Activation Of Human Monocytes Research Articles

Silver nanoparticles induce pro-inflammatory gene expression and inflammasome activation in human monocytes.

A complete cytotoxic profile of exposure to silver (AgNP) nanoparticles investigating their biological effects on the innate immune response of circulating white blood cells is required to form a complete understanding of the risk posed. This was explored by measuring AgNP-stimulated gene expression of the pro-inflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in THP-1 monocytes. A further study, on human monocytes extracted from a cohort of blood samples, was carried out to compare with the AgNP immune response in THP-1 cells along with the detection of pro-IL-1β which is a key mediator of the inflammasome complex. The aims of the study were to clearly demonstrate that AgNP can significantly up-regulate pro-inflammatory cytokine gene expression of IL-1, IL-6 and TNF-α in both THP-1 cells and primary blood monocytes thus indicating a rapid response to AgNP in circulation. Furthermore, a role for the inflammasome in AgNP response was indicated by pro-IL-1β cleavage and release. These results highlight the potential inflammatory effects of AgNP exposure and the responses evoked should be considered with respect to the potential harm that exposure may cause. Copyright © 2016 John Wiley & Sons, Ltd.

The effect of the accessory proteins, soluble CD14 and lipopolysaccharide-binding protein on Toll-like receptor 4 activity in human monocytes and adipocytes.

There is a growinge body of evidence pointing towards an important role for Toll-like receptors (TLR) especially TLR4 in obesity and metabolic syndrome. Owing to the paucity of data on the effect of the accessory proteins, lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14) on TLR4 activation, the present study was undertaken to examine the effect of sCD14 and LBP on TLR4 activation in pivotal cells of meta-inflammation, monocytes and adipocytes. The dose-response effects of sCD14 and LBP on TLR4 protein abundance in monocytes obtained from normal human volunteers was determined by flow cytometry and in human-differentiated adipocytes by western blotting. Additionally, the nuclear factor-kappaB (NF-κB) p65 and downstream biomediators interleukin (IL)-1β, IL-8, IL-6 and tumor necrosis factor (TNF)-α were measured in the cell culture supernatants by ELISA (enzyme-linked immunosorbent assay). In LPS-primed monocytes, sCD14 but not LBP, augments both TLR4 abundance and inflammatory biomediators (IL-1β, IL-8, IL-6 and TNF-α).sCD14 also showed a similar effect in LPS-primed human adipocytes by augmenting TLR4 protein expression and activity in terms of NF-κB p65 and downstream biomediators (IL-1β, IL-8, IL-6 and TNF-α). LBP at the highest concentration only promoted secretion of IL-8 and TNF-α. However in both monocytes and adipocytes, the effect of sCD14 was superior to LBP. In the present report, we make the novel observation that sCD14 compared with LBP, offers a preferred target to ameliorate TLR especially TLR4-induced inflammation and insulin resistance in human obesity and metabolic syndrome.

Citrus bergamia Juice Extract Attenuates β-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways.

Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer’s disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1–42 (Aβ1−42) -mediated inflammation. Exposure of THP-1 cells to Aβ1−42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1−42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes.

Influence of PPH dendrimers' surface functions on the activation of human monocytes: a study of their interactions with pure lipid model systems

The influence of surface functions on the interactions between Poly(PhosphorHydrazone) PPH dendrimers and human monocytes is discussed on the basis of complementary biological and physicochemical studies on membrane models (monolayers and multi-lamellar vesicles). The studies were performed on both an active and non-toxic phosphonic acid capped dendrimer and a non-active but toxic carboxylic acid capped one. On the one hand, comparative studies of the behaviour of DPPC monolayers in the presence or absence of PPH dendrimers in the subphase showed differences in the phase transitions, highlighting interactions between both dendrimers and phospholipid monolayers, with a larger incidence for the carboxylic acid capped dendrimer (negative control), validating its cellular toxicity. On the other hand, comparative biological studies (activation of human monocytes and binding of fluorescent dendrimers on human monocytes) show the pre-eminence of phosphonic acid capped dendrimers towards specific binding and subsequent activation of human monocytes.

