Sarcoidosis activates diverse transcriptional programs in bronchoalveolar lavage cells

Abstract

Background: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease.

Methods: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs.

Controls: To better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles.

Results: Sarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis.

Conclusions: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.

Keywords: Microarray; Network analysis; Proteasome; Sarcoidosis.

Fig. 1

Correspondence analysis of transcriptional profiles between sarcoidosis patients (magenta) and normal controls (cyan). Each axis captures a proportion of the observed whole-genome expression variability across all subjects. Note that this unbiased analysis segregated the cohort into two groups based on disease status, implying that sarcoidosis induces global changes in BAL cell transcriptome

Fig. 2

Cluster analysis of differentially expressed genes in BAL cells between sarcoidosis patients and normal controls. Subject numbers correspond to Table 1 descriptions. Significantly over-represented processes among differentially upregulated and downregulated genes in sarcoidosis were identified using Gene Ontology (GO) annotation and are shown on the right panel

Fig. 3

Integrative network analysis of enriched pathways in sarcoidosis as identified using GSEA. A gene set was considered enriched for a given phenotype if most of its member genes were upregulated in that condition. 83 gene sets were upregulated in patients with sarcoidosis whereas very few pathways were enriched in the controls (not shown). In the figure, each sphere designates an enriched pathway in sarcoidosis and the size of each sphere (i.e., gene set) is proportional to the number of its gene members. Only a selected number of pathways have been labeled due to space restriction, but full list is available at Additional file 3: Table S2. Since pathways can share common genes, connectivity lines have been drawn to link these inter-pathway relationships and define the topology of the enrichment network by identifying larger aggregates of functionally associated pathways known as modules. The most prominent module associated with sarcoidosis was “immunity” and incorporated T-cell signaling (T helper and CD8), graft vs. host disease, IL12, IL23, and IL17 pathways among others, but also note the presence of other modules such as “oxidative phosphorylation” and “cell cycle/protein degradation”. Importantly, some pathways from different modules were also linked together, such as the inter-modular connectivity of “adaptive immune system” with “proteasome” as shown in the figure. This relationship indicates that these two gene sets share common functional characteristics and implicates proteasomal processes as potential modulators of adaptive immunity in sarcoidosis

Fig. 4

Heatmap display (a) and gene product interaction network (b) of the enriched proteasome pathway in sarcoidosis. Subject numbers correspond to Table 1 descriptions. Note the prominent role of IFNG in integrating key immunoproteasome components, such as PSMB8 and PSMB9

Fig. 5

BAL levels of IL6 (a) and CCL5 (b) are elevated in sarcoidosis patients. Cell-free bronchoalveolar fluid (BALF) was analyzed by Mesoscale Discovery in normal controls (N = 12), sarcoidosis subjects who underwent the microarray analysis of their BAL cells (sarcoidosis 1, N = 15) and a validation group of sarcoidosis patients that were not part of the original microarray study (sarcoidosis 2, N = 10). P-values were calculated using two-tailed Mann–Whitney test

Fig. 6

Immunoproteasome components are overexpressed in BAL cells from patients with sarcoidosis. PSMB8, PSMB9, and IFNG gene expression levels were determined by qPCR from RNA isolated from BAL cells of normal controls and sarcoidosis patients. RNA expression was normalized to GAPDH as described in Methods section. Data are presented as Log₂ [fold change] in mRNA expression between sarcoidosis patients versus control subjects. P-values were calculated using two-tailed Student’s t-test of log₂-transformed data