The Peripheral Blood Transcriptome Is Correlated With PET Measures of Lung Inflammation During Successful Tuberculosis Treatment

Abstract

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [¹⁸F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [¹⁸F]FDG activity as a surrogate for lung inflammation. Our linear mixed-effects models required that for each individual the slope of the line of [¹⁸F]FDG data in the outcome and the slope of the peripheral blood transcript expression data correlate, i.e., the slopes of the outcome and explanatory variables had to be similar. Of 10,295 genes that changed as a function of time, we identified 639 genes whose expression profiles correlated with decreasing [¹⁸F]FDG uptake levels in the lungs. Gene enrichment over-representation analysis revealed that numerous biological processes were significantly enriched in the 639 genes, including several well known in TB transcriptomics such as platelet degranulation and response to interferon gamma, thus validating our novel approach. Others not previously associated with TB pathobiology included smooth muscle contraction, a set of pathways related to mitochondrial function and cell death, as well as a set of pathways connecting transcription, translation and vesicle formation. We observed up-regulation in genes associated with B cells, and down-regulation in genes associated with platelet activation. We found 254 transcription factor binding sites to be enriched among the 639 gene promoters. In conclusion, we demonstrated that of the 10,295 gene expression changes in peripheral blood, only a subset of 639 genes correlated with inflammation in the lungs, and the enriched pathways provide a description of the biology of resolution of lung inflammation as detectable in peripheral blood. Surprisingly, resolution of PTB inflammation is positively correlated with smooth muscle contraction and, extending our previous observation on mitochondrial genes, shows the presence of mitochondrial stress. We focused on pathway analysis which can enable therapeutic target discovery and potential modulation of the host response to TB.

Introduction

Tuberculosis (TB) is among the leading causes of mortality due to infectious diseases worldwide, with 1.5 million deaths recorded in 2018 (1). TB is caused by Mycobacterium tuberculosis and transmitted via inhalation of air droplets expelled by a person with active TB. The primary site of M. tuberculosis infection is the lung, which leads to pulmonary TB (PTB).

Lung inflammation seen in PTB is a result of the effector functions of host immune cells at the site of infection. In the absence of outright eradication of M. tuberculosis at the site, an accumulation of immune cells around invading bacteria leads to granuloma formation in an attempt to control infection. During disease progression, persistent lung inflammation results in the coalescence of granulomas into larger lung lesions with concomitant necrosis and formation of cavities (2, 3). Cavities can be detected using radiological methods such as chest X-ray and computed tomography (CT) (4, 5).

Positron emission tomography-computed tomography (PET-CT) is a medical imaging method, in which PET provides functional and CT structural information (6–10). ¹⁸F-labeled fluorodeoxyglucose (FDG) is a common PET tracer, and its accumulation in tissues of the body indicates enhanced glucose metabolism (11–17), which, in inflammatory diseases, can be used as a surrogate for the extent of inflammation (18). PET-CT is, however, a very expensive procedure (19) that is not readily available in resource-limited settings with high TB incidence and exposes subjects to radiation (20, 21) while not providing any information about underlying pathophysiological changes.

A number of prior studies have identified gene expression signatures from whole blood which reflect changes in transcript levels in response to TB as well as response to treatment (22–30). Some studies aimed at developing blood transcriptomic signatures for diagnosis of active (31, 32) or incipient (28, 33) TB showed that the signatures correlated with [¹⁸F]FDG uptake levels. Most of these studies focused primarily on biomarker discovery by analyzing group differences between timepoints in peripheral blood (24, 25, 28, 31). In contrast to previous studies (31, 33, 34), we focused on elucidation of biology by constraining the transcriptional changes to resolution of lung inflammation, i.e., correlation with the PET metrics, within subject over time. Previous studies have suggested correlation between peripheral blood transcriptional changes and the resolution of lung inflammation (28, 31–33, 35). Here we used a linear mixed models approach to model the response of each subject separately using the [¹⁸F]FDG decrease over time as an outcome variable and requiring that the slope of the gene expression over time as an explanatory variable correspond with that of the [¹⁸F]FDG. That is, there had to be within-subject correlation between outcome and explanatory variable, and then we modeled to overall groupwise trends. This modeling constrains the gene response to be tightly correlated with the [¹⁸F]FDG metric and therefore with resolution of lung inflammation. Gene expression and PET-CT measurements were analyzed from 75 patients who were considered cured based on microbiological tests during the course of successful treatment (16). Using our models, we were able to account for the inherent intra- and inter-subject correlation and accommodate missing data as well as the hierarchical data structure. Linear mixed-effect models are statistically more powerful than merely modelling the means of groups, as is done in more conventional statistical analyses (36). In contrast to the 10,295 genes that changed as a function of time, our method identified 639 genes whose expression levels were consistent with decreasing [¹⁸F]FDG activity in the lung, during PTB treatment. These genes could reveal more about the inflammation-related biological processes involved in the lung disease than models that do not account for within subject variation and do not constrain the transcriptional changes to the changes in PET metrics. We also performed cellular deconvolution with the transcriptomics data to determine the influence of cell type on the modeling, and found that variations in cell proportions change the genes and pathways identified as correlated with PET metrics.

