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Published in final edited form as: Biol Blood Marrow Transplant. 2012 Sep 13;19(1):164–168. doi: 10.1016/j.bbmt.2012.09.001

Influence of donor microbiota on the severity of experimental graft-versus-host-disease

Isao Tawara 1, Chen Liu 2, Hiroya Tamaki 1, Tomomi Toubai 1, Yaping Sun 1, Rebecca Evers 1, Evelyn Nieves 1, Nathan Mathewson 1, Gabriel Nunez 3, Pavan Reddy 1,*
PMCID: PMC3529780  NIHMSID: NIHMS407655  PMID: 22982686

Abstract

The link between microbial flora and the shaping of immune responses is being increasingly appreciated and recent data have uncovered a role for recipient microbiota on the severity of graft-versus-host disease (GVHD). However the impact of donor microbiota on T cell mediated allo-responses and GVHD is not known. Utilizing multiple clinically relevant murine models we analyzed the effect of donor microbiota on the severity of GVHD induced by T cells from specific pathogen free (SPF) and germ-free (GF) donors and found that donor microbiota does not alter the expansion, differentiation of alloreactive T cells or the severity of GVHD.

Keywords: germ-free, microbial flora, allogeneic BMT, GVHD

INTRODUCTION

Development of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) is the major obstacle for an effective utilization of this potentially curative therapy against many hematological diseases such as leukemia. The composition of the allo-antigens, antigen presenting cells, inflammatory milieu, age and several other host specific factors play important roles in the biology of GVHD. These factors notwithstanding, the frequency of the allo-reactive donor T cells and their type of response to allo-antigens is fundamental for the induction and severity of GVHD (Jenq et al, 2010).

Countless microbes colonize the intestine, skin and form part of the host microbial ecology. Cross talk of the microbiota with various immune cell subsets of the host immune system is known to modulate the immune responses and affect autoimmunity, metabolic diseases and response to pathogens (Jarchum et al, 2011; Chinen et al, 2012). In line with this, past and recent data have uncovered a role for host (recipient) microbiome in the severity of GVHD. Studies in mice have shown reduction of GVHD with gut-decontaminating antibiotics (van Bekkum et al, 1974) and transplantation in germ-free conditions (Jones et al, 1971). Data have demonstrated shift in the gut flora in GVHD recipients (Eriguchi et al, 2012; Jenq et al, 2012). Furthermore, breaching of the epithelial barriers and translocation of PAMPs have been shown to enhance pro-inflammatory cytokines released from host damaged tissue, enhance donor T cell alloreactivity and aggravate GVHD (Panack et al, 2010). Resident microbiota plays a key role in the development/maturation of T cells. CD4+ T cells from GF donors caused more severe or similar colitis compared with T cells from conventionally housed donors (Bleich et al, 2009; Liu et al, 2011). However, the influence of the cross-talk between microbiota in shaping donor T cell allo-responses and GVHD has heretofore not been reported. Utilizing well characterized murine models, we compared the allo-reactivity of T cells from GF donors with conventionally housed (SPF) donors to induce GVHD and found that donor microbiota does not alter the severity of GVHD in the hosts.

Materials and methods

Mice

Specific pathogen free (SPF) C57BL/6J (SPF-B6, H-2b) mice were purchased from the Jackson Laboratories (Bar Harbor, ME) and BALB/c (H-2d) and B6D2F1 (H-2b/d) from the National Cancer Institute-Frederick (Frederick, MD). Germ-free C57BL6/J (GF-B6) mice were maintained in a germ-free mouse facility in the University of Michigan, where they were housed in soft-sided plastic isolators in which they remain free of all bacteria, exogenous virus, fungi and parasites until just before euthanasia. All animal studies were performed according to approved protocol by the University of Michigan Committee on Use and Care of Animals.

Antibodies and flow cytometry

FITC-, PE-, PerCP-Cy5.5- or APC-conjugated monoclonal antibodies (mAbs) to mouse CD4, CD8a, CD25, H-2Kb, H-2Kd, FoxP3 (from eBioscience, San Diego, CA), anti-mouse TCRβ (from BD Pharmingen San Diego, CA). Flow cytometry was performed as outlined before (Tawara et al, 2010) and the cells were analyzed on a C6 flowcytometer (BD Accuri Cytometers, Ann Arbor, MI).

