Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon-α via toll-like receptor 9

Background High‐affinity IgE receptor (Fc?RI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro‐inflammatory cytokines (IL‐6, TNF‐α) following Fc?RI stimulation – a mode of activation that simultaneously reduces expression of Toll‐like receptor 9 (TLR9). Whether or not TLR9 and/or Fc?RI levels and their function on dendritic cells relate to allergic status is unknown. Objective The aim of this study is to compare the innate (TLR9‐mediated) immune response of human pDCs to TLR9 and Fc?RIα receptor expression in allergic and non‐allergic subjects. Methods Basophil‐depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non‐allergic subjects. Intracellular TLR9 and surface Fc?RIα expression in blood dendritic cell antigen‐2‐positive cells were determined by flow cytometry. Activating anti‐IgE antibody, anti‐Fc?RIα antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA. Results No difference in the frequency of pDCs was detected among allergic (n=9) vs. non‐allergic (n=11) subjects (P=0.261). While there was also no difference in the baseline expression of TLR9, pDCs from allergic subjects produced sixfold less IFN‐α when stimulated with CpG (P=0.002). Conversely, there was higher Fc?RIα expression (P=0.01) on the pDCs of allergic subjects. Conclusions Impaired TLR9‐dependent immune responses in human pDCs are associated with allergic status and inversely correlated with Fc?RIα expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG‐based immunotherapy.     

Subcutaneous allergen immunotherapy restores human dendritic cell innate immune function

Background We recently reported that human blood dendritic cells from allergic subjects have impaired IFN‐α production following toll‐like receptor 9 (TLR9)‐dependent innate immune stimulation. It is not known how subcutaneous allergen immunotherapy (SCIT) affects dendritic cell immune responses. Objective The aim of this study is to determine how SCIT affects human dendritic cell function. Methods Peripheral blood mononuclear cell (PBMC) and plasmacytoid dendritic cells (pDCs) were isolated from the blood of seven dust mite allergic subjects at baseline and upon reaching a standard SCIT maintenance dose that included dust mite and other aeroallergens. Cells were stimulated with various adaptive and innate immune receptor stimuli, or media alone for 20 h with secreted cytokine levels determined by ELISA. A portion of the cells were used to measure intracellular signalling proteins by flow cytometry. Humoral immune responses were measured from plasma. Results SCIT resulted in a threefold increase in PBMC production of IFN‐α in response to CpG at 100 nm (P=0.015) and at 500 nm (P=0.015), n=7. The predominant cell type known to produce IFN‐α in response to CpG (CpG ODN‐2216) and other TLR9 agonists is the pDC. As expected, a robust innate immune response from isolated pDCs was re‐established among allergic subjects undergoing SCIT resulting in a fivefold increase in IFN‐α production in response to CpG at 500 nm (P=0.046), n=7. In contrast, IL‐6 production was unaffected by SCIT (P=0.468). Consistent with published reports, IgG4 blocking antibody increased 10‐fold with SCIT (P=0.031), n=7. There was no significant increase in the frequency of pDCs or the expression of TLR9 that would account for the rise in IFN‐α production. Conclusions Allergen immunotherapy increases dendritic cell TLR9‐mediated innate immune function, which has previously been shown to be impaired at baseline in allergic subjects. Cite this as: J. R. Tversky, A. P. Bieneman, K. L. Chichester, R. G. Hamilton and J. T. Schroeder, Clinical & Experimental Allergy, 2010 (40) 94–102.     

Evaluation of the effects of probiotic supplementation from the neonatal period on innate immune development in infancy

BACKGROUND: Activation of the innate immune system by microbial stimulation is believed to be critical for normal immune maturation, and there has been speculation that these pathways are important for inhibiting allergic-immune responses. OBJECTIVE: To assess innate immune function following a 6-month supplementation with probiotic bacteria. METHODS: Two hundred and thirty-one allergic, pregnant women were recruited into a randomized, controlled trial. The infants received either a probiotic (Lactobacillus acidophilus LAVRI-A1; Probiomics) or placebo (maltodextrin alone) daily for the first 6 months of life. Mononuclear cell samples were available from 118 infants. Functional responses to toll-like receptor (TLR) were assessed using ligands for TLR2 (Pansorbin) and TLR4/CD14 [lipopolysaccharide (LPS)] and measuring cytokine responses in the supernatants. Antigen-presenting cell function, as well as capacity for cytokine production (IL-12p70 and IL-10) was assessed. RESULTS: Infants in the probiotic group did not demonstrate differences in innate immune function compared with those in the control group. No differences were seen when cytokine responses were examined following stimulation with Pansorbin (TLR2) or LPS (TLR4). Similarly, no differences were seen in the antigen-presenting capacity of these infants. The mean fluorescence intensities of human leucocyte antigen-DR (HLA-DR) on monocytes, B cells and dendritic cells (DC) subsets were not affected, nor were the percentage of circulating DC subsets affected by a 6-month supplementation with L. acidophilus LAVRI-A1. CONCLUSIONS: Probiotic supplementation with L. acidophilus for the first 6 months of life did not alter early innate immune responses in this population at high risk of developing allergic disease.     