Immune effects and antiacetylcholinesterase activity of Polygonum hydropiper L.

To determine the potential utility of Polygonum hydropiper (tade) as an anti-dementia functional food, the present study assessed the acetylcholinesterase inhibitory and anti-inflammatory activities of tade crude extracts in human cells. Crude extracts of tade were obtained by homogenizing tade in distilled water and then heating the resulting crude extracts. The hot aqueous extracts were purified by centrifugation and freeze-dried. The inhibition of acetylcholinesterase (AChE) by tade was investigated quantitatively by Ellman’s method. Furthermore, the in vitro effects on human leukocytes (phagocytic activity, phagosome-lysosome fusion, and superoxide anion release) of coating inactive Staphylococcus aureus cells with tade crude extracts were studied. The tade crude extracts inhibited AChE activity. Furthermore, they increased phagocytic activity and phagosome-lysosome fusion in human neutrophils and monocytes in a nominally dose-dependent manner. However, the tade crude extracts did not alter superoxide anion release (O2−) from neutrophils. Our results confirmed that crude extracts of P. hydropiper exhibit antiacetylcholinesterase and immunostimulation activities in vitro. P. hydropiper thus is a candidate functional food for the prevention of dementia.

DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients.

ABSTRACTHigh levels of circulating immunocomplexes (ICs) are found in patients with either infectious or sterile inflammation. We report that patients with either Plasmodium falciparum or Plasmodium vivax malaria have increased levels of circulating anti-DNA antibodies and ICs containing parasite DNA. Upon stimulation with malaria-induced ICs, monocytes express an NF-κB transcriptional signature. The main source of IC-induced proinflammatory cytokines (i.e., tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β])in peripheral blood mononuclear cells from acute malaria patients was found to be a CD14+ CD16 (FcγRIIIA)+ CD64 (FcγRI)high CD32 (FcγRIIB)low monocyte subset. Monocytes from convalescent patients were predominantly of the classical phenotype (CD14+ CD16−) that produces high levels of IL-10 and lower levels of TNF-α and IL-1β in response to ICs. Finally, we report a novel role for the proinflammatory activity of ICs by demonstrating their ability to induce inflammasome assembly and caspase-1 activation in human monocytes. These findings illuminate our understanding of the pathogenic role of ICs and monocyte subsets and may be relevant for future development of immunity-based interventions with broad applications to systemic inflammatory diseases.

Therapeutic targeting of BET protein BRD4 delays murine lupus

BRD4 is a member of the BET (bromodomain and extraterminal domain) family proteins that can bind acetylated histones and influence transcription, which are considered as potential therapeutic targets in many distinct diseases. And the BET inhibitor JQ1 has been proven to be effective in suppressing multiple inflammatory and autoimmune diseases. This study aimed to examine the therapeutic potential of JQ1 on a lupus model, MRL-lpr mice. Ten-week-old MRL-lpr mice were treated with JQ1 (oral administration of 200mg/kg) or vehicle for 8weeks. The proteinuria, nephritic damage, serum biochemistry, autoantibodies and cytokines were examined. Splenocytes of MRL-lpr mice were isolated for in vitro experiments. Treatment with JQ1 significantly attenuated the progression of proteinuria and nephritis. The serum concentrations of anti-dsDNA antibody as well as B-cell activating factor (BAFF), interleukin (IL)-1β, IL-6, IL-17 and INF-γ were inhibited, and IL-10 augmented by JQ1. Importantly, JQ1 improved the survival of lupus mice. In vitro, BAFF, IL-1β, IL-6, IL-17 and INF-γ were inhibited, and IL-10 augmented by JQ1 (500nM) in the cultures of splenocytes from diseased MRL-lpr mice, which was further supported by a significant reduction in immune complex-mediated activation of human monocytes in vitro by JQ1. Taken together, JQ1 effectively alleviates lupus in MRL-lpr mice by suppressing BAFF, pro-inflammatory cytokines and autoimmunity, supporting the therapeutic value of JQ1 in lupus disease.