Bone marrow transplantation

BMT were performed as described before (Tawara et al, 2011). Briefly, recipient mice were irradiated (137Cs source) with 8 Gy (BALB/c recipients) or 11 Gy (B6D2F1 recipients) TBI 1 day before bone marrow transplant (BMT). Seven-eight week old SPF-B6 mouse bone marrow cells were harvested from femurs and tibias and were depleted of T cells with MACS CD90.2-microbeads and LS column (Miltenyi Biotec, Auburn, CA). T cells were harvested from the spleens of 7–8 week old SPF-B6 or GF-B6 mice. GF-B6 donor mice were euthanized immediately after transferred into SPF environment and then T cell isolation was done. BM cells and T cells were transplanted to allogeneic experimental mice and were housed in sterilized microisolator cages under SPF condition and maintained on acidified water (pH < 3) for 3 weeks (Reddy et al, 2001). Survival was monitored daily and clinical GVHD was assessed weekly (Cooke et al, 1996).

Antibiotic treatment

SPF C57BL/6J mice were provided with cocktail of broad spectrum oral antibiotics metronidazole 0.5mg/ml, neomycin 0.5mg/ml, ampicillin 0.5mg/ml (Sigma, St. Louis, MO), and vancomycin 0.5mg/ml along with Splenda (McNeil Nutritional, Fort Washington, PA) in autoclaved drinking water for 10–14 days prior to being utilized as source of donor T cells.

In vitro proliferation assay

CD90+ T cells were isolated from spleen cells of SPF-B6 and GFB6 mice (4×105) and incubated with syngeneic (C57BL/6J) or allogeneic (BALB/c) irradiated (30 Gy) spleen cells for 72 h proliferation was measured by the incorporation of 3H-thymidine (1 μCi/well).

In vivo donor T cell expansion

B6D2F1 recipient mice were irradiated and transplanted with SPF-B6 TCD BM and 2×106 CD90+T cells from either SPF-B6 or GF-B6 mice. On day 14 post-BMT, spleen cells were harvested and analyzed for donor T cells (H-2Kd−) by flow cytometry.

Cytokine analysis

Levels of IFN-γ, IL-5, IL-17A and IL-10 in sera and culture supernatant were determined by ELISA (BD Pharmingen) per manufacturer's instructions.

Histology

Formalin-preserved gut and liver were embedded in paraffin, cut into 5-μm thick sections, and stained with haematoxylin and eosin for histologic examination. Slides were coded and examined in a blinded fashion by a pathologist (C. Liu). A semiquantitative scoring system was used to assess the following abnormalities known to be associated with GVHD (Reddy et al, 2001).

Statistical analysis

Student t test was used for the statistical analysis of in vitro data while the Log-rank test was used to analyze survival data. P< 0.05 was considered statistically significant.

RESULTS and DISCUSSION

We first determined the in vitro allo-responses and Treg frequencies of T cells harvested from the spleens of germ-free (GF) animals. The CD90+ T cells from SPF-B6 and GF-B6 mice were analyzed for the frequency of CD4+FoxP3+ regulatory T cells. The frequency of Foxp3+ Tregs in the CD90+ T cell fraction was similar in both SPF-B6 and GF-B6 mice (Figure 1A). We next analyzed the allo-responses of the T cells in vitro with mixed leukocyte reaction (MLR) against allogeneic BALB/c irradiated spleen cells at different responder/stimulator ratios. As shown Figure 1B, both SPF-B6 and GF-B6 T cells demonstrated equivalent proliferation. The levels of IFN-γ, IL-5 and IL-17A (signature cytokines for Th1, Th2 and Th17) in the supernatants were also similar (Figure 1C).

Figure 1. Germ-free mouse T cells respond against allogeneic stimulator as same as specific pathogen-free mouse T cells in vitro and in vivo.

