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. 2009 Sep 28;206(10):2101-10.
doi: 10.1084/jem.20090623. Epub 2009 Sep 8.

NOD2 regulates hematopoietic cell function during graft-versus-host disease

Olaf Penack  1 , Odette M Smith, Amy Cunningham-Bussel, Xin Liu, Uttam Rao, Nury Yim, Il-Kang Na, Amanda M Holland, Arnab Ghosh, Sydney X Lu, Robert R Jenq, Chen Liu, George F Murphy, Katharina Brandl, Marcel R M van den Brink
Affiliations

NOD2 regulates hematopoietic cell function during graft-versus-host disease

Olaf Penack et al. J Exp Med. .

Abstract

Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2(-/-) hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.

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Figures

Figure 1.
Figure 1.
NOD2 deficiency in donor BM cells and/or donor T cells does not affect the development of GVHD. (A–C) Lethally irradiated (8.5 Gy) BALB/c allo-BMT recipients received 5 × 106 CFSE-labeled B6 WT or B6 NOD2−/−CD5+ T cells. Recipient spleens were harvested 96 h after transfer of T cells; n = 8/group; combined data from two independent experiments are shown. (A) Identical CFSE histogram overlays of WT and NOD2−/− donor T cells (left), as well as similar total number of CFSEhigh and CFSElow donor T cells. (B) Similar up-regulation of the alloactivation marker CD25 on WT B6 donor T cells versus NOD2−/− donor T cells. (C) Similar expression of the gut-homing marker LPAM on WT B6 donor T cells versus NOD2−/− donor T cells. (D) MLR with WT versus NOD2−/−CD5+ T cells and irradiated BALB/c stimulators. Combined data of three independent experiments are shown; n = 12/group. (E) Lethally irradiated BALB/c (8.5 Gy) or LP (11Gy) allo-BMT recipients were transplanted with 5 × 106 B6 WT TCD-BM versus B6 NOD2−/− TCD-BM and 106 B6 WT T cells versus B6 NOD2−/− T cells. Combined data from four independent experiments are shown; n = 15–45/group. Error bars indicate SEM.
Figure 2.
Figure 2.
NOD2−/− allo-BMT recipients have increased systemic and target organ GVHD because of enhanced activation and proliferation of alloreactive donor T cells. (A) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− allo-BMT recipients received 5 × 106 B10BR TCD-BM or 5 × 106 LP TCD-BM with or without 106 B10BR T cells or 2 × 106 LP T cells. Combined data from four independent experiments are shown; n = 45/group. (B-E) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− allo-BMT recipients received 5 × 106 B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day 21 after allo-BMT. Combined data from two independent experiments are shown; n = 10/group. (B) Histopathologic GVHD scores in target organs showing increased GVHD in NOD2−/− allo-BMT recipients. (C) CD3+ histochemistry images showing increased infiltration of T cells (brown) in liver tissue of NOD2−/− allo-BMT recipients. Bars, 50 µm. (D) Quantification of T cell infiltration in GVHD target organs by CD3+ histochemistry. (E) Increased thymic GVHD in NOD2−/− allo-BMT recipients demonstrated by reduced numbers of CD4+/CD8+ double-positive thymocytes. Shown are flow cytometric plots and the absolute numbers of DP (CD4+/CD8+), DN (CD4−/CD8−), CD4+, and CD8+ thymocytes. (F) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− allo-BMT recipients received 5 × 106 CFSE-labeled B10BR CD5+ T cells. Recipient spleens were harvested 96 h after transfer of T cells; n = 8/group; combined data from two independent experiments are shown. CFSE histogram overlays of donor T cells in WT allo-BMT recipients versus NOD2−/− allo-BMT recipients (left); ratio of proliferating donor T cells (right, top) and absolute numbers of donor T cells (right, bottom) showing increased donor T cell proliferation in NOD2−/− allo-BMT recipients. (G and H) Lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− allo-BMT recipients were transplanted with 5 × 106 B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown; n = 10/group. (G) Absolute numbers and activation status (CD25+) of donor CD3+ T cells in spleen and MLNs. Percentage of FoxP3+ cells in CD4+ splenocytes (top right) and expression of the gut homing marker LPAM in MLNs (bottom right) are shown. (H) Absolute numbers of donor B cells, NK cells, monocytes, DCs, and granulocytes in spleen. Error bars indicate SEM.
