GRAPEVINE, TEXAS—Intestinal bacteria can offer protection from death related to graft-vs-host disease (GVHD), according to research presented at the 2014 BMT Tandem Meetings.

Experiments showed that Blautia, commensal bacteria found in the intestinal tract, can protect against GVHD-related mortality in mice and in humans.

So efforts to support Blautia survival—such as restricting the use of antibiotics and promoting better nutrition—may

prevent GVHD-related death, according to researchers.

Robert Jenq, MD, of Memorial Sloan-Kettering Cancer Center in New York, discussed this possibility when presenting this research, which was designated one of the “Best Abstracts” at the meeting (abstract 1*).

Dr Jenq noted that researchers have been trying for decades to determine whether the intestinal flora impact GVHD. Clinical studies have suggested that prophylaxis against anaerobes and gram-positive bacteria can reduce GVHD.

And murine studies have indicated that prophylaxis against gram-negative bacteria can reduce GVHD, that Lactobacillus can reduce GVHD, and that donor microbiota do not impact GVHD.

“If you’re confused, so are we,” Dr Jenq said. “It seems like it’s a mixed picture.”

So he and his colleagues conducted a series of experiments in an attempt to determine if any bacterial subgroups impact the risk of gut GVHD in mice and humans.

Bacteria seem to impact GVHD

The researchers first studied 76 adult transplant patients, analyzing stool samples taken at roughly 10 days after transplant (+/- 4 days). The team performed 16S gene sequencing using the Roche 454 platform.

This revealed the presence of several types of bacteria, including 6 gram-positive Firmicutes, 2 gram-negative Proteobacteria, and 2 gram-negative Bacteroidetes.

The researchers then used a computational assay to determine which of these bacteria might be associated with protection from GVHD. And they identified 2 possibilities—Lactobacillus and Blautia.

Additional analyses revealed that Blautia and Lactobacillus were significantly associated with GVHD-related mortality at 1500 days after transplant (P=0.03 and 0.01, respectively). But there was no significant association with Bacteroides (P=0.6), Enterobacteriales (P=0.2), or Enterococcus (P=0.3).

Blautia appears to affect GVHD-related mortality

To confirm their initial findings, Dr Jenq and his colleagues analyzed a second cohort of 50 adult transplant patients. The team analyzed stool samples for the abundance of bacterial subgroups using a different sequencing platform, Illumina miseq.

This time, they found that Blautia abundance predicted GVHD-related mortality at more than 500 days after transplant, but the abundance of Lactobacillus did not (P=0.01 and 1, respectively).

“Not enough Blautia in your gut seems to lead to an increase in GVHD-related mortality,” Dr Jenq said. “So what does this do to overall survival? In the first cohort, there’s a big difference in overall survival between the ‘haves’ and ‘have nots’ with Blautia [P=0.0008]. And this also holds up in the second cohort [P=0.04].”

Further analyses of data from both cohorts suggested that Blautia abundance was associated with GVHD-related mortality (P=0.004) and relapse-related mortality (P=0.01) but not non-relapse- and non-GVHD-related mortality (P=0.4).

“I don’t have a good explanation for [the relationship between Blautia and relapse-related death],” Dr Jenq said. “This was a surprise finding.”

The researchers also looked at Blautia’s ability to predict GVHD-related mortality. They found that, around day 10 after transplant, Blautia abundance predicts “very strongly” for GVHD-related death.

Another question was whether known GVHD risk factors—such as donor type, race, gender, and performance status—impact Blautia abundance. But an analysis revealed that Blautia is an independent risk factor for GVHD-related mortality.

A possible mechanism

To gain more insight into the association between Blautia and GVHD-related death, Dr Jenq and his colleagues decided to study it in mice.

The team killed off Blautia in mice using vancomycin and ampicillin, then introduced either murine Blautia or murine Enterococcus, transplanted the mice with MHC-disparate T cells, and monitored them for GVHD.

Mice that received Blautia had significantly better overall survival (at more than 80 days after transplant) than mice that received Enterococcus (P<0.001).

“So how is this happening?” Dr Jenq asked. “We think, potentially, it might be due to short-chain fatty acids . . . butyric acid, propionic acid, and acetic acid. These are metabolites that bacteria produce when they ferment glucose and other sugars.”

To test this theory, the researchers treated mice with antibiotics and introduced Blautia or Enterococcus.

Blautia increased the level of short-chain fatty acids (butyrate and propionate) when compared to Enterococcus, although levels were not as high as those observed in mice that did not receive antibiotics. Nevertheless, these results point to a possible mechanism, according to Dr Jenq.

Explaining Blautia reduction

Dr Jenq also noted that antibiotics may contribute to the decrease in Blautia observed in transplant patients. When patients come in for transplant, they often have more than 25% Blautia in their stool. But the bacteria decrease to negligible levels by day 2 after transplant.

To determine the role of antibiotics, the researchers treated mice with 4 different antibiotics and looked at the levels of different bacteria.

They found that aztreonam and cefepime increased the levels of Bacteroidales and Clostridiales (the family to which Blautia belongs), but imipenem and metronidazole decreased bacteria levels.

So antibiotics do affect Blautia levels, Dr Jenq said, but they’re only part of the problem. He noted that patients’ Blautia levels start to decrease before antibiotics are administered. So he and his colleagues believe nutrition might also play a part.

The team found a significant difference in Blautia abundance between patients who received total parenteral nutrition and those who did not (P<0.001).

The researchers also discovered that reduced caloric intake led to a loss of Blautia and other Clostridiales. They analyzed 50 samples from 5 patients and found that patients who consumed fewer than 500 calories had a marked reduction in Blautia (P<0.0001).

And experiments in mice confirmed this association. A week of calorie restriction significantly reduced the abundance of Blautia and other Clostridiales (P=0.0002).

“In GVHD, as we all know, patients and mice eat less because of the nausea,” Dr Jenq said. “And we found that GVHD itself can also lead to a reduction in Clostridiales, both in humans [P=0.02] and in mice [P=0.01].”

Protecting Blautia to prevent GVHD

Having confirmed the role of nutrition in Blautia reduction, the researchers set out to identify a nutrition-based intervention to support Blautia in transplant recipients.

They settled on a sugar called raffinose, which is found in beans, cruciferous vegetables, and whole grains. It passes undigested through the upper intestine but is fermented in the lower intestine and metabolized to produce short-chain fatty acids.

The team tested raffinose in mice by introducing it into their drinking water. At 100 days after transplant, mice that received raffinose had significantly better overall survival than controls (P<0.001).

Based on these results, Dr Jenq and his colleagues believe nutritional intervention can protect Blautia and, therefore, may prevent GVHD and related death. The team thinks encouraging eating, gastric nutritional supplementation, and flora-targeted nutritional supplements might all prove effective.

But other interventions might work as well, such as reintroducing endogenous flora (via autologous fecal microbiota transplant), reintroducing select bacteria with beneficial potential, selecting antibiotics that spare bacteria with beneficial potential, and identifying and introducing bacterial metabolites that mediate anti-inflammatory effects.

*Data in the abstract differ from data presented.

Bacteria may protect against GVHD-related mortality