Figure 1

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(A) CD90+ T cells were isolated from SPF-B6 (left) or GF-B6 (right) mice and frequencies of CD4+FoxP3+ regulatory T cells were analyzed by flow cytometry. (B) Isolated CD90+ T cells from SPF-B6 (filled bar) or GF-B6 (open bar) mice were plated at 4×105/well in 96 well flat-bottom plate and co-cultured with different number syngeneic B6 or allogeneic BALB/c irradiated (30 Gy) spleen cells for 72 hours. 3H-Thymidine (1 μCi/well) incorporation during last 6 hours of culture was measured. (C) Cytokine levels in the supernatant of 66 hour-culture was determined by ELISA. (D, E) B6D2F1 recipients were irradiated (11 Gy) on day −1 and injected with allogeneic 5×106 SPF-B6 T cell-depleted bone marrow (TCDBM) + 2×106 SPF-B6 CD90+ T cells (filled bar, n=3 for each time point) or allogeneic 5×106 SPF-B6 TCDBM + 2×106 GF-B6 CD90+ T cells (open bar, n=3 for each time point). Spleen cells, sera were collected from recipients on day 7 or day 14. (D) Spleen cells were counted, stained with anti-H-2Kd, CD4 and CD8 mAbs and analyzed by flowcytometry. Donor CD4 and CD8 T cell expansion was determined based on spleen cell count and the percentages of CD4 and CD8 positivity in gated (H-2Kd−) cells. (E) Cytokine levels in the sera were determined by ELISA.

We next evaluated the in vivo allo-responses of SPF-B6 and GF-B6 T cells. We utilized MHC mismatched B6→B6D2F1 and performed allo-BMT with donor T cells from either GF-B6 or SPF-B6 animals. The recipient sera and spleens were harvested on days on day 7 or day 14 after BMT and analyzed for allogeneic T cell expansion and differentiation. Donor T cell expansion was similar between the allogeneic recipients for both CD4 (P=0.1220) and CD8 (P=0.0594) subsets (Figure 1D). Serum levels of IFN-γ, IL-5, IL-17A and IL-10 (Th1, Th2, Th17 and Tr1) cytokines were also equivalent (Figure 1E). These data thus demonstrate that absence of cross-talk with microbial flora in donors did not alter T cell allo-reactivity in vitro and in vivo.

To further determine the functional and clinical relevance of the microbiome on donor T cell alloreactivity we analyzed the severity of GVHD induced by T cells isolated from SPF-B6 mice and GF-B6 mice utilizing well established MHC mismatched B6→BALB/c GVHD model. We transplanted 5×105 T cells from either SPF-B6 or GF-B6 donors along with 5×106 SPF-B6 T TCD BM. The clinical severity and mortality from GVHD was similar in both groups (P=0.9500, Figure 2A). To rule potential strain and model dependent artifacts we analyzed the induction of GVHD in a second well-established B6→B6D2F1 model. There was no statistically significant difference in GVHD mediated mortality between the SPF-B6 and the GF-B6 T cell transplanted allo-recipients animals (P=0.1061) (Figure 2B). To further determine whether the absence of donor microbiota might affect the ability of donor T cells to cause differential GVHD target organ damage we analyzed GVHD specific histo-pathology scores of small intestine and liver from the allogeneic B6D2F1 recipients on day 7 or 14 after BMT (Figure 2C) and found no significant difference in GVHD severity. We next analyzed whether treatment of SPF donor mice with a cocktail of broad spectrum oral antibiotics that is known to reduce and alter gut microbial flora would alter donor T cell mediated GVHD mortality in the recipient animals (Hill et al, 2012) (Abt et al, 2012). Treatment of B6 donor mice with broad spectrum antibiotics induced GVHD mortality at a rate similar to that control SPF donor mice. These data suggest reducing and altering the microbial flora in the donors did not impact on their T cell alloreactivity and induction of GVHD after allogeneic BMT.

Figure 2. Germ-free mouse T cells and specific pathogen-free mouse T cells induce similar extent of GVHD.

