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. 2018 Jul 1;350:64-77.
doi: 10.1016/j.taap.2018.05.006. Epub 2018 May 9.

Increased Butyrate Priming in the Gut Stalls Microbiome Associated-Gastrointestinal Inflammation and Hepatic Metabolic Reprogramming in a Mouse Model of Gulf War Illness

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Free PMC article

Increased Butyrate Priming in the Gut Stalls Microbiome Associated-Gastrointestinal Inflammation and Hepatic Metabolic Reprogramming in a Mouse Model of Gulf War Illness

Ratanesh Kumar Seth et al. Toxicol Appl Pharmacol. .
Free PMC article

Abstract

Most of the associated pathologies in Gulf War Illness (GWI) have been ascribed to chemical and pharmaceutical exposures during the war. Since an increased number of veterans complain of gastrointestinal (GI), neuroinflammatory and metabolic complications as they age and there are limited options for a cure, the present study was focused to assess the role of butyrate, a short chain fatty acid for attenuating GWI-associated GI and metabolic complications. Results in a GWI-mouse model of permethrin and pyridostigmine bromide (PB) exposure showed that oral butyrate restored gut homeostasis and increased GPR109A receptor copies in the small intestine (SI). Claudin-2, a protein shown to be upregulated in conditions of leaky gut was significantly decreased following butyrate administration. Butyrate decreased TLR4 and TLR5 expressions in the liver concomitant to a decrease in TLR4 activation. GW-chemical exposure showed no clinical signs of liver disease but a significant alteration of metabolic markers such as SREBP1c, PPAR-α, and PFK was evident. Liver markers for lipogenesis and carbohydrate metabolism that were significantly upregulated following GW chemical exposure were attenuated by butyrate priming in vivo and in human primary hepatocytes. Further, Glucose transporter Glut-4 that was shown to be elevated following liver complications were significantly decreased in these mice after butyrate administration. Finally, use of TLR4 KO mice completely attenuated the liver metabolic changes suggesting the central role of these receptors in the GWI pathology. In conclusion, we report a butyrate specific mechanistic approach to identify and treat increased metabolic abnormalities in GWI veterans with systemic inflammation, chronic fatigue, GI disturbances, metabolic complications and weight gain.

Keywords: Claudin-2; Cytokines; Gut dysbiosis; Permethrin; Pyridostigmine bromide; TLR4.

