CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

Abstract Background Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1β. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1β in murine neutrophils through its ITIM receptor. Results We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1 −/− mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G−) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and β-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and β-actin from RP105. Conclusion This novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS

A Humanized UGT1 Mouse Model Expressing the UGT1A1*28 Allele for Assessing Drug Clearance by UGT1A1-Dependent Glucuronidation

Humanized mice that express the human UDP-glucuronosyltransferase (UGT) 1 locus have been developed in a Ugt1-null background as a model to improve predictions of human UGT1A-dependent drug clearance. Enzyme kinetic parameters (Km and Vmax) and pharmacokinetic properties of three probe drugs were compared using wild-type and humanized UGT1 mice that express the Gilbert’s UGT1A1*28 allele [Tg(UGT1A1*28) Ugt1(−/−) mice]. The well characterized substrate for UGT1A1, 7-ethyl-10-hydroxy-camptothecin (SN-38), showed the greatest difference in parent drug exposure (∼3-fold increase) and clearance (∼3-fold decrease) in Tg(UGT1A1*28) Ugt1(−/−) mice after intravenous administration compared with wild-type and phenobarbital-treated animals. In contrast, the clearance of the UGT2B7 substrate (−)-17-allyl-4, 5α-epoxy-3, 14-dihydroxymorphinan-6-one (naloxone) was not altered in Tg(UGT1A1*28) Ugt1(−/−) mice. In addition, pharmacokinetic parameters with 1-(4-fluorophenyl)3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone (ezetimibe, Zetia; Merck & Co., Whitehouse Station, NJ), considered to be a major substrate for UGT1A1, showed small to no dependence on UGT1A1-directed glucuronidation. Enzyme kinetic parameters assessed for SN-38, ezetimibe, and naloxone using liver microsomes prepared from wild-type and Tg(UGT1A1*28) Ugt1(−/−) mice showed patterns consistent with the in vivo pharmacokinetic data. For SN-38 glucuronidation, Vmax decreased 5-fold in Tg(UGT1A1*28) Ugt1(−/−) mouse liver microsomes compared with microsomes prepared from wild-type mice, and decreased 10-fold compared with phenobarbital-treated Tg(UGT1A1*28) Ugt1(−/−) mice. These differences are consistent with SN-38 glucuronidation activities using HLMs isolated from individuals genotyped as UGT1A1*1 or UGT1A1*28. For ezetimibe and naloxone the differences in Vmax were minimal. Thus, Tg(UGT1A1*28) Ugt1(−/−) mice can serve as a pharmacokinetic model to further investigate the effects of UGT1A1 expression on drug metabolism

Transmission analysis of <i>MEFV</i> in cohort of 120 fibromyalgia trios¹.

<p>Transmission analysis of <i>MEFV</i> in cohort of 120 fibromyalgia trios<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198625#t002fn001" target="_blank">¹</a>.</p

Wild type versus varCCL4 in the down-regulation of CCR5 in a 9 day mixed lymphocyte culture.

<p>Purified CD4 T-cells from mismatched donors were co-incubated for nine days and treated with WT or varCCL4 (50ng/mL) for 3 hours, surface stained with anti-CCR5 antibody and analyzed by flow cytometry (** P<0.01, *** P<0.001 in comparison with untreated control; # P<0.05 in comparison with WT CCL4).</p

Human skeletal muscles cells secrete CCL4, CCL3 and CCL5 after treatment with cytokines and chemokines.

<p>Confluent skeletal muscle myoblasts were treated with LPS (10 ng/mL), CCL2 (20 nM), CCL11 (20 nM), IL-13 (20 nM) or IFN<b>γ</b> (100 ng/mL) for 48hrs and cytokines levels were measured using cytokine multiplex analysis. Ctrl: untreated control, n = 4 (** P<0.01, *** P<0.001 in comparison with controls).</p

SNPs in inflammatory genes <i>CCL11</i>, <i>CCL4</i> and <i>MEFV</i> in a fibromyalgia family study

