Cytokine Production In Monocytes Research Articles

Identification of Novel Synthetic Toll-like Receptor 2 Agonists by High Throughput Screening

Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.

Platelet Factor 4 Regulation of Monocyte KLF4 in Experimental Cerebral Malaria

Cerebral malaria continues to be a difficult to treat complication of Plasmodium falciparum infection in children. We have shown that platelets can have major deleterious immune functions in experimental cerebral malaria (ECM). One of the platelet derived mediators we have identified as particularly important is platelet factor 4/CXCL4. Our prior work demonstrated that PF4−/− mice are protected from ECM, have reduced plasma cytokines, and have reduced T-cell trafficking to the brain. We now show that PF4 drives monocyte cytokine production in a Kruppel like factor 4 (KLF4) dependent manner. Monocyte depleted Plasmodium berghei infected mice have improved survival, and KLF4 is greatly increased in control, but not monocyte depleted mice. PF4−/− mice have less cerebral monocyte trafficking and no change in KLF4 expression. These data indicate that PF4 induction of monocyte KLF4 expression may be an important step in the pathogenesis of ECM.

Epigenetic regulation of high glucose‐induced proinflammatory cytokine production in monocytes by curcumin

Diabetes is a pro‐inflammatory state. We have shown increased monocyte proinflammatory cytokines in patients with diabetes. High glucose (HG) induces inflammatory cytokines via epigenetic changes. Curcumin is anti‐inflammatory and may regulate chromatin remodeling by inhibiting histone acetylation. In this study, we tested the effect of curcumin on histone acetylation and inflammatory cytokine secretion under HG conditions in monocytes. THP‐1 cells were cultured in presence of normal glucose (NG, 5.5 mmol/L) or HG(25 mmol/L) conditions in absence or presence of curcumin (1.5–12.5μM) for 72h. Cytokine level, NFκB transactivation, histone deacetylases (HDACs) activity, and histone acetylases (HATs) activity were measured by immunoblotting, ELISA and immunofluorescence. HG activates HAT which in turn acetylates p65 and suppresses HDAC2 resulting in NFκB activation and increased transcription of IL‐6 and TNF‐α in monocytes. Curcumin induces HDAC2 and decreases HAT (p300) activity, thereby resulting in decreased NFκB activation and decreased inflammatory cytokine release (p<0.001) in HG conditions. These results indicate that curcumin decreases HG‐induced cytokine production in monocytes via epigenetic changes involving NFκB. In conclusion, curcumin supplementation by reducing vascular inflammation may prevent diabetic complications.Grant Funding Source: N/A

A novel Highly Specific Small Molecule Inhibitor for BTK Suppresses Fc Receptor Function in Macrophages and Prevents Inflammatory Arthritis (96.18)

Abstract Activating Fc receptors and inflammatory cytokine production by macrophages have been implicated in the development and pathology of arthritis. However, the molecular components that mediate these events are not well characterized. Here we identify Bruton’s tyrosine kinase (BTK) as an essential player not only in B-cell dependent but also in myeloid-cell dependent inflammatory arthritis using a novel highly specific small molecule inhibitor of BTK (BTK SMI). In rodent models of collagen-induced arthritis, treatment with BTK SMI inhibits disease by at least two mechanisms: preventing autoantibody production and suppressing inflammatory cytokine production. Specifically, BTK SMI treatment prevents signaling and inflammatory cytokine production in response to immune complex activation in macrophages. Pathways found in rodents translate to human as BTK SMI prevents Fc receptor induced cytokine production in human monocytes. These data indicate that BTK SMI may be beneficial for the treatment of arthritis and that BTK contributes substantially to myeloid cell-dependent disease pathogenesis.

Non-T cells are required for a poised Th17 phenotype and elevated IL-17 secretion by T cells from type 2 diabetes patients. (44.3)

