Immune Netw.  2015 Jun;15(3):125-134. 10.4110/in.2015.15.3.125.

Attenuation of Hepatic Graft-versus-host Disease in Allogeneic Recipients of MyD88-deficient Donor Bone Marrow

• Lim JY
• Lee YK
• Lee SE
• Ju JM
• Park G
• Choi EY
• Min CK

• KMID: 2168038
• DOI: http://doi.org/10.4110/in.2015.15.3.125

Abstract

Acute graft-versus-host-disease (GVHD) is characterized by selective damage to the liver, the skin, and the gastrointestinal tract. Following allogeneic hematopoietic stem cell transplantation, donor bone marrow (BM) cells repopulate the immune system of the recipient. We previously demonstrated that the acute intestinal GVHD (iGVHD) mortality rate was higher in MyD88-deficient BM recipients than that in the control BM recipients. In the present study, the role of MyD88 (expressed by donor BM) in the pathophysiology of hepatic GVHD (hGVHD) was examined. Unlike iGVHD, transplantation with MyD88-deficient T-cell depleted (TCD) BM attenuated hGVHD severity and was associated with low infiltration of T cells into the liver of the recipients. Moreover, GVHD hosts, transplanted with MyD88-deficient TCD BM, exhibited markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the liver. Adoptive injection of the MDSC from wild type mice, but not MyD88-deficient mice, enhanced hepatic T cell infiltration in the MyD88-deficient TCD BM recipients. Pre-treatment of BM donors with LPS increased MDSC levels in the liver of allogeneic wild type BM recipients. In conclusion, hGVHD and iGVHD may occur through various mechanisms based on the presence of MyD88 in the non-T cell compartment of the allograft.

Keyword

Myeloid differentiation factor 88 (MyD88); Myeloid-derived suppressor cells (MDSC); Hepatic graft-versus-host disease (GVHD); Allogeneic hematopoietic stem cell transplantation

MeSH Terms

Allografts
Animals
Bone Marrow*
Gastrointestinal Tract
Graft vs Host Disease*
Hematopoietic Stem Cell Transplantation
Humans
Immune System
Liver
Mice
Mortality
Skin
T-Lymphocytes
Tissue Donors*

Figure

• 

  Figure 1 Allogeneic recipients of MyD88KO donor TCD-BM exhibit less severe hepatic. Lethally irradiated F1 recipients were given 5×106 WT or MyD88KO TCD-BM cells plus 1×106 purified WT T cells from allogeneic B6 donors (n=10 in each group, allo WT and allo KO). Control mice were given only TCD-BM from WT or MyD88KO mice (thus without co-transplantation of B6 T cells; n=10 in each group, WT TCD-BM and KO TCD-BM). (A) Liver histology in representative F1 recipients transplanted with WT or MyD88KO TCD-BM plus WT T cells. The extent of mononuclear cell infiltration into portal triads was assessed, and the pathological status was scored semi-quantitatively; 20 portal areas of two liver sections from each animal were evaluated. The areas within the black squares in the ×50 images of the upper panel were magnified to ×200, as shown at the bottoms of the images. The extent of pathological damage to liver was evaluated using the semi-quantitative scoring system described above, in both the allo WT and allo KO groups (n=5 in each group). (B) Serum alanine transaminase (ALT) level. Data are presented as means ± SEMs. Statistics: **p<0.01 and ***p<0.001. Data shown are representative of two independent experiments.

• 

  Figure 2 Effect of MyD88 deficiency in TCD-BM on the expansion and infiltration of donor-type T cells in liver and peripheral lymph nodes (PLN). Lethally irradiated F1 recipients were given 5×106 WT or MyD88KO TCD-BM cells plus 1×106 purified WT T cells isolated from congenic B6 donors (CD45.1+) (n=5, each group, allo WT and allo KO). (A) The expansion of donor T cells in the liver and PLN were assessed on day 13 post-transplantation. The frequencies and absolute numbers of CD45.1+ cells analyzed via flow cytometry are shown. Data are presented as means±SEMs and are representative of duplicate experiments. Statistics: ***p<0.001. (B and C) The frequencies and numbers of donor-type CD4+ (B) and CD8+ (C) T cells were examined on day 13. Data are presented as means±SEMs. Statistics: **p<0.01 and ***p< 0.001. (D) Infiltration of CD45.1+ cells in the liver during GVHD. Livers were harvested from F1 recipient mice (n=5) on day 13 and sectioned for staining with anti-CD45.1 Ab as described in "Materials and Methods". Magnification: ×50 (upper) and ×100 (lower). Data from one of two replicate experiments that yielded similar results are shown.

• 

  Figure 3 Effect of MyD88 deficiency in TCD-BM on the expansion of CD11b+Gr-1+ MDSC. Lethally irradiated F1 recipients were given 5×106 WT or MyD88KO TCD-BM cells plus 1×106 purified WT T cells isolated from WT B6 donors (n=5, each group, allo WT and allo KO). (A) On day 13 thereafter, the liver and peripheral lymph nodes (PLN) were harvested and the expression levels of CD11b+Gr-1+ were measured via flow cytometric analysis. The proportions and absolute numbers of CD11b+Gr-1+ cells are shown. Data are presented as means ± SEMs and are representative of duplicate experiments. Statistics: ***p<0.001. (B) Peripheral blood mononuclear cells (PBMC) from GVHD hosts that received WT or MyD88KO TCD-BM (n=3 in each group, allo WT and allo KO, respectively) were stained with anti-LAMP2 antibody. The numbers of LAMP2-positive neutrophils in immunofluorescence fields were compared between the allo WT and allo KO groups. The data shown are representative of two independent experiments.

• 

  Figure 4 Injection of CD11b+Gr-1+ MDSC from WT mice, but not MyD88KO mice, enhances donor T-cell infiltration into the livers. Lethally irradiated F1 recipients were given 5×106 MyD88KO TCD-BM cells plus 1×106 purified WT T cells from allogeneic B6 donors. Mice exhibiting GVHD that had received MyD88KO TCD-BM cells were injected with 1.0×106 CD11b+Gr-1+ MDSC isolated from WT or MyD88KO mice at days 3, 5, and 7 post-transplantation. (A~D) The frequencies and absolute numbers of donor T cells (A) with donor-type CD4+ (B) and CD8+ (C) T cells analyzed via flow cytometry are shown. (D) Expression of CD11b+Gr-1+ was measured using FACS analysis. The frequencies and absolute numbers of CD11b+Gr-1+ cells are shown. Data are presented as means±SEMs. Statistics: *p<0.05, **p<0.01 and ***p<0.001. Data are representative of duplicate experiments (n=6 per experiment).

• 

  Figure 5 Recipients of TCD BM from WT donor mice injected with LPS show high MDSC levels in the liver with decreased frequency of donor T cells. WT or MyD88KO B6 donor mice were subjected to i.p. injection of LPS at 2 mg/kg for 3 days. Mice not receiving LPS received an equal amount of the LPS vehicle (PBS i.p.). Lethally irradiated F1 recipients were given 5×106 LPS- or PBS-treated donor TCD-BM cells plus 1×106 purified WT T cells from allogeneic B6 donors (n=6 per group). (A~D) The frequencies and absolute numbers of donor T cells (A), and donor-type CD4+ (B) and CD8+ (C) T cells analyzed via flow cytometry are shown. (D) The expression levels of CD11b+Gr-1+ were measured via FACS. The frequencies and absolute numbers of CD11b+Gr-1+ cells are shown. The data are presented as means±SEMs. Statistics: **p<0.01 and ***p<0.001. Data are representative of duplicate experiments.

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