Materials and Methods

Study Design

We analyzed [¹⁸F]FDG PET-CT scan metrics and whole blood RNA-sequencing (RNA-seq) data available from study subjects (Figures 1 and S1, and Table 1). In previously published studies (16, 28, 37), 99 patients were followed up during TB treatment from diagnosis (Dx), week 1 (W01), week 4 (W04), to week 24 (W24). Approval was obtained from the Health Research Ethics Committee (HREC) of Stellenbosch University (registration number N10/01/013), to recruit patients and collect specimens. For the current study to re-analyze the PET-CT and mRNA expression data, we received a separate HREC approval (X18/09/029).

Figure 1

PTB patients for the original study were recruited from the local communities or at Tygerberg Academic Hospital in Cape Town, South Africa. They were 16 – 70 years old, had no other lung disease, were non-diabetic, and were HIV-negative at diagnosis. They had also not been on any steroid medication in the past 6 months, were not pregnant and did not have cancer (for a detailed list of inclusion and exclusion criteria, see Supplementary Methods). The study patients received treatment as prescribed by the South African National Tuberculosis Programme, based on WHO guidelines. This consisted of 6 months of combination therapy: 2 months of intensive phase with rifampicin, isoniazid, pyrazinamide and ethambutol daily, followed by 4 months of continuation phase with rifampicin and isoniazid daily (for details, see Supplementary Methods) (1, 16). All subjects were diagnosed with active TB by microbiological sputum culture examination at Dx. After 2 years of follow-up, 76 patients were designated “cured”; 8 “failed treatment”; 12 “recurrent TB”; and 3 “unevaluable” by microbiological sputum culture at the end of treatment (W24).

RNA-seq was performed on specimens from time points: Dx, W04 and W24. We used 75 cured subjects out of the 76, who had [¹⁸F]FDG PET-CT scans and RNA-seq data available at a minimum of 2 time points (Table 1). We chose to use all available data, i.e., perform an incomplete-case data analysis since 85% of the cases were complete and would stabilize the estimates against wild swings from the incomplete cases. Additionally, another 6% of the data consisted of start and end timepoints which should result in less divergence of the slope estimates than cases missing either the first or the last timepoint. There is extensive literature that indicates that performing a complete-case analysis will lead to greater bias than performing an incomplete-case analysis (i.e., all available data, cases with complete data plus those with missing data) (38–50). One approach to incomplete data is imputation. In this instance imputation was not possible. Fortunately, linear mixed models as implemented in generalized linear mixed models are robust against missingness even under less restrictive assumptions (i.e., missing at random as opposed to missing completely at random), thus using incomplete-case data is plausible for the current analyses (48, 51).

We downloaded log₂ transformed RNA-seq read counts from Gene Expression Omnibus (GSE89403) (28). Read pairs were previously aligned and mapped to human genome hg19 with STAR version 2.3.1d (52), and htseq version 0.6.0 (53) was used for counting the overlaps of reads with genes. Read counts were normalized with the cpm function in edgeR package (54) in R (55), and transformed to log₂ values, hence no further pre-processing or quality control steps were performed on the data in this study. Reads were mapped to transcripts identified by Ensembl IDs and were mapped to Entrez Gene IDs using biomaRt (56).

PET-CT Metrics

We had PET-CT scan data at time points Dx, W04 and W24 for 75 “Cured” subjects, whose RNA-seq data were available (Table 1). In previous studies (57, 58), mean standardized lesion activity (MSLA) was one of the quantified PET metrics. MSLA is a normalized mean intensity of [¹⁸F]FDG uptake in the area of the lung above a background threshold, computed as Z-score (58). It describes mean lesion intensity in a specified area of the lung. MSLA decreased nearly linearly over time, when time was treated as an ordinal (ordinal time in weeks corresponds approximately to the cube root of weeks). MSLA was also characterized by large variance (Figure S2), hence we included it in our models as an outcome variable. MSLA is a PET metric, previously used to calculate [¹⁸F]FDG lung lesion activity, and was reported to correlate with changes in lung inflammation (16, 57). It should be noted that few subjects had MSLA of 0 at the end of treatment, and that most (67 of 75) of the PET-CT scans were considered not resolved at the end of treatment. For statistical modeling, [¹⁸F]FDG PET-CT scan data were merged with whole blood RNA-seq data, using R version 3.6.1 (55).

Estimation of Cell Proportion by Deconvolution

To estimate the proportions of lymphocyte types in the peripheral blood samples, we performed deconvolution using the gene expression counts and CIBERSORT (59) and the immunoStates expression matrix (60). The CIBERSORT deconvolution uses linear support vector regression together with an expression matrix of genes that are differential for one or more cell types. The immunoStates matrix comprises expression values for 317 genes and 20 cell types. We obtained estimates of cell proportions for all 20 cell types for each subject and timepoint for which data were available. For the cell types with non-trivial proportions and variance we performed a repeated measures ANOVA using lme4 (61) in R (55) to determine if there was evidence for change in proportion over time.

Statistical Modeling

We fit a linear mixed-effects, multi-level model (61, 62) that accounts for intra- and inter-subject variation. Mathematically the model is written as:

where i represents multiple values of the variable, y_i is the outcome variable for the i-th individual at level one, in j-th group at level two, α_ij is the varying-intercept, x_i is level one predictor variable, β is the slope, and ϵ_i is the level one random error. β represents the rate of change in y per unit in x. The predictor variables are the independent variables.

We fit a varying-intercept, and varying slope model; treating the subjects and gene expression levels, as random effects; and time, as a fixed effect (Figure S1B).