Copy number of the X‐linked genes TLR7 and CD40L influences innate and adaptive immune responses

The number of the X chromosome–linked genes has been previously suggested to influence immune responses and the development of autoimmune diseases. In the present study, we aimed at evaluating the level of expression of CD40L (an X‐linked gene involved in adaptive immunity) and TLR7 (an X‐linked gene involved in innate immunity) in a variety of different karyotypes. Those included males, females and patients with X chromosome aneuploidy. Healthy females (46, XX; n = 10) and healthy males (46, XY; n = 10) were compared to females with Turner syndrome (TS) (45, X; n = 11) and males with Klinefelter syndrome (KS) (47, XXY; n = 5). Stimulation of peripheral blood mononuclear cells (PBMCs) with PMA and ionomycin resulted in higher percentage of CD3 + CD40L+ T cells (P < 0.001) and higher level expression of CD40L in T cell (P < 0.001) in female and KS patients compared with male and TS patients. TLR7‐mediated IFN‐alpha production by HLADR + CD3? CD19? cells was significantly upregulated in healthy women compared with healthy males, TS and KS patients (P < 0.001). TLR7 agonist‐stimulated PBMCs from healthy females and KS patients expressed significantly higher levels of TLR7 mRNA than those from male and TS patients (P < 0.05). The increased expression of the X‐linked genes TLR7 and CD40L in healthy females and KS patients suggests that the presence of two X chromosomes plays a major role in enhancing both innate and adaptive immune responses. These results may contribute to the explanation of sex‐based differences in immune biology and the sex bias in predisposition to autoimmune diseases.     

Maternal allergy influences p38-mitogen-activated protein kinase activity upon microbial challenge in CD14+ monocytes from 2-year-old children

Background The development of allergic diseases is dependent on genetic and environmental factors. It has been shown previously that cord blood mononuclear cells (CBMCs) from infants with parental allergy have altered cytokine profiles upon bacterial encounter; it might be possible that such impairment persists during the early years of childhood. Objective The aim of this study was to investigate anti‐microbial responses with regard to p38‐mitogen‐activated protein kinase (MAPK) activity in CD14⁺ monocytes and IL‐6 release from mononuclear cells in the same group of children at birth and at 2 years of age. Methods Paired samples of CBMCs and peripheral blood mononuclear cells (PBMCs) were stimulated with either lipopolysaccharide (LPS) or peptidoglycan in vitro. CD14⁺ monocytes were analysed for p38‐MAPK activity by flow cytometry, and soluble IL‐6 receptor, soluble glycoprotein130 and IL‐6 release from PBMC cultures were quantified by ELISA. Results CBMCs from newborns with allergic mothers tended to have a lower IL‐6 response following an LPS (P=0.09) challenge compared with the group without maternal allergy while p38‐MAPK activation levels did not differ between the groups. PBMCs from 2‐year‐olds with allergic mothers released significantly less (P<0.05) IL‐6 upon peptidoglycan stimuli compared with age‐matched infants with non‐allergic mothers. Infants with allergic mothers displayed markedly reduced CD14⁺ monocyte p38‐MAPK phosphorylation after LPS (P<0.05) and peptidoglycan (P<0.01) challenge. This altered anti‐microbial response was attributed to maternal allergy rather than to being IgE‐sensitized at 2 years of age. Conclusion Monocytes from children with allergic mothers are less responsive to bacterial challenge than monocytes from children with non‐allergic mothers, and this impairment persists during the first 2 years of infancy.     

