Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis.

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Immune Checkpoint Inhibitor-Induced Thyroiditis Is Associated with Increased Intrathyroidal T Lymphocyte Subpopulations.

Background: Immune checkpoint inhibitors (ICIs) frequently cause thyroid dysfunction but their underlying mechanism remains unclear. We have previously demonstrated increased circulating natural killer (NK) cells and human leukocyte antigen (HLA)-DR surface expression on inflammatory intermediate CD14+CD16+ monocytes in programmed cell death protein-1 (PD-1) inhibitor-treated patients. This study characterizes intrathyroidal and circulating immune cells and class II HLA in ICI-induced thyroiditis. Methods: This is a single-center prospective cohort study of 10 patients with ICI-induced thyroiditis by flow cytometry of thyroid fine needle aspirates (n = 9) and peripheral blood (n = 7) as compared with healthy thyroid samples (n = 5) and healthy volunteer blood samples (n = 44); HLA class II was tested in n = 9. Results: ICI-induced thyroiditis samples demonstrated overall increased T lymphocytes (61.3% vs. 20.1%, p = 0.00006), CD4-CD8- T lymphocytes (1.9% vs. 0.7%, p = 0.006), and, as a percent of T lymphocytes, increased CD8+T lymphocytes (38.6% vs. 25.7%; p = 0.0259) as compared with healthy thyroid samples. PD-1 inhibitor-induced thyroiditis had increased CD4+PD1+ T lymphocytes (40.4% vs. 0.8%; p = 0.021) and CD8+PD1+ T lymphocytes (28.8% vs. 1.5%; p = 0.038) in the thyroid compared with the blood. Circulating NK cells, certain T lymphocytes (CD4+CD8+, CD4-CD8- T, gamma-delta), and intermediate monocytes were increased in ICI-induced thyroiditis. Six patients typed as HLA-DR4-DR53 and three as HLA-DR15. Conclusions: ICI-induced thyroiditis is a T lymphocyte-mediated process with intra-thyroidal predominance of CD8+ and CD4-CD8- T lymphocytes. The HLA haplotypes may be involved but need further evaluation. These findings expand the limited understanding of ICI-induced thyroiditis, which could be further translated to guide immunomodulatory therapies for advanced thyroid cancer.

SIRPα expression delineates subsets of intratumoral monocyte/macrophages with different functional and prognostic impact in follicular lymphoma.

Signal regulatory protein-α (SIRPα) is a key member of the "do-not-eat-me" signaling pathway, but its biological role and clinical relevance in B-cell NHL is relatively unknown. Using biopsy specimens from follicular lymphoma (FL), we identified three subsets (CD14+SIRPαhi, CD14-SIRPαlow, and CD14-SIRPαneg) of monocyte/macrophages (Mo/MΦ) based on CD14 and SIRPα expression. CD14+SIRPαhi cells expressed common Mo/MΦ markers; exhibited characteristic differentiation, migration, and phagocytosis; and suppressed T-cell function. CD14-SIRPαlow cells expressed fewer typical Mo/MΦ markers; migrated less and phagocytosed tumor cells less efficiently; and stimulated rather than suppressed T-cell function. Interestingly, the CD14-SIRPαneg subset expressed distinct Mo/MΦ markers compared to the other two subsets; had limited ability to migrate and phagocytose; but stimulated T-cell function. When using SIRPα-Fc to block the interaction between SIRPα and CD47, alone or in combination with rituximab, phagocytosis of tumor cells was differentially increased in the three Mo/MΦ subsets. Clinically, increased numbers of CD14+SIRPαhi cells were associated with an inferior survival in FL. In contrast, increased numbers of the CD14-SIRPαlow subset appeared to correlate with a better survival. Taken together, our results show that SIRPα expression delineates unique subsets of intratumoral Mo/MΦs with differing prognostic importance.

Soluble ST2 links inflammation to outcome after subarachnoid hemorrhage.