The models were formulated as follows:

where G is Gene, T is Time, S is Subject

where M is MSLA, G is Gene, T is Time, S is Subject, C is Cell-type

In the equations, i is the subscript for time point (Dx, W04, W24) as an ordinal value; and j is the subscript for subject (n=75). Model 1 was used to extract the slope of each gene in each subject, while Model 2 constrains the expression levels of each gene to correlate with MSLA levels, over time, in each subject (Figure S1). In Model 2, we identified genes with significant model fit, using Satterthwaite’s method (63, 64), and Type III ANOVA F statistic test, implemented in Anova function in the car package (65) in R version 3.6.1 (55, 66, 67). We corrected for multiple testing using false discovery rate (FDR) (68), with the p.adjust function in R, and FDR < 0.05 was considered significant. The linear mixed-effects model imposes strict constraints on the data (RNA-seq, PET), such that the expression levels of the genes are correlated with PET levels, over time. Traditional differential gene expression analysis does not account for intra- and inter-subject variation.

Adjusting for cell type proportion entails adding variables to the model and incurs a penalty in terms of the degrees of freedom. Increasing the model complexity can result in false negatives due to this penalty. Correction for a single cell type alone leaves the result poorly interpretable, since significance can be driven by changes in cell proportion in any of the remaining cell types. We therefore selected a set of 4 cell types, i.e., naïve B cells, CD8⁺ αβ T cells, CD14⁺ monocytes and neutrophils and modeled these using a leave-one-out approach.

We generated results for 5 different analyses based on Model 2:

• 2.1) Base, a model without correction for cell type (C_ij omitted)

• 2.2) B cell, a model correcting for CD8⁺ αβ T cells, CD14⁺ monocytes, and neutrophils

• 2.3) CD8 T cell, a model correcting for naïve B cells, CD14⁺ monocytes, and neutrophils

• 2.4) Monocyte, a model correcting for naïve B cells, CD8⁺ αβ T cells, and neutrophils

• 2.5) Neutrophil, a model correcting for naïve B cells, CD8⁺ αβ T cells, and CD14⁺ monocytes.

Gene Set Enrichment Analysis

We performed three different gene set enrichment analyses, using the following tools: (a) gene set enrichment analysis (GSEA) (69) using the SetRank package in R (70, 71) and the Reactome pathway database (72) for the gene set definitions; and (b) over-representation analysis (ORA) using the tmod package (73) in R with the Reactome pathway database (72) and (c) ORA using WebGestalt (74). For ORA we used Fisher’s exact test to estimate the significance of enrichment for a category. We used the weighted set cover for gene set redundancy reduction, which finds a minimum subset of gene sets that include the maximum number of genes by iteratively adding sets based on a marginal benefit for adding the set. The marginal benefit is the number of genes that will be added (distinct and not yet present among all included sets) multiplied by −log₁₀(p) for the set (74). SetRank produces a network representation of the enriched pathways that was visualized using Cytoscape (75).

We merged the networks from all 5 models (2.1 to 2.5) into a single network to create a master layout of all enriched pathways. The network graph of each model was then represented using the master layout to generate per-model graphs with identical positioning of pathways for ease of comparison. Analyses controlled for false discovery using either the Benjamini and Hochberg (68) or Holm (76) approach. The SetRank output includes two rounds of correction with the Holm procedure. Since this second correction is dependent on the collection of pathways identified as enriched after applying the first correction, it is specific to the results for a given model. We therefore present the results for the comparison of models using only the p-values adjusted with the first correction.

Identification of Transcription Factors (TFs) and Their Binding Sites in the Promoter Regions

We downloaded 485 experimental transcription regulators (TRs) from ReMap, which includes transcription factors, transcriptional co-activator and chromatin regulators (77), generated from previous chromatin immunoprecipitation sequencing (ChIP-seq) experiments, across 346 cell types. We used ReMap to obtain the co-ordinates of the TF binding sites (TFBS). For each gene, we then extracted the TF binding site start (TFStart) and end (TFEnd), chromosome, TF, track, location, strand, gene transcription start site (GeneTSS), and symbol for a promoter of 4,000 bases upstream of the transcription start site of the target genes from human genome GRCh38.p13, using in-house scripts. To estimate over-representation of the TFs in PETGenes, we first extracted the TFBS and their genomic features from the list of PETGenes. Next, we calculated the counts of gene promoters containing the TFs. The counts are for gene promoters that have at least one TFBS for the TFs. Finally, we computed the TF over- or under- representation for a set of TFs comparing the frequencies in the query set (PETGenes) to that of the remaining genes in the reference (Human Genome), using Fisher’s exact test and an FDR < 0.05 (68).

Construction of Gene Regulatory Network

We used the induced network module analysis in ConsensusPathDB (78, 79) to build a regulatory network from genes of interest. ConsensusPathDB is a database that integrates gene regulatory interaction, physical protein interactions, genetic interactions, metabolic and signaling reactions, and drug-target interactions, in human, mouse and yeast, from 30 public resources. The induced network module analysis uses regulatory information from public databases to connect genes based on their regulatory interaction. We exported the built network from ConsensusPathDB, into Cytoscape (75) for visualization. We integrated the RNA-seq expression data, to describe the functionality of the gene regulatory network at different time points. The interaction among genes in this network suggest transcriptional regulatory interaction, and their expression profiles suggest up-regulation. The feedback loops indicate gene-protein synthesis, influenced by transcription factors, i.e., a transcription factor regulates its target gene to synthesize its protein.

