP‐induced cytokine production by human blood‐derived immune cells (PBMCs and whole blood) was inhibited by silent agonists. Taken together, our results suggest that nAChRs modulate pro‐inflammatory monocyte numbers and function via receptor desensitization rather than channel opening. nAChR silent agonists may thus be a novel source of anti‐inflammatory compounds that could be used for the treatment of inflammatory disorders.Support or Funding InformationThis work was supported by the MS Society of Canada, the New Brunswick Health Research Foundation and NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aberrant Cytokine Production in Myelofibrosis Is Not Rectified By Ruxolitinib and Is Differentially Sensitive to Inhibition of JAK/STAT, MAP Kinase, and NFκB Signaling

Inflammatory cytokine production is characteristic of myeloproliferative neoplasms including myelofibrosis (MF). Clonal dominance and bone marrow fibrosis have been attributed in part to cytokine overproduction. Elevated plasma levels of several cytokines have been associated with decreased survival in MF patients; notably IL-8/CXCL8, which has been associated with transformation to secondary acute myeloid leukemia (sAML; Tefferi et al., 2011, J. Clin. Oncol.).In MF patients, JAK2 inhibition with ruxolitinib ameliorates constitutional symptoms and splenomegaly; but improvements in anemia, fibrosis, and malignant clonal burden are infrequent, and survival benefit is modest. We have measured blood plasma cytokines in MF patients prior to and on treatment with ruxolitinib, versus healthy controls and sAML patients. Cytokines significantly elevated in MF patients versus controls (VEGF, TNF, IL-6, IL-10, IL-16) showed minimal differences in pre-versus-on ruxolitinib levels. Indeed, both pre- and on ruxolitinib cytokine levels in MF were similar to those observed in sAML. Therefore, overall cytokine-producing activity in MF is of similar magnitude to that in leukemic hematopoiesis, and persists despite ruxolitinib treatment.To identify cell populations responsible for cytokine production, MF blood cells were studied using mass cytometry (CyTOF). In MF versus healthy control samples, 14/15 cytokines measured were found to be constitutively overproduced in myeloid cell populations. IL-8 was highly expressed in CD34+ hematopoietic stem and progenitor cells (HSPC), which are prevalent in MF blood. The prognostic impact of IL-8 may therefore reflect overproduction of phenotypic HSPC, which could indicate incipient transformation to sAML. For the majority of MF overproduced cytokines, however, the largest source population was monocytes. This indicates that cytokines produced by monocytes may impact disease-propagating HSPC in a non-cell-autonomous fashion.To evaluate mechanisms driving cytokine overproduction, MF patient and control samples were exposed to 4-hour incubations with TPO, TNF, and/or TLR ligands PAM3CSK4 and R848. These compounds activate either JAK/STAT or NFκB signaling. The majority of cytokines elevated in MF exhibited hypersensitivity to these agents in MF versus normal monocytes. TPO induced cytokines in monocytes via its receptor MPL, which was expressed in a subset of both normal and MF monocytes. Cytokine induction by TPO was observed in normal and MF monocytes isolated by flow sorting (CD14+CD34-CD61-), confirming that MPL and JAK2 can induce cytokine overproduction in monocytes cell-autonomously. TPO induced cytokine production in monocytes could be suppressed either by ruxolitinib or pevonedistat, a NEDD8-activating enzyme inhibitor that blocks NFκB activation. This implies that both JAK2 and NFκB signaling pathways must be intact for maximal cytokine induction downstream of MPL.In contrast to TPO induced cytokine production, basal constitutively elevated cytokine production in MF monocytes was not consistently reduced by ruxolitinib. Among the TPO/TNF/TLR inducible cytokines, a subset (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TNF/TLR-sensitive cytokines, however, were at least partially sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling, as observed from incubation with pevonedistat, trametinib (MEK inhibitor), VX-745 (p38 MAPK inhibitor), and/or JNKi8 (JNK inhibitor). In contrast, these inhibitors did not suppress basal production of non-TPO-inducible cytokines (e.g. TGF-β, IFNγ), which are also overproduced in MF.These results indicate that overproduction of one set of cytokines in MF is driven by a combination of JAK/STAT, NFκB, and MAP kinase signaling; while that of another set is not directly driven by any of these pathways. Cytokines whose production is refractory to ruxolitinib may underlie features of MF that resist ruxolitinib therapy. A combined inhibitory approach may provide therapeutic benefit beyond that observed with ruxolitinib. Based on this hypothesis, our group has initiated a Phase 1 clinical trial combining pevonedistat with ruxolitinib for MF treatment (NCT03386214). Further results will indicate whether inhibition of NFκB confers an additional benefit to that observed from ruxolitinib alone. DisclosuresZhou:Incyte: Speakers Bureau. Oh:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Takeda: Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding.

Pentoxifylline Alone or in Combination with Gentamicin or Vancomycin Inhibits Live Microbe-Induced Proinflammatory Cytokine Production in Human Cord Blood and Cord Blood Monocytes In Vitro.

Neonatal sepsis and its accompanying inflammatory response contribute to substantial morbidity and mortality. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses transcription and production of proinflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that PTX decreases live microbe-induced inflammatory cytokine production in newborn blood. Cord blood was stimulated with live microorganisms commonly encountered in newborn sepsis (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, or Candida albicans) and simultaneously treated with antimicrobial agents (gentamicin, vancomycin, or amphotericin B) and/or clinically relevant concentrations of PTX. Microbial colony counts were enumerated by plating, supernatant cytokines were measured by multiplex assay, intracellular cytokines and signaling molecules were measured by flow cytometry, and mRNA levels were measured by quantitative reverse transcription-PCR. PTX inhibited concentration-dependent E. coli-, S. aureus-, S. epidermidis-, and C. albicans-induced tumor necrosis factor (TNF) and E. coli-induced interleukin-1β (IL-1β) production in whole blood, with greater suppression of proinflammatory cytokines in combination with antimicrobial agents. Likewise, PTX suppressed E. coli-induced monocytic TNF and IL-1β, whereby combined PTX and gentamicin led to significantly greater reduction of TNF and IL-1β. The anti-inflammatory effect of PTX on microbe-induced proinflammatory cytokine production was accompanied by inhibition of TNF mRNA expression and was achieved without suppressing the production of the anti-inflammatory IL-10. Of note, microbial colony counts in newborn blood were not increased by PTX. Our findings demonstrated that PTX inhibited microbe-induced proinflammatory cytokine production, especially when combined with antimicrobial agents, without enhancing microbial proliferation in human cord blood in vitro, thus supporting its utility as candidate adjunctive agent for newborn sepsis.

IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant.

Intravenous Immunoglobulin (IVIg) is used to treat autoimmune or inflammatory diseases, but its mechanism of action is not completely understood. We asked whether IVIg can induce interleukin-10 (IL-10) and reduce pro-inflammatory cytokine production in human monocytes, and whether this response is reduced in monocytes from people with an Fcγ receptor IIA (FcγRIIA) gene variant, which is associated with increased risk of inflammatory diseases and poor response to antibody-based biological therapy. IVIg increased IL-10 production and reduced pro-inflammatory cytokine production in response to bacterial lipopolysaccharide (LPS), which required FcγRI and FcγRIIB and activation of MAPKs, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38. IL-10 production was lower and pro-inflammatory cytokine production was higher in monocytes from people with the FcγRIIA risk variant and the risk variant prevented IL-10 production in response to (IVIg+LPS). Finally, we show that IVIg did not induce MAPK activation in monocytes from people with the risk variant. Our results demonstrate that IVIg can skew human monocytes to an anti-inflammatory, IL-10-producing activation state, which is compromised in monocytes from people with the FcγRIIA risk variant. This research has profound implications for the use of IVIg because 25% of the population is homozygous for the FcγRIIA risk variant and its efficacy may be reduced in those individuals. In addition, this research may be useful to develop new therapeutic strategies to replace IVIg by cross-linking FcγRIs and FcγRIIBs to promote anti-inflammatory macrophage activation, independent of the FcγRIIA genotype.

In vitro and in vivo Effects of Lactate on Metabolism and Cytokine Production of Human Primary PBMCs and Monocytes.

