Fig 1.
Normalized AmpliSeq read count (RPM) distribution from 36 control and infected human macrophage (MDM and HAM) samples (A), compared to RNA-Seq counts from 36 randomly selected whole blood samples in GTEx (B). Note the large divergence at high RPM (high expression).
Fig 2.
Comparison of transcript counts at read number >5 (RPM) and ≥1.5-fold (0.7 log2) differential expression ratio (DER) M.tb/control.
Blue bars represent the total number of transcripts detected at 5 RPM or greater in individual 24 h control samples. Red bars indicate the number of transcripts differentially expressed at 0.7 log2 (≥1.5 fold in either direction) after 24 h exposure to M.tb. Note the increased number of DE transcripts in MDM compared to HAM.
Fig 3.
(A) Volcano plot (p value versus fold change ratio M.tb/control) and MA plot (fold change ratio versus average expression in RPM) of HAM at 24 h after M.tb infection. Red dots are significant at p = ≤0.05. A few genes are identified as shown, both up- and down-regulated. (B) The same plots for MDM at 24 h. Note the substantially greater response to M.tb compared to HAM. The number of unique DE genes is greater in MDM than HAM at all 3 time points. Fig 3A and 3B were generated from an interactive web site designed to display Volcano and MA plots of the HAM and MDM AmpliSeq data at all 3 time points; producing results tables with selectable expression, FDR and p-values. The site is accessible at https://dataportal.bmi.osumc.edu/macrophagedb/.
Table 1.
Highly expressed (>2,000 RPM) DE genes in HAM and MDM at 2, 24, and 72 h post M.tb infection.
The common and unique DE genes are separated out. DE genes are defined as those with ≥1.5-fold change of mRNA expression in either direction upon M.tb infection. These genes also meet the criterion of adjusted p = ≤0.05 by edgeR. ‘Common to all’ indicates that these genes are DE genes >2,000 RPM in both HAM and MDM at all three time points, while ‘common to all HAM’ and ‘Common to all MDM’ indicates overlap at all time-points only for HAM or MDM.
Fig 4.
Top scoring network of DE genes (IPA results) after 2 h upon M.tb infection in HAM (A) and MDM (B). Red indicates mRNA up-regulation and green down-regulation. Intensity of color is proportional to log2 differential expression ratio (DER) of average paired infected/control RNA expression. DE ratios used are the mean of three HAM and three MDM donor samples.
Fig 5.
IPA heat map of canonical pathway activation in HAM and MDM upon M.tb infection at 2, 24 and 72 h, derived from DE genes with adjusted p≤0.05.
Colors represent activation z scores. TREM1 signaling is the top pathway in both MDM and HAM. Columns left to right are: 2, 24, 72 h MDM, 2, 24, 72 h MDM infected.
Fig 6.
TREM1 pathway gene activation in individual HAM and MDM samples at 2 h post M.tb infection.
The heatmap color and intensity indicate the ratio of mRNA expression M.tb/control cells. Red indicates a positive activation z-score and blue is a negative z-score. IL-6 and GM-CSF (CSF2) expression stand out in all 3 HAM samples at 2 h whereas multiple other genes are more highly differentially expressed in MDM.
Fig 7.
Linkage disequilibrium or LD heatmap of SNPs around the IL-10 gene locus that are either recognized as eQTLs in GTEx or as GWAS hits in dbGaP (for any phenotype), or both.
LD maps are constructed from Caucasian (CEU) and Yoruban (YRI) genomic SNP data, showing different haplotype configurations. The colored bars on top mark SNPs with eQTLs for IL-10 mRNA (blue), and dbGaP hits (red). The three marker SNPs highlighted (rs1518111, rs6686931, rs3024498) are marked by a * if recognized as an eQTL (black) or a dbGaP hit (red). Only rs1518111has both associations along with other variants in its LD block (white squares), while rs6686931and rs3024498 are only eQTLs. rs6686931 is in partial LD with rs1518111 in CEUs but in lower LD in Yorubans (YRIs). GWAS variants are part of two separate blocks—one separate from all eQTLs and one part of the LD block marked by rs1518111.
Table 2.
Association of IL-10 SNPs with persistent TST negative (PTST–) phenotype.
The most significant SNPs in the IL-10 gene plus 100 kb upstream and downstream are listed.