Results

To identify biological processes underlying the resolution of lung inflammation, during PTB treatment, we first identified genes with significant changes in expression, over time, using Model 1. We refer to these as TimeGenes. Biological processes enriched in these genes reflect changes in transcript levels, over time, in response to PTB treatment. Next, using different forms of Model 2 (Models 2.1 to 2.5), we identified genes with expression levels significantly correlated with the changes in the PET metric MSLA, in patients who were considered cured at the end of the 6-month PTB treatment. Using GSEA, we could identify the biological processes and pathways associated with these genes that undergo changes in transcript levels in peripheral blood, and which correlated with inflammation in the lungs during PTB treatment.

Deconvolution

We filtered cell proportions to remove cell types that did not reach a mean proportion > 0.05 at any time point. Seven cell types remained, namely CD14⁺ monocytes, CD16⁺ monocytes, CD4⁺ αβ T cells, CD8⁺ αβ T cells, CD56^bright NK cells, naïve B cells and neutrophils. The results from the ANOVA are shown in Figure 2. Four of the cell types, naïve B cells, CD8⁺ αβ T cells, CD14⁺ monocytes and neutrophils, showed changes in proportions over time in a manner that suggested that they could influence the statistical model.

Figure 2

Model 1: Changes in Transcript Levels, Over Time, in Peripheral Blood During PTB Treatment (TimeGenes)

As a baseline analysis with which to compare our constrained analysis, we applied a simple model that identified transcripts that change over time. We identified 11,229 transcripts (10,295 genes, with Entrez Gene ID) with significant changes in expression levels, over time, using Model 1 (Table S1). We used WebGestalt and ORA to identify gene ontology biological process annotations enriched in these genes. Among the 289 significantly overrepresented gene ontology biological process annotations, we observed many related to immune response as well as annotations like “response to molecule of bacterial origin” and “cellular response to drug” (Table S2) emphasizing that Model 1, by not being constrained, detected all changes over time including numerous processes that do not contribute to understanding the pathophysiology of TB.

Model 2: Changes in Transcript Levels in Peripheral Blood, Correlated With Lung [¹⁸F]FDG Uptake Levels (PETGenes)

We found a significant decrease in the average [¹⁸F]FDG uptake levels as represented by MSLA, over time, during PTB treatment (Figure S2). MSLA is a PET metric, previously used to calculate [¹⁸F]FDG lung lesion activity, and was reported to correlate with changes in lung inflammation (16, 57). To identify genes with expression levels in correlation with MSLA levels, over time, we used Model 2. We found 693 transcripts (639 with Entrez Gene ID) with significant changes in expression levels, in correlation with MSLA levels, over time (Table S3) using the Base model (2.1), and refer to these as PETGenes.

As expected, most of the PETGenes were among the TimeGenes, with an overlap of 591 (p=1.7e-130; odds ratio=13.8) genes between TimeGenes and PETGenes (Table 2 and Table S4). Only 48 genes were new to the PETGenes and not amongst the TimeGenes, a result of intra-subject correlation between the gene expression and PET levels. The 591 overlapping genes represent changes in transcript levels in peripheral blood, associated with the resolution of inflammation in the lungs, during PTB treatment.

We performed GSEA using the model fit results per gene and SetRank with the Reactome pathways (72). We identified 47 pathways that were enriched for the Base model (2.1) (Table 3 and Figure S3). Models 2.2 to 2.5 identified 48, 35, 35 and 33 pathways, respectively, as enriched (Table 4). Overall, we identified 103 Reactome pathways (72) as enriched in one or more of the models (Figures 3, S3–S7 and Table 4). Many of the well-known pathways in TB biology were represented among the enriched pathways (80), but the results also included some novel observations. Nine pathways were identified in all models: “Interferon gamma signaling”, “Response to elevated platelet cytosolic Ca²⁺”, “Smooth muscle contraction”, “G alpha I signaling events”, “Platelet activation signaling and aggregation”, “Cell surface interactions at the vascular wall”, “rRNA modification in the nucleus and cytosol”, “PD-1 signaling” and “Interferon signaling” (Table 4).

Figure 3

Many pathways are related by sharing of genes and therefore function, but some of the other pathways can be grouped together by related function as shown in Figure 3. Approximately half of the pathways represented are connected (outlined in long black dashed line, Figure 3). The inter-related pathways include “platelet activation, signaling and aggregation”, “extracellular matrix reorganization”, “cell surface interactions at the vascular wall” and “response to elevated cytosolic Ca²⁺” (grey dot-dash Figure 3) (80, 81), the interferon and interleukin signaling cluster (grey dotted Figure 3) (80, 81), and a cluster of phospholipid acyl chain metabolism that includes unconnected small clusters of lipid metabolism pathways (red medium dash Figure 3) (82). Smaller clusters of pathways include Golgi trafficking (83) and N-glycosylation (dotted dark blue Figure 3) (84), and “smooth muscle contraction” (light green dot-dash, Figure 3).

Among the 639 PETGenes identified in Model 2.1, we found 3 distinct clusters of genes, with similar expression patterns over time (co-expression) (Figure 4A, Table S5). The expression levels of PETGenes in Cluster 1 started high at Dx, and decreased over time (378 genes; Figure 4B) whereas genes in Cluster 2 (191 genes; Figure 4C) and Cluster 3 (66 genes; Figure 4D), started low at Dx and increased over time (Figure 4D), thus suggesting up-regulation in Clusters 2 and 3 (Figures 4C, D), and down-regulation in Cluster 1 (Figure 4B).

