Significance of PD1 Alternative Splicing in Celiac Disease as a Novel Source for Diagnostic and Therapeutic Target

Abstract

Background: We have focused on the alteration of the PD-1/PD-L1 pathway in celiac disease and discussed the roles of the PD1 pathway in regulating the immune response. We explored the idea that the altered mRNA splicing process in key regulatory proteins could represent a novel source to identify diagnostic, prognostic, and therapeutic targets in celiac disease.

Methods: We characterized the PD1 mRNA variants' profile in CD patients and in response to gluten peptides' incubation after in vitro experiments. Total RNA from whole blood was isolated, and the coding region of the human PD-1 mRNA was amplified by cDNA PCR.

Results: PCR amplification of the human PD-1 coding sequence revealed an association between the over-expression of the sPD-1 protein and the PD-1Δex3 transcript in celiac disease. Thus, we have found three novel alternative spliced isoforms, two of which result in a truncated protein and the other isoform with a loss of 14 aa of exon 2 and complete exon 3 (Δ3) which could encode a new soluble form of PD1 (sPD-1).

Conclusions: Our study provides evidence that dietary gluten can modulate processes required for cell homeostasis through the splicing of pre-mRNAs encoding key regulatory proteins, which represents an adaptive mechanism in response to different nutritional conditions.

Keywords: PD1/PDL; alternative splicing; celiac disease; gluten peptides; immune checkpoint.

Figure 1

(A, B) Genotyping of the (G/A) PD-1.3 polymorphism of the PD1 gene. (A) Schematic representation of the 331 bp PCR amplified fragment containing the SNP. The presence of A in the SNP generates a target for the restriction endonuclease PstI that generates two fragments of 55 and 276 bp. (B) Electrophoresis of the PstI digested PCR products on a 2% agarose gel, showing the different banding patterns obtained for each genotype (AA, GG and GA). Lane 1 = DNA size marker. Lane 2 = undigested plasmid used as an internal control of the PstI digestion. The numbers on the left indicate the size of DNA fragments in bp. Asterisk marks linearized form of the plasmid used as internal control for the PstI digestion. (C, D) Genotyping of the (C/T) PD-1.5 polymorphism of the PD1 gene. (C) Schematic representation of the 333 bp PCR amplified fragment containing the SNP. The presence of T in the SNP generates an additional target for the restriction endonuclease AluI that generates three fragments of 69, 180 and 84 bp. (D) Electrophoresis of the AluI digested PCR products on a 3% agarose gel, showing the different banding patterns obtained for each genotype (CC, CT and TT). First lane is the DNA size marker. The numbers on the left indicate the size of DNA fragments in bp. Asterisk marks the two fragments generated by the DNA used as an internal control for the AluI digestion.

Figure 2

IFN-gamma release by PBMCs of CD patients stimulated with different peptides and PHA as positive control and of healthy controls stimulated and unstimulated with PHA. In axis X the patients and in axis Y the levels of IFN-gamma expressed in pg/ml.

Figure 3

Gel electrophoresis of PD-1 cDNA amplification products from PHA stimulated PBMCs of celiac patients as control positive of T cell stimulation.

Figure 4

Detection of PD1 in Human PBMCs from healthy controls by flow cytometry either unstimulated (A) and stimulated with PHA (B).

Figure 5

Immunohistochemistry analysis: (A) Negative immunostaining for PD1 in celiac disease patients. (B) Immunostaining for PDL1 in celiac disease patients showing expression in lamina propria cells and in Lieberkhün crypts. (C) Immunostaining for PDL2 in CD patients showing expression in epithelial cells. Magnification 200×. Arrows: cells with immunoreaction.

Figure 6

PD-1/PD-L1 expression in the serum of celiac disease patients and healthy controls (pg/ml). PD-1 and PD-L1 are highly expressed in serum from celiac disease patients (n = 25) in relation to healthy controls (n = 5). Significant difference at *p < 0.05 is shown.

Figure 7

Schematic diagram for PD1 splicing variants found in this study. flPD-1, PD-1Dex3, PD-1Dex2, PD-1Dex2,3 with sizes about 687, 531, 327, and 171 bp, respectively. New alternative spliced isoforms that retained part of different introns, with sizes of 781 and 715 bp, and isoform with a loss of 42 bp from exon 2 and a complete exon 3 (Δ3) with a size of 489 bp. Asterisks indicate the new isoforms found in this study.

Figure 8

Gel electrophoresis of PD-1 cDNA amplification products from PBMCs stimulated with gluten peptides in healthy controls (A) and in celiac patients (B). With the used primers in the present study, the sizes of the respective PCR products were 687 bp for the full-length PD-1 transcript, 531 bp for PD-1Dex3, 327 bp for PD-1Dex2, 171 bp for PD-1Dex2,3, and three new isoforms with 781, 715, and 489 bp. Asterisks indicate the new isoforms found in this study.