p16^INK4a in the Gut Mucosa Associates with NFKB1 Gene Polymorphisms and Decreased Serum Levels of Inflammation-Associated Proteins in Patients with Gastrointestinal Acute Graft Versus Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Cellular senescence is characterized by irreversible cell cycle arrest and pro-inflammatory secretion, mostly regulated by NF-kB. Expression of p16INK4a - a cell cycle inhibitor, is considered a hallmark of cellular senescence. Based on overlapping features observed in inflammatory signaling of both GVHD and cellular senescence, we have postulated a hypothesis whether genetic variants of the NFKB1 gene and expression of p16INK4a in intestinal mucosa of the recipients have impact on transplant-related inflammatory complications.

Here we present 52 consecutive patients with symptoms of lower gastrointestinal GVHD after allogeneic stem cell transplantation. The patients were genotyped for GVHD-prone polymorphisms of the NFKB1 gene, rs3774937 C/T and rs3774959 A/G. All patients underwent diagnostic proctosigmoidoscopy with gut mucosa biopsy. Immunohistochemical detection of p16INK4a expression was evaluated in normal intestinal crypts and stroma. On day +14 after graft infusion serum levels of inflammation - associated proteins were analyzed.

Increased p16INK4a expression in intestinal crypts was independently associated with late manifestation of lower gastrointestinal symptoms. Patients with and without p16INK4a expression in intestinal crypts developed lower gastrointestinal symptoms in median of 43 days (range,12-140) and 24 days (range,13-80), respectively. The effect was observed in the whole study cohort (HR 2.3 [95%CI 0.99-5.47];p=0.006) as well as in a subgroup of patients who fulfilled the clinical diagnostic criteria of GVHD only (HR 3.1 [95%CI 0.95-10.01];p=0.002).

Increased risk of histological grade 3-4 aGVHD was observed in patients without stromal p16INK4a expression (HR 3.45 [95%CI 1.26-9.42];p=0.01). The impact was confirmed by means of multivariate Cox regression (p=0.009).

No clinical variable had effect on p16INK4a expression but NFKB1 genotype. The GVHD-prone genotypes rs3774937 CC and rs3774959 AA were significantly less frequent in patients with increased p16INK4a expression (p=0.04 and p=0.02, respectively).

In 13 patients, measurement of serum levels of 92 inflammation-associated proteins from the Proseek Multiplex lnflammation - I kit on day +14 after graft infusion was performed. Patients with increased expression of p16INK4a in intestinal crypts at the time of gut biopsy had significantly lower serum levels of proinflammatory proteins (IL-17A, IL-18, IL-4, CXCL10 and TNFRSF9) on day +14. Using the STRING web engine we performed the enrichment analysis with statistically significant confirmation of interactions among these proteins suggesting various T-cell cytokine production or Type -2 immune response from the gene ontology analysis.

Cellular senescence is considered a program of innate immunity. In GVHD, innate immunity becomes seriously decontrolled. Cellular senescence may thus bring novel approaches towards GVHD diagnostics and therapy.

Supported by Ministry of Health, Czech Republic - conceptual development of research organization (FNOl, 00098892)

Papajík:Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Minarik:Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; BSM: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; EUSA Pharma: Consultancy, Honoraria; GSK: Consultancy, Honoraria; JANSSEN: Consultancy, Honoraria; SANOFI: Consultancy, Honoraria; TAKEDA: Consultancy, Honoraria.