Regulatory cells, cytokine pattern and clinical risk factors for asthma in infants and young children with recurrent wheeze

Background Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. Objectives This study was designed to assess and compare the immunological status during the first 2 years in steroid‐naïve young children with three episodes of physician‐confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age‐matched healthy controls (n=30). Methods Peripheral blood CD4⁺CD25⁺ and CD4⁺CD25^high T cells and their cytotoxic T‐lymphocyte‐associated antigen‐4 (CTLA‐4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN‐γ, TGF‐β and IL‐10), CTLA‐4 and Foxp3 mRNA expression were evaluated (real‐time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12‐myristate 13‐acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). Results Flow cytometry results showed a significant reduction in the absolute number of CD4⁺CD25^high and the absolute and percentage numbers of CD4⁺CD25⁺CTLA‐4⁺ in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4⁺CD25⁺ (P=0.01) and CD4⁺CD25^high (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA‐4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA‐4 (P=0.03) and IFN‐γ (P=0.04) expression was diminished in wheezy children compared with healthy children. High‐risk children had lower expression of IFN‐γ (P=0.03) compared with low‐risk and healthy children and lower expression of CTLA‐4 (P=0.01) compared with healthy children. Conclusions Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.     

The role of T-regulatory cells and toll-like receptors 2 and 4 in atopic dermatitis

Regulatory T cells (Tregs), toll‐like receptors (TLRs) and interleukin‐17 (IL‐17) play important role in inflammatory diseases; however, their relevance in atopic dermatitis (AD) pathogenesis is not clear. The aim of study was to evaluate the number of circulating Tregs and peripheral blood mononuclear cells (PBMC) expressing TLR2 and TLR4 receptors in patients with AD. PBMC and CD4+/CD25^high+ Tregs were isolated from the whole blood of 32 AD patients and 36 healthy volunteers. Expression of CD4+CD25+, TLR2 and TLR4 receptors and IL 17+ was assessed with the flow cytometry. In the peripheral blood of AD patients, the percentage of Tregs was significantly higher when compared with the controls (P = 0.0003). The number of TLR2+PBMC and TLR4+ PBMC in AD patients was significantly lower than in the controls (P = 0.035; P = 0.001, respectively). Also the percentages of Tregs with expression of both TLR2+ and TLR4+ in AD patients were significantly lower than in the control (3.85 versus 21.6, P < 0.0001; 2.2 versus 17.6, P < 0.0001, simultaneously). The percentage of CD4+/CD25high+/FOXP3+ Treg lymphocytes with expression of IL‐17 was significantly higher in AD group than in healthy subjects (0.3% versus 0.06%; P = 0.011). Distinct number of Tregs and various distribution of TLR2 and TLR4 expression on PBMC in AD patients suggest their contribution in the pathogenesis of AD.     

Children with atopic histories exhibit impaired lipopolysaccharide‐induced Toll‐like receptor‐4 signalling in peripheral monocytes

Background The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll‐like receptor (TLR)‐4 on CD4⁺ cells in nasal mucosa. Objective To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4⁺ leucocytes. Methods Peripheral blood mononuclear cells from children (aged 2–18) and adults with or without a history of atopic conditions were cultured with/without IL‐4 or LPS for up to 24 h. Expression of surface TLR‐4, CD14, CD4, CD3, as well as of intracellular phosphorylated ^p42/p44ERK and p38 mitogen‐activated protein kinase (MAPK) were assessed by flow cytometry. Results A history of atopy in children was associated with impaired LPS‐induced TLR‐4‐dependent phosphorylation of ^p42/44ERK and p38 MAPK by CD4⁺ monocytes. Decreased LPS signalling was reproduced by pre‐incubation of control cells with recombinant IL‐4. LPS stimulation also decreased TLR‐4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4⁺ T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non‐atopic children, TLR‐4 expression on monocytes of children with atopic histories decreased as a function of age. Conclusions This study provides evidence for defective LPS recognition on circulating CD4⁺ leucocytes of subjects with atopic histories compared with those from non‐atopic children. CD4⁺TLR4⁺ monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR‐4‐mediated innate immune function compared with non‐atopic children. Cite this as: D. Préfontaine, A.‐A. Banville‐Langelier, P.‐O. Fiset, J. Guay, J. An, M. Mazer, Q. Hamid and B. D. Mazer, Clinical & Experimental Allergy, 2010 (40) 1648–1657.     

