A Double-Humanized Mouse Model for Studying Host Gut Microbiome-Immune Interactions in Gulf War Illness

Abstract

Unraveling the multisymptomatic Gulf War Illness (GWI) pathology and finding an effective cure have eluded researchers for decades. The chronic symptom persistence and limitations for studying the etiologies in mouse models that differ significantly from those in humans pose challenges for drug discovery and finding effective therapeutic regimens. The GWI exposome differs significantly in the study cohorts, and the above makes it difficult to recreate a model closely resembling the GWI symptom pathology. We have used a double engraftment strategy for reconstituting a human immune system coupled with human microbiome transfer to create a humanized-mouse model for GWI. Using whole-genome shotgun sequencing and blood immune cytokine enzyme linked immunosorbent assay (ELISA), we show that our double humanized mice treated with Gulf War (GW) chemicals show significantly altered gut microbiomes, similar to those reported in a Veteran cohort of GWI. The results also showed similar cytokine profiles, such as increased levels of IL-1β, IL-6, and TNF R-1, in the double humanized model, as found previously in a human cohort. Further, a novel GWI Veteran fecal microbiota transfer was used to create a second alternative model that closely resembled the microbiome and immune-system-associated pathology of a GWI Veteran. A GWI Veteran microbiota transplant in humanized mice showed a human microbiome reconstitution and a systemic inflammatory pathology, as reflected by increases in interleukins 1β, 6, 8 (IL-1β, IL-6, IL-8), tumor necrosis factor receptor 1 (TNF R-1), and endotoxemia. In conclusion, though preliminary, we report a novel in vivo model with a human microbiome reconstitution and an engrafted human immune phenotype that may help to better understand gut-immune interactions in GWI.

Keywords: IL-6; NSG; TNF R-1; bacteriome; gut–immune axis; humanized mice.

Figure 1

Establishment of human gut bacteria in NSG-CD34+ mice. (A) Box plots showing α-diversities (Chao 1) of gut bacteriome in NSG_Control group (mice administered with the vehicle), NSG_ABX Treatment (mice administered with antibiotic cocktail via oral gavage for 12 days), and NSG_Hu-FMT (mice administered with human fecal microbiota transfer after gut bacteriome depletion with antibiotic cocktail). (B) β-Diversity analysis (Bray–Curtis) of NSG_Control, NSG_ABX Treatment, and NSG_Hu-FMT groups. (C) Stacked bar representation of relative abundance of gut bacteriome at the phylum level in NSG_Control, NSG_ABX Treatment, and NSG_Hu-FMT groups; p < 0.05 was considered as statistically significant.

Figure 2

Bar graph representation of relative abundances of gut bacteriome at the genus level in NSG_Control, NSG_ABX Treatment, and NSG_Hu-FMT groups; p < 0.05 was considered as statistically significant.

Figure 3

Altered expressions of systemic proinflammation biomarkers during establishment of human gut bacteria in NSG-CD34+ mice. Bar graph representation of systemic cytokines (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TNF R-1 in NSG_Control, NSG_ABX Treatment, and NSG_Hu-FMT groups. (E) Bar graph representation of serum endotoxemia, measured by LAL Assay in NSG_Control, NSG_ABX Treatment, and NSG_Hu-FMT groups; p < 0.05 was considered as statistically significant. NS denotes non-significant change.

Figure 4

Exposure to representative GW chemicals altered gut bacteriome profile in NSG-CD34+ mice with established human gut bacteria. (A) Box plots showing α-diversities of gut bacteriome (Chao 1) in NSG_Hu-FMT (mice administered with human fecal microbiota transfer after gut bacteriome depletion with antibiotic cocktail) and NSG_Hu-FMT+GWI (mice administered with representative GW chemicals pyridostigmine bromide and permethrin for 15 days after gut bacteriome depletion with antibiotic cocktail and human fecal microbiota transfer). (B) β-Diversity analysis (Bray–Curtis) of NSG_Hu-FMT and NSG_Hu-FMT+GWI groups. (C) Stacked bar representation of relative abundance of gut bacteriome at the phylum level in NSG_Hu-FMT and NSG_Hu-FMT+GWI groups; p < 0.05 was considered as statistically significant.

Figure 5

Bar graph representation of relative abundances of gut bacteriome at the genus level in NSG_Hu-FMT and NSG_Hu-FMT+GWI groups; p < 0.05 was considered as statistically significant.

Figure 6

Exposure to representative GW chemicals altered gut resistome profile in NSG-CD34+ mice with established human gut bacteria. (A) Box plots showing α-diversities (Chao 1) of gut resistome in NSG_Hu-FMT (mice administered with human fecal microbiota transfer after gut bacteriome depletion with antibiotic cocktail) and NSG_Hu-FMT+GWI (mice administered with representative GW chemicals pyridostigmine bromide and permethrin for 15 days after gut bacteriome depletion with antibiotic cocktail and human fecal microbiota transfer). (B) β-Diversity analysis (Bray–Curtis) of NSG_Hu-FMT and NSG_Hu-FMT+GWI groups. (C) Bar graph representation of relative abundances of altered antibiotic resistance genes in NSG_Hu-FMT and NSG_Hu-FMT+GWI groups; p < 0.05 was considered as statistically significant.

Figure 7

Altered expression of systemic proinflammation biomarkers after administration with representative GW chemicals in NSG-CD34+ mice with established human gut bacteria. Bar graph representation of systemic cytokines (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TNF R-1 in NSG_Hu-FMT and NSG_Hu-FMT+GWI groups. (E) Bar graph representation of serum endotoxemia, measured by LAL assay in NSG_Hu-FMT and NSG_Hu-FMT+GWI groups; p < 0.05 was considered as statistically significant.

Figure 8

Administration with human fecal microbiota transfer from GWI Veteran’s stool sample altered gut bacteriome profile in NSG-CD34+ mice. (A) Box plots showing α-diversities of gut bacteriome (Chao 1) in NSG_Hu-FMT (mice administered with human fecal microbiota transfer after gut bacteriome depletion with antibiotic cocktail) and NSG_GWIV (mice administered with human fecal microbiota transfer from GWI Veteran’s stool sample after gut bacteriome depletion with antibiotic cocktail). (B) β-Diversity analysis (Bray–Curtis) of NSG_Hu-FMT and NSG_GWIV groups. (C) Stacked bar representation of relative abundances of gut bacteriome at the phylum level in NSG_Hu-FMT and NSG_GWIV groups; p < 0.05 was considered as statistically significant.

Figure 9

Bar graph representation of relative abundances of gut bacteriome at the genus level in NSG_Hu-FMT and NSG_GWIV groups; p < 0.05 was considered as statistically significant.

Figure 10

Administration with human fecal microbiota transfer from GWI Veteran’s stool sