[The influence of different collagenases on the analysis of immune cells in salivary glands of mice with chronic graft-versus-host disease].

Objective: To preliminarily explore the influence of different collagenases on the analysis of immune cells in salivary glands of mice with chronic graft-versus-host disease (cGVHD) . Methods: A major histocompatibility complex mismatch model was established (bone marrow: 5×10(6) cells; spleen: 0.25×10(6) cells), including the non-graft-versus-host disease (GVHD) control group (BALB/c to BALB/c, n=15) and cGVHD group (C57BL/6c to BALB/c, n=15). Body weight, clinical scores, and survival rates of the mice were observed after transplantation. On day 45 after transplantation, salivary glands were collected (n=6-12), and hematoxylin and eosin and Masson trichrome staining were performed to assess the degree of injury and fibrosis. Flow cytometry analysis was conducted to compare immune cell populations and phenotypes among samples without collagenase digestion, with collagenase Ⅱ digestion, and with collagenase D digestion. Results: Compared with the non-GVHD group, mice in the cGVHD group showed significant weight loss [original body weight percentage on day 60 after transplantation: (66.77±11.72) % vs (101.20±4.19) %, P<0.0001], higher symptom scores (2.11±0.59 vs 0±0, P<0.0001), and significantly lower survival rates (survival rate on day 60 after transplantation: 50% vs 100%, P=0.0011). The salivary glands of cGVHD mice showed acinar atrophy, cellular infiltration (3.67±0.52 vs 2.00±0, P<0.0001), and fibrosis [ (14.54±4.05) % vs (4.11±0.87) %, P=0.0001]. Collagenase digestion significantly increased the yield and viability of mononuclear cells (P<0.05). Among the tested protocols, digestion with collagenase Ⅱ resulted in the greatest recovery of mononuclear cells (P<0.01). However, collagenaseⅡdigestion significantly reduced the proportion of CD4(+) T cells [ (5.64±2.05) % vs (31.98±12.23) %, P=0.0052], whereas collagenase D had no significant effect on CD4(+) T cells[ (31.51±16.37) % vs (31.98±12.23) %, P=0.9973]. In the cGVHD group, the method containing collagenase D recovered more CD4(+) T cells, whereas the method containing collagenase Ⅱ recovered more CD8(+) T cells (all P value<0.05). In addition, the number of natural killer (NK) cells was significantly increased in both groups after collagenase digestion (all P value<0.05). Among myeloid cells, collagenaseⅡdigestion significantly reduced the proportion of Ly6C(low)CD11b(+)Ly6G(+) neutrophils [ (0.05±0.01) % vs (0.34±0.20) %, P=0.0171], whereas collagenase D had no significant effect [ (0.49±0.19) % vs (0.34±0.20) %, P=0.2800]. In cGVHD tissues, the number of CD11b(+)Ly6C(+)Ly6G(-) monocytes rcovered with collagenase D digestion was significantly higher than that recovered with collagenase Ⅱ digestion [ (0.06±0.05) ×10(5) vs (0.01±0) ×10(5), P=0.0384], whereas the number of macrophages rcovered with collagenase Ⅱ digestion was significantly higher than that recovered with collagenase D digestion [ (1.42±0.56) ×10(5) vs (0.57±0.36) ×10(5), P<0.0001]. Conclusion: Collagenase digestion significantly increased the number and viability of mononuclear cells. The method containing collagenase Ⅱ yielded the highest number of mononuclear cells as well as CD8(+) T cells, NK cells, and macrophages, whereas collagenase D was more suitable for recovering CD4(+) T cells, neutrophils, and monocytes.