Cd14 SNPs regulate the innate immune response☆
Highlights
► We discovered a novel genetic mechanism affecting murine CD14 translation and secretion. ► Variation in human CD14 transcript length also affects its translational efficiency. ► Soluble CD14 altered human dendritic cell cytokine production and augmented T cell responses. ► This data provide a novel context for evaluating the role of CD14 in human disease susceptibility.
Introduction
Since only ∼1% of the human genome is located within exons (Venter et al., 2001), the vast majority of polymorphisms are single nucleotide polymorphisms (SNPs) located within non-coding regions. Approximately 40% of the ∼1200 SNPs identified in human genome-wide disease association studies are not located within exons and thus are regulatory SNPs (Visel et al., 2009). Several regulatory SNPs (rSNPs) have been identified that cause human genetic diseases by known mechanisms, including: including: thalassaemias, preaxial polydactyly, and Hirschsprung's disease (reviewed in Brem et al., 2002, Buckland, 2004, Visel et al., 2009). A small number of rSNPs are located within transcription factor binding sites, and may affect disease susceptibility through a cis-genetic effect on gene expression (Helms et al., 2003, Knight et al., 1999, Ozaki et al., 2002, Prokunina et al., 2002, Tokuhiro et al., 2003). However, the functional effects of most rSNPs have not been characterized, including their effects on gene regulation. Genetic analyses in organisms ranging from yeast (Brem et al., 2005, Yvert et al., 2003) to mice (Schadt et al., 2003) have demonstrated that most gene expression differences arise from genetic variation located outside of a pre-selected interval containing the differentially expressed gene, which is referred to as a trans-acting genetic effect. Moreover, the levels of expression of most mRNAs are regulated by more than one genetic locus (Brem et al., 2005), and each locus may have a small individual tissue or context-specific effect (Dimas et al., 2009, Knight, 2005).
In order to understand how the vast majority of our genetic differences contribute to phenotypic differences, we must identify and characterize the effects that trans-acting rSNPs have on gene expression, but this is currently a complex and time-consuming undertaking (Buckland, 2006, Knight, 2005). We previously demonstrated that haplotype-based computational genetic mapping (HBCGM) (Liao et al., 2004, Wang and Peltz, 2005) could be used to analyze microarray-generated gene expression data obtained from a panel of inbred mouse strains, and a novel cis-acting enhancer element contributing to the tissue-specific pattern of differential H2-Ea mRNA expression was identified (Liao et al., 2004). Other analysis methods have been used to analyze gene expression differences in rodents (Tesson and Jansen, 2009), humans (Franke and Jansen, 2009) or plants (Jansen et al., 2009). Genetic changes exerting a local effect on target gene expression (in cis) often have a strong influence that is replicable. However, distal (trans) genetic associations that were identified by these methods have more subtle effects, which have proven to be more difficult to validate (Majewski and Pastinen, 2010). Therefore, we explored whether a different approach for the identification of genetic factors with a trans effect on gene expression would be productive. We wanted to determine if we could identify groups of genes with a common pattern of differential expression in lymphocytes, and then use HBCGM to investigate whether there was a common genetic basis for the differential expression of a gene cluster. This approach was used because a genetic factor affecting the expression of a cluster of downstream genes is more likely to impact phenotypes related to immunological diseases. Since B cells play an important role in immune responses, the genetic basis for an expression cluster of 40 genes with a similar expression profile in murine B cells was investigated. rSNPs located near the Cd14 transcriptional start site were shown to alter basal CD14 protein secretion and macrophage type I IFN production by a novel mechanism. Because the variable length of human CD14 transcripts also affected human CD14 protein translation efficiency, we investigated the impact that sCD14 has on cytokine production by human DCs and on human CD4T cell responses.
Section snippets
B cell and chondrocyte isolation
Mice were obtained from Jackson Labs, and were used in the experiments at 8–10 weeks of age. Spleen cells were independently prepared from three female mice of the following 11 inbred strains: 129/SvJ, A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, DBA/2J, MRL/MpJ, NZB/BinJ, NZW/LaC, SMJ. Single-cell suspensions of spleen cells were prepared by lysis in ACK buffer (Cambrex, East Rutherford, N.J.). B cells were positively selected using B220 magnetic beads (Miltenyi Biotec, Auburn, CA) on LS cell
A trans-genetic locus regulates an IFN-responsive gene expression cluster
Microarrays were used to measure the level of mRNA expression in un-stimulated splenic B cells purified from 11 different inbred mouse strains that were cultured for 24 h. Using highly stringent criteria (ANOVA p-value < 10−10 and a fold change >10 across the 11 strains analyzed), we identified 183 differentially expressed genes that could be separated into 40 groups with similar expression profiles across the inbred strains using K-mean cluster analysis, which is as an un-supervised (non-biased)
Discussion
The genetic and experimental analyses in mice demonstrate that Cd14 rSNPs affect basal sCD14 secretion through a coupled effect on the Cd14 transcript length and protein translation efficiency. This novel genetic effecter mechanism has substantial downstream consequences for the innate immune response; it altered type I IFN production by macrophages, and consequently, the level of expression of at least 40 IFN-responsive mRNAs in B cells, many of which (e.g. Irf7, Mx1, and Mx2) play important
Acknowledgement
We thank Dr. Robert Lewis for helpful discussions.
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Cited by (0)
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G.P. was partially supported by funding from a transformative RO1 award (1R01DK090992-01) from the NIDDK. This work was also supported in part by the Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health (R.S. and W.J.L.).
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Current address: Pediatrics Department of EnChuKong Hospital, New Taipei City, Taiwan.