OBJECTIVE: To investigate whether soluble growth stimulation expressed gene 2 (sST2), a prognostic marker in cardiovascular and inflammatory disorders, is associated with neurological injury after aneurysmal subarachnoid hemorrhage (SAH). METHODS: We studied SAH patients from 2 independent cohorts. Outcome assessments included functional status at 90 days using the modified Rankin Scale (mRS), mortality, and delayed cerebral ischemia (DCI). The relationships between sST2 plasma level and outcome measures were assessed in both cross-sectional and longitudinal analysis. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry. RESULTS: In the discovery cohort, sST2 predicted 90-day mRS 3-6 (C index = 0.724, p < 0.001) and mortality in Kaplan-Meier analysis (p < 0.001). The association with functional status was independent of age, sex, World Federation of Neurosurgical Societies score, modified Fisher score, treatment modality, and cardiac comorbidities (adjusted odds ratio = 2.28, 95% confidence interval = 1.04-5.00, p = 0.039). Higher sST2 concentration was observed in those patients with DCI (90.8 vs 53.7ng/ml, p = 0.003). These associations were confirmed in a replication cohort. In patients with high sST2, flow cytometry identified decreased expression of CD14 (4.27 × 105 ± 2,950 arbitrary unit (AU) vs 5.64 × 105 ± 1,290 AU, p < 0.001), and increased expression of CD16 (39,960 ± 272 AU vs 34,869 ± 183 AU, p < 0.001). INTERPRETATION: Plasma sST2 predicts DCI, functional outcome, and mortality after SAH, independent of clinical and radiographic markers. Elevated sST2 is also associated with changes in CD14+ CD16+ monocytes. ANN NEUROL 2019;86:384-394.

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.

Presepsin As a Biomarker for Evaluating Prognosis and Early Innate Immune Response of Out-of-Hospital Cardiac Arrest Patients After Return of Spontaneous Circulation.

OBJECTIVES: After return of spontaneous circulation, patients who experienced out-of-hospital cardiac arrest present an impaired innate immune response that resembles sepsis. Presepsin, a new biomarker for sepsis, has not been studied in out-of-hospital cardiac arrest patients. This study explored the role of presepsin in evaluating the prognosis and early innate immune alteration of out-of-hospital cardiac arrest patients after return of spontaneous circulation by observing presepsin levels, CD14, and human leukocyte antigen-DR expression on monocytes. DESIGN: Retrospective analysis. SETTING: The emergency department of an urban university tertiary hospital. PARTICIPANTS: One hundred sixty-five out-of-hospital cardiac arrest patients with return of spontaneous circulation more than 12 hours, and 100 healthy individuals. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma presepsin and procalcitonin levels were tested after resuscitation (day 0) and on days 1 and 3 after return of spontaneous circulation. Presepsin levels were higher in out-of-hospital cardiac arrest patients than in healthy individuals. In the first 3 days, presepsin and procalcitonin levels were persistently lower in 28-day survivors and patients with favorable neurologic outcome patients than in 28-day nonsurvivors and patients with unfavorable neurologic outcome. On days 0, 1, and 3, different cut-off values of presepsin showed prognostic value for 28-day mortality and favorable neurologic outcomes similar to procalcitonin. CD14 and human leukocyte antigen-DR expression on monocytes were analyzed by flow cytometry. Compared with controls, CD14 expression in out-of-hospital cardiac arrest patients increased on day 1 and began to decrease on day 3, whereas human leukocyte antigen-DR+ monocyte percentages decreased on days 1 and 3. Presepsin and procalcitonin had a low positive correlation with CD14 expression and a strong negative correlation with human leukocyte antigen-DR+ monocyte percentages on day 1. CONCLUSIONS: Plasma presepsin concentrations are independent prognostic factors for out-of-hospital cardiac arrest patients after return of spontaneous circulation and are correlated with abnormal CD14 and human leukocyte antigen-DR expression on monocytes. Monitoring presepsin levels may be helpful for evaluating the prognosis and impaired innate immune response in the early period after return of spontaneous circulation.