Impact of genetic variants of CD14 and TLR4 on subgingival periodontopathogens

CD14 and toll‐like receptor 4 (TLR4) are involved in host's immune response to bacterial pathogens including periodontal bacteria. Functional important gene polymorphisms are described for both genes. The aim of this study was to evaluate links between genetic polymorphisms of CD14 and TLR4 and risk markers of periodontitis in a multivariate model. One hundred and thirty‐three periodontitis patients (chronic: n = 60, aggressive: n = 73) and 80 healthy controls without periodontitis were included in the study. Polymorphisms in CD14 c.–159C>T and in TLR4 Asp299Gly, Thr399Ile were determined by restriction fragment length polymorphism analyses. The clinical investigation included smoking status, plaque and bleeding indexes, pocket depth and attachment loss. Subgingival bacterial colonization was analysed molecularbiologically using the micro‐Ident®test. Prevotella intermedia occurred less frequently in individuals positive for the TT genotype of CD14 in bivariate analysis (odds ratio = 0.36%, confidence interval: 0.14–0.91, P = 0.045). In binary logistic regression analyses, the occurrence of this bacterium was significantly decreased in TT carriers (odds ratio = 0.31%, confidence interval: 0.81–0.12, P = 0.017) considering age, smoking and maximum clinical attachment loss at microbial test site as confounding factors. However, no significant association with chronic and or aggressive periodontitis and polymorphisms in CD14 and TLR4 could be proven. Although the CD14 c.–159C>T polymorphism could be shown to be associated with subgingival colonization with P. intermedia, there is no evidence that CD14 and TLR4 polymorphisms investigated are independent risk factors for chronic or aggressive periodontitis in German periodontitis patients.     

乙型肝炎e抗原、s抗原对Toll样受体及共刺激分子表达的调控和影响

目的：探讨乙型肝炎e抗原、s抗原（HBeAg、HBsAg）对健康人外周血单个核细胞（PBMC）Toll样受体（TLRs）和共刺激分子表达的影响。方法：分别用HBeAg、HBsAg、空质粒以及卵清蛋白（OVA）作为无关蛋白刺激健康人外周血单个核细胞（PBMC）,采用流式细胞技术检测各刺激组CD14＋细胞TLR2、TLR4、CD86、PD-L1的表达水平。结果：健康人PBMC经HBeAg或HBsAg刺激后,CD14＋细胞表面TLR2、TLR4的表达明显低于未刺激组（P〈0.05）,CD14＋细胞PD-L1的表达则明显增高（P〈0.05）;HBeAg刺激后同时可见CD14＋细胞表面共刺激分子CD86的表达水平明显降低（P〈0.02）。结论 HBeAg和HBsAg通过上调单核细胞表面PD-L1的表达,下调CD14＋单个核细胞表面TLR2、TLR4和共刺激分子CD86的表达,削弱机体固有免疫系统的应答能力,降低启动特异性免疫的活化信号,从而导致免疫清除逃避。因此,HBeAg和HBsAg可能是促进HBV感染慢性化的重要因素。     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

Expression of adhesion molecules on mononuclear cells from individuals with stable atopic asthma

Activated mononuclear cells (lymphocytes and monocytes) (MNCs) circulate in peripheral blood and accumulate in the airways of individuals with steady-state asthma. The expression of adhesion molecules on MNCs of 10 patients with steady-state atopic asthma and 10 non-atopic control subjects was measured by flow cytometry. The mean fluorescence intensity of CD23 (P= 0.0003), CDl la (P= 0.034), and very late antigen-4 (VLA-4) (P= 0.016) was increased on lymphocytes of asthmatic patients relative to those of controls. Although the expression of CD16 (P= 0.002) and CD23 (P= 0.002). which are associated with differentiation into macrophages, was increased on monocytes of patients relative to those of controls, monocyte expression of VLA-4 (P= 0.006) and sialyl Lewis X (P= 0.005) was reduced. The concentration of interleukin-4 (lL-4) in serum was significantly higher in asthmatic patients than in normal subjects (P= 0.023), and a significant correlation was apparent between the serum concentration of lL-4 and the expression of VLA-4 on lymphocytes (p= 0.71, P= 0.03) or on monocytes (p=?0.72, P= 0.03) in asthmatic patients. Results suggest that lL-4 may contribute to the priming of adhesion molecule expression on lymphocytes even in steady-state conditions in individuals with chronic allergic inflammation.     