Figure 3.
Figure 3.
NOD2 deficiency of the hematopoietic system of the allo-BMT recipient aggravates GVHD. (A–D) Chimeric mice with NOD2 deficiency either in the hematopoietic system or in the nonhematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2−/− or B6 NOD2−/−→B6 WT). After 90 d, lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− versus chimeric allo-BMT recipients were transplanted with 5 × 106 B10BR TCD-BM or 5 × 106 LP TCD-BM + 2 × 106 B10BR T cells or 3 × 106 LP T cells. (A) Survival curves. Combined data from two independent experiments are shown; n = 16/group. (B-E) Lethally irradiated (11 Gy) B6 WT→B6 NOD2−/− or B6 NOD2−/−→B6 WT chimeric allo-BMT recipients were transplanted with 5 × 106 B10BR TCD-BM + 106 B10BR T cells. Organs were harvested at day 14 after allo-BMT. Combined data from two independent experiments are shown; n = 8/group. (B) Histopathologic GVHD scores in GVHD target organs. (C) Quantification of T cell infiltration in GVHD target organs by CD3+ histochemistry. (D) Absolute number and activation status (CD25+) of donor CD3+ T cells in spleen and MLNs, percentage of FoxP3+ regulatory T cells (top right), and expression of the gut-homing marker LPAM in MLNs (bottom right). Error bars indicate SEM.
Figure 4.
Figure 4.
NOD2−/− DCs have a higher activation status and increased ability to induce T cell proliferation during GVHD. Lethally irradiated (11 Gy) B6 WT versus B6 NOD2−/− allo-BMT recipients were transplanted with 5 × 106 B10BR TCD-BM + 2 × 106 B10BR T cells. Organs were harvested at day 7 after allo-BMT. Donor and host-derived DCs were distinguished by MHC class I disparity in flow cytometry (H2k vs. H2b). Combined data from two independent experiments are shown; n = 8/group. (A) Quantification of host DC subsets by flow cytometry. (B) Quantification of activation marker expression of host DCs by flow cytometry before allo-BMT and at day 7 after allo-BMT. (C) Histogram overlays of activation marker expression of host DCs during GVHD. (D) MLR using 105 splenic DCs (CD11+ selection) from B6 WT allo-BMT recipients versus B6 NOD2−/− allo-BMT recipients as stimulators and 105 B10BR T cells (CD5+ selection). Shown is the thymidine H3 uptake (cpm) between 48 and 72 h of MLR. Error bars indicate SEM.
Figure 5.
Figure 5.
NOD2 deficiency in the hematopoietic system regulates experimental colitis. (A) TNBS colitis (5 mg TNBS in 50% ethanol) was induced in B6 WT or B6 NOD2−/− mice. Higher weight loss of NOD2−/− mice during colitis. Combined data from three independent experiments are shown; n = 12/group. (B–D) First, chimeric mice with NOD2 deficiency either in the hematopoietic system or in the nonhematopoietic system were created by syngeneic BMT (B6 WT→B6 NOD2−/− or B6 NOD2−/−→B6 WT). After 90 d, TNBS colitis (5 mg TNBS in 50% ethanol) was induced. Colons were harvested at day 3 after induction of colitis. Combined data from two independent experiments are shown; n = 8/group. (B) Higher weight loss of NOD2−/−→WT chimera versus WT→NOD2−/− chimera during colitis. (C) Hematoxylin and eosin–stained images of the colon at day 3 after induction of colitis. Bar, 200 µm. (D) Histological scoring showing increased colitis in the hematopoietic NOD2-deficient chimera. Error bars indicate SEM.
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