Gut Dysbiosis Increases Gut Barrier Damage during Anti-Leukemia Chemotherapy: Implications for Acute Graft-Versus-Host Disease

Abstract

We recently reported an association between low pre-conditioning serum citrulline, reflecting unresolved gut barrier injury from prior chemotherapy, and higher incidence of grade II-IV acute graft-versus-host disease (aGVHD). Citrulline is an amino acid exclusively synthesized by enterocytes and its serum level is a marker of total functional intestinal mass. We hypothesized that gut microbiota changes during prior anti-leukemia chemotherapy determine the extent of gut barrier damage. We performed 16S rRNA gene profiling of 207 stool samples collected thrice weekly from 20 acute leukemia patients (16 AML, 4 ALL; 13 first induction, 7 re-induction or salvage chemotherapy; 9 men, 11 women; median [range] age: 53 [22-74] years) between admission and day 28 of chemotherapy. The only inclusion criterion was receiving intensive chemotherapy with planned 1-month inpatient hospitalization. Patients received prophylactic levofloxacin with the initiation of chemotherapy and cefepime upon neutropenic fever. Serum samples, concurrent with stool samples, were analyzed by ELISA for citrulline. The lower level of quantification in this assay was 0.04 ng/mL. We classified patients to 2 groups based on detectability of citrulline at its nadir. We compared the two groups for Shannon's diversity index and taxa relative abundance (5 most abundant phyla and 10 most abundant genera) with a false discovery rate-based q value of Antibiotic exposure was intense, with 43 anti-bacterial antibiotic doses per 30 patient-days (for antibiotics given >1 dose/day, only 1 dose/day was considered). During their inpatient stay, 18 (90%), 5 (25%), and 4 (20%) patients developed neutropenic fever, bloodstream infection, and C. difficile diarrhea, respectively, and 5 (25%) received total parenteral nutrition. Serum citrulline declined, reached undetectable levels in 13 patients, and partially recovered by day 28 in 7 (Fig. 1A). The group with consistently detectable citrulline had a significantly higher diversity (q=0.03; Fig. 1B) and relative abundance of Actinobacteria (q=0.005; Fig. 1C). Other taxonomic comparisons were not significant. Members of the phylum Actinobacteria have key roles in gut barrier homeostasis. Our findings in patients receiving highly mucotoxic chemotherapy suggest that patients who experience more severe contraction of Actinobacteria also develop more severe gut barrier damage. This may result in higher levels of residual gut barrier damage in patients proceeding to allo-HCT. Our results support novel microbiota protection/restoration approaches during/after intensive chemotherapy to augment the gut barrier and potentially limit future risks of aGVHD.