Association of CD14 and macrophage migration inhibitory factor gene polymorphisms with inflammatory microRNAs expression levels in ankylosing spondylitis and polyarthralgia.

This study aimed to investigate the genetic basis of ankylosing spondylitis (AS) and polyarthralgia (PA) conditions among Indian subjects through genotyping two immune regulatory genes CD14 (-159C>T) and MIF (-173G>C) and find their association with the expression levels of three circulating inflammatory miRNAs. This investigation may provide early genetic cause of these two forms of arthritis and more optimal biological targets to predict early therapeutic outcomes. A total of 140 patients (AS: 70 and PA: 70) and 156 controls were recruited from Indian population. CD14 and MIF genotyping was performed using ARMS-PCR. Expression level of three inflammatory miRNAs (miRNA-146a, miRNA-155 and miRNA-181) was quantified using RT-qPCR. C/T genotype of CD14 gene was found to cause 2.06-fold risk of developing AS (CI 1.06-5.98, p = .04) as compared to others and G/C genotype in MIF also shown significant variation between AS and control subjects. In PA subjects, CD14 genotypes (C/T) was found to be associated with disease susceptibility and G/C genotype of MIF gene polymorphism showed 4.71-fold risk of developing PA (CI 2.58-8.62, p = .0001). The study also revealed significant upregulation of miRNA-155 expression in AS subjects (p = .0001) with more than 1.3-fold difference between AS and PA as compared to the control subjects. miRNA-155 had strong association with AS patients with CD14 genotypes (p < .05) than PA and control subjects. This study provides better understanding of the mechanisms and disease susceptibility for MIF and CD14 genetic variants and inflammatory miRNAs networks involved in AS and PA.

Human colostrum action against Giardia lamblia infection influenced by hormones and advanced maternal age.

Children are more susceptible to Giardia lamblia infection. Cells and hormones contained in human colostrum have an immunoprotective action against giardiasis, but the effects of advanced maternal age on these components are poorly understood. This study analyzed the colostrum of older women to determine melatonin and cortisol levels besides the participation of these hormones on the functional activity of phagocytes against G. lamblia. Colostrum samples were collected from younger (18 to 35 years old) and older (over 36 years old) lactating women. Colostrum samples were subjected to melatonin and cortisol determination, immunophenotyping, quantification of superoxide release, and assessment of phagocytic rate and microbicidal activity of phagocytes treated with hormones and in the presence of G. lamblia. Colostrum from mothers of advanced age contained higher melatonin and cortisol levels and a lower rate of cells expressing CD14+ and CD15+. In the colostru of these older mothers, melatonin increased superoxide release by phagocytes. In both groups, superoxide release by phagocytes treated with cortisol was higher in the presence of G. lamblia. In colostrum from mothers of advanced age, mononuclear (MN) phagocytes treated with melatonin showed higher phagocytosis of G. lamblia and higher microbicidal index. In younger mothers, MN and polymorphonuclear (PMN) colostrum phagocytes exhibited higher rates of G. lamblia elimination when treated with both melatonin and cortisol. In older mothers, cortisol and melatonin regulation for the functional activity of colostrum phagocytes against G. lamblia may represent an additional defense mechanism, relevant for the protection and treatment of parasitic infections in breastfed children.

The CD14 (-159 C/T) SNP is associated with sCD14 levels and allergic asthma, but not with CD14 expression on monocytes.

