Distinct blood transcriptomic signature of treatment in latent tuberculosis infected individuals at risk of developing active disease

Abstract

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Keywords: Blood transcriptomics; Latent tuberculosis; Prophylaxis treatment.

Figure 1:. Identification of LTBI participants at higher risk progression based on the expression of a previously published ‘risk of progression to ATB’ signature prior the start of treatment.

A) Receiver operating characteristic (ROC) curves and B) individual z-scores for the Zak16 gene signature in blood samples collected prior to treatment in ATB (n=17), LTBI (n=69) and TBneg (n=11) individuals. The upper value in the TBneg cohort was used to stratify the LTBI cohort into LTBI-Risk (n=10), and LTBI-Other (n=59) groups. C) Heatmap representing the normalized expression of individuals genes of the Zak16 gene signature in blood samples collected prior to treatment in LTBI-Risk and LTBI-Other individuals. If a gene was mapped to several probes, average expression of all probes was shown. D) Differences in full blood counts parameters in blood samples collected prior to treatment in ATB (n=15), TBneg (n=7), LTBI-Risk (n=9) and LTBI-Other (n=55). Statistical differences between cohorts were defined with the non-parametric unpaired Mann-Whitney test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2:. Blood transcriptomic signature of treatment in the ATB cohort is associated with NK cells, monocytes and IFN signaling.

A) Heatmap representing the log fold change expression upon treatment of the upregulated (red) and downregulated (green) gene-mapped probes in the ATB_Δ signature derived from differential expression analysis (adjusted p value < 0.1) between post-and pre-treatment paired blood samples in the ATB cohort (n=8) (Table S3). Asterisks show microbiologically confirmed cases. B) Individual z-scores pre and post treatment for the upregulated and downregulated genes in the ATB_Δ signature in B) all ATB participants (n=8) and C) microbiologically confirmed (n=3) vs suspected (n=5) ATB participants. D) Immune cell type-specific expression and E) top-10 biological pathways enriched in the upregulated genes of the ATB_Δ signature. F) Immune cell type-specific expression and G) top-10 biological pathways enriched in the downregulated genes of the ATB_Δ signature. (D,F) For immune cell type-specific expression, each bar consists of stacked sub-bars showing the TPM normalized expression of every gene in corresponding cell type, extracted from the DICE database (http://dice-database.org/). (E,G) Biological pathways were ranked with increasing adjusted p value and dotted line represents significance threshold (adjusted p value < 0.05). H) Overlap between our newly identified ATB_Δ treatment signature (Burel238) and previously reported signatures associated with anti-TB therapy ^{, ,} .

Figure 3:. Blood transcriptomic signature of treatment in the LTBI-Risk cohort is associated with activated T cells and IFN signaling.

A) Heatmap representing the log fold change expression upon treatment and B) individual z-scores pre and post treatment for the upregulated (red) and downregulated (green) gene-mapped probes in the LTBI-Risk_Δ signature derived from differential expression analysis (non-adjusted p value < 0.01, absolute log fold change > 0.2) between post-and pre-treatment paired blood samples in the LTBI-Risk cohort (n=6) (Table S5). C) Immune cell type-specific expression and D) top-10 biological pathways enriched in the upregulated genes of the LTBI-Risk_Δ signature. E) Immune cell type-specific expression and F) top-10 biological pathways enriched in the downregulated genes of the LTBI-Risk_Δ signature. (C,E) For immune cell type-specific expression, each bar consists of stacked sub-bars showing the TPM normalized expression of every gene in corresponding cell type, extracted from the DICE database (http://dice-database.org/). (D,F) Biological pathways were ranked with increasing adjusted p value and dotted line represents significance threshold (adjusted p value < 0.05).

Figure 4:. Blood transcriptomic signature of treatment in the LTBI-Other cohort is associated with NK cells, B cells and platelet-related pathways.

A) Heatmap representing the log fold change expression upon treatment and B) individual z-scores pre and post treatment for the upregulated (red) and downregulated (green) gene-mapped probes in the LTBI-Other_Δ signature derived from differential expression analysis (non-adjusted p value < 0.01, absolute log fold change > 0.2) between post-and pre-treatment paired blood samples in the LTBI-Other cohort (n=36) (Table S6). C) Immune cell type-specific expression and D) top-10 biological pathways enriched in the upregulated genes of the LTBI-Other_Δ signature. E) Immune cell type-specific expression and F) top-10 biological pathways enriched in the downregulated genes of the LTBI-Other_Δ signature. (C,E) For immune cell type-specific expression, each bar consists of stacked sub-bars showing the TPM normalized expression of every gene in corresponding cell type, extracted from the DICE database (http://dice-database.org/). (D,F) Biological pathways were ranked with increasing adjusted p value and dotted line represents significance threshold (adjusted p value < 0.05).

Figure 5:. Blood transcriptomic signatures of treatment in the LTBI-Risk cohort significantly overlaps with the ATB cohort.

A) Venn-diagram representing overlaps between individual genes from the ATB_Δ, LTBI-Risk_Δ and LTBI_Δ-Other treatment signatures as defined in Figures 2, 3 and 4. B) Heatmap representing the log fold change expression upon treatment and C) individual z-scores pre and post treatment for the 21 overlapping genes between the LTBI-Risk_Δ and ATB_Δ treatment signatures across LTBI-Risk, LTBI-Other and ATB cohorts. For B), if a gene was mapped to several probes, average expression of all probes was shown. D) Immune cell type-specific expression in the 21 overlapping genes between the LTBI-Risk_Δ and ATB_Δ treatment signatures. Each bar consists of stacked sub-bars showing the TPM normalized expression of every gene in corresponding cell type, extracted from the DICE database (http://dice-database.org/). E) top-10 biological pathways enriched in the 21 overlapping genes between the LTBI-Risk_Δ and ATB_Δ treatment signatures. Biological pathways were ranked with increasing adjusted p value and dotted line represents significance threshold (adjusted p value < 0.05).

Figure 6:. Genes from ‘risk of progression to ATB’ signatures that are associated with IFN signaling are downregulated upon anti-TB treatment in LTBI-Risk but not LTBI-Other participants.

A) Individual classification of genes within the ‘risk of progression to ATB’ signatures (reported in references ^{, , ,} ) based on whether they are differentially expressed in the LTBI-Risk or the LTBI-Other group upon treatment (LTBI-Risk_Δ and LTBI-Other_Δ treatment signatures defined in Figures 3 and 4). None of the genes were differentially expressed in the LTBI-Other group upon treatment. B) top-10 biological pathways enriched and C) functional protein-protein association network in the downregulated genes upon treatment in the LTBI-Risk group. D) top-10 biological pathways enriched and E) functional protein-protein association network in genes that are not significantly dysregulated upon treatment in either the LTBI-Risk or LTBI-Other groups. (B,D) Biological pathways were ranked with increasing adjusted p value and dotted line represents significance threshold (adjusted p value < 0.05). (C,E) Functional protein-protein association networks were defined by querying the online STRING database (version 11.0).