Figure 4

Pathways related to platelets in Cluster 1 and B cells in Cluster 3 were significantly overrepresented (Table 5, Table S5). Some new pathways in Cluster 1 included “hemostasis”, “adaptive immune system”, “innate immune system”, “p130Cas linkage to MAPK signaling for integrins”, and “GRB2 SOS provides linkage to MAPK signaling for integrins”. The modules enriched in B cells included the following PETGenes in Cluster 3: CD19, VPREB3, CD72, CD22, FCRLA, CD79B, CD79A, HLA-DOB, P2RX5, FAM129C, FCRL5, PCDH9, BCL11A, PNOC, and BLNK. The expression profiles of PETGenes in Cluster 3, suggest up-regulation in B cells during treatment response, whereas Cluster 1, suggests down-regulation in platelet activation during treatment response. Curiously, there were no pathways overrepresented in Cluster 2 (Figure 4C).

To validate the PETGenes, we compared our results against published gene signatures of TB treatment response and transcriptional profiling of lung biopsies from PTB subjects, using Fisher’s exact test. We found 63 PETGenes overlapped (p=7.3e-34; odds ratio=8.34) with the treatment-specific signature from Bloom et al. (24) (320 microarray probes mapping to 267 distinct protein-coding genes; Table S6), and 99 PETGenes overlapped (p=3.8e-136; odds ratio=576.1) with the 393-probe, 307-gene, signature from Berry et al. (22) (Table S7). Three PETGenes, ITGB3, MMP1, and STAT1 were found to overlap with “top 15 most highly differentially expressed regulator genes in lung TB granulomas” from Subbian et al. (85), 29 genes overlapped (p=7.1e-21; odds ratio=13.7) with “TB blood biomarkers in the lung” from Subbian et al. (85) (Table S8), and although 164 overlapped with the “list of significantly differentially expressed genes common to fibrotic nodules and cavitary lesions” from Subbian et al. (85) (Table S9), this was not significant due to the size of the set (4,417 genes) (p=0.99; odds ratio=0.79). Additionally, we investigated if there was an overlap between the 639 genes identified here and prior small signatures (27). Due to the size of the signatures, we did not perform tests for significance of overlap. We identified 17 prior signatures (Table S10). Of these 3 were for conditions that are not relevant to the current study, i.e., prediction of failure, response to treatment, and diagnosis of active TB from latent TB. The subjects in this study were not failures, were all cured at end of treatment, and there were no latent TB cases. After removing these three signatures, the mean proportion of genes shared was 0.60 (Table S10).

Identification of Transcriptional Regulatory Factors in PETGenes

We found 254 TFs to be significantly over-represented in the list of PETGenes, with FDR < 0.05 (Table S11). Among the over-represented TFs in PETGenes (Table S11), NF-KB1, CREB, STAT, FOS, and JUN, have been reported to play a critical role in regulating inflammation in lung diseases (86), and we extracted their TFBS in the promoter region of their target genes (Table S12).

Further, we identified an induced regulatory network among Cluster 3 genes enriched for B cell genes and pathways. The expression level of genes in this network was low at Dx (Figure S8), and high at W24 (Figure S9). The induced gene network suggests transcriptional regulation of B cell genes to synthesize proteins (Figures S8, S9). The genes in this network were enriched in “B cell activation” and “B cell receptor signaling pathway”, thus suggesting up-regulation in B cell activation. Among Cluster 3 genes, we identified genes coding for TFs UBTF, MITF, ATF3, GRHL1, SPIB, STAT1, and STAT2. We extracted their TFBS (see Methods), as well as their target genes (Table S12). The TFs PAX5, EBF1, and SPIB are included in Cluster 3, and CD19, CD79A, and BLK are direct regulatory targets of PAX5 (Figure S8, Table S12). PAX5 and EBF1 have been reported to regulate the expression of B cell genes and play an important role in B cell development (87, 88). Gene set enrichment revealed that B cell receptor signaling pathway is significantly enriched in Cluster 3 genes: PAX5, SPIB, EBF1, CD19, CD79A, BLK, BLNK, CD79B, CD22, and CD72 (31).

We identified a regulatory network among Cluster 1 genes enriched in genes related to platelet activation and signaling. The expression profiles of the genes included in this network were high at Dx and low at W24 (Figure S10, and Figure S11, respectively). The following genes were significantly enriched in platelet degranulation: F13A1, ITGA2B, ITGB3, LY6G6F, PF4, PPBP, RAB27B, SERPINE1, and STTL4. The KEGG pathway “platelet activation” was significantly enriched in Cluster 1 genes: F2RL3, GP1BA, GP6, GP9, ITGA2B, ITGB3, PRKG1, and PTGS1. Also, we observed high expression level in platelet surface markers: GP9, GP1BA, GP6, PF4, GP1BB, ITGB3, ITGA2B, F2RL3, and ITGB5 at Dx (Figure S10), and low expression at W24 (Figure S11), thus suggesting down-regulation of platelet genes. We identified a complex of interacting platelet surface receptors, which exhibited high expression at Dx (Figure S10), suggesting functional activation of platelets at Dx, and low expression at W24 (Figure S11), suggesting down-regulation of platelet activation during treatment.

In addition, we observed interaction between tissue factor pathway inhibitor (TFPI) and MMP1, MMP8, and MMP12, which were enriched with the biological processes: “coagulation” and “regulation in response to wounding”, respectively. These genes exhibited high expression at Dx (Figure S10), and low expression at W24 (Figure S11), suggesting down-regulation of MMP-driven coagulation.

