1,25-Dihydroxyvitamin D3 Facilitates M2 Polarization and Upregulates TLR10 Expression on Human Microglial Cells.

OBJECTIVE: To explore the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, on M1/M2 polarization of human microglia and the expression of Toll-like receptor 10 (TLR10) on these cells, which has been suggested to play an inhibitory role in inflammation previously. METHODS: Microglial HMO6 cells were treated with 1,25(OH)2D3, and mRNA or protein levels of M1 and M2 cytokines and TLR10 were examined. RESULTS: 1,25(OH)2D3 upregulated TLR10 in HMO6 cells at both mRNA and protein level. 1,25(OH)2D3 enhanced basal mRNA expression of M2 cytokines, such as IL-10 and CCL17, but did not affect the expression of M1 cytokines, including IL-12 and TNF-α. 1,25(OH)2D3 downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 cytokines IL-12 and TNF-α. Concomitantly, it upregulated not only the M2 cytokines IL-10 and CCL17, but also TLR10 in microglial cells treated with LPS, in a concentration-dependent manner. CONCLUSIONS: Our results suggest that 1,25(OH)2D3 may exert anti-inflammatory action by facilitating the M2 polarization of human microglial cells.

The host response: Toll-like receptor expression in periprosthetic tissues as a biomarker for deep joint infection.

BACKGROUND: Toll-like receptors (TLRs) 1 and 6 are consistent molecular indicators of the host inflammatory response against bacterial infection. Our aims were to determine whether TLR elevation could be detected in infected periprosthetic tissues and to assess the utility of these biomarkers as tests for detecting a periprosthetic joint infection. METHODS: Fifty-nine patients undergoing revision total joint arthroplasty (twenty-seven hips and thirty-two knees) were prospectively evaluated for periprosthetic joint infection according to currently recommended diagnostic criteria. Nine patients were excluded because of insufficient work-up, leaving fifty available for study. Of these, twenty-one were categorized as infected and twenty-nine as noninfected. Periprosthetic tissues were collected intraoperatively, and total RNA was extracted by standard techniques. Expression of TLR messenger RNAs was assessed by first-strand complementary DNA synthesis from 1 µg of total RNA followed by real-time PCR (polymerase chain reaction). Results were normalized relative to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Expression of TLRs 1, 6, and 10 in the infected and noninfected groups was compared with use of the Student t test. The receiver operating characteristic curve, area under the curve (AUC), sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated to determine the accuracy of each TLR for predicting periprosthetic joint infection at its optimal diagnostic threshold. RESULTS: Mean TLR1 mRNA expression was significantly elevated in infected compared with noninfected samples (0.600 compared with 0.005, p = 0.0003); the same was true of TLR6 (0.208 compared with 0.0165, p = 0.0059) but not of TLR10 (0.00019 compared with 0.00014, p = 0.6238). The AUC was 0.995 for TLR1, 0.883 for TLR6, and 0.546 for TLR10. The optimal threshold for diagnosing periprosthetic joint infection was 0.0924 for TLR1 (sensitivity = 95.2%, specificity = 100%, LR+ = 13.80, LR- = 0.91) and 0.0215 for TLR6 (sensitivity = 85.7%, specificity = 82.8%, LR+ = 4.98, LR- = 0.83). CONCLUSIONS: In our pilot study, TLR1 expression in periprosthetic tissues most accurately predicted periprosthetic joint infection. This measure of the host response may be particularly helpful in detecting culture-negative infections and avoiding false positives resulting from contamination. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Reduced expression of TLR3, TLR10 and TREM1 by human macrophages in Chronic cavitary pulmonary aspergillosis, and novel associations of VEGFA, DENND1B and PLAT.

