Transcriptomic signatures reveal immune dysregulation in human diabetic and idiopathic gastroparesis

Abstract

Background: Cellular changes described in human gastroparesis have revealed a role for immune dysregulation, however, a mechanistic understanding of human gastroparesis and the signaling pathways involved are still unclear.

Methods: Diabetic gastroparetics, diabetic non-gastroparetic controls, idiopathic gastroparetics and non-diabetic non-gastroparetic controls underwent full-thickness gastric body biopsies. Deep RNA sequencing was performed and pathway analysis of differentially expressed transcripts was done using Ingenuity®. A subset of differentially expressed genes in diabetic gastroparesis was validated in a separate cohort using QT-PCR.

Results: 111 genes were differentially expressed in diabetic gastroparesis and 181 in idiopathic gastroparesis with a log2fold difference of | ≥ 2| and false detection rate (FDR) < 5%. Top canonical pathways in diabetic gastroparesis included genes involved with macrophages, fibroblasts and endothelial cells in rheumatoid arthritis, osteoarthritis pathway and differential regulation of cytokine production in macrophages and T helper cells by IL-17A and IL-17F. Top canonical pathways in idiopathic gastroparesis included genes involved in granulocyte adhesion and diapedesis, agranulocyte adhesion and diapedesis, and role of macrophages, fibroblasts and endothelial cells in rheumatoid arthritis. Sixty-five differentially expressed genes (log2fold difference | ≥ 2|, FDR < 5%) were common in both diabetic and idiopathic gastroparesis with genes in the top 5 canonical pathways associated with immune signaling. 4/5 highly differentially expressed genes (SGK1, APOLD1, CXCR4, CXCL2, and FOS) in diabetic gastroparesis were validated in a separate cohort of patients using RT-PCR. Immune profile analysis revealed that genes associated with M1 (pro inflammatory) macrophages were enriched in tissues from idiopathic gastroparesis tissues compared to controls (p < 0.05).

Conclusions: Diabetic and idiopathic gastroparesis have both unique and overlapping transcriptomic signatures. Innate immune signaling likely plays a central role in pathogenesis of human gastroparesis.

Keywords: Diabetes mellitus; Macrophages; Next generation sequencing; RNA; Signaling.

Conflict of interest statement

Ethics approval and consent to participate

IRB approval obtained at all clinical sites and at Mayo Clinic as follows: Temple University Office for Human Subjects Protections (FWA00004964); California Pacific Medical Center Research Institute (FWA00000921); Texas Tech University Health Sciences Center (El Paso) (FWA 00006767); University of Mississippi Medical Center (FWA00003630) and Mayo Clinic (IRB 07–003371). Written informed consent was obtained for gastroparesis cases and verbal consent was obtained for controls as per approval by respective IRBs.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1

Heat maps of differentially expressed genes (Log2fold change | ≥ 2|, FDR < 0.05) in (a) Diabetic gastroparesis and (b) Idiopathic gastroparesis

Fig. 2

a Venn diagram showing overlapping differentially expressed genes between diabetic gastroparetics and diabetic controls and idiopathic gastroparetics and idiopathic controls (Log2fold change | ≥ 2|, FDR < 0.05). b Heat map demonstrating these overlapping, differentially expressed genes between diabetic gastroparetics and diabetic controls and idiopathic gastroparetics and idiopathic controls (Log2fold change | ≥ 2|, FDR < 0.05). c Top canonical pathways linking the overlapping genes involved between the two comparisons. The horizontal bars represent total number of genes present in the pathway, scaled to 100%. The orange dots indicate the ratio of the overlapping differentially expressed genes that map to the pathway divided by the total number of genes present in the same pathway, e.g. > 25% for Granulocyte Adhesion and Diapedesis

Fig. 3

RT-PCR validation of 5 genes differentially expressed in diabetic gastroparetics and diabetic controls by RNA seq in a different set of diabetic gastroparesis patients: Significant downregulation of APOLD1, apolipoprotein L domain containing 1; CXCR4, C-X-C motif chemokine receptor 4; CXCL2, C-X-C motif chemokine ligand 2; and FOS, Fos proto-oncogene in diabetic gastroparesis, as observed in the RNA seq analysis. SGK1, serum/glucocorticoid regulated kinase 1 expression was not statistically different on RT-PCR in the validation cohort, but had significantly lower log2fold changes in expression on RNA seq (1.77)

Fig. 4

CIBERSORT analysis displaying distribution of patients with M1 and M2 macrophage associated genes in (a) Diabetic controls and diabetic gastroparesis: No differences were seen in % genes/subject associated with M1 or M2 macrophage phenotype (b) Idiopathic controls and idiopathic gastroparesis: Significantly greater number of idiopathic gastroparesis patients express % genes associated with an M1 (proinflammatory) macrophage phenotype (Mean (SD): 0.04 (0.03) % vs 0.004 (0.007) % M1 associated genes in idiopathic gastroparesis and idiopathic controls respectively, p = 0.02). No differences were seen in % genes/subject associated with M2 macrophage phenotype