<div><p>Background</p><p>Fibromyalgia (FM) is a chronic pain syndrome with a high incidence in females that may involve activation of the immune system. We performed exome sequencing on chemokine genes in a region of chromosome 17 identified in a genome-wide family association study.</p><p>Methods and findings</p><p>Exome sequence analysis of 100 FM probands was performed at 17p13.3-q25 followed by functional analysis of SNPs found in the chemokine gene locus. Missense SNPs (413) in 17p13.3-q25 were observed in at least 10 probands. SNPs rs1129844 in <i>CCL11</i> and rs1719152 in <i>CCL4</i> were associated with elevated plasma chemokine levels in FM. In a transmission disequilibrium test (TDT), rs1129844 was unequally transmitted from parents to their affected children (p< 0.0074), while the <i>CCL4</i> SNP was not. The amino acid change (Ala23Thr), resulting from rs1129844 in <i>CCL11</i>, predicted to alter processing of the signal peptide, led to reduced expression of CCL11. The variant protein from <i>CCL4</i> rs1719152 exhibited protein aggregation and a potent down-regulation of its cognate receptor CCR5, a receptor associated with hypotensive effects. Treatment of skeletal muscle cells with CCL11 produced high levels of CCL4 suggesting CCL11 regulates CCL4 in muscle. The immune association of FM with SNPs in <i>MEFV</i>, a chromosome 16 gene associated with recurrent fevers, had a p< 0.008 TDT for a combined 220 trios.</p><p>Conclusions</p><p>SNPs with significant TDTs were found in 36% of the cohort for <i>CCL11</i> and 12% for <i>MEFV</i>, along with a protein variant in CCL4 (41%) that affects CCR5 down-regulation, supporting an immune involvement for FM.</p></div

Expression of wild type and variant (Ala23Thr) CCL11.

<p>Plasmids encoding the genes for wild type and variant (Ala23Thr) CCL11 were expressed in HEK cells and the expression levels determined by (<b>A</b>) EIA (triplicates; ***, p< 0.001) and (<b>B</b>) Coomassie Blue staining (triplicates). Control media (<b>A</b>: Ctrl) had undetectable levels of CCL11. Recombinant CCL11 standards (<b>B</b>: left) were used to quantitate protein expression of wild type (WT) and variant (A>T) CCL11 (<b>B</b>: right). Control media (Ctr) had <0.05 µg expression of CCL11. The difference in migration of recombinant standards vs expressed protein on SDS gel electrophoresis is due to glycosylation (confirmed by mass spectrometry, data not shown). <b>C</b>: Control monocytes expressing either wild type (n = 12) or varCCL11 (heterozygous, n = 8) were incubated in media for 18 hrs and CCL11 levels were measured (*, p<0.026). <b>D</b>: Eosinophils isolated from a donor were stained for CCR3 expression before (blue) or after incubation with equal amounts of WT (green) or varCCL11 (orange). The green and orange plots are identical. Isotype control (red).</p

SNPs of immune related genes in chromosome 17 from FM cohorts and transmission analysis from FM trios¹^,²^,³.

<p>SNPs of immune related genes in chromosome 17 from FM cohorts and transmission analysis from FM trios<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198625#t001fn001" target="_blank">¹</a>^,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198625#t001fn002" target="_blank">²</a>^,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198625#t001fn003" target="_blank">³</a>.</p

Functional analyses of WT vs varCCL4 in immature dendritic cells or CEM.NK^R CCR5 T-cells.

<p><b>A-B</b>. WT CCL4 and varCCL4 both stimulate and cross-block calcium mobilization in CCR5 positive immature dendritic cell (<b>A</b>) or CEM.NK^R CCR5 T-cells (<b>B</b>). Ionomycin and CCL3 were used as positive controls. <b>C-D</b>. varCCL4 induces greater down-regulation of cell surface CCR5 than WT CCL4 on immature dendritic cells (<b>C</b>) and CEM.NK^R CCR5 T-cell line (<b>D</b>). Cells were treated with 80 ng/mL of each CCL4 species for 3 hours, surface CCR5 was stained with anti-CCR5 antibody and analyzed by flow cytometry (** P<0.01, *** P<0.001 in comparison with untreated control; # P<0.05 in comparison with WT CCL4).</p

Plasma levels of CCL11, CCL4 and CCL3 in female FM patients with varCCL11 or varCCL4.

<p><b>A-C</b>, CCL11, CCL4, and CCL3 levels in female healthy controls (Ctrl, n = 48) vs female FM patients with wild type CCL11 (n = 19) or var CCL11 (n = 14). <b>D-F</b>, CCL4, CCL3 and CCL11 levels in female healthy controls (Ctrl, n = 48) vs wild type CCL4 (n = 49) or varCCL4 (n = 24) in female FM patients. (* P<0.05, ** P<0.01, *** P<0.001 in comparison with female healthy controls; # P<0.05 in comparison with female FM patients with wild type CCL4.</p