Abstract
Introduction Infection and immune-driven inflammation of the gut often result in severe intestinal dysfunction or even end-organ disease. Intestinal dysfunction negatively affects outcome of allogeneic hematopoietic stem cell (HSCT) recipients. Cellular senescence (CS) represents a stress response mechanism. It is considered as a program of innate immunity and exhibits immunomodulatory characteristics. The study of DNA-damage and CS markers in nucleated cells of the intestinal stroma including innate and adaptive immunity cellular effectors infiltrating the intestinal stroma was performed. Correlations between DNA-damage and immune responses and the effect of common pre- and peri-transplant risk factors responses were analyzed.
Methods Using the multiplex immunofluorescence (Lunaphore CometTM) we analyzed a tissue microarray marked with 34 antibodies targeting immune cells, CS, apoptosis and inflammation from 27 colonic biopsies of 24 patients who developed lower gastro-intestinal (GI) symptoms after allogeneic HSCT. CD3 and CD68 markers were used to identify T-lymphocytes and macrophages infiltrating intestinal stroma, respectively. Promyelocytic leukemia protein (PML) and p53-binding protein 1 (53BP1) nuclear bodies as DNA-damage markers and CS marker p16INK4a were analyzed in all nucleated cells and in CD3 and CD68 cellular subsets of the intestinal stroma. Nuclear markers were quantified per nuclei count and per nuclear area. Immune cells were quantified per cell count and per stromal area. Mean fluorescence intensity was measured for every marker.
Results Median time of GI symptoms onset was 64 days (12-903). GI symptoms stage III-IV were present in 71 % of recipients. The median time of onset of GI symptoms stage III-IV was 45 days (range 12-134), whereas the time of onset of GI symptoms stage I-II was 126 days (range 64-903) (p<0.01). Acute graft versus host disease (GVHD) was diagnosed in 71 % of patients. Overlap syndrome and chronic GVHD developed in 21 and 30 % of patients, respectively.
Nuclear PML foci count positively correlated with the count of nuclear 53BP1 foci (R=0.53, p<0.01). Importantly, positive correlation was found between the count of 53BP1 foci and p16INK4a positivity (R=0.50, p<0.01).
Increased PML nuclear foci count correlated with augmented infiltration with CD68+macrophages and CD3+lymphocytes (R=0.46, p=0.02, R=0.45, p=0.02), respectively. Also, increased 53BP1 nuclear foci correlated with enriched infiltration with CD68+macrophages and CD3+lymphocytes (R=0.61, p<0.01, R=0.39, p=0.04), respectively.
Increased counts of 53BP1 foci were observed in patients who developed GI symptoms earlier than the median time onset (p=0.07). Also, patients presenting with severe GI symptoms (stage III-IV) had increased counts of 53BP1 nuclear foci (p=0.02).
There was a positive correlation found between the time of lower GI symptoms onset and the number of CD3+ T-cells positive for p16INK4a (p=0.03). Patients classified as late onset acute GVHD/overlap syndrome had increased expression of p16INK4a in the intestinal stromal cells (p=0.08).
Combined immunosuppression was associated with augmented counts of PML (p=0.03) and 53BP1 (p=0.06) nuclear foci in CD3+ lymphocytes, respectively. Moreover, in CD3+ lymphocytes, 53BP1 nuclear foci were increased in HLA-mismatched recipients (p<0.01).
Conclusion Organ-intrinsic immune reconstitution favors successful HSCT outcome. DNA-damage response and CS elicit immunomodulatory effects implying innate immune responses that may provide transitional substitute for restoring adaptive immunity but may become deleterious in long time perspective. Deciphering their roles in the context of HSCT-related complications may unravel novel treatment venues.
Supported by Prchal Foundation for Molecular and Cellular Hematology.
