Effect of Anti-Fungal Prophylaxis on the Fungal Mycobiome after Allogeneic Bone Marrow Transplantation

Background:  Intestinal bacteria shape the immune system and have been shown to contribute to GI tract inflammation. Modification of the lower GI tract bacterial microbiota has been correlated with decreased acute graft-versus-host disease (aGvHD) [1]. Additionally, decreased intestinal bacterial diversity has been associated with increased nonrelapse mortality following allogeneic stem cell transplantation (alloSCT), largely due to an association with severe aGvHD[2].  However, the role of the fungal mycobiome during aGvHD has been largely ignored. Antifungal prophylaxis with fluconazole has been found to decrease long-term transplant mortality following alloSCT. We wanted to determine (1) effect of anti-fungal prophylaxis on the fungal mycobiome and (2) whether enteric fungi contributes to aGvHD by promoting a pro-inflammatory environment in the GI tract, similar to certain bacteria.

Methods: B6D2 recipient mice were lethally irradiated, and the following day, recipient mice received 3 x10⁶ T cell depleted bone marrow cells and 4 x 10⁶ naïve T cells from B6 donor mice. Recipient mice were followed for survival and scored for clinical GvHD using a standard scoring system.  Mice were treated daily with the antifungal agent fluconazole at 5mg/kg/day.  Stool samples were collected on days 0, 7, and 14 post-transplant, and DNA was isolated from stool.  The fungal ribosomal internal transcribed spacer region (specifically ITS-1) was amplified by PCR and sequencing libraries generated using Illumina chemistry and evaluated on a MiSeq sequencer. Additionally, real-time PCR was performed on DNA from stool samples to quantitate fungi in the colon. Colons were harvested, immune cells isolated and evaluated for flow cytometric analysis after bone marrow transplantation.  Intestinal permeability was assessed by oral administration of FITC-labeled dextran 4000, followed by peripheral blood collection to assess plasma FITC-dextran levels.     

Results:  In B6D2 mice, the most common fungal species found in the colon before and after bone marrow transplantation was Candida tropicalis.  We found a 50-75% decrease in the quantity of fungi present in the colon in fluconazole treated mice.  However there was no alteration in the percentages of fungi found in the GI tract indicating that anti-fungal therapy alter the quantity and not the quality of the fungal microbiome.  Specifically, we did not find that the administration of fluzonazole was associated with an increase in fluconazole-resistant Candida species such as C kruseii or with fungi intrinsically resistant to fluconazole such as Aspergillus organisms. While there was no difference in clinical scoring of GVHD or weight loss in fluconazole treated mice, there was a modest improvement in overall survival in the treated group. We found no differences in IFN-γ or IL-17 production by colonic T cells from mice treated with fluconazole as compared to control mice evaluated 14 days after transplant. Additionally, the percentage of Foxp3+ T cells was also similar between treated and control mice.  We also found no change in aGvHD mediated intestinal permeability suggesting that fluconazole treatment has no effect on epithelial barrier function. 

Conclusion:  These studies demonstrate that fluconazole decreases total colonic fungi without altering the relative frequency of fungal organisms and is not associated in mice with an increase in the presence of fluconazole-resistant fungi.  Fluconazole treatment was associated with a modest improvement in overall survival.  However, no differences were found in the percentage/number of donor TH1, TH17 or Treg cells in the colon. These data suggest that, in the B6->B6D2 model, decreasing fungal burden in the colon is associated with a modest improvement in overall survival without substantially altering the generation of pro-inflammatory T cells or Tregs. 

1. Jenq RR et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J Exp Med. 2012 May 7;209(5):903-11. doi: 10.1084/jem.20112408. Epub 2012 Apr 30.

2. Taur Y, et al. The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantation. Blood. 2014 Aug 14;124(7):1174-82. doi: 10.1182/blood-2014-02-554725. Epub 2014 Jun 17.