LPS-ligation to CD14/TLR-4 on monocytes/macrophages triggers the production of IL-12-family cytokines. IL12/18 promote TH1-differentiation, counteracting the TH2-driven asthma. Therefore, CD14 modulation could alter the TH2-differentiation and should be taken into account when studying asthma. To analyse the alteration in CD14 levels and its association with CD14 (-159 C/T) SNP (rs2569190) in Caucasian adults with stable allergic asthma, we performed a cross-sectional study (277 healthy subjects vs. 277 patients) where clinical parameters, CD14 values and the CD14 (-159 C/T) SNP were studied. Apart from typical biomarkers, we found an increment of neuron-specific enolase (NSE) in allergic asthma, probably linked to monocyte activity. Indeed, we evidenced increased monocyte numbers, but lower CD14 expression and normalised sCD14 values in patients. Moreover, we noticed an association of the T allele (P = 0.0162) and TT genotype (P = 0.0196) of the CD14 SNP with a decreased risk of allergic asthma and augmented sCD14 levels. In conclusion, monocyte CD14 expression and normalized sCD14 values were reduced in stable state asthmatics, and this could be related to the presence of an expanded CD14low monocyte subset. This study also demonstrates that the CD14 (-159 C/T) polymorphism is a risk factor for moderate-severe allergic asthma in adult Caucasians.

Anti-inflammatory effect of diterpenes-enriched fractions from Pterodon polygalaeflorus through inhibition of macrophage migration and cytokine production.

OBJECTIVES: To evaluate the anti-inflammatory potential of Pterodon polygalaeflorus hexane extract (HE) and its fractions on macrophage migration in vitro and in vivo. METHODS: Hexane extract from P. polygalaeflorus fruits was fractionated and yielded four fractions. RAW 264.7 cells were treated with samples to evaluate cell viability (MTT assay), cell migration (wound healing and transwell assays), CD14 expression (flow cytometry), iNOS and cytokine mRNA expression (RT-qPCR), NO (Griess reaction) and cytokine (ELISA) production. In vivo migration was evaluated on the thioglycollate-induced peritonitis model. Qualitative analysis was performed by GC-MS. KEY FINDINGS: All fractions inhibited the NO production by LPS-stimulated RAW 264.7 cells. Fr3 and Fr4 presented the lowest IC50 values. The expressions of iNOS and IL-1ß, TNF-α and IL-10 cytokines were inhibited by Fr3 and Fr4, whereas the CD14 expression was only inhibited by Fr3. All the samples inhibited RAW 264.7 migration in the wound healing and transwell assays. Fr3 and Fr4 reduced the migration of Mac-1+ Gr-1- cells to the peritoneum and presented in their compositions: 6α-hydroxy-7ß-acetoxyvouacapan-17ß-oate, methyl 6α,7ß-dihydroxyvouacapan-17ß-oate, methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate, geranylgeraniol and 14,15-epoxy-geranylgeraniol. CONCLUSIONS: The anti-inflammatory effects of Fr3 and Fr4 involve inhibition of cell migration, iNOS expression and NO production, cytokine expression (mRNA and proteins) and CD14 expression (Fr3).

Acanthoic acid suppresses lipin1/2 via TLR4 and IRAK4 signalling pathways in EtOH- and lipopolysaccharide-induced hepatic lipogenesis.

OBJECTIVES: In alcoholic liver disease, alcohol and lipopolysaccharide (LPS) are major stimulation factors of hepatic lipogenesis. Our objective was to determine the protective mechanism of acanthoic acid (AA) in EtOH- and LPS-induced hepatic lipogenesis. METHODS: HSC-T6 cells were treated with ethanol (200 mm) plus LPS (1 µg/ml) for 1 h, followed by AA (10 or 20 µm) for another 6 h. C57BL/6 mice were pretreated with of AA (20 and 40 mg/kg) or equal volume of saline and then exposed to three doses of ethanol (5 g/kg body weight) within 24 h. The mice were sacrificed at 6 h after the last ethanol dosing. KEY FINDINGS: Acanthoic acid significantly decreased the expressions of α-SMA, collagen-I, SREBP-1, and lipin1/2 induced, also decreased fat droplets caused by EtOH/LPS. AA treatment decreased the protein expressions of TLR4, CD14, IRAK4, TRAF3, p-TAK1 and NF-κB increased by EtOH/LPS on HSC cells. Results in vivo were consistent with results in vitro. CONCLUSIONS: Our data demonstrated that AA might modulate hepatic fibrosis and lipid deposition in HSC-T6 cell stimulated with ethanol combined with LPS by decreasing lipin1/2 via TLR4 and IRAK4 signalling pathways, and AA might be considered as a potential therapeutic candidate for alcoholic liver disease.