Association between levels of Toll-like receptors 2 and 4 and CD14 mRNA and allergy in pregnant women and their offspring

The microbial environment in early infancy or even in utero may modulate the risk to develop allergic disease. Since Toll-like receptors (TLR) recognize microbial products, we hypothesized that maternal allergies may be associated with decreased levels of TLR2, TLR4 and CD14 mRNA in mothers and their offspring. 185 healthy pregnant women from Germany (n = 48), Hungary (n = 50) and Spain (n = 87) were enrolled in a European multicenter study. Levels of TLR2, TLR4 and CD14 mRNA were quantified in maternal peripheral blood samples taken at delivery and placental cord blood samples. Numbers of TLR2+, TLR4+ and CD14+ monocytes were quantified by flow cytometry in 42 cord blood samples obtained from the German participants. Bivariate and multivariate regression analyses were performed. Maternal allergies were associated with significantly lower levels of TLR2/4/CD14 mRNA in maternal blood and cord blood samples. Maternal and fetal TLR2/4/CD14 mRNA levels were significantly correlated with each other (TLR2 r = 0.42; TLR4 r = 0.58; CD14 r = 0.54). The results suggest that maternal allergy status may affect allergic risk in offspring through a decreased expression of fetal TLR2/4/CD14.     

Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model

The in vitro Peripheral Tissue Equivalent (PTE) module is a three‐dimensional tissue‐engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll‐like receptor (TLR) agonists, including polyinosine‐polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1α/β, 6, 8 and 10 and tumour necrosis factor‐α. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100‐fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up‐regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE‐derived DC treated with Gardiquimod, a TLR‐7 agonist, significantly augmented anti‐tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33⁺) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen‐presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.     

Curcumin induces maturation-arrested dendritic cells that expand regulatory T cells in vitro and in vivo

Dendritic cells (DC) and regulatory T cells (T_regs) are vital to the development of transplant tolerance. Curcumin is a novel biological agent extracted from Curcuma longa (turmeric), with anti‐inflammatory and anti‐oxidant activity mediated via nuclear factor (NF)‐κB inhibition. We investigated the immunomodulatory effects of curcumin on human monocyte‐derived and murine DC. Human monocyte‐derived DC (hu‐Mo‐DC) were generated in the presence (CurcDC) or absence (matDC) of 25 µM curcumin, and matured using lipopolysaccharide (1 µg/ml). DC phenotype and allostimulatory capacity was assessed. CD11c⁺ DC were isolated from C57BL/6 mice, pretreated with curcumin and injected into BALB/c mice, followed by evaluation of in vivo T cell populations and alloproliferative response. Curcumin induced DC differentiation towards maturation‐arrest. CurcDC demonstrated minimal CD83 expression (<2%), down‐regulation of CD80 and CD86 (50% and 30%, respectively) and reduction (10%) in both major histocompatibility complex (MHC) class II and CD40 expression compared to matDC. CurcDC also displayed decreased RelB and interleukin (IL)‐12 mRNA and protein expression. Functionally, CurcDC allostimulatory capacity was decreased by up to 60% (P < 0·001) and intracellular interferon (IFN‐γ) expression in the responding T cell population were reduced by 50% (P < 0·05). T cell hyporesponsiveness was due to generation of CD4⁺CD25^hiCD127^loforkhead box P3 (FoxP3)⁺ T_regs that exerted suppressive functions on naïve syngeneic T cells, although the effect was not antigen‐specific. In mice, in vivo infusion of allogeneic CurcDC promoted development of FoxP3⁺ T_regs and reduced subsequent alloproliferative capacity. Curcumin arrests maturation of DC and induces a tolerogenic phenotype that subsequently promotes functional FoxP3⁺ T_regsin vitro and in vivo.     

Macrophage migration inhibitory factor, Toll-like receptor 4, and CD14 polymorphisms with altered expression levels in patients with ulcerative colitis

Ulcerative colitis is a multifactorial disease in which genetic factors play a major role. Functional mutations in the genes related to innate immune response exacerbate mucosal damage coupled with persistent inflammation. The cytokine macrophage migration inhibitory factor (MIF), CD14, and Toll-like receptor 4 (TLR4) are the central players with clearly defined roles in inflammation. The aim of this study was to investigate the association between MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms and mononuclear cell expression in patients with ulcerative colitis (UC). Genotyping of MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms was performed by amplification refractory mutation system-polymerase chain reaction and allele-specific amplification in 139 and 176 patients with UC and controls, respectively. Simultaneously, the expression levels of intracellular MIF, mCD14, and mTLR4 were determined in mononuclear cells using a flow cytometer. Polymorphisms in CD14-159C > T and TLR4-299A > G significantly affected mCD14 and mTLR4 expression levels and also increased susceptibility to UC. Although intracellular MIF expression levels differed among patient and control groups, the polymorphism in MIF 173G > C was not observed to be associated with a risk of UC.     