Human caspase-4 and caspase-5 regulate the one-step non-canonical inflammasome activation in monocytes.

Monocytes promote the early host response to infection releasing key pro-inflammatory cytokines, such as IL-1β. The biologically inactive IL-1β precursor is processed to active form by inflammasomes, multi-protein complexes activating caspase-1. Human monocytes exhibit an unconventional one-step pathway of inflammasome activation in response to lipopolysaccharide (LPS) alone. Although this lineage-restricted mechanism is likely to contribute to the pathology of endotoxin shock, signalling pathways regulating this mechanism are currently unknown. Here we report that caspase-4 and caspase-5 mediate IL-1α and IL-1β release from human monocytes after LPS stimulation. Although caspase-4 remains uncleaved, caspase-5 undergoes rapid processing upon LPS treatment. We also identify an additional caspase-5 cleavage product in LPS-stimulated monocytes, which correlates with IL-1 secretion. This one-step pathway requires Syk activity and Ca2+ flux instigated by CD14/TLR4-mediated LPS internalization. Identification of caspase-4/5 as the key determinants of one-step inflammasome activation in human monocytes provides potential targets for therapeutic intervention in endotoxin shock.

Mannoside Glycolipid Conjugates Display Anti-inflammatory Activity by Inhibition of Toll-like Receptor-4 Mediated Cell Activation.

Inhibition of excessive Toll-like receptor 4 (TLR4) signaling is a therapeutic approach pursued for many inflammatory diseases. We report that Mannoside Glycolipid Conjugates (MGCs) selectively blocked TLR4-mediated activation of human monocytes and monocyte-derived dendritic cells (DCs) by lipopolysaccharide (LPS). They potently suppressed pro-inflammatory cytokine secretion and maturation of DCs exposed to LPS, leading to impaired T cell stimulation. MGCs did not interfere with LPS and could act in a delayed manner, hours after LPS stimulation. Their inhibitory action required both the sugar heads and the lipid chain, although the nature of the sugar and the structure of the lipid tail could be modified. They blocked early signaling events at the cell membrane, enhanced internalization of CD14 receptors, and prevented colocalization of CD14 and TLR4, thereby abolishing NF-κB nuclear translocation. When the best lead conjugate was tested in a mouse model of LPS-induced acute lung inflammation, it displayed an anti-inflammatory action by suppressing the recruitment of neutrophils. Thus, MGCs could serve as promising leads for the development of selective TLR4 antagonistic agents for inflammatory diseases.

Induction of IL-12 from human monocytes after stimulation with Androctonus crassicauda scorpion venom

The objective of this study was to evaluate the capacity of venom from Androctonus crassicauda to induce expression/production of interleukin (IL)-12 by isolated human monocytes. For this purpose, isolated human monocytes were exposed to different concentrations of the venom (0.16–20 μg/ml) for varying periods (6, 12, and 24 h). Apart from measures of venom cytotoxicity (i.e., lactase dehydrogenase activity [LDH] release), measures of IL-12 p40 mRNA (by Real-time PCR) of IL-12 release (by ELISA) were performed. The results showed that the venom produced significant concentration- and duration of incubation-dependent cytotoxicity. Expression of IL-12 p40 mRNA was significantly increased at all exposure timepoints relative to that in unexposed cells, but was maximal after 6 h of exposure. At that timepoint, the effect from a dose of 2.5 μg venom/ml provided the maximal increase among all doses tested. At the level of the protein itself, IL-12 production remained almost consistently elevated (vs. unexposed control values) across all exposure timepoints, with the greatest formation again occurring after 6 h of incubation at a dose of 2.5 μg venom/ml. The findings from this study demonstrated that venom from the A. crassicauda scorpion contained active constituents that could induce a sustained activation of human monocytes that was manifested, in part, as promotion of the expression/production of IL-12.

Antifungal antibiotics modulate the pro-inflammatory cytokine production and phagocytic activity of human monocytes in an in vitro sepsis model.

The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes. THP-1 monocytes were incubated with L-AMB, ITC or ANI and LPS. Gene expression levels of cytokines (TNF-<alpha>, IL-1<beta>, IL-6, and IL-10) were measured after 2h, 6h, and 24h. Cytokine protein levels were evaluated after 24h and phagocytic activity was determined following co-incubation with Escherichia coli. All antimycotics differentially modulated the gene and protein expression of cytokines in sepsis-like conditions. In the presence of LPS, we identified L-AMB as immunosuppressive, whereas ITC demonstrated pro-inflammatory properties. Both compounds induced remarkably less phagocytosis. Our study suggests that antimycotics routinely used in septic patients alter the immune response in sepsis-like conditions by modulating cytokine gene and protein expression levels and phagocytic activity. Future treatment strategies should consider the immune status of the host and apply antimycotics accordingly in bacterial septic patients with secondary fungal infections.

Abstract 4279: A new class of immunomodulators derivatized from the Tat protein of HIV-1 in a murine breast cancer model

Abstract Introduction: Immunotherapies such as immune checkpoint inhibitors have had limited success in treating breast cancers. We observed that women with HIV infection and even AIDS have an unusually low incidence of breast cancer. The Tat protein of HIV-1 modulates innate immunity through activation of antigen presenting cells (APCs), and thus could be the reason for this finding. A Tat protein derivative designed to eliminate elements contributing to HIV-1 mediated immunosuppression was expressed as a recombinant protein in E. coli and found to stimulate monocytes to differentiate into antigen presenting cells. This protein, designated PIN-2, was tested in the syngeneic 4T1 murine breast cancer model, a poorly immunogenic and invasive triple negative breast cancer (TNBC) cell line. Methods: In vitro activity of PIN-2 was confirmed using a human monocyte activation assay that determined cell surface expression of CD80 and CD86 by flow cytometry after 72h of incubation with PIN-2, compared with controls. BALB/c mice were implanted orthotopically in the mammary fat pad with 10000 4T1 cells (ATCC No. CRL-2593). Treatment was initiated 7 days after implantation. Mice were administered 100ng PIN-2 intravenously 3x/week for 3 weeks. Tumors were measured 2x/week starting 12 days after implantation using a standard caliper method. Mice were sacrificed 29 days after implantation and examined macroscopically for spontaneous pulmonary metastases. Results: PIN-2 significantly increased human monocyte cell surface expression of costimulatory molecules CD80 and CD86 in vitro at concentrations as low as 30ng/mL culture compared to the negative control (P&amp;lt;0.01). The activation was dose dependent. In vivo, PIN-2 treatment reduced tumor volume to less than 60% of the tumor volume of untreated mice 29 days after tumor implantation (686 mm3 tumor size in placebo vs. 407mm3 in treated mice, p&amp;lt;0.01). Moreover, PIN-2 significantly inhibited the development of spontaneous 4T1 lung metastases (p&amp;lt; 0.05 when compared to placebo). Preliminary examination indicates that 90% of the mice in the control group had visible nodules on the surface of the lungs. Macroscopically visible metastases were reduced to only 50% of the PIN-2 treated mice. There was no evidence of weight loss or apparent toxicity by PIN-2. Conclusion: PIN-2 stimulates cell surface expression of costimulatory molecules that serve as markers for activated antigen presenting cells and are essential for cytotoxic T-cell activation. It reduces primary and metastatic tumor burden in an orthotopic murine model of TNBC. These data suggest that PIN-2 functions as an innate immune agonist to facilitate adaptive anti-tumor immune responses in a poorly immunogenic model of breast cancer. Moreover, PIN-2 appears to be well tolerated, supporting further evaluation of PINS in human breast cancer clinical trials. Citation Format: Christoph M. Hotz-Behofsits, Sophie J. Hanscom, Joshua B. Goldberg, Colin B. Bier. A new class of immunomodulators derivatized from the Tat protein of HIV-1 in a murine breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4279. doi:10.1158/1538-7445.AM2015-4279