Chronic cavitary pulmonary aspergillosis (CCPA) is an uncommon but serious pulmonary disease of humans, with an annual mortality rate of 10-30%. It is caused by the fungus Aspergillus fumigatus. Patients are overtly immunocompetent; however, some immunogenetic defect is likely. To investigate this, we performed a genetic association study analysing biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls, and investigated gene expression in monocyte-derived macrophages from patients and controls at baseline and during stimulation with A. fumigatus. Single-nucleotide polymorphisms (SNPs) associated with CCPA were found in TLR1, CLEC7A (dectin-1), PLAT (n=2), VEGFA, and DENND1B. Macrophages from CCPA patients showed low TLR3 and TLR10 expression and high TREM1 expression at baseline, as compared with macrophages from healthy subjects, with major expression differences being seen in most Toll-like receptors (TLRs) during 9 h of co-culture with A. fumigatus. The differences in baseline expression between the healthy and CCPA groups suggest roles for TLR3 and TLR10 in susceptibility to CCPA, and the association of SNPs in PLAT (n=2), VEGFA and DENND1B supports novel roles for plasminogen activation and angiogenesis and of these genes specifically in susceptibility to CCPA.

TLRs innate immunereceptors and Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) CIDR1α-driven human polyclonal B-cell activation.

Chronic malaria severely affects the immune system and causes polyclonal B-cell activation, as evidenced by the presence of hypergammaglobulinemia, elevated levels of autoantibodies, loss of B-cell memory and the frequent occurrence of Burkitt's lymphomas (BL) in children living in malaria endemic areas. Previous studies have shown that the cysteine-rich interdomain region 1α (CIDR1α) of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) of the FCR3S1.2 strain, subsequently named CIDR1α, interacts with B cells partially through the binding to the B-cell receptor (BCR). This interaction leads to an activated phenotype, increased survival, and a low degree of proliferation. CIDR1α preferentially activates the memory B-cell compartment, therefore PfEMP1 is considered to act as a polyclonal B-cell activator and its role in memory maintenance has been suggested. In this report, we extend the analysis of the PfEMP1-CIDR1α B-cell interaction and demonstrate that PfEMP1-CIDR1α increases the expression of TLR7 and TLR10 mRNA transcripts and sensitizes B cells to TLR9 signalling via the MyD88 adaptor molecule. Furthermore, despite its ability to bind to surface Igs, PfEMP1-CIDR1α-induced B-cell activation does not seem to proceed through the BCR, since it does not induce Lyn and/or phospho-tyrosine mediated signalling pathways. Rather PfEMP1-CIDR1α induces the phosphorylation of downstream kinases, such as ERK1/2, p38 and IKBα, in human B cells. These findings indicate that PfEMP1-CIDR1α induces a persistent activation of B cells, which in turn can contribute to the exhaustion and impairment of B-cell functions during chronic malaria infection.

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

FOXP3 regulates TLR10 expression in human T regulatory cells.

Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4(+)CD25(-)FOXP3(-) T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.

Menstrual cycle-dependent changes of Toll-like receptors in endometrium.

BACKGROUND: Rapid innate immune defences against infection usually involve the recognition of invading pathogens by specific pattern recognition receptors recently attributed to the family of Toll-like receptors (TLRs). Reports from our laboratory and others have demonstrated the existence of TLRs 1-6 in the female reproductive tract. However, little has been done to identify TLRs 7-10 in the female reproductive tract, particularly in the uterus. Also little information exists regarding variation in TLRs in the female reproductive tract during the menstrual cycle. METHOD: The distribution of TLR7-10 protein was detected by immunostaining in timed endometrial biopsies from normal women. RT-PCR was used to show the existence of TLR1-10 genes in endometrial tissue and real-time PCR analysis to investigate the relative expression of these genes during the menstrual cycle in normal human endometrium. RESULTS: TLR7-10 proteins were detected in endometrial epithelium and stroma. TLR1-10 genes were expressed in human endometrial tissue, and the mean relative expression of TLR2-6, 9 and 10 genes was significantly higher during the secretory phase compared with other phases of the menstrual cycle. CONCLUSIONS: TLR7-10 localization is not limited to endometrial epithelium but is also present in the stroma of the endometrial tissue. Endometrial TLR2-6, 9 and 10 genes are cyclically expressed during the menstrual cycle.