Figure 2

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(A) BALB/c recipients were irradiated (8 Gy) on day −1 and injected with syngeneic 5×106 BALB/c bone marrow (BM) (▼, n=3), allogeneic 5×106 specific pathogen-free C57BL/6J (SPF-B6) T cell-depleted bone marrow (TCDBM) + 5×105 SPF-B6 CD90+ T cells (•, n=6) or allogeneic 5×106 SPF-B6 TCDBM + 5×105 germ-free C57BL/6J (GF-B6) CD90+ T cells (○, n=6). Survival was monitored daily, and GVHD clinical score and body weight change were monitored weekly. (B) B6D2F1 recipients were irradiated (11 Gy) on day −1 and injected with syngeneic 5×106 B6D2F1 bone marrow (BM) (▼, n=5), allogeneic 5×106 SPF-B6 TCDBM + 2×106 SPF-B6 CD90+ T cells (•, n=11) or allogeneic 5×106 SPF-B6 TCDBM + 2×106 GF-B6 CD90+ T cells (○, n=11). Survival was monitored daily, and GVHD clinical score and body weight change were monitored weekly. (C) B6D2F1 recipients were irradiated (11 Gy) on day −1 and injected with allogeneic 5×106 SPF-B6 TCDBM + 2×106 SPF-B6 CD90+ T cells (filled bar, n=3 for each time point) or allogeneic 5×106 SPF-B6 TCDBM + 2×106 GF-B6 CD90+ T cells (open bar, n=3 for each time point). GVHD target tissue (small intestine and liver) were collected from recipients on day 7 or day 14. HIstopathological score of small intestine (left) and liver (right) was determined. (D) Antibiotic treatment of donors does not mitigate GVHD in the recipients. B6 donor mice were treated with a cocktail of oral antibiotics (open circles, n=10) or autoclaved water alone (filled circles, n=11), as in the methods. The donor T cells were harvested 10–14 days later and transplanted into allogeneic BALB/c or syngeneic B6 animals (inverted triangle) after conditioning with 10 Gy along with TCDBM from SPF B6 donors. o vs.•, P=NS. Data shown are combined from two similar experiments.

Emerging data demonstrate an important role for recipient microbiota on the outcome of GVHD following allogeneic BMT from conventional donors. The role of donor microbiota on the induction of alloreactivity and GVHD in conventional recipients has not been studied. We found that T cells from GF-donors demonstrated similar magnitude of proliferation, differentiation and GVHD as the T cells from conventional or SPF mice. These data demonstrate that in contrast to host microbiota, donor microbial flora do not affect the induction and severity of GVHD. Our data are also consistent with the notion that following adoptive transfer, the T cells from GF mice can induce pathogenic autoimmune responses such as colitis and collectively suggest that either the donor microbial status does not affect the T cell responses or that once transferred into conventional settings, the prior lack of interaction with microbiota do not influence their subsequent responses (Liu et al, 2011).

Our donor mice were housed under stringent GF conditions and fed with normal chow. They were, however, not controlled for the potential LPS content of the diet. Nonetheless, Hrncir et al demonstrated there was no significant impact on the systemic immune-phenotype regardless of LPS contamination of the chow in GF mice (Hrncir et al, 2008). It is possible that specific components or the type of microbiota dysbiosis in the donors may have differential effects on the ability of donor T cells to respond to allo-antigens. As such, it is important to note that our data did not address the impact of any unique microbial genus on the T cells in causing GVHD, but instead analyzed the impact of lack of all microbial floras in the donors.

Acknowledgements

Supported by NIH grants: AI-075284, HL-090775 and CA-143379 to P.R.

IT: designed and performed research, analyzed data and wrote the paper

CL: performed research and analyzed data

HT: performed research

TT: performed research

YS: performed research

RE: performed research

EC: performed research

NM: performed research

GN: intellectual in-put, contributed new reagents and mice

PR: conceived project, designed experiments, analyzed data, wrote the paper

Footnotes

itawara@umich.edu (I.T.), liu@pathology.ufl.edu (C.L.), htamaki@med.umich.edu (H.T.), tomomit@umich.edu (T.T.), yapings@umich.edu (Y.S.), raevers@med.umich.edu (R.E.), ecnieves@med.umich.edu (E.N.), nmathew@med.umich.edu (N.M.), bclx@med.umich.edu (G.N.)

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