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. 2015 Feb 2;10(2):e0118079.
doi: 10.1371/journal.pone.0118079. eCollection 2015.

Disease severity and mortality can be independently regulated in a mouse model of experimental graft versus host disease

Rômulo G Galvani  1 , Ramon Lemos  1 , Rômulo B Areal  2 , Pollyanna A Salvador  2 , Dario S Zamboni  3 , João Luiz M Wanderley  4 , Adriana Bonomo  5
Affiliations

Disease severity and mortality can be independently regulated in a mouse model of experimental graft versus host disease

Rômulo G Galvani et al. PLoS One. .

Abstract

Graft versus host disease (GVHD) is the major limitation of allogeneic hematopoietic stem cell transplantation (HSCT) presenting high mortality and morbidity rates. However, the exact cause of death is not completely understood and does not correlate with specific clinical and histological parameters of disease. Here we show, by using a semi-allogeneic mouse model of GVHD, that mortality and morbidity can be experimentally separated. We injected bone marrow-derived dendritic cells (BMDC) from NOD2/CARD15-deficient donors into semi-allogeneic irradiated chimaeras and observed that recipients were protected from death. However, no protection was observed regarding clinical or pathological scores up to 20 days after transplantation. Protection from death was associated with decreased bacterial translocation, faster hematologic recovery and epithelial integrity maintenance despite mononuclear infiltration at day 20 post-GVHD induction with no skew towards different T helper phenotypes. The protected mice recovered from aGVHD and progressively reached scores compatible with healthy animals. Altogether, our data indicate that severity and mortality can be separate events providing a model to study transplant-related mortality.

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Conflict of interest statement

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

Figures

Fig 1
Fig 1. NOD2KO bone marrow myeloid cells protect mice from GVHD-related mortality.
F1 (bxd) mice were lethally irradiated and received BM cells (5x106) along with splenocytes (corrected to 5x106 CD3+ cells) derived from F1, WT or NOD2KO as indicated (A-B). F1 mice (C57BL/6 x BALB/c) were lethally irradiated and received F1 WT BM cells and splenocytes as syngeneic control or (C) WT BM cells along with WT or NOD2KO purified T cells, (D) WT or NOD2KO BM cells along with WT purified T cells (E), WT BM cells and purified T cells along with B6 WT or NOD2KO non-T, non-B and non-NK spleen cells (non-LØ), (F) WT BM cells and purified T cells along with WT or NOD2KO non -CD11b, -CD11c, -CD4 and -CD8 spleen cells (non-APC). The results are represented as percent of surviving animals. Pooled results from 2 experiments; n = 10 animals per group. Log-rank test for trend. *p<0,05 and **p<0,01.
Fig 2
Fig 2. NOD2KO DCs impairs aGVHD mortality.
F1 (bxd) mice were lethally irradiated and received F1 WT BM cells and splenocytes as syngeneic control or WT BM cells and purified T cells, along with WT or NOD2KO BMDCs. (A) Survival and (B) overall GVHD clinical scores are depicted. (C) Weight loss and individual scores for fur, diarrhea, activity and hunching. Log Rank test. *p<0.05. Pooled results from 3 experiments; n = 16 animals per group.
Fig 3
Fig 3. Mice that received NOD2KO BMDC developed histopathological progressive protection in skin, colon and liver.
F1 (bxd) mice were lethally irradiated and received F1 WT BM and splenocytes as syngeneic control or B6 WT BM cells and B6 WT purified T cells, along with B6 WT or B6 NOD2KO BMDCs. Histopathology score of colon, liver and skin of animals after (A) 20, (B) 40 and (C) 180 days. (D) Parametric score of skin, colon and liver histopathology. One-way ANOVA with Bonferroni. *p<0.05, **p<0.01 and ***p<0.001. Pooled results from 3 experiments; n = 15 animals per group.
Fig 4
Fig 4. NOD2KO BMDC express lower levels of MHC-II when activated.
(A) WT or NOD2KO BMDC, generated as indicated in material and methods, were treated with LPS, heat-killed bacteria or peptidoglycan and stained for evaluation of membrane expression of I-A/I-E, CD80, CD86 and CD40. One-way ANOVA with Bonferroni post-test, ***p<0.001. Pooled results from 2 experiments; n = 14 animals per group. (B) BMDC from WT and NOD2KO mice were stained for CD11b, CD11c and Gr1. (C) Real-time quantitative PCR for Arginase1 and iNOS in WT and NOD2KO BMDC. (D) F1 mice were lethally irradiated and received F1 WT BM cells and splenocytes as syngeneic control or WT BM cells and WT purified T cells, along with WT or NOD2KO BMDCs or purified CD11c cells from WT or NOD2KO. *p<0.05. Pooled results from 2 experiments; n = 10 animals per group.
Fig 5
Fig 5. Mice that received WT or NOD2KO BMDC have the same frequency of Treg cells.
F1 (bxd) mice were lethally irradiated and received F1 WT BM cells and splenocytes as syngeneic control or WT BM cells and WT purified T cells, along with WT or NOD2KO BMDC. After 21 days, spleen, mesenteric lymph nodes, colon, liver and skin of transplanted F1 animals were stained for CD4, CD25 and Foxp3 for the evaluation of the (A) percentage of Treg cells (CD4+CD25+Foxp3+) and (B) the Treg:Teff ratio. One-way ANOVA with Bonferroni post-test, *p<0.05 and ***p<0.001. One experiment representative of 2; n = 5 mice per group.
Fig 6
Fig 6. Mice that received WT or NOD2KO BMDC had lower levels of CCL5 and LPS and higher levels of G-CSF.
F1 (bxd) mice were lethally irradiated and received F1 WT BM cells and splenocytes as syngeneic control or WT BM cells and purified T cells, along with WT or B6 NOD2KO BMDC. Serum of transplanted animals was collected 21 days post transplantation. (A) CCL5 and G-CSF were analyzed by multiplex ELISA. (B) Bone marrow and blood from transplanted animals were collected on day 6 and 20, cytocentrifuged and stained with H&E for differential cell counts, (C) LPS from individual mice was quantified on days 21 and 40 after transplantation using the LAL assay. T test for A and One-way ANOVA with Bonferroni post test for B. *p<0.05, **p<0.01 and ***p<0.001. Pooled results from 2 experiments; n = 10 mice per group.
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This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnològico (CNPq) and the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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