Fig 1.
Microglia expresses all the members of CLM family.
A) Genomic organization of CLM genes cluster at chromosomal region 11D. B) Sequence alignment of the Ig, transmembrane (cursive) and cytoplasmic domains of CLM members. Conserved cysteine residues are marked with asterisks. Transmembrane charged residues and cytoplasmic tyrosines are in bold type and ITIM-like sequences are in grey background. C) RT-PCR of CLM members was performed using specific primers on primary microglia total RNA (MG). Amplification from RAW264.7 (R) and NIH3T3 (N) cells lines was included as positive and negative controls, respectively. The image is representative 3 independent experiments.
Fig 2.
CLM-1 mRNA and protein levels in microglial cells under basal conditions.
A) QT-PCR analysis of CLM-1 transcript was performed on primary microglia total RNA. RAW264.7 and NIH3T3 cell lines were used as positive and negative controls respectively. CLM-1 transcript was normalized with 18S RNA levels. B) Surface expression of CLM-1 on primary microglia was monitored by flow cytometry using anti-CLM-1 mAb (white histogram) and an isotypic mAb as a negative control (gray histrogram). C) Immunohistochemistry on microglial primary cultures was performed to analyze the expression of CLM-1 in basal conditions, using an anti-CLM-1 monoclonal antibody. Nuclei were stained with DAPI. No staining was observed in the absence of primary antibody. Scale bar, 20 μM.
Fig 3.
CLM-1 receptor expression levels are modified during microglial activation by TLRs agonists.
A) Microglia was treated with LPS (100 ng/mL) at different time points (left) and with TLRs agonists LPS (TLR4 agonist, 100 ng/mL), PGN (TLR2/6 heterodimeragonist, 1 μg/mL) and poly I:C (TLR3 agonist, 10 μg/mL) for 24 hours (right). CLM-1 mRNA levels were quantified by QT-PCR. B) Surface expression of CLM-1 in microglial cells was monitored by flow cytometry 24 and 48 hours after treatment with LPS (100 ng/mL) or PGN (1μg/mL). Isotypic antibody (grey histograms), anti-CLM1 (white histograms). C) Immunostaining with an antibody for CLM-1 of microglia 24 hours after treatment with LPS (100 ng/mL) or PGN (1 μg/mL). Bar, 20 μM. Data are presented as mean ± SEM of 3 independent experiments. Statistically significant differences between treatments were determined by a one-way Anova followed by Newman Keules post-test. *P < 0.05 compared to IgG.
Fig 4.
Microglia expresses a soluble isoform of CLM-1.
A) Genomic organization and splicing pattern of CLM-1. A diagram representing CLM-1 splice variants 1, 2 and 3 is shown. Exons are represented by solid boxes (with their respective lengths in base pairs) and introns by the connecting lines. B) Comparison of the predicted amino acid sequences of CLM-1 variant 1 (NM_001169153) and variant 3 (JX073136). C) mRNA levels of the full length (variant 1) and soluble (variant 3) CLM-1 isoforms in microglia. Total RNA was extracted from primary microglia and RAW264.7 cells and RT-PCR was performed. As controls, cDNAs of both transcripts cloned in the vector pSPARK were used. The image is representative 3 independent experiments. D) Microglia was treated with the TLR agonists LPS (TLR4 agonist, 100 ng/mL), PGN (TLR2/6 heterodimer agonist, 1 μg/mL) and poly I:C (TLR3 agonist, 10 μg/mL) for 24 hours and RT-PCR analysis of the full length and the soluble isoforms of CLM-1 was performed. cDNAs were resolved in 2% agarose gels. The image is representative of 3 independent experiments. Actin mRNA levels were used as a loading control. Signal quantification is graphed bellow. E) Levels of soluble CLM-1 were measured in the supernatants of microglia after treatment with LPS (100 ng/mL) for 24 hours. Data are presented as mean ± SEM of 4 independent experiments. T-test was performed to determine significance and p-value was 0.1953.
Fig 5.
CLM-1 receptor enhances LPS-induced pro-inflammatory mediators production.
Microglia was stimulated either with anti-CLM-1 Ab or an isotypic control in combination with LPS (100 ng/mL) or IL-4 (10 ng/mL). After 24 hours, total RNA was extracted and QT-PCR analysis was performed. Relative quantification was performed using the condition IgG+LPS as the calibrator condition for NOS-2, COX-2, TNFα, IL-1β, IL-6 and CCL17. For the quantification of RELMα, IgG+mock condition was used as the calibrator condition. Data are presented as mean ± SEM of 3 independent experiments. Statistically significant differences between treatments were determined by one-way ANOVA followed by Newman Keuls post-test, or Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. *P < 0.05, **P<0.01 and ***P<0.001 compared to IgG.
Fig 6.
CLM-1 receptor amplifies LPS-induced TNFα protein levels.
Microglia was stimulated either with anti-CLM-1 Ab or an isotypic control together with LPS (100 ng/mL). Supernatants were recovered 24 hours later and protein levels were measured by ELISA. Data are presented as mean ± SEM of 4 independent experiments. Statistically significant differences between treatments were determined by one-way ANOVA followed by Newman Keuls post-test. *P < 0.05 compared to IgG.