Deciphering genetic regulation of CD14 by SP1 through characterization of peripheral blood mononuclear transcriptome of P. faiciparum and P. vivax infected malaria patients

Abstract

Background: Plasmodium falciparum and Plasmodium vivax are two major parasites responsible for malaria which remains a threat to almost 50% of world's population despite decade-long eradication program. One possible reason behind this conundrum is that the bases of clinical variability in malaria caused by either species are complex and poorly understood.

Methods: Whole-genome transcriptome was analyzed to identify the active and predominant pathways in the PBMC of P. falciparum and P. vivax infected malaria patients. Deregulated genes were identified and annotated using R Bioconductor and DAVID/KEGG respectively. Genetic and functional regulation of CD14, a prioritized candidate, were established by quantitative RT-PCR, genotyping using RFLP and resequencing, mapping of transcription factor binding using CONSITE and TFBIND, dual luciferase assay, western blot analysis, RNAi- mediated gene knockdown and chromatin-immunoprecipation.

Findings: The study highlighted that deregulation of host immune and inflammatory genes particularly CD14 as a key event in P. falciparum malaria. An abundance of allele-C of rs5744454, located in CD14 promoter, in severe malaria motivated us to establish an allele-specific regulation of CD14 by SP1. An enhancement of SP1 and CD14 expression was observed in artemisinin treated human monocyte cell line.

Interpretation: Our data not only reinstates that CD14 of TLR pathway plays a predominant role in P. falciparum malaria, it establishes a functional basis for genetic association of rs5744454 with P. falciparum severe malaria by demonstrating a cis-regulatory role of this promoter polymorphism. Moreover, the study points towards a novel pharmacogenetic aspect of artemisinin-based anti-malarial therapy. FUND: DST-SERB, Govt. of India, SR/SO/HS-0056/2013.

Keywords: CD14; Host Polymorphism; Malaria; Plasmodium falciparum; SP1; Transcriptome.

Fig. 1

Description of microarray based global gene expression profiles. Volcano plots representing pairwise comparison of data pertaining to patients with (a) P. falciparum and P. vivax infections (b) acute phase of P. falciparum infection and at their convalescence stage (c) P. falciparum infection and GSE 45919 dataset. Coordinates of Volcano plot represent log odds of P value from a modified t-test vs. log ratio of fold change. (d) Venn diagram showing the number of genes differentially expressed in datasets namely F, V, AF and N. (e) Bar chart showing significantly enriched top ten functional categories of genes up regulated in P. falciparum infection compared to P. vivax infection. (f) Bar chart showing all eight significant GO categories for genes down regulated in P. falciparum infection compared to that with P. vivax infection. Absolute values of the –log10 (P value) of the enrichment analysis were used for plotting. Only categories with P value <0.05 threshold were shown. (g) Bar diagram depicting comparison of F-V and F-N datasets with respect to major GO enriched categories composed of genes up regulated in acute phase of P. falciparum infection. F, V, AF and N indicate P. falciparum infection, P. vivax infection, P. falciparum convalescent and malaria naïve US adults from GSE 45919 dataset respectively.

Fig. 2

Differentially regulated immune and inflammatory responsive genes associated with P. falciparum. (a) List of genes differentially regulated in F compared to V and their respective fold changes. Hierarchical clustering using 45 differentially regulated immune genes classifying samples pertaining to (b) F and V and (c) F, V and N datasets. Heat map was constructed based on Z-Score transformed expression values. A color code of orange from darker shade to light represents negative to positive distribution of Z score.

Fig. 3

Immune and inflammatory pathways with special reference to TLR signaling in P. falciparum malaria. (a) KEGG pathways pertaining to differentially regulated immune response genes in F compared to V. X axis denotes the enrichment score -log10(P value). n denotes number of genes. (b) Bar plot showing relative mRNA expression levels of candidate genes from TLR pathway as part of validation of microarray results. (c) Bar diagram representing relative mRNA expression of candidate genes in U937 cell line stimulated with 1,25α dihydroxy vitamin D3 and treated with LPS. (d) Representative result of Western blot for CD14 in U937 cell line treated with LPS. β-actin was used as an endogenous control. Histogram depicting relative expression of CD14 in treated and control U937 cells. * denotes P value <0.05 which was considered significant.

Fig. 4

Analysis of CD14 promoter polymorphisms. (a) Diagram of 1.0 kb promoter sequence showing positions of rs2569190, rs2569191 and rs5744454 with respect to CD14 transcriptional start site. (b) Schematic representation of altered transcription factor binding due to allelic (A or C) variation of rs5744454. (c) Consensus binding sequence and scores of Irf1, HLF and SP1 for the invariant locations. (d) Quantitative PCR showing significant up regulation of SP1 in P. falciparum infected samples compared to P. vivax. Graphical representation of (e) genotype and (f) allele proportions of rs5744454 in P. falciparum infected uncomplicated and severe malaria patients. Frequency differences were tested using χ2 statistics. P value <0.05 was considered significant.

Fig. 5

SP1 dependent expression and promoter activity of CD14. (a) Western blot in U937 cell line showing siRNA mediated knock down of SP1 and corresponding down regulation of CD14 and (b) Western blot showing artemisinin mediated increase of SP1 and CD14 protein levels in U937 cell line. Bar diagrams representing normalized relative light units obtained from U937 and HepG2 cell lines following (c) transfection with empty vector or promoter construct containing rs5744454: A/C allele and (d) SP1 specific siRNA treatment and transfection with empty vector or rs5744454: A/C allele. (e) Electropherograms showing AA, AA and CC genotypes at −281 position in CD14 in U937, HepG2 and PC3 cell lines respectively. (f) ChIP assay conducted to compare SP1 binding efficiencies in HepG2 and PC3 cell lines on CD14 promoter using quantitative PCR. A higher binding efficiency of SP1 in PC3 compared to HepG2 cells with respect to IgG control was shown. Binding efficiencies of SP1 for a positive control locus in CD14 gene and a negative control locus in DHFR gene were demonstrated. * denotes P value <0.05 which was considered significant.