Smooth muscle contraction was identified by GSEA as well as by ORA among Cluster 1 genes. Cluster 1 genes decrease in expression from Dx to W24 (Figure 4B), suggesting that the pathway is downregulated during resolution of inflammation. We verified that 15 of the 33 genes in the pathway have mean transcript levels in excess of 100 transcripts per million, and most of the genes are transcribed above the nominal 10 transcripts per million (after adjusting for library size). Additionally, it should be noted that the transcription level is expected to be much higher in the sub-population of cells in peripheral blood that express the smooth muscle contraction phenotype. We constructed a gene regulatory network using the genes in the smooth muscle contraction pathway as well as the subset of transcription factors identified as enriched that also occurred in the smooth muscle contraction promoters (Figure 5). The genes enriched in Cluster 1 were: CALD1, GUCY1A1, GUCY1B1, ITGB5, MYL9, TPM1, TPM4, VCL. To these we added the genes whose nominal p-value contributed to the GSEA enrichment score: ACTA2, ANXA6, DYSF, MEF2A, MEF2C, MYH11, MYL12B, MYL6, MYLK, SORBS1, SORBS3, TLN1, TPM2, TPM3, as well as the transcription factors: CREB1, CREBBP, GATA1, GATA2, GATA4, GATA6, SRF, TEAD1, TEAD4. All but SORBS1, SORBS3, TPM2, and VCL decreased expression from Dx to W24. The network demonstrates that a highly interconnected cluster of contraction proteins connected to another interconnected cluster of transcription factors (Figure 5).

Figure 5

Discussion

In this study using linear mixed-effects models accommodating intra-subject correlation between outcome and explanatory variables, we have identified 639 genes which exhibit changes in blood transcript levels that correlate with [¹⁸F]FDG uptake changes in lung lesions during successful PTB treatment. These 639 genes are almost entirely a subset of the 10,295 genes that change over time during treatment. Since all modelled subjects were clinically cured at the end of treatment, the genes identified by Model 2 are those correlated with resolution of lung inflammation during PTB treatment. The changes are, however, those in the peripheral blood that reflect the resolution of lung inflammation, and not the changes in situ. These PETGenes also allowed us to identify a number of biological processes associated with inflammation.

We identified 47 enriched pathways (Table 3) using a model without correction for cell proportion (Base, model 2.1), and a total of 103 enriched pathways when accounting for the influence of cell proportions, of which the 47 are a subset, in one or more of the 5 models (models 2.1 to 2.5). The correlation of expression of genes identified by models correcting for cell proportions could then be due to: a) the change in proportion of the remaining cell type; b) change in regulation of gene expression in any of the cell types; c) a change in proportion of another cell type not estimated by deconvolution; or d) a combination of the above. Since the proportions of cell types were estimated by deconvolution, the adjustment might be neither efficient, in statistical terms, nor as accurate as differential counts. Therefore, caution needs to be used in the interpretation of the results obtained here.

We identified several pathways, “CD22 mediated BCR regulation”, “antigen activates B cell receptor (BCR) leading to generation of second messengers”, and “RUNX1 regulates transcription of genes involved in BCR signaling,” whose function is restricted to B cells (Table 4) in model 2.2 “B Cell”. This supports the premise of the approach that adjusting for the proportions of several other cell types permits cautious attribution of at least some of the gene expression changes to corresponding changes in the proportions of the remaining cell type (B cells).

Eleven pathways, all consistent with different aspects of lipid and phospholipid metabolism are enriched in the model 2.3 “CD8 T cell.” The pathways are: synthesis of phosphatidic acid (PA); synthesis of dolichyl phosphate; fatty acid metabolism; synthesis of phosphatidylethanolamine (PE); beta oxidation of very long chain fatty acids; triglyceride metabolism; phospholipid metabolism; synthesis of phosphatidylinositol phosphates (PIPs) at the early endosome membrane; synthesis of PIPs at the late endosome membrane; PI metabolism; and triglyceride biosynthesis. Lipid metabolism has been established as important to the function of T cells (89), again supporting attribution of some of the effect to changes in proportions of the remaining cell type (CD8⁺ αβ cells). There are also several pathways identified here as potentially relevant to T cells that have not previously been identified. These include some of the phospholipid metabolism pathways and the PIP pathways.

For CD14⁺ monocytes and neutrophils no pathway or collection of pathways provided support in the same way as for naïve B cells and CD8⁺ αβ cells. The pathways identified are generally consistent with inflammatory processes, but not characteristic of the cell types.

Nine pathways were identified in all models: Interferon gamma signaling; Response to elevated platelet cytosolic Ca²⁺; Smooth muscle contraction; G alpha I signaling events; Platelet activation signaling and aggregation; Cell surface interactions at the vascular wall; rRNA modification in the nucleus and cytosol; PD-1 signaling; and Interferon signaling (Table 4). It seems therefore that the proportions of the 4 cell types are not relevant to their detection. These pathways could be driven by regulation in all 4 cell types, or by cells of another type, or both. Seven of the nine pathways, “Interferon gamma signaling”, “Response to elevated platelet cytosolic Ca²⁺”, “G alpha I signaling events”, “Cell surface interactions at the vascular wall”, “Platelet activation signaling and aggregation”, “PD-1 signaling”, and “Interferon signaling”, are well-established inflammatory response pathways and it is reasonable that regulation in all 4 cell types occurs during response to treatment in TB.