Different cytokine production and toll‐like receptor expression induced by heat‐killed invasive and carrier strains of Neisseria meningitidis

Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA‐DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat‐killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up‐regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA‐DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains.     

Expression of NK Cell and Monocyte Receptors in Critically Ill Patients – Potential Biomarkers of Sepsis

Sepsis is characterized by activation of both the innate and adaptive immune systems as a response to infection. During sepsis, the expression of surface receptors expressed on immune competent cells, such as NKG2D and NKp30 on NK cells and TLR4 and CD14 on monocytes, is partly regulated by pro‐ and anti‐inflammatory mediators. In this observational study, we aimed to explore whether the expression of these receptors could be used as diagnostic and/or prognostic biomarkers in sepsis. Patients with severe sepsis or septic shock (n = 21) were compared with critically ill non‐septic patients (n = 15). Healthy volunteers (n = 15) served as controls. To elucidate variations over time, all patients were followed for 4 days. Cell surface expression of NKG2D, NKp30, TLR4 and CD14 and serum levels of IL‐1β, IL‐6, IFN‐γ, TNF‐α, IL‐4 and IL‐10 was estimated by flow cytometry. We found that NK cell expression of NKG2D and monocyte expression of CD14 were lower in the septic patients compared with the non‐septic patients, both at ICU admission and during the observation period (P < 0.01 for all comparisons). Both at ICU admission, and during the observation period, levels of IL‐6 and IL‐10 were higher in the septic patients compared with the non‐septic patients (P < 0.001 for all comparisons). Conclusion: As both NKG2D and CD14 levels appear to distinguish between septic and non‐septic patients, both NKG2D and CD14 may be considered potential diagnostic biomarkers of severe sepsis and septic shock.     

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll‐like receptors (TLRs) and C‐type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c⁺, CD141⁺ and CD16⁺ myeloid DCs and CD123⁺ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR‐directed targeting. Whole blood was obtained from 13 allergic and six non‐allergic individuals. Mononuclear cells were purified and multi‐colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141⁺ DCs from allergic compared with non‐allergic donors. The subset‐specific PRR expression profiles suggests individual responsiveness to PRR‐targeting and supports functional specialization.     

The Synergy of ‐260T T CD14 and ‐308GG TNF‐α Genotypes in Survival of Critically Ill Patients

Literature suggests that the analysis of several polymorphic genetic markers is more informative than the analysis of a single polymorphism. In this study, we tested whether the shared inheritance of TLR2 and TLR4 and TNF‐α allelic variants may act in synergy with ‐260C>T CD14 SNP on the outcome from critical conditions. We monitored 524 critically ill patients from South Brazilian, daily from the ICU admission to their discharge from hospital, or death. Our results revealed that TLR2, TLR4 or TNF‐α SNPs alone did not show a significant role in the outcome from critical illness. However, when we performed a combined analysis with the CD14 inheritance, we detected a significant higher survivor rate in ‐260TT CD14/‐308GG TNF‐α individuals (P = 0.037). In the adjusted analysis including the main clinical predictors to mortality, we observed that ‐260TT/‐308GG double‐genotype was a significant protective factor towards survival (P = 0.046). An increased probability for survival of ‐260TT/‐308GG was also observed by ‘pathway genetic load’ analysis (unweighted: P = 0.041; weighted: P = 0.036). When we applied a hazard function analysis with the ‐260TT/‐308GG variable as a discriminating factor, ‐260TT/‐308GG patients group had, in fact, a higher survivor rate (P = 0.024). Connected to the beneficial effect of ‐260TT CD14, the ‐308GG TNF‐α genotype was protective against the reported over expression of TNF‐α caused by ‐308A rare allele. Results support the hypothesis that the interaction between ‐260C>T CD14 and ‐308G>A TNF‐α functional SNPs may be synergistically influencing the outcome of critically ill patients.