Lactate, the end product of anaerobic glycolysis, is produced in high amounts by innate immune cells during inflammatory activation. Although immunomodulating effects of lactate have been reported, evidence from human studies is scarce. Here we show that expression of genes involved in lactate metabolism and transport is modulated in human immune cells during infection and upon inflammatory activation with TLR ligands in vitro, indicating an important role for lactate metabolism in inflammation. Extracellular lactate induces metabolic reprogramming in innate immune cells, as evidenced by reduced glycolytic and increased oxidative rates of monocytes immediately after exposure to lactate. A short-term infusion of lactate in humans in vivo increased ex vivo glucose consumption of PBMCs, but effects on metabolic rates and cytokine production were limited. Interestingly, long-term treatment with lactate ex vivo, reflecting pathophysiological conditions in local microenvironments such as tumor or adipose tissue, significantly modulated cytokine production with predominantly anti-inflammatory effects. We found time- and stimuli-dependent effects of extracellular lactate on cytokine production, further emphasizing the complex interplay between metabolism and immune cell function. Together, our findings reveal lactate as a modulator of immune cell metabolism which translates to reduced inflammation and may ultimately function as a negative feedback signal to prevent excessive inflammatory responses.

Blood manufacturing methods affect red blood cell product characteristics and immunomodulatory activity

AbstractTransfusion of red cell concentrates (RCCs) is associated with increased risk of adverse outcomes that may be affected by different blood manufacturing methods and the presence of extracellular vesicles (EVs). We investigated the effect of different manufacturing methods on hemolysis, residual cells, cell-derived EVs, and immunomodulatory effects on monocyte activity. Thirty-two RCC units produced using whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD), and whole blood–derived (WBD) methods were examined (n = 8 per method). Residual platelet and white blood cells (WBCs) and the concentration, cell of origin, and characterization of EVs in RCC supernatants were assessed in fresh and stored supernatants. Immunomodulatory activity of RCC supernatants was assessed by quantifying monocyte cytokine production capacity in an in vitro transfusion model. RCF units yielded the lowest number of platelet and WBC-derived EVs, whereas the highest number of platelet EVs was in AD (day 5) and in WBD (day 42). The number of small EVs (<200 nm) was greater than large EVs (≥200 nm) in all tested supernatants, and the highest level of small EVs were in AD units. Immunomodulatory activity was mixed, with evidence of both inflammatory and immunosuppressive effects. Monocytes produced more inflammatory interleukin-8 after exposure to fresh WBF or expired WBD supernatants. Exposure to supernatants from AD and WBD RCC suppressed monocyte lipopolysaccharide-induced cytokine production. Manufacturing methods significantly affect RCC unit EV characteristics and are associated with an immunomodulatory effect of RCC supernatants, which may affect the quality and safety of RCCs.

A high-resolution mass cytometry analysis reveals a delay of cytokines production after TLR4 or TLR7/8 engagements in HIV-1 infected humans

HIV infection is associated with chronic inflammation in both non-treated and treated patients. TLR-dependent mechanisms are strongly involved in the maintenance of this inflammation. Indeed, the residual replication of HIV, the potential viral co-infections, or the products issued from microbial translocation provide TLR ligands, which contribute to trigger innate immune responses. Maintaining this chronic inflammation leads to an exhaustion of the immune system. Therefore, the TLR-dependent responses could be altered in HIV-infected patients. To investigate this hypothesis, we performed high-resolution phenotyping using a mass cytometry panel of 34 cell markers. Whole blood cells from healthy, non-treated HIV-infected and ART-treated HIV-infected subjects were stimulated with LPS, R848 or Poly(I:C). We observed the immune responses induced in T-cells, B-cells, polymorphonuclear cells, NK cells, basophils, monocytes and dendritic cells. We observed that, for either LPS or R848 stimulations, the production of cytokines in monocytes and conventional dendritic cells was delayed in treated or non-treated HIV-infected patients, compared to healthy individuals. These results suggest that leukocytes from chronic HIV-infected patients are slower to respond following the sensing of pathogens and danger signals, which may be an important feature of HIV infection.

CD46 activation induces distinct CXCL-10 response in monocytes and monocyte-derived dendritic cells

CD46 is an important immune regulatory receptor with dual functions, however, the CD46 isoform distribution and the effect of CD46 activation on the cytokine production in monocytes and monocyte-derived dendritic cells (moDCs) is unclear. Here, we show that CD46 activation of moDCs downregulates LPS-induced CXCL-10 expression, while the expression of CXCL-10 in monocytes is unaffected. Furthermore, the differentiation of moDCs induces a switch towards dominance of CYT-2 isoforms of CD46. These data indicate that CD46 activation exerts different functions in monocytes and moDCs and this correlates with a switch in CD46 isoform expression upon differentiation of moDCs.