The remaining two pathways are: “Smooth muscle contraction,” and “rRNA modification in the nucleus and cytosol.” Although smooth muscle contraction has been identified as a pathway enriched in TB transcriptomics, it was in the context of lymph node M. tuberculosis infection (90). rRNA modification in the nucleus and cytosol has not been identified previously. Here we see both pathways in all models, suggesting that these are important in the biological changes that occur during response to treatment.

“Smooth muscle contraction” is an interesting pathway to detect in peripheral blood. Most of the genes characteristic of this pathway are not expressed in leukocytes. Nevertheless, it is one of the most enriched pathways in our analysis (P_corr < 1.3 x 10⁻⁷; Table 4). Although it has been observed before in the context of TB (90, 91), it was not interpreted or discussed. In the mouse study (91) it was observed in peripheral blood, but in the human study (90) it was in a TB-infected lymph node. Prolonged and coordinated expression of smooth muscle contraction genes in peripheral blood is unusual. This raises the question about the origin of the cells that give rise to this expression. One possibility is proliferation of smooth muscle progenitor cells (SPCs), which have been observed in peripheral blood, in response to vascular or tissue injury, for tissue repair and remodeling. SPCs have previously been identified in human and animal peripheral blood (92–94). Among the responses to injury and inflammation are signals that stimulate SPC division and migration from tissue reservoirs or the bone marrow to the site of injury (95). Platelets have been shown to interact with SPCs in peripheral blood, where platelets express adhesion receptors that enable them to interact with endothelial cells, leukocytes and SPCs (92). This interaction triggers the following in SPCs: mobilization of progenitor cells from either the bone marrow to peripheral circulation or from local tissue reservoirs, e.g., pericytes, chemotaxis to target tissue, adhesion on vascular wall, survival and engraftment into local tissue, differentiation into mature functional cell types, such as endothelial cells, and proliferation, which are essential steps of progenitor cell-mediated tissue repair (96). The extensive activation of platelets and platelet related pathways observed in TB (Figure 3 and Figures S3–S7) (80, 81) suggest that mobilization of SPCs is likely. Almost all the genes in the “smooth muscle contraction” pathway are down-regulated during response to treatment consistent with overall wound-healing and a decreased requirement for angiogenesis, neo- or revascularization. A critical question for TB is whether the angiogenesis around granulomas is as dysregulated as around cancer tumors (97). The dysregulated angiogenesis around tumors prevents immune cells from crossing into the tumor and contributes to the inability of the immune system to attack the tumor (97). This could have implications for TB treatment.

Another enriched pathway “rRNA modification in the nucleus and cytosol” has previously been reported in peripheral blood of Friedrich’s Ataxia patients (98), but not in TB. It can be involved in at least two aspects. First, modification of rRNA plays a role in stability of ribosomes (99) and their efficiency in translation (99, 100). In part it is likely a response to the increased biosynthesis necessary for the immune cells in lung inflammation and wound healing. Second, modification of rRNA is also involved in progenitor cell differentiation (101). The “rRNA modification in the nucleus and cytosol” pathway could therefore tie back in to the SPC differentiation. The origins of the signal for rRNA modification can also arise in other cell types and the alterations are important for ribosome stability and efficiency (100). It is conceivable that rRNA modifications are necessary during the chronic lung inflammation of PTB and that the modifications return to homeostatic conditions when the inflammation subsides.

The cluster of mitochondrion-related pathways is also interesting. The genes are mostly downregulated during response to treatment. The pathway of “cristae” formation includes genes for proteins that induce the folding of the inner mitochondrial membrane (MICOS10, MICOS13, DNAJC11) and mitochondrial morphogenesis (TMEM11), but also includes many genes for mitochondrial complex V, the ATP synthase complex (ATP5F1A, ATP5F1B, ATP5F1C, ATP5F1D, ATP5F1E, ATP5MC1, ATP5MC2, ATP5MC3, ATP5ME, ATP5MF, ATP5MG, ATP5PB, ATP5PD, ATP5PF, ATP5PO, as well as the mitochondrially encoded genes ATP6 and ATP8), which are all downregulated during treatment. Downregulation of these genes is consistent with the requirement of extensive oxidative phosphorylation during the peak of inflammation. Cells can increase oxidative phosphorylation by mitochondrial division or by increasing the density of cristae in mitochondria, which facilitates a higher density of oxidative phosphorylation complexes and enables mitochondria to be more efficient (102). Interestingly, enrichment of the pathways “SMAC XIAP regulated apoptotic response” and “rRNA modification in the mitochondrion,” which are connected via numerous protein-protein interactions, further suggests that the demand placed on the mitochondria has led to some mitochondrial dysfunction.

We used the pathways related to B cells and platelet activation to validate our approach. First, we demonstrated an increase in the expression of genes enriched in B cells, B cell activation and B cell receptor signaling during treatment (Figures 4A, D) and decrease in expression of genes enriched in platelet activation (Figure 4B). Second, we constructed induced regulatory pathways that incorporated TF with enriched TFBS. The regulatory interaction among PETGenes and their TFs demonstrates transcriptional regulation among PETGenes. High expression of genes enriched in “B cell activation”, at the end of treatment suggests up-regulation in B cells activation (Figures S8 and S9) and low expression of genes enriched in “platelet activation” suggests down-regulation (Figures S10, S11).

Previous studies on TB treatment response (24, 25, 28–30) have only reported general changes in transcript levels in the peripheral blood, which are likely to be a collection of responses to drugs, reduction in bacterial load and inflammation. Four previous studies on human TB also reported a correlation between peripheral blood signatures and PET-CT [¹⁸F]FDG uptake measurements (28, 31, 33, 34). The uniqueness of our findings is that we directly correlated changes in transcripts levels in peripheral blood with [¹⁸F]FDG uptake in the lungs—a measure of inflammation—over time during TB treatment, taking into account the inherent intra- and inter-subject correlation structure of repeated measurements, thus our results describe more of the biology of lung inflammation in TB, as well as the temporal dynamics of transcriptional changes, and with statistical accuracy. Our models (linear mixed-effect models) identified subtle significant changes in transcript levels that are ignored in traditional gene expression analyses and provide robust results for downstream differential expression analysis and clustering.

Limitations

Our study had some limitations. We used a linear-mixed effect model to analyze gene expression levels, which only considers linear correlation or relationship of gene expression with time. The linear-mixed effect model accounts only for the linear characteristics of measurements, ignoring other dynamics or patterns of gene expression and PET measurements which may be characteristic of TB. Additionally, transcriptomics only reveals changes in regulation of RNA levels which do not necessarily correspond to functional changes in proteins; nevertheless, it does provide insight into the regulatory responses, especially in longitudinal analyses. Lastly, the models correcting for cell proportions rely on computational deconvolution and not on differential counts, and the choice of which cell types to include in the models is difficult. Including too many variables (too many cell proportions) can weaken the inferential power of the models, and including cell types with little effect reduces power without any benefit. For future studies, we suggest using mixed-effect models with splines or other non-linear models with more time points. Another limitation is that PET-CT measures only glucose metabolism and not oxidative phosphorylation, nor does it provide a measure of bacterial burden; a direct measure of TB load would be ideal.

Conclusion

In summary we have demonstrated that the resolution of inflammation in the lungs during TB treatment, measured with changes in PET-CT [¹⁸F]FDG uptake in the lungs, is positively correlated with down-regulation of genes enriched in “platelet activation”, “interferon and interleukin signaling”, and negatively correlated with up-regulation of genes enriched in “B cell activation” as well as many other pathways consistent with prior literature. These results validate our overall approach. In addition, we have shown that correcting for major cell type proportions using a leave-one-out approach allows identification of processes consistent with the remaining cell type. Lastly, we have identified “smooth muscle contraction” and pathways related to mitochondrial stress and dysfunction as highly enriched pathways. The extent of coordinated smooth muscle contraction gene expression suggests that the signal is derived from non-leukocyte origins, such as SPCs. The results obtained from our comprehensive pathway analyses provide important new insight into the pathobiology of TB. In future studies they could contribute to therapeutic target discovery and potential modulation of the host response to TB.

The Catalysis TB-Biomarker Consortium

André G. Loxton, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Annare Ellman, Division of Nuclear Medicine, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Bronwyn Smith, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Caroline G. G. Beltran, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Clifton E. Barry III, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa, Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Science, University of Cape Town, Observatory 7925, South Africa; David Alland, Center for Emerging Pathogens, Department of Medicine, Rutgers-New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ, United States; Friedrich Thienemann, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Science, University of Cape Town, Observatory 7925, South Africa, Department of Medicine, Groote Schuur Hospital, Faculty of Health Science, University of Cape Town, Cape Town, South Africa; Gerard Tromp, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Gerhard Walzl, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; James M. Warwick, Division of Nuclear Medicine, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Jill Winter, Catalysis Foundation for Health, San Ramon, CA, United States; Katharina Ronacher, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Kim Stanley, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Ilse Kant, Division of Nuclear Medicine, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Lani Thiart, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Lance A. Lucas, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Laura E. Via, Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Science, University of Cape Town, South Africa; Lori E. Dodd, Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health Bethesda, Maryland, United States; Magdalena Kriel, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Nelita Du Plessis, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Patrick Dupont, Laboratory for Cognitive Neurology, Department of Neurosciences, KU Leuven, Belgium, Division of Nuclear Medicine, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Ray Y. Chen, Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States; Robert J. Wilkinson, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Science, University of Cape Town, South Africa, Department of Medicine, Groote Schuur Hospital, Faculty of Health Science, University of Cape Town, Cape Town, South Africa, The Francis Crick Institute, London, United Kingdom, Department of Medicine, Imperial College London, United Kingdom; Shubhada Shenai, Center for Emerging Pathogens, Department of Medicine, Rutgers-New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ, United States; Stephanie Griffith-Richards, Division of Radiodiagnosis, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Stephanus T. Malherbe, Department of Science and Innovation/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

Funding

TO, SM, GW, and GT were supported by the South African Tuberculosis Bioinformatics Initiative (SATBBI), a Strategic Health Innovation Partnership grant from the South African Medical Research Council and South African Department of Science and Technology. SMa received funding from the EDCTP2 program supported by the European Union (grant number CDF1576). GW received funding from the South African National Research Foundation (SARChI TB Biomarkers #86535) and the South African Medical Research Council. AL is supported by the NRF-CSUR (Grant Number CSUR60502163639) and by the Centre for Tuberculosis Research from the South African Medical Research Council (SAMRC). HK was supported by the Faculty of Medicine and Health Sciences, Stellenbosch University, South Africa. The Catalysis Biomarker Consortium was funded by the Catalysis Foundation for Health, the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, and the National Institute of Allergy and Infectious Diseases, International Collaborations in Infectious Disease Research.

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