Differential blood transcriptome modules predict response to corticosteroid therapy in alcoholic hepatitis

Abstract

Background & aims: In patients with severe alcoholic hepatitis (SAH), little is known about the profile of peripheral blood mononuclear cells (PBMCs) at baseline and during corticosteroid therapy, among those who can be treated successfully with steroids (steroid-responders [R] and those who cannot (steroid-non-responders [NR]); 2 groups with different outcomes.

Methods: We performed RNA-seq analysis in PBMCs from 32 patients with definite SAH, at baseline and after 7 days of corticosteroids. The data were sorted into R and NR (n = 16, each group) using the Lille model and 346 blood transcription modules (BTMs) were identified. BTMs are predefined modules of highly co-expressed PBMC genes, which can determine specific immune cell types and cellular functions. The activity of each BTM was taken as the mean value of its member genes.

Results: At baseline, 345 BTMs had higher activity (i.e. were upregulated) in NR relative to R. The 100 most upregulated BTMs in NR, included several modules related to lymphoid lineage (T, B, and natural killer [NK] cells), modules for cell division and mitochondrial respiratory electron transport chain (ETC, relating to energy production), but only a few modules of myeloid cells. Correlation studies of BTM activities found features of significantly greater activation/proliferation and differentiation for T and B cells in NR relative to R. After 7 days of corticosteroids, NR had no significant changes in BTM activities relative to baseline, whereas R had downregulation of BTMs related to innate and adaptive immunity.

Conclusions: At baseline and during corticosteroid therapy, increased activity in the PBMCs of gene modules related to activation/proliferation and differentiation of T and B cells, NK cells, and mitochondrial ETC, is a hallmark of SAH patients who are steroid-non-responders.

Lay summary: Patients with severe alcoholic hepatitis receive steroid therapy as the main line of treatment; however, this treatment is ineffective in some patients. This only becomes apparent after 7 days of steroid therapy. We have developed an approach where it can be estimated if a patient is going to respond or not to steroid therapy using the gene expression information of blood cells. This method will allow clinicians to assess the response of patients to steroids earlier, and will help them in adopting alternate strategies if the treatment is found to be ineffective in a particular patient.

Keywords: Alcoholic liver disease; BTM, blood transcription module; CTP score, Child-Turcott-Pugh score; DEGs, differentially expressed genes; ETC, electron transport chain; Glucocorticoid receptor; MDF, Maddrey’s discriminant function; MELD, model for end-stage liver disease; NK cells, natural killer cells; NR, non-responders; NR3C1; NR3C1, nuclear receptor subfamily 3 group c gene member 1; OxPhos, oxidative phosphorylation; PBMCs, peripheral blood mononuclear cells; R, responders; RNA-seq, RNA sequencing; SAH, severe alcoholic hepatitis; Steroid; Transcriptome.

Graphical abstract

Fig. 1

Blood transcription modules (BTMs) are able to differentiate responders (R) and non-responders (NR) to steroid therapy at baseline. (A) Principal component analysis of 346 BTMs using their activity scores showed that BTMs can segregate the NR and R, at baseline. The colour of the dots is only to identify the R or NR group. (B) Unsupervised clustering also confirmed the ability of the 346 BTMs to segregate NR and R, at baseline, with the exception of 1 sample (NR). The brown colour shows an increase and blue colour shows a decrease in gene expression in different samples (in columns). (C) Heatmap showing unsupervised clustering of the top 207 differentially expressed (>2-fold and p <0.05) BTMs between NR and R, which were able to segregate robustly NR and R, at baseline. The red colour shows an increase and green colour shows a decrease in gene expression in different samples (in columns).

Fig. 2

Correlation of cell type and function of blood transcription molecules (BTMs). Dendrogram of unsupervised clustered top 100 BTMs to determine the correlations between the BTMs. The BTMs aggregated into 15 clusters, with BTMs belonging to B- and T-cell types clustering together. The colour of the edges in the dendrogram is to identify the clusters and do not refer to expression values. TBA, to be announced, i.e. those BTMs whose exact cellular/functional characterisation is pending; ZZ, break in dendrogram branches to make it fit in one page.

Fig. 3

Baseline peripheral blood mononuclear cell flow cytometry confirms cell types identified by blood transcription modules. Comparative estimates of cell frequency (%) for B, T, and NK cells types at baseline. Significant difference between transitional, activated and regulatory B cells, and CD4+ and CD8+ T-cells and NK cells were observed (Mann-Whitney U test). NR, non-responder; R, responder.

Fig. 4

Flow cytometry-based assessment confirms variations in blood transcription module (BTM) identified cells associated with 7-days of steroid therapy. (A) Comparison of B and T cell type frequencies between baseline and day 7 shows a significant reduction in naive, transitional, activated and regulatory B cells in responders. A similar trend is observed in non-responders, with the exception of activated B-cells, which were not significantly different post-therapy from baseline. (B) An assessment of T-cells showed an increase in CD4+ and CD8+ cells post-therapy in R but not in NR, suggesting defective cell types in NR, which could be the reason for patients not responding to therapy. (C) Baseline cell frequency of NK cells corroborates with significant increase in BTM 61.1 (NK cell related KIR cluster activity; Table S4) among NR. (Mann-Whitney U test.)

Fig. 5

Variations between responders and non-responders in blood transcription module (BTM) scores after 7 days of steroid therapy. (A) Heatmap for unsupervised clustering of BTM activity scores of NR and R, at day 7 post-steroid therapy. Note the persistent increase in BTM activity among NR and reduction among R post-therapy. The red colour shows an increase and green colour shows a decrease in gene expression in different samples (in columns). (B) Principal components analysis of BTMs post-therapy confirmed their ability to robustly segregate NR and R, at day 7 also. The colour of the dots is only to identify the R or NR group. NR, non-responder; R, responder.