Conflict of interest statement

Conflict of interest

The authors declare that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Gut microbiome alteration in mice model of Gulf War Illness (GWI). A. The proportional abundance of microbial genera: Graphical representation of the most abundant taxa of bacteria at the genus level. Groups compared are gulf war illness group (wild-type mice exposed to gulf war chemicals) (GWI, n = 3) and control group fed with vehicle (Veh, n = 3) (p-value: < 0.05). B. Percentage abundance of gut bacteria Bifidobacterium (i), Roseburia (ii), and Lactobacillus (iii) in a group of mice co-exposed with Gulf war chemicals and Sodium butyrate (GWI + NaBT, n = 3) as compared with GWI mice (n = 3) (p-value: < 0.05).
Fig. 2.
Fig. 2.
Change in gut microbiome in GWI alter niacin receptor (GPR109A) and tight junction proteins in the intestine. A. The expression pattern of butyrate and niacin receptor GPR109A was assessed by immunofluorescence microscopy. The representative images showed immunoreactivity of GPR109A in the distal part of the small intestine of veh control group of mice (veh, n = 3), gulf war illness group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3). B and C. The expression pattern of Claudin-2 and Occludin (tight junction proteins) was assessed by immunofluorescence microscopy. Tissue levels of Claudin-2 (B) and Occludin (C) in Vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3) was assessed by immunofluorescent microscopy after labeling the protein with the red fluorescent secondary antibody and counterstained with DAPI (blue). D–F. The bar diagram shows the quantitative morphometric analysis of fluorescence intensities of GPR109A (D), Claudin-2 (E), and Occludin (F) immunoreactivity in the region of interest (ROI) in the small intestine. *(p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Fig. 3.
Sodium butyrate priming in a rodent model of GWI attenuates TLR4 activation in the small intestine. A. Immunofluorescence microscopy of small intestine showing TLR4 (red) trafficking to the lipid rafts (green) of the small intestine tissue, an essential process for TLR4 activation causing a co-localization of TLR4 in flotillin-rich rafts (yellow). Representative images of TLR4-flotillin co-localization in the small intestine of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3) shown by white circles covering the yellow spots created by an overlay of red (TLR4) and green (Flotillin). Images were taken at higher magnification (40× oil). B. Graphical representation of the quantitative morphometric analysis of colocalization events in the region of interest (ROI) in the small intestine. Images for analysis were randomly chosen in different microscopic fields. Data is represented as Mean ± SEM and *(p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4.
Fig. 4.
Sodium butyrate priming in a rodent model of GWI improves proinflammatory phenotype in small intestine mediated by the TLR4 pathway. A. Small intestine tissue slices were probed for IL-1β immunoreactivity in vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3) using immunohistochemistry. Specific immunoreactivity to IL-1β is evident by dark brown spots. B. Graphical representation of morphometric analysis of the IL-1β immunoreactivity in tissue slices. Data normalized against vehicle control (veh) *(p < 0.05). C. Quantitative real-time PCR (qRTPCR) analysis of inflammatory markers in the small intestine. mRNA expression of IL-1β, MCP-1, and TNF-α was analyzed in the samples of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3). Normalized mRNA expression is represented as a fold change of vehicle control (veh) on Y-axis. Data points represented with Mean ± SEM *(p < 0.05). D. Graphical representation of serum IL-1β in pg/mL of the samples of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5.
Fig. 5.
Sodium butyrate treatment in GWI improves circulatory DAMPs. Similar to the pathogen-associated molecular pattern (PAMPs), the endogenous molecules called damage-associated molecular patterns or DAMPs (such as HMGB1) are linked with proinflammatory responses in distal organs. A. Western blot analysis of serum high mobility group box 1 protein (HMGB1) and serum adipokine leptin from samples of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3). Ponceau S staining was done to see the equal loading of serum proteins and used for normalization of protein expression. B–C. Graphical representation of morphometric analysis of HMGB1 and leptin western blot bands. The data was normalized to a total serum protein (Ponceau S). Y-axis depicts the HMGBl/Ponceau S ratio (B) and leptin/Ponceau S ratio (C) from Veh, GWI and GWI + NaBT groups. *p < 0.05 is considered statistically significant. D-E. Graphical representation of serum HMGB1 (D) and serum leptin (E) in ng/mL of the samples of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3).
Fig. 6.
Fig. 6.
Sodium butyrate treatment in a rodent model of GWI attenuates TLR4 activation in Liver. A. Immunofluorescence microscopy of liver slices showing TLR4 (red) trafficking to the lipid rafts (green), an essential process for TLR4 activation causing a co-localization of TLR4 in flotillin-rich rafts (yellow). Representative images of TLR4-flotillin co-localization in the liver of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3) shown by white circles covering the yellow spots created by an overlay of red (TLR4) and green (Flotillin). Images were taken at higher magnification (60× oil). B. Graphical representation of the quantitative morphometric analysis of colocalization events in the liver. Images for morphometric analysis were randomly chosen in different microscopic fields. Data is represented as Mean ± SEM *(p < 0.05). C. Tissue levels of TLR5 immunoreactivity in vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3) mouse liver samples as observed by immunofluorescent microscopy after labeling the TLR5 protein with the green fluorescent secondary antibody and counterstained by DAPI (blue). D. The bar diagram shows the quantitative morphometric analysis of fluorescence intensities of TLR5 immunoreactivity in the liver tissue. Images for morphometric analysis of TLR5 were randomly chosen in different microscopic fields. Data is represented as Mean ± SEM *(p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7.
Fig. 7.
TLR4 activation is associated with metabolic changes and inflammatory response in the liver but the phenotypic liver injury is predominantly absent. A. Quantitative real-time PCR (qRTPCR) analysis principle carbohydrate metabolic markers (PFK, GLUT-1, and GLUT-4) and fat metabolic markers (SREBP1c, PPAR-α, and PPAR-γ) in the liver tissue. mRNA expression of SREBP1c, PPAR-α, PPAR-γ and PFK, GLUT-1, GLUT-4 and B. mRNA expression analysis of inflammatory marker IL-1β, MCP-1, TNF-α, and Kupffer cell activation marker CD68 were analyzed in the liver sample of vehicle control group of mice (Veh, n = 3), gulf war chemical treated group of mice (GWI, n = 3) and a group of mice co-exposed with GWI and sodium butyrate (GWI + NaBT, n = 3). Normalized mRNA expression is represented as a fold change of Vehicle control (veh) on Y-axis. Data points represented with Mean ± SEM *(p < 0.05). C. mRNA expression of SREBP1c, PPAR-γ were analyzed in the primary human hepatocytes cells treated with lipopolysaccharide (LPS) and Co-treated with LPS and sodium butyrate (LPS + NaBT). Normalized mRNA expression is represented as a fold change of Vehicle control (Veh Cont) on Y-axis. Data points represented with Mean ± SEM *(p < 0.05). D. Representative Hematoxylin and Eosin stained (H&E) images of liver sections showed liver pathophysiology of vehicle control group of mice (Veh, n = 3), gulf war chemical treated a group of mice (GWI, n = 3). Images were taken at 10× magnification.
Fig. 8.
Fig. 8.
TLR4 drives the metabolic alterations in GW-chemical exposed liver. A. Tissue levels of TLR5 in Gulf War chemical treated a group of wild-type mice (GWI, n = 3) and a group of TLR4 knockout mice (TLR4 KO, n = 3). Mouse liver samples as observed by immunofluorescence microscopy after labeling the TLR5 protein with green fluorescent secondary antibody and nuclear counterstaining by DAPI (blue). Images were taken at 60× (oil) magnification. B. The bar diagram showed the quantitative morphometric analysis of fluorescence intensities of TLR5 immunoreactivity in the liver tissue. *(p < 0.05). C. mRNA expression analysis of principle carbohydrate metabolic markers (PFK, GLUT-1, and GLUT-4) and fat metabolic markers (SREBP1c, PPAR-α) in the liver tissue of vehicle, GWI and TLR4 KO mice. Normalized mRNA expression is represented as a fold change of vehicle control (veh) on Y-axis. Data points represented with Mean ± SEM *(p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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