Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model

Abstract

The gut microbiota has recently been proposed as a novel component in the regulation of host homeostasis and immunity. We have assessed for the first time the role of the gut microbiota in a mouse model of leukemia (transplantation of BaF3 cells containing ectopic expression of Bcr-Abl), characterized at the final stage by a loss of fat mass, muscle atrophy, anorexia and inflammation. The gut microbial 16S rDNA analysis, using PCR-Denaturating Gradient Gel Electrophoresis and quantitative PCR, reveals a dysbiosis and a selective modulation of Lactobacillus spp. (decrease of L. reuteri and L. johnsonii/gasseri in favor of L. murinus/animalis) in the BaF3 mice compared to the controls. The restoration of Lactobacillus species by oral supplementation with L. reuteri 100-23 and L. gasseri 311476 reduced the expression of atrophy markers (Atrogin-1, MuRF1, LC3, Cathepsin L) in the gastrocnemius and in the tibialis, a phenomenon correlated with a decrease of inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1, interleukin-4, granulocyte colony-stimulating factor, quantified by multiplex immuno-assay). These positive effects are strain- and/or species-specific since L. acidophilus NCFM supplementation does not impact on muscle atrophy markers and systemic inflammation. Altogether, these results suggest that the gut microbiota could constitute a novel therapeutic target in the management of leukemia-associated inflammation and related disorders in the muscle.

Figure 1. Transformed mouse proB BaF3 cells infiltrate the liver and spleen.

A. Liver and spleen weights. N = 8. *p<0.05 vs. control. B. Bcr-Abl mRNA levels in liver and spleen, ND: not detected. C. Histological analysis of liver tissue (hematoxylin-eosin staining). Bar  = 50 µm. White arrows indicate BaF3 cells.

Figure 2. Changes in gut microbiota composition occur in mice transplanted with BaF3 cells.

A. DGGE profiles of the bacterial DNA isolated from the cecal content. Each profile corresponds to one animal. UPGMA dendogram with Dice coefficient. B. Levels of total bacteria, Bacteroides spp. and Lactobacillus spp. (quantified with two different sets of primers). C. Lactobacillus johnsonii/gasseri, Lactobacillus murinus/animalis and Lactobacillus reuteri levels. D. Relative proportions of each Lactobacillus species, expressed as a percentage of the Lactobacillus spp. levels (second primer set). N = 8, *p<0.05 vs. control. Percentages of each species of lactobacilli are significantly different (p<0.05 with Student’s t-test) between control mice (CT) and mice transplanted with BaF3 cells (BaF3 mice).

Figure 3. Supplementation with Lactobacillus reuteri 100-23 and Lactobacillus gasseri 311476 mixture restores the lactobacilli levels.

A–D. Lactobacillus spp., L. reuteri, L. jonhsonii/gasseri and L. murinus/animalis levels in control mice (CT), in mice receiving lactobacilli (Lrg), in mice transplanted with BaF3 cells (BaF3) and in mice transplanted with BaF3 cells and receiving lactobacilli (BaF3-Lrg). E. Daily food intake. N = 7–8. Data with different superscript letters are significantly different (p<0.05). For the food intake, n = 4 and *p<0,05 BaF3 and BaF3-Lrg mice versus CT and Lrg mice.

Figure 4. Supplementation with Lactobacillus reuteri 100-23 and Lactobacillus gasseri 311476 mixture reduces inflammatory cytokines.

A–F. Plasma levels of interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), and granulocyte colony-stimulating factor (G-CSF) in control mice (CT), in mice receiving lactobacilli (Lrg), in mice transplanted with BaF3 cells (BaF3) and in mice transplanted with BaF3 cells and receiving lactobacilli (BaF3-Lrg). Data with different superscript letters are significantly different (p<0.05).

Figure 5. Supplementation with Lactobacillus reuteri 100-23 and Lactobacillus gasseri 311476 mixture reduces muscle atrophy markers.

A–D. Atrophy marker expression (Atrogin-1, MurF1, LC3 and Cathepsin L) measured in the gastrocnemius muscle of control mice (CT), mice receiving lactobacilli (Lrg), mice transplanted with BaF3 cells (BaF3) and mice transplanted with BaF3 cells and receiving lactobacilli (BaF3-Lrg). E–H. Atrophy marker expression (Atrogin-1, MurF1, LC3 and Cathepsin L) measured in the tibialis muscle. F. Muscle weights. N = 7–8. Data with different superscript letters are significantly different (p<0.05).