The effect of alcohol on the differential expression of cluster of differentiation 14 gene, associated pathways, and genetic network.

Alcohol consumption affects human health in part by compromising the immune system. In this study, we examined the expression of the Cd14 (cluster of differentiation 14) gene, which is involved in the immune system through a proinflammatory cascade. Expression was evaluated in BXD mice treated with saline or acute 1.8 g/kg i.p. ethanol (12.5% v/v). Hippocampal gene expression data were generated to examine differential expression and to perform systems genetics analyses. The Cd14 gene expression showed significant changes among the BXD strains after ethanol treatment, and eQTL mapping revealed that Cd14 is a cis-regulated gene. We also identified eighteen ethanol-related phenotypes correlated with Cd14 expression related to either ethanol responses or ethanol consumption. Pathway analysis was performed to identify possible biological pathways involved in the response to ethanol and Cd14. We also constructed a genetic network for Cd14 using the top 20 correlated genes and present several genes possibly involved in Cd14 and ethanol responses based on differential gene expression. In conclusion, we found Cd14, along with several other genes and pathways, to be involved in ethanol responses in the hippocampus, such as increased susceptibility to lipopolysaccharides and neuroinflammation.

CD14brightCD16+ intermediate monocytes are induced by interleukin-10 and positively correlate with disease activity in rheumatoid arthritis.

BACKGROUND: Three different subsets of circulating human monocytes, CD14brightCD16- (classical), CD14brightCD16+ (intermediate), and CD14dimCD16+ (non-classical) have been recently identified. It has been reported that CD14brightCD16+ monocytes are increased in rheumatoid arthritis (RA). However, the role of each monocyte subset in the pathogenesis of RA is still unclear. The purpose of this study was to investigate the association of CD14brightCD16+ monocytes with RA. METHODS: The study enrolled 35 patients with RA and 14 healthy volunteers. The three subsets of peripheral blood monocytes were analyzed by flow cytometry. Serum cytokines were measured at baseline in patients with RA and in healthy volunteers. CD14brightCD16- monocytes were isolated and cultured in vitro with different cytokines for 14 hours, and CD16 induction was assessed. RESULTS: The proportion of CD14brightCD16+ monocytes, and serum interleukin (IL)-6, IL-8, and IL-10 were increased in patients with RA compared to healthy controls. The proportion of CD14brightCD16+ monocytes correlated with the disease activity of RA positively, whereas the proportion of CD14brightCD16- monocytes correlated negatively. When isolated CD14brightCD16- monocytes were stimulated with IL-6, IL-8, and IL-10, the only cytokine that significantly induced CD16 expression on the cells was IL-10. CONCLUSIONS: The proportion of CD16brightCD14+ monocytes was positively correlated with RA disease activity. The expression of CD16 in monocytes was induced by IL-10 but not IL-6, and IL-8 was enhanced in the sera of patients with RA. Our results suggest that CD16brightCD14+ monocytes are involved in the pathogenesis of RA and that IL-10 is a key cytokine that regulates CD16 expression in monocytes.

GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes.

Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding-and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.

Etomidate Mitigates Lipopolysaccharide-Induced CD14 and TREM-1 Expression, NF-κB Activation, and Pro-inflammatory Cytokine Production in Rat Macrophages.

