Studies of allogeneic BMT using mouse models have characterized exaggerated inflammatory mechanisms that lead to acute GVHD in target organs, including the intestine (Reddy and Ferrara, 2008
). MHC-disparate donor/host combinations, including those used in this study, typically result in robust GVHD with full penetrance and rapid kinetics (Schroeder and DiPersio, 2011
). The B10.BR→B6 model (H2k
) used in most of our experiments is well-established and has been used in the past by us and others (Blazar et al., 1997
; Penack et al., 2009
). On day 14, we evaluated histologically for evidence of GVHD and found villous shortening, increased lymphocytic cell infiltration, crypt regeneration, crypt destruction, and epithelial apoptosis. The number of Paneth cells was also decreased, whereas goblet cells appear to be minimally affected (). We then quantified copies of 16S rRNA genes to determine bacterial load. After BMT, we noticed an increase in bacterial load in the ileum (), but not in the cecum (unpublished data). This occurred both in the absence and presence of GVHD, suggesting that bacterial expansion may result from reduced host-defense mechanisms in the post-BMT setting. Indeed, we found that levels of IgA in the ileal lumen were decreased after BMT, regardless of GVHD ().
Figure 1. GVHD in mice produces marked changes in the microbiota. (A) B6 mice were lethally irradiated and transplanted with 5 × 106 B10.BR T cell–depleted BM supplemented with or without 1 × 106 splenic T cells. Features of GVHD are indicated (more ...)
We evaluated for effects on the microbiota by performing 16S rRNA gene sequencing and evaluating microbial diversity, as measured by the Shannon index (Magurran, 2004
). Loss of diversity has been found to occur with antibiotic use (Dethlefsen et al., 2008
; Ubeda et al., 2010
) and increasing age (Woodmansey, 2007
), and may predispose mice to disease. Mice undergoing BMT without GVHD showed little change in diversity (unpublished data), but phylogenetic classification of 16S rRNA sequences did show some expansion of unclassified Firmicutes and Barnesiella
and mild contraction of unclassified Porphyromonadaceae (), demonstrating that radiation does produce some changes in the intestinal flora composition.
In contrast, mice with GVHD showed a dramatic loss of bacterial diversity during the first 2 wk after BMT (). To quantify changes in the composition of the flora, we used unweighted UniFrac (Lozupone et al., 2006
) analyzed by the principal coordinate analysis (PCoA). We found that ileal floras of mice with GVHD were distinct from both those of untreated mice and those of mice after BMT without GVHD (). Mice after BMT without GVHD clustered apart from untreated mice inconsistently (one of three experiments); however, comparing the floras using the Bray-Curtis index, we found that GVHD increases dissimilarity from baseline more than BMT alone ().
We then evaluated for changes in bacterial subpopulations in the setting of GVHD and found large shifts within the phylum Firmicutes, with a dramatic increase in Lactobacillales and decreases in Clostridiales and other Firmicutes in the ileum (). Previously, we have shown that the flora from the murine ileum is quite distinct from that of the large intestine, whereas samples within different compartments of the large intestine, including cecum and fresh stool pellets, are similar, although there are some minor changes in representation (Ubeda et al., 2010
). Thus, we also evaluated for changes in the cecum with GVHD and found changes similar to those in the ileum, but of lesser magnitude (). At the genus level, we found marked ileal expansion of Lactobacillus
, the dominant member of Lactobacillales (). Housing mice individually from the day of transplant to address the possibility of individual mice influencing the flora of cagemates produced identical results (). Within the 16S sequences assigned to the genus Lactobacillus
, nearly all had identical sequence homology with Lactobacillus johnsonii
, a species found as a commensal in humans (Pridmore et al., 2004
) and rodents (Buhnik-Rosenblau et al., 2011
), and also in probiotic preparations.
Figure 2. GVHD in mice produces marked changes in the microbiota. (A) B6 mice were transplanted with B10.BR donor BM and T cells as in . Comparison of representation by Lactobacillales, Clostridiales, and other Firmicutes from ileal samples. Combined results (more ...)
We asked if GVHD-associated changes could be secondary to increased gut motility. We evaluated the effects of an osmotic laxative, as well as enteritis caused by dextran sodium sulfate, and found changes with both agents that were distinct from GVHD (). Together, these results suggest that GVHD changes the flora in a unique, reproducible pattern.
The flora of mice can vary widely from colony to colony. Our data presented thus far used B6 recipient mice from The Jackson Laboratory; we also performed BMT experiments using additional strains and vendors. With BALB/c host mice from The Jackson Laboratory, we found an abundance of Lactobacillus
in mice with GVHD, although the floras of BALB/c mice are dominated by Lactobacillus
at baseline (unpublished data). In the CD4-driven MHC II–disparate B6→BM12 model, we also found characteristic expansion of Lactobacillus
with GVHD (). This indicated that alloreactive CD4 T cells are sufficient and do not require CD8 T cells to produce changes in the flora. Interestingly, in two additional models with B10.BR hosts from The Jackson Laboratory and B6 hosts from Charles River Laboratories, we noted expansion of Enterobacteriales with GVHD (). Enterobacteriales from both strains appears to be of the same type, an unclassified Enterobacteriaceae that, in our experience, is rarely detectable in B6 mice from The Jackson Laboratory (3 of 71 mice). Expansion of Enterobacteriaceae has been reported before in Japanese (Eriguchi, Y., S. Takashima, N. Miyake, Y. Nagasaki, N. Shimono, K. Akashi, and T. Teshima. 2010. ASH Annual Meeting Abstracts. Abstr. 244) and German (Heimesaat et al., 2010
) mouse colonies. Collectively, these data suggest that Lactobacillales and Enterobacteriales (both capable of surviving in aerobic environments) may populate a niche that expands with GVHD at the expense of obligate anaerobes, including Clostridiales and other Firmicutes. Whether Lactobacillales or Enterobacteriales expand appears to depend on the presence of these organisms in the baseline flora. The potential impact of these expanding populations on GVHD has not been well-described.