Unfractionated Heparin Modulates Lipopolysaccharide-Induced Cytokine Production by Different Signaling Pathways in THP-1 Cells.

Sepsis is a complex syndrome resulting from the innate host response to infection. Apart from the well-known anticoagulant effects of heparin, it also possesses various immunomodulatory properties. Thus heparin seems to be a therapeutic drug in sepsis. We have demonstrated that unfractionated heparin (UFH) can inhibit lipopolysaccharide (LPS)-induced inflammatory responses in endothelial cells. The monocyte/macrophage system is a major contributor to host immunity and immune surveillance against infection. The aim of the study is to determine the inhibitory effect of UFH on cytokine production in THP-1 monocytes induced by LPS and to define the possible signaling pathways. The THP-1 cells were treated with UFH (0.1-10 U/mL) for 15 min before exposure to LPS (100 ng/mL). After 1 h, nuclear factor-κB (NF-κB) and phosphorylated inhibitor κB-α (IκB-α), c-Jun, c-fos, ERK1/2, JNK, and p38 mitogen-activated protein kinase (MAPK) expression levels were evaluated by Western blot. After 6 h, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, IL-8, and IL-18 protein concentrations were measured by enzyme-linked immunosorbent assay. Cell viability was determined by methyl thiazoyltetrazolium (MTT) assay. UFH inhibited LPS-induced phosphorylation of IκB-α, c-Jun, ERK1/2, JNK, and p38 MAPK but not c-fos. UFH also suppressed LPS-induced nuclear translocation of NF-κB. As expected, UFH decreased LPS-induced IL-1β, TNF-α, IL-6, IL-8, and IL-18 protein levels, suggesting that UFH has an anti-inflammatory effect on THP-1 cells by interrupting the MAPK, NF-κB, and c-Jun signaling pathways. UFH could potentially contribute to treatments for sepsis.

Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells

Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.

Emergence of Polyfunctional Cytotoxic CD4+ T Cells in Mycobacterium avium Immune Reconstitution Inflammatory Syndrome in Human Immunodeficiency Virus-Infected Patients.

Immune reconstitution inflammatory syndrome (IRIS) is an aberrant inflammatory response in individuals with advanced human immunodeficiency virus (HIV) infection, after antiretroviral therapy (ART) initiation. The pathogenesis of Mycobacterium avium complex (MAC)-associated IRIS has not been fully elucidated. We investigated monocyte and CD4+ T-cell responses in vitro, tumor necrosis factor (TNF) expression in tissues, and plasma cytokines and inflammatory markers, in 13 HIV-infected patients with MAC-IRIS and 14 HIV-uninfected patients with pulmonary MAC infection. Prior to ART, HIV-infected compared with HIV-uninfected patients, had reduced TNF+ monocytes (P = .013), although similar cytokine (interferon gamma [IFN-γ], TNF, interleukin 2 [IL-2], and interleukin 17 [IL-17])-expressing CD4+ T cells. During IRIS, monocyte cytokine production was restored. IFN-γ+ (P = .027), TNF+ (P = .004), and polyfunctional CD4+ T cells (P = 0.03) also increased. These effectors were T-betlow, and some expressed markers of degranulation and cytotoxic potential. Blockade of cytotoxic T-lymphocyte associated protein 4 and lymphocyte activation gene-3 further increased CD4+ T-cell cytokine production. Tissue immunofluorescence showed higher proportions of CD4+ and CD68+ (monocyte/macrophage) cells expressed TNF during IRIS compared with HIV-uninfected patients. Plasma IFN-γ (P = .048), C-reactive protein (P = .008), and myeloperoxidase (P < .001) levels also increased, whereas interleukin 10 decreased (P = .008) during IRIS. Advanced HIV infection was associated with impaired MAC responses. Restoration of monocyte responses and expansion of polyfunctional MAC-specific T-betlow CD4+ T cells with cytotoxic potential after ART initiation may overwhelm existing regulatory and inhibitory mechanisms, leading to MAC-IRIS.

Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.