This study was aimed at investigating the effect of etomidate on the viability of rat macrophages and the function of lipopolysaccharide (LPS)-stimulated macrophages as well as the potential mechanisms. Rat macrophages were isolated and treated with different doses of etomidate for 24 h, and their viability was determined by the CCK-8 assay. Furthermore, macrophages were treated with, or without, 1 µg/ml of LPS, and/or 2.5 or 5 µM etomidate in the presence or absence of a TREM-1 inhibitor (LP17, 100 ng/ml), and the levels of TNF-α, IL-6, CD14, and TREM-1 in the different groups of cells were determined by quantitative RT-PCR, ELISA, and Western blot assays. The levels of NF-κB activation in the different groups of cells were analyzed by an electrophoretic mobility shift assay (EMSA). Etomidate at 31.25 µM or a low dose did not affect the viability of rat macrophages, while etomidate at higher doses reduced the viability of macrophages in vitro. Treatment with 2.5 or 5 µM etomidate or with LP17 alone did not affect the levels of TNF-α, IL-6, CD-14, and TREM-1 in macrophages. Treatment with etomidate significantly mitigated LPS-stimulated TNF-α, IL-6, CD-14, and TREM-1 expression (p < 0.05 for all) and inhibited LPS-induced NF-κB activation in macrophages in vitro. However, treatment with both etomidate and LP17 did not enhance the inhibitory effects in macrophages. Hence, etomidate mitigates LPS-up-regulated pro-inflammatory cytokine production and inhibits LPS-enhanced CD14 and TREM-1 expression and NF-κB activation in macrophages.

TNF-α-mediated microRNA-136 induces differentiation of myeloid cells by targeting NFIA.

Immune cell-lineage specification and function are influenced by progenitor origin and environmental factors. The mechanism of differentiation of immune cells, such as neutrophils, monocytes, and myeloid-derived suppressor cells, in inflammatory environments has not been elucidated completely. In this study, we have identified human microRNA-136 as a positive regulator of the differentiation of granulocytes and monocytes. Ectopic microRNA-136 induced cells to express higher levels of CD11b, CD14, and C/EBPε, secrete more cytokines, and synthesize higher levels of reactive oxygen species and H(2)O(2). microRNA-136 was shown to target and degrade multiple differentiation-associated molecules, such as the transcription factor NFIA, which induced the release of another microRNA, microRNA-223, with the ability to promote CD11b expression. Furthermore, microRNA-136 expression was remarkably increased by TNF-α, which activated NF-κB to bind to the DNA-promoter region controlling microRNA-136 expression. Additionally, TNF-α may alter NFIA expression through its modulation of microRNA-136 expression. Thus, TNF-α-mediated microRNA-136 may play a critical role in the generation and differentiation of inflammatory immune cells.

Mannan-binding lectin at supraphysiological concentrations inhibits differentiation of dendritic cells from human CD14+ monocytes.

Mannan-binding lectin (MBL), a circulating C-type lectin, is an important member of the defense collagen family. It exhibits a high potential for recognizing broad categories of pathogen-associated molecular patterns and initiating complement cascade responses. DCs are well-known specialist antigen-presenting cells that significantly trigger specific T cell-mediated immune responses. In our previous study, it was observed that high concentrations of MBL significantly attenuate LPS-induced maturation of monocyte-derived DCs (MoDCs). In the current study, it was postulated that MBL at similar supraphysiological concentrations would affect early differentiation of MoDCs in some way. CD14(+) monocytes from human peripheral blood mononuclear cells were cultured with granulocyte-macrophage colony-stimulating factor and IL-4 in the presence or absence of physiological (1 µg/mL) and supraphysiological concentrations (20 µg/mL) of MBL protein, respectively. Phenotypic analysis indicated that the differentiated DCs incubated with high concentrations of MBL expressed MHC class II and costimulatory molecules (e.g., CD80 and CD40) more weakly than did control groups. The secretion of IL-10 and IL-6 increased markedly, whereas their mixed lymphocyte reaction-stimulating capacity decreased. Members of the signal transducer and activator of transcription family were also found to be differentially regulated. Thus, beyond the role of MBL as an opsonin, our data reveal a possible inhibitory effect of MBL at high concentrations in monocyte-DC transition, which probably provides one way of regulating adaptive immune responses by strict regulation of DCs, making MBL a better prospect for controlling relevant pathological events such as autoimmune diseases.

Impact of Detachment Methods on M2 Macrophage Phenotype and Function.