Abstract Type 2 Diabetes (T2D) has recently been identified as a chronic inflammatory disease with changes in immune cell function. Th17 cells and their major product, IL-17, have been implicated in multiple chronic inflammatory diseases. However, the relationship between Th17s and T2D has not been reported. Pro-inflammatory cytokines hyper-secreted by T2D patient monocytes promote Th17 activation. These data suggest a functional link between Th17s and monocytes in T2D. To identify such a link, we first showed that fresh ex vivo T cells from T2D patients have molecular signatures of Th17 cells. T2D T cells stimulated in the context of peripheral blood mononuclear cells (PBMCs) activated Th17 signature genes and elevated IL-17 secretion. In contrast, LPS stimulation of PBMCs from T2D patients failed to elevate IL-17 secretion, indicating that increased monocyte cytokine production, characteristic of T2D, does not further stimulate Th17 cells. Unexpectedly, continuous co-culture of T cells with the non-T cell fraction was required to maintain elevated Th17 function. These data indicate a crucial role for non-T cells in maintaining the Th17 phenotype and elevating IL-17 production in T2D. We conclude the interaction between T cells and non-T cells, most likely monocytes, promotes inflammation in T2D through previously unappreciated mechanisms.

Reciprocal Regulation of Activating and Inhibitory Fcγ Receptors by TLR7/8 Activation: Implications for Tumor Immunotherapy

Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcgamma receptor (FcgammaR) function, as they are critical mediators of antibody therapy. The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcgammaR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcgammaR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model. Overnight incubation with R-848 increased FcgammaR-mediated cytokine production and antibody-dependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcgammaRI, FcgammaRIIa, and the common gamma-subunit was increased. Surprisingly, expression of the inhibitory FcgammaRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the gamma-subunit in TLR7(-/-) nor MyD88(-/-) cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody. These results show an as-yet-undiscovered regulatory and functional link between the TLR7/8 and FcgammaR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy.

Xanthohumol and Related Prenylated Flavonoids Inhibit Inflammatory Cytokine Production in LPS-Activated THP-1 Monocytes: Structure-Activity Relationships andIn SilicoBinding to Myeloid Differentiation Protein-2 (MD-2)

Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops and beer. Our objective of this study was to determine the anti-inflammatory activities of XN, isoxanthohumol (IX), and 15 related prenylated chalcones and flavanones, as well as their structure-activity relationships. The anti-inflammatory activities of the flavonoids were measured by their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production in human monocytic THP-1 cells. The position, number, and length of the prenyl groups had a marked influence on the inhibitory activity of the prenylfavonoids towards MCP-1 and IL-6 production. The α,β-unsaturated carbonyl moiety present in chalcones such as XN was not an absolute requirement for inhibitory activity, as the saturated XN derivative, tetrahydroxanthohumol (TX), showed inhibitory activity comparable to XN. With the aim to determine the mechanism of the observed anti-inflammatory effects, cellular protein levels of Toll-like receptor 4 (TLR4) were measured by Western blot 24 h following coexposure of THP-1 cells to LPS and either XN, TX, or IX. Only XN reduced the cellular TLR4 protein content. Therefore, an additional hypothesis was developed for an anti-inflammatory mechanism that involves the TLR4 coreceptor myeloid differentiation protein-2 (MD-2), which provides the actual binding site for LPS. Molecular docking studies showed that the complementarity of prenylated flavonoids with the hydrophobic MD-2 pocket (indicating goodness of fit) directly predicted their relative ability to inhibit MCP-1 and IL-6 production. In conclusion, prenylated flavonoids may suppress LPS-induced TLR4 activation at least partly by interfering with LPS binding to the TLR4 coreceptor MD-2, and XN (but not other prenylflavonoids) exerts an additional anti-inflammatory effect by downregulating the cellular TLR4 protein content.

Transient Receptor Potential Melastatin 2 Is Required for Lipopolysaccharide-Induced Cytokine Production in Human Monocytes

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable nonselective cation channel that is stimulated by oxidative stress and specifically activated by intracellular ADP-ribose. Because TRPM2 is highly expressed in immunocytes, a role of this channel in inflammation processes has been proposed. The aim of the current study was to determine the function of TRPM2 in LPS-induced cytokine production of human monocytes. Incubation of human primary monocytes with LPS resulted in an upregulation of TRPM2 mRNA, protein, and of ADP-ribose-induced membrane currents. By using short hairpin RNA to downregulate TRPM2 expression in THP-1 monocytes, we demonstrate that TRPM2 is required for the LPS-induced production of IL-6, IL-8, IL-10, and TNF-alpha. Application of LPS led to a time-dependent increase in intracellular Ca(2+) concentrations in THP-1 cells that was clearly reduced by downregulation of TRPM2. Omission of extracellular Ca(2+) strongly decreased TNF-alpha production in TRPM2-expressing cells. Thus, TRPM2-mediated Ca(2+) entry is a central mechanism for LPS-induced cytokine production in monocytic cells. The identification of TRPM2 as a major player in this LPS-dependent process makes it a promising tool in modulating monocyte functions.

Blockade of T Cell Contact-Activation of Human Monocytes by High-Density Lipoproteins Reveals a New Pattern of Cytokine and Inflammatory Genes

BackgroundCellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.Methodology/Principal FindingsTo further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNγ, CCL2, CCL5, TNF, IL-1β, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1β, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E2—both involved in inflammatory response—were triggered by T cell contact and inhibited in the presence of HDL.Conclusions/SignificanceThese results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.

C1q enhances microglial clearance of apoptotic neurons and neuronal blebs, and modulates subsequent inflammatory cytokine production

The expression of C1q, a recognition molecule of the complement system, is up-regulated following neuronal injury and is detected early in neurodegenerative disorders such as Alzheimer's disease. This multimeric protein triggers an enhancement of phagocytosis of suboptimally opsonized targets by microglia, the phagocytic cells of the CNS, similar to other phagocytes, enhances the uptake of apoptotic cells in peripheral phagocytes, and suppresses inflammatory cytokine production in human monocytes, macrophages and dendritic cells in the absence of activation of the entire complement cascade. The goal of this study was to determine if C1q could influence the inflammatory response to injury in the CNS, using primary rat microglia and neurons. The data show that microglia preferentially ingest apoptotic cells in comparison to live cells, like other professional phagocytes, that microglial ingestion of apoptotic neurons and neuronal blebs is enhanced by the presence of normal serum and that these enhanced levels of uptake are diminished in serum depleted of C1q. In addition, purified C1q bound to apoptotic neurons and neuronal blebs in a dose dependent manner, and alone triggered a significant enhancement of uptake by microglia. Microglia added to C1q coated wells or fed apoptotic neurons or neuronal blebs coated with C1q suppressed the lipopolysaccharide-induced production of proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and TNF-alpha, while the presence of C1q enhanced levels of the chemokine MCP-1/CCL2. The data are consistent with a protective role for C1q in the CNS during early stages of cell death by enhancing microglial clearance of apoptotic cells and suppressing proinflammatory cytokines.

Prostacyclin Inhibits IFN-γ-Stimulated Cytokine Expression by Reduced Recruitment of CBP/p300 to STAT1 in a SOCS-1-Independent Manner

Increasing evidence indicates that pulmonary arterial hypertension is a vascular inflammatory disease. Prostacyclin (PGI(2)) is widely used to treat pulmonary arterial hypertension and is believed to benefit patients largely through vasodilatory effects. PGI(2) is also increasingly believed to have anti-inflammatory effects, including decreasing leukocyte cytokine production, yet few mechanistic details exist to explain how these effects are mediated at the transcriptional level. Because activated monocytes are critical sources of MCP-1 and other cytokines in cardiovascular inflammation, we examined the effects of iloprost on IFN-gamma- and IL-6-stimulated cytokine production in human monocytes. We found that iloprost inhibited IFN-gamma- and IL-6-induced MCP-1, IL-8, RANTES, and TNF-alpha production in monocytes, indicating wide-ranging anti-inflammatory action. We found that activation of STAT1 was critical for IFN-gamma-induced MCP-1 production and demonstrated that iloprost inhibited STAT1 activation by several actions as follows: 1) iloprost inhibited the phosphorylation of STAT1-S727 in the transactivation domain, thereby reducing recruitment of the histone acetylase and coactivator CBP/p300 to STAT1; 2) iloprost selectively inhibited activation of JAK2 but not JAK1, both responsible for activation of STAT1 via phosphorylation of STAT1-Y701, resulting in reduced nuclear recruitment and activation of STAT1; and 3) SOCS-1, which normally terminates IFN-gamma-signaling, was not involved in iloprost-mediated inhibition of STAT1, indicating divergence from the classical pathway for terminating IFN-gamma-signaling. We conclude that PGI(2) exerts anti-inflammatory action by inhibiting STAT1-induced cytokine production, in part by targeting the transactivation domain-induced recruitment of the histone acetylase CBP/p300.

MicroRNA-155 Regulates Inflammatory Cytokine Production in Tumor-associated Macrophages via Targeting C/EBPβ

Macrophages (Mphi) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mphi to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPbeta was markedly elevated in tumor-associated Mphi both in vitro and human tumors in situ. The expression of C/EBP protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPbeta expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 (miR-155) in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPbeta is a potential target of miR-155 and luciferase assay confirmed that C/EBPbeta translation is suppressed by miR-155 through interaction with the 3'UTR of C/EBPbeta mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPbeta protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPbeta. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mphi, which in turn regulates the functional activities of monocytes/Mphi by releasing the translational inhibition of transcription factor C/EBPbeta.

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF‐κB signal transduction pathways

Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti-inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes. We have used human THP-1 cells as an in vitro monocyte model. The effect of BGMP on the secretion of tumour necrosis factor (TNF), interleukin (IL)-1beta and IL-8 was assessed, as well as the involvement of the NF-kappaB and MAP kinase signalling pathways. The stimulatory effect of BGMP was also tested in human peripheral blood monocytes. BGMP up-regulated the secretion of TNF, IL-1beta and IL-8 in a concentration-dependent fashion. The biological activity was exerted by the intact peptide, because cytokine secretion was not affected by protease inhibitors. The secretion of IL-8 and specially TNF and IL-1beta was blocked by PD98059, SP600125, SB203580 and Bay11-7082, suggesting the involvement of the MAP kinases p38, c-Jun N-terminal kinase and ERK and particularly the NF-kappaB pathway, although IL-8 secretion was independent of p38. BGMP was shown to elicit the phosphorylation of IkappaB-alpha and the nuclear translocation of the NF-kappaB subunits p50 and p65. The effect of BGMP on cytokine secretion was validated in human primary blood monocytes. BGMP stimulates human monocytes, operating via MAP kinase and NF-kappaB pathways. BGMP may exert an indirect intestinal anti-inflammatory effect by potentiating host defences against invading microorganisms.

Granulocyte–Macrophage Colony-stimulating Factor to Reverse Sepsis-associated Immunosuppression

Sustained sepsis-associated immunosuppression is associated with uncontrolled infection, multiple organ dysfunction, and death. In the first controlled biomarker-guided immunostimulatory trial in sepsis, we tested whether granulocyte-macrophage colony-stimulating factor (GM-CSF) reverses monocyte deactivation, a hallmark of sepsis-associated immunosuppression (primary endpoint), and improves the immunological and clinical course of patients with sepsis. In a prospective, randomized, double-blind, placebo-controlled, multicenter trial, 38 patients (19/group) with severe sepsis or septic shock and sepsis-associated immunosuppression (monocytic HLA-DR [mHLA-DR] <8,000 monoclonal antibodies (mAb) per cell for 2 d) were treated with GM-CSF (4 microg/kg/d) or placebo for 8 days. The patients' clinical and immunological course was followed up for 28 days. Both groups showed comparable baseline mHLA-DR levels (5,609 +/- 3,628 vs. 5,659 +/- 3,332 mAb per cell), which significantly increased within 24 hours in the GM-CSF group. After GM-CSF treatment, mHLA-DR was normalized in 19/19 treated patients, whereas this occurred in 3/19 control subjects only (P < 0.001). GM-CSF also restored ex-vivo Toll-like receptor 2/4-induced proinflammatory monocytic cytokine production. In patients receiving GM-CSF, a shorter time of mechanical ventilation (148 +/- 103 vs. 207 +/- 58 h, P = 0.04), an improved Acute Physiology and Chronic Health Evaluation-II score (P = 0.02), and a shorter length of both intrahospital and intensive care unit stay was observed (59 +/- 33 vs. 69 +/- 46 and 41 +/- 26 vs. 52 +/- 39 d, respectively, both not significant). Side effects related to the intervention were not noted. Biomarker-guided GM-CSF therapy in sepsis is safe and effective for restoring monocytic immunocompetence. Use of GM-CSF may shorten the time of mechanical ventilation and hospital/intensive care unit stay. A multicenter trial powered for the improvement of clinical parameters and mortality as primary endpoints seems indicated. Clinical trial registered with www.clinicaltrials.gov (NCT00252915).

Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes

Objectives Serum amyloid A (SAA) is a biomarker of inflammation. Elevated blood levels in cardiovascular disease and local deposition in atheroma implies a role of SAA as a mediator rather than just a marker of inflammation. This study explored SAA-induced cytokine production and secretion by mononuclear cells. Methods and results RT-PCR showed that SAA time-dependently induced cytokine mRNAs in peripheral blood mononuclear cells (PBMC) and THP-1 monocytoid cells, and dramatically increased IL-1β, MCP-1, IL-6, IL-8, IL-10, GM-CSF, TNF, and MIP-1α secretion by PBMC to levels 28 to 25,000 fold above baseline, as measured with Bio-Plex kits; monocytes were the principle source. SAA induction of cytokines in monocyte-derived macrophages (MDM) was significantly higher than from monocytes from the same donors. SAA time-dependently induced transient and significant upregulation of NF-κB1 mRNA; inhibitor studies indicate that activation of NF-κB through the ERK1/2, p38 and JNK MAPKs and the PI3K pathway was involved. PBMC from 10 patients with coronary artery disease (CAD) spontaneously secreted higher levels of IL-6 and MIP-1α after 24 h incubation than PBMC from normal controls, whereas SAA-induced levels of all cytokines were similar to controls. Aortic and coronary sinus sampling in 23 CAD patients indicated significant SAA release into the coronary circulation, not evident in 11 controls. Conclusions SAA can increase monocyte and macrophage cytokine production, possibly at sites of atherosclerosis, thereby contributing to the pro-inflammatory state in coronary artery disease.

The Selective α7 Agonist GTS-21 Attenuates Cytokine Production in Human Whole Blood and Human Monocytes Activated by Ligands for TLR2, TLR3, TLR4, TLR9, and RAGE

The cholinergic antiinflammatory pathway modulates inflammatory cytokine production through a mechanism dependent on the vagus nerve and the alpha7 subunit of the nicotinic acetylcholine receptor. GTS-21 [3-(2,4-dimethoxybenzylidene) anabaseine], a selective alpha7 agonist, inhibits inflammatory cytokine production in murine and human macrophages and in several models of inflammatory disease in vivo, but to date its antiinflammatory efficacy in human monocytes has not been characterized. We report here our findings that GTS-21 attenuates tumor necrosis factor (TNF) and interleukin 1beta levels in human whole blood activated by exposure to endotoxin. GTS-21 inhibited TNF production in endotoxin-stimulated primary human monocytes in vitro at the transcriptional level. The suppressive effect of GTS-21 was more potent than nicotine in whole blood and monocytes. Furthermore, GTS-21 attenuated TNF production in monocytes stimulated with peptidoglycan, polyinosinic-polycytidylic acid, CpG, HMGB1 (high-mobility group box 1 protein), and advanced glycation end product-modified albumin. GTS-21 decreased TNF levels in endotoxin-stimulated whole blood obtained from patients with severe sepsis. These findings establish the immunoregulatory effect of GTS-21 on human monocytes, and indicate the potential benefits of further exploration of GTS-21's therapeutic uses in human inflammatory disease.

Synergistic regulation of Pseudomonas aeruginosa‐induced cytokine production in human monocytes by mannose receptor and TLR2

The immune response to pathogen is regulated by a combination of specific PRR, which are involved in pathogen recognition. Pseudomonas aeruginosa, a bacterium that causes life-threatening disease in immuno-compromised host, is recognized by distinct members of the TLR family. We have previously shown that viable P. aeruginosa bacteria are recognized by human monocytes mainly through TLR2. Using ligand-specific blocking antibodies, we herein show that the mannose receptor (MR), a phagocytic receptor for unopsonized P. aeruginosa bacteria, contributes equally to TLR2 in proinflammatory cytokine production by human monocytes in response to P. aeruginosa infection. Synergy of both receptors totally controls the immune response. Viable P. aeruginosa bacteria activate NF-kappaB and MAPK pathways and enhance TLR2-mediated signaling in MR-transfected human embryonic kidney 293 cells. Moreover, MR follows the same kinetics and colocalizes with TLR2 in the endosome during in vivo infection of human macrophages with P. aeruginosa. The studies provide the first demonstration of a significant role for MR, synergistic with TLR2, in activating a proinflammatory response to P. aeruginosa infection.

Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes

Abstract Objectives Serum amyloid A (SAA) is a biomarker of inflammation. Elevated blood levels in cardiovascular disease and local deposition in atheroma implies a role of SAA as a mediator rather than just a marker of inflammation. This study explored SAA-induced cytokine production and secretion by mononuclear cells. Methods and results RT-PCR showed that SAA time-dependently induced cytokine mRNAs in peripheral blood mononuclear cells (PBMC) and THP-1 monocytoid cells, and dramatically increased IL-1β, MCP-1, IL-6, IL-8, IL-10, GM-CSF, TNF, and MIP-1α secretion by PBMC to levels 28 to 25,000 fold above baseline, as measured with Bio-Plex kits; monocytes were the principle source. SAA induction of cytokines in monocyte-derived macrophages (MDM) was significantly higher than from monocytes from the same donors. SAA time-dependently induced transient and significant upregulation of NF-κB1 mRNA; inhibitor studies indicate that activation of NF-κB through the ERK1/2, p38 and JNK MAPKs and the PI3K pathway was involved. PBMC from 10 patients with coronary artery disease (CAD) spontaneously secreted higher levels of IL-6 and MIP-1α after 24 h incubation than PBMC from normal controls, whereas SAA-induced levels of all cytokines were similar to controls. Aortic and coronary sinus sampling in 23 CAD patients indicated significant SAA release into the coronary circulation, not evident in 11 controls. Conclusions SAA can increase monocyte and macrophage cytokine production, possibly at sites of atherosclerosis, thereby contributing to the pro-inflammatory state in coronary artery disease.

Short-Term Amiodarone Treatment Attenuates the Production of Monocyte Cytokines and Chemokines by C-Reactive Protein and Improves Cardiac Function in Patients With Idiopathic Dilated Cardiomyopathy and Ventricular Tachycardia

Increased expression of cytokines and chemokines has been observed in chronic heart failure (CHF). Amiodarone reduces circulating cytokine levels, so it may attenuate the production of monocyte cytokines and chemokines by C-reactive protein (CRP) and thus improve the left ventricular ejection fraction (LVEF) in dilated cardiomyopathy (DCM) patients with ventricular tachycardia (VT). Peripheral blood mononuclear cells (PBMCs) were stimulated by 25 microg/ml CRP in 23 patients with DCM, who were divided into 2 groups based on whether or not amiodarone was included in their treatment (Amiodarone group n=8; No amiodarone group n=15). Tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 on monocytes at baseline and after 4 weeks of treatment was measured by ELISA and expressed as mean+/-SD (pg.ml(-1)x10(-6) PBMCs). The LVEF and the CRP-induced monocyte cytokine and chemokine production were unchanged in the No amiodarone group after 4 weeks; however, LVEF in the Amiodarone group was increased (32.7+/-6.9 to 39.2+/-6.9%; P=0.005), and TNF-alpha and MCP-1 production in the Amiodarone group were decreased (P=0.012, respectively). Amiodarone attenuates the production of monocyte cytokines and chemokines by CRP, and improves LVEF in CHF patients with VT.

Effects of tropism and virulence ofLeishmaniaparasites on cytokine production by infected human monocytes

The nature of early interactions between Leishmania and macrophages which determine the outcome of infection can be related directly to parasite biological properties. Here we compared the capacity of L. major (Lm) strains, reported to be high (LmHV) and low virulent and (LmLV) in the mouse model and L. infantum (Li) strains, dermotropic (LiD) and viscerotropic (LiV), to infect and modulate cytokine production in human peripheral blood derived monocytes. Monocytes were infected with metacyclic promastigotes for 24, 48 and 72 h. Parasite burden was significantly higher in Lm- than in Li-infected monocytes. LmHV and LiD induced a significantly higher parasite burden than LmLV and LiV respectively. Cytokine production was evaluated in monocytes infected for 24 h. Contrary to interleukin (IL)-12p70, monocyte chemotactic protein-1 and transforming growth factor-beta production was increased significantly in infected monocytes with no differences between strains. Lm isolates induced significantly higher quantities of tumour necrosis factor (TNF)-alpha than Li isolates. Low levels of IL-10 were induced by all Leishmania strains and, interestingly, co-stimulation with lipopolysaccharide (LPS) was accompanied by a dramatic increase in IL-10 production by infected monocytes. In conclusion, Lm isolates displaying different levels of virulence in mice exhibited significant differences in parasite burden but similar abilities to modulate cytokine production in human monocytes. Li strains showed weaker infectivity and TNF-alpha inducing-capacity compared with Lm strains. The dramatic increase of IL-10 production in infected monocytes co-stimulated by LPS may play a role in disease progression considering the presence of LPS during bacterial superinfections observed during human leishmaniasis.