Expression of adenosine receptors in monocytes from patients with bronchial asthma

Adenosine is generated from adenosine triphosphate, which is released by stressed and damaged cells. Adenosine levels are significantly increased in patients with bronchial asthma (BA) and mediate mast cell degranulation and bronchoconstriction. Over the last decade, increasing evidence has shown that adenosine can modulate the innate immune response during monocytes differentiation towards mature myeloid cells. These adenosine-differentiated myeloid cells, characterized by co-expression of monocytes/macrophages and dendritic cell markers such as CD14 and CD209, produce high levels of pro-inflammatory cytokines, thus contributing to the pathogenesis of BA and chronic obstructive pulmonary disease. We found that expression of ADORA2A and ADORA2B are increased in monocytes obtained from patients with BA, and are associated with the generation of CD14posCD209pos pro-inflammatory cells. A positive correlation between expression of ADORA2B and IL-6 was identified in human monocytes and may explain the increased expression of IL-6 mRNA in asthmatics. Taken together, our results suggest that monocyte-specific expression of A2 adenosine receptors plays an important role in pro-inflammatory activation of human monocytes, thus contributing to the progression of asthma.

Controlled Release of Dexamethasone from Organosilicone Constructs for Local Modulation of Inflammation in Islet Transplantation.

Inflammation is a significant detriment to the engraftment of cells and tissues, particularly for islet transplantation, where a low tolerance for the inflammatory milieu results in significant graft loss. Local treatment with anti-inflammatories, such as glucocorticoids, provides the benefits of site-targeted delivery with minimization of the broad side effects associated with systemic delivery. Polydimethylsiloxane (PDMS) is a flexible platform that is capable of providing sustained delivery of hydrophobic drugs. Here, we evaluated the capacity of PDMS constructs loaded with the anti-inflammatory glucocorticoid dexamethasone (Dex) to locally mitigate inflammation in islet grafts. Dex-PDMS constructs, fabricated in rod or disk geometries, demonstrated prolonged and sustained release at therapeutically relevant levels. In vitro, Dex-PDMS constructs inhibited endotoxin-induced human monocyte and macrophage activation, but they did not impair islet viability or function. Dex-PDMS rods, co-transplanted with islet-seeded scaffolds in a murine model, demonstrated suppression of host inflammatory responses during early- and late-phase engraftment, without significantly altering islet graft potency. The facile nature of these glucocorticoid-doped PDMS constructs allows for the optimization of targeted dose delivery with wide applicability in cell and tissue transplantation.

MPLA shows attenuated pro-inflammatory properties and diminished capacity to activate mast cells in comparison with LPS.

Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen-specific immunotherapy. Nevertheless, its cell-activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side-by-side comparison of the immune-modulating properties of MPLA and LPS on different immune cells. Immune-activating properties of MPLA and LPS were compared in human monocytes and mast cells (MCs), a mouse endotoxin shock model (ESM), and mouse bone marrow (BM)-derived myeloid dendritic cells (mDCs), Tcells (TCs), Bcells, and MCs. In a mouse invivo ESM and a human exvivo monocyte activation test (MAT), MPLA induced the same cytokine secretion pattern as LPS (ESM: IL-6, IL-12, TNF-α; MAT: IL-1β, IL-6, TNF-α), albeit at lower levels. Mouse mDCs and exvivo isolated Bcells stimulated with MPLA required a higher threshold to induce TRIF-dependent cytokine secretion (IL-1β, IL-6, IL-10, and TNF-α) than did LPS-stimulated cells. In mDC:DO11.10 CD4 TC cocultures, stimulation with MPLA, but not with LPS, resulted in enhanced OVA-specific IL-4 and IL-5 secretion from DO11.10 CD4 TCs. Unexpectedly, in both human and mouse MCs, MPLA, unlike LPS, did not elicit secretion of pro-inflammatory cytokines. Compared to LPS, MPLA induced a qualitatively similar, but less potent pro-inflammatory immune response, but was unable to activate human or mouse MCs.

Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi.

Background Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease.Methodology/Principal FindingsWe evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain.Conclusion/SignificanceOur study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

Phenomenon of individual difference in human monocyte activation

Macrophages play an important role in the pathogenesis of atherosclerosis, including the early pre-clinical stages of the disease development. We have explored the possibility that the disease onset could be associated with altered monocyte/macrophage response to activating pro- and anti-inflammatory stimuli. We evaluated the susceptibility of circulating monocytes from healthy individuals and patients with asymptomatic carotid atherosclerosis to M1 and M2 activation. The obtained data indicated the existence of a remarkable individual difference in susceptibility to activation among monocytes isolated from the blood of different subjects, regardless of the presence or absence of atherosclerosis. The identified differences in susceptibility to activation between monocytes may explain the individual peculiarities of the immune response in different subjects.

Evidence for TLR4 and FcRγ–CARD9 activation by cholera toxin B subunit and its direct bindings to TREM2 and LMIR5 receptors

Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX–TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88−/− or TLR4−/− macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB–TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3-O-sulfo-β-d-galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ–CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin.

Induction of tissue-resident TFH-like cells in human cancer links monocyte activation to M2b macrophage polarization (TUM10P.1021)

Abstract The existence, regulation, and functions of IL-21+ immune cells are poorly defined in human cancers. Here we identified a subset of protumorigenic IL-21+ TFH-like cells in human hepatocellular carcinoma. These cells were the major source of IL-21 in tumors and represented about 10% of the CD4+ T cell population at levels comparable to amounts of TFH cells present in lymph nodes. However, the TFH-like cells displayed a unique CXCR5−PD-1−BTLA−CD69high tissue-resident phenotype with substantial IFN-γ production, which differed from the phenotype of TFH cells. TLR4-elicited innate monocyte inflammation was essential for IL-21+ TFH-like cell induction in tumors, and activation of STAT1 and STAT3 was critical for TFH-like cell polarization in this process. Importantly, the TFH-like cells operated in IL-21-IFN-γ-dependent pathways to induce plasma cell differentiation and thereby create conditions for M2b macrophage polarization. Thus induction of TFH-like cells links innate inflammation to immune privilege in tumors.

C1q-based tolerance mechanism in HMGB1-mediated activation of human monocytes (IRM5P.649)

Abstract C1q, a component of the classical complement pathway, is required for clearance of apoptotic cells. C1q also regulates activation of innate immune cells through engagement of C1q receptors present on the immune cell surface. C1q deficiency is highly associated with the development of systemic lupus erythematosus (SLE). High Mobility Group Box 1 (HMGB1), a pro-inflammatory mediator, is secreted by inflammatory cells and passively released from necrotic cells. HMGB1 interacts with TLR4 and receptor for advanced glycation end products (RAGE), to augment the production of type 1 interferon and proinflammatory cytokines. Here we show that C1q inhibits HMGB1-mediated monocyte activation through cross-linking RAGE and Leukocyte-associated Ig-like receptor-1 (LAIR-1), which is an inhibitory receptor for collagen and C1q. This crosslinking causes LAIR-1 to enter lipid rafts following HMGB1 and C1q stimulation. C1q suppress HMGB-1 mediated induction of type I-interferon and pro-inflammatory cytokines including IL-12 and TNF-α. Moreover, exposure of monocytes to HMGB1 and C1q together leads to upregulation of a number of anti-inflammatory molecules that might function to induce phagocytosis and to maintain monocyte quiescence. Our results suggest that C1q regulates the activation of innate immunity by HMGB1 and maintains immune homeostasis. These data are consistent with the established requirement for C1q in the prevention of autoimmunity.