The methods of cell detachment influence phenotype and function of human macrophages cultured in vitro. However, comparative studies defining the influence of cell detachment techniques on secondary characterization of M1 or M2 polarized macrophages are largely absent from the literature. In this study we evaluated the impact of trypsin, accutase, EDTA, PBS, and cell scraping on: A. cell recovery, B. phenotype and C. function of in vitro polarized macrophages. Our data demonstrate that while exposure to trypsin or accutase yields highly efficient recovery of viable cells, such chemical cleavage results in loss of select M2 cell surface markers with correlative changes in cell function. In contrast, phenotype and function are maintained following detachment by EDTA on ice. Our data suggest that seemingly "trivial" changes in methodologies for macrophage detachment induce both variable and profound changes on cell phenotype and function which can dramatically impact the results of polarization experiments.

Polychlorinated Biphenyls (PCB 101, 153, and 180) Impair Murine Macrophage Responsiveness to Lipopolysaccharide: Involvement of NF-κB Pathway.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are persistent organic pollutants, associated with a range of adverse health effects, including interference with the immune system. In this study, we investigate the capability of NDL-PCBs 101, 153, and 180, 3 of the 6 NDL-PCBs defined as indicators, to impair the immune response in lipopolysaccharide (LPS)-activated J774A.1 and primary murine macrophages. Our results clearly demonstrate that the exposure of J774A.1 and primary macrophages to NDL-PCB 153 or 180 or all NDL-PCBs mixtures causes a significant reduction in LPS-induced cytokine/chemokine synthesis, such as tumor necrosis factor-α and interleukin-6, together with monocyte chemoattractant protein-1, involved in cell recruitment. Moreover, PCBs were found to suppress LPS-stimulated NO production, and to reduce cyclooxygenase-2 and inducible nitric oxide synthase expression in J774A.1 and primary macrophages. At mechanistic level, PCBs significantly counteract the LPS-driven toll-like receptor (TLR) 4 and CD14 upregulation, therefore inhibiting downstream nuclear factor-κB (NF-κB) activation in J774A.1. Furthermore, PCBs determine a significant loss of macrophage endocytic capacity, a prerequisite for efficient antigen presentation. Taken together, these data indicate that NDL-PCBs reduce macrophage responsiveness, particularly when they are combined at concentrations per se inactive, impairing the capability to orchestrate a proper immune response to an infectious stimulus, disrupting TLR4/NF-κB pathway.

Heterogeneity in the Locomotory Behavior of Human Monocyte Subsets over Human Vascular Endothelium In Vitro.

Human monocytes comprise three distinct subsets, defined by their relative expression of CD14 and CD16. These subsets appear to have different functional roles within homeostasis and inflammation, but little is known about the manner in which they interact with macro- and microvascular endothelial cells, a key enabling component for the fulfillment of their functional roles. In the present study, we examined the locomotory behavior of the three major human monocyte subsets over human endothelial monolayers subjected to physiologically relevant levels of shear flow in vitro. Each subset was shown to preferentially perform different types of locomotory behavior in a resting state. A long-range crawling behavior, similar to the "patrolling" behavior of murine Ly6C(-) monocytes, was observed in CD14(+)CD16(-) and CD14(dim)CD16(+) monocytes, but not in CD14(+)CD16(+) monocytes. CD14(dim)CD16(+) and CD14(+)CD16(+) monocytes showed a preference for adhering to microvascular over macrovascular endothelium, whereas CD14(+)CD16(-) monocytes showed the opposite. Transendothelial migration was not observed in CD14(dim)CD16(+) monocytes during the 30-min observation period. Long-range crawling behavior in CD14(dim)CD16(+) monocytes was abrogated by blockade of ICAM1, VCAM1, or CX3CL1, in contrast with CD14(+)CD16(-) monocytes, which only required ICAM1 for this behavior. These studies indicate the existence of subtype-specific human monocyte migratory behavior patterns with distinct adhesion molecule dependence, which may assist in elucidating their physiological function and relevance to disease.