Induction of RET dependent and independent pro-inflammatory programs in human peripheral blood mononuclear cells from Hirschsprung patients

Marta Rusmini, Paola Griseri, Francesca Lantieri, Ivana Matera, Kelly L Hudspeth, Alessandra Roberto, Joanna Mikulak, Stefano Avanzini, Valentina Rossi, Girolamo Mattioli, Vincenzo Jasonni, Roberto Ravazzolo, William J Pavan, Alessio Pini-Prato, Isabella Ceccherini, Domenico Mavilio, Marta Rusmini, Paola Griseri, Francesca Lantieri, Ivana Matera, Kelly L Hudspeth, Alessandra Roberto, Joanna Mikulak, Stefano Avanzini, Valentina Rossi, Girolamo Mattioli, Vincenzo Jasonni, Roberto Ravazzolo, William J Pavan, Alessio Pini-Prato, Isabella Ceccherini, Domenico Mavilio

Abstract

Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Phenotypic distribution and levels of…
Figure 1. Phenotypic distribution and levels of expression of RET receptor on circulating immune cell subsets.
(A) Flow cytometric dot plot (upper line) and histogram (lower line) graphs showing a representative example from an healthy donor of CD14pos monocytes, CD56pos (NK cells), CD3pos (T cells) and CD20pos (B cells) lymphocytes expressing RET receptor (black line) compared to isotype control (gray shaded histograms). (B) Summary graph of dot plots with medians (horizontal black bars) showing the mean fluorescence intensities (MFIs) of RET receptor on immune cells (black symbols) compared to that of isotype controls (open white symbols) from 17 healthy donors.
Figure 2. Correlation between the RET receptor…
Figure 2. Correlation between the RET receptor expression and RET transcripts on four different cell lines.
(A) Flow cytometric histogram graphs showing the MFI levels of expression of RET receptor (black line) of 4 different cell lines. Gray shaded histograms represent the isotype controls. (B) Histogram bar graph showing the number of RET mRNA copies produced by the same cell lines displayed in panel A and analyzed within the same time-frame in culture. Values are normalized on SK-N-MC cell line of one experiment and are reported as fold increased in expression (2−ΔCt) as mean of three independent experiments. Of note, the level of RET receptor expressed on cell membrane significantly correlated with the amount of RET transcripts, as assessed by the Spearman rank test for correlation.
Figure 3. Expression of RET receptor on…
Figure 3. Expression of RET receptor on immune cells from HSCR patients associated with a single nucleotide polymorphism at exon 2.
Summary graph of statistical dot plots showing MFI values of RET receptor expressed on lymphocytes and monocytes from 50 HSCR patients with medians (horizontal black bars) either in the absence (A) or in the presence (B) of an association analysis stratification based on the genotype at the exon 2 of RET gene (SNP rs1800858). We did not detect any statistically significant association between the phenotypic distribution of RET receptor on immune cells with RET genotype at exon 2 SNP.
Figure 4. Expression of RET receptor on…
Figure 4. Expression of RET receptor on immune cells from HSCR patients associated with pathogenic mutations of RET gene.
Summary graph of statistical histogram bars (upper panel) and dot plots (lower panel) showing MFI values of RET expressed on monocytes, T, B and NK lymphocytes from 46 HSCR patients stratified on the basis of individuals either carrying (gray histogram bars in upper panel and black symbols in the lower panel) or not carrying (empty histogram bars in upper panel and empty symbols in the lower panel) pathogenic RET mutation. 4 HSCR patients were excluded from the analysis because they were carrying mutations of the RET gene with uncertain effects.
Figure 5. Modulation of RET-dependent genes.
Figure 5. Modulation of RET-dependent genes.
Colorimetric scale graph showing the fold increase (red) and decrease (green) of mRNA transcripts for those genes modulated in both PBMCs from healthy donors and HSCR patients following treatment with GDNF and GFRα1. The differences in fold changes of RET-dependent genes between the PBMCs of three healthy donors and three HSCR patients analyzed are indicated by means of 2−ΔΔCt values included in each square and by the ranges of color tonality of the same square.
Figure 6. Modulation of RET-independent genes.
Figure 6. Modulation of RET-independent genes.
Histogram bar graph showing the relative transcript levels of genes that, regardless of treatment with GDNF and GFRα1, are differently modulated in PBMCs from healthy donors and HSCR patients. The amounts of mRNA either in freshly purified (left part of the graph) or treated (right part of the graph) of PBMCs of healthy donors (white bars) and HSCR patients (black bars) were detected by Taqman Low Density Array (TLDA) card.

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