Treatment of B6 mice with ampicillin, followed by a recovery period, results in loss of Lactobacillus
from the flora, with expansion of other commensal bacteria (Ubeda et al., 2010
) such as Blautia
(order Clostridiales; ). We cultured the predominant L. johnsonii
endogenous to B6 mice from The Jackson Laboratory and found that reintroduction after ampicillin treatment restores representation (). We then used ampicillin and L. johnsonii
reintroduction as tools to test if expansion of Lactobacillales with GVHD could have clinical repercussions. Surprisingly, upon development of GVHD, mice treated with ampicillin before BMT showed loss of Blautia
and emergence of Enterococcus
(order Lactobacillales; ). Mice that received L. johnsonii
reintroduction after ampicillin showed domination with L. johnsonii
and no expansion of Enterococcus
(). We found similar results in the B6→BM12 model, though BM12 mice after ampicillin treatment demonstrated expansion of both Enterococcus
and Enterobacteriaceae with GVHD (). BM12 mice that received L. johnsonii
reintroduction after ampicillin also showed domination with L. johnsonii
and no expansion of Enterococcus
or Enterobacteriaceae. This occurred even when using monoclonal BM12-specific donor T cells from TCR transgenic ABM (Sayegh et al., 2003
) RAG-1 deficient mice, suggesting that a broad alloreactive T cell repertoire is not required to produce changes in the microbiota with GVHD.
Figure 3. Composition of intestinal flora can impact on severity of intestinal GVHD. (A) Schematic of treatment: B6 mice received ampicillin for 1 wk, followed by a 2-wk recovery period with unmodified drinking water; some were gavaged every 2 d with L. johnsonii (more ...)
We then evaluated effects of flora manipulation on GVHD severity, focusing on the B10.BR→B6 model. Notably, ampicillin treatment before BMT resulted in worsened GVHD survival. Histologically, these mice had evidence for increased GVHD pathology in the small and large intestines, including epithelial damage and increased inflammation. Remarkably, L. johnsonii
reintroduction prevented increased GVHD lethality and pathology (). Enterococcus
has not been described as a potential contributor to gut GVHD, though enterococcal bacteremia occurs often in patients with GVHD (Dubberke et al., 2006
). In mouse models, Enterococcus
can contribute to gut inflammation by compromising epithelial barrier integrity (Steck et al., 2011
) and stimulating TNF production from macrophages (Kim et al., 2006
). Thus, one mechanism by which L. johnsonii
may reduce GVHD severity could be prevention of Enterococcus
expansion which may exacerbate GVHD-associated intestinal damage and inflammation.
We then studied the relationship between the flora and GVHD in humans. We collected weekly stool samples from allogenic BMT patients during transplant hospitalization at our center. Of 9 patients who developed gut GVHD during hospitalization, 8 developed symptoms early, with GVHD onset clustering between days 18 and 21; these 8 were selected for our GVHD cohort. 18 additional patients provided weekly samples through day 21; of these, 10 met our prospective eligibility for inclusion in our non-GVHD cohort, with survival to at least day 30 and absence of GVHD in any target organ through day 100. Clinical parameters for included patients are summarized in . Importantly, non-GVHD and GVHD patients had similar exposures to antibiotics during the period of stool collection.
Figure 4. GVHD produces marked changes in the microbiota of humans, and the microbiota may affect risk of developing GVHD. (A) Summary of clinical parameters of non-GVHD and GVHD patients. (B) Flora diversity, by Shannon index, of stool samples after BMT. Individual (more ...)
We first examined the effects of GVHD on flora diversity. We found that before GVHD, patients had flora diversity similar to controls but lost diversity over time, particularly after GVHD onset (). Thus, GVHD is associated with loss of flora diversity in humans, similar to in mice.
We then looked for bacterial populations that changed with the onset of GVHD. Interestingly, we discovered increases in Lactobacillales and decreases in Clostridiales, a pattern identical to our findings in mice. Other populations, as well as classifications at the family or genus level, were otherwise not significantly changed (unpublished data). Importantly, we did not identify these shifts in non-GVHD patients (), suggesting that these flora changes were indeed a result of GVHD rather than BMT or antibiotic exposure.
Our sample size did not identify specific populations as potential risk factors for subsequent GVHD. Patients who later developed GVHD, however, did have significantly greater microbial chaos early after BMT (before our observed GVHD-associated changes), which we quantified using the Bray-Curtis dissimilarity index (Magurran, 2004
) over time (). This suggests that large fluctuations in the microbiota early on may lead to an increased risk of GVHD.
In conclusion, our findings demonstrate the influence of inflammation on the structure of the intestinal microbiota after allogenic BMT in both mice and humans. The flora, in turn, can modulate severity of intestinal inflammation. Our mouse experiments indicate that antibiotic exposure before BMT, which occurs commonly in patients with hematologic malignancies, may be a risk factor for subsequent intestinal GVHD. This may be remedied with targeted flora reintroduction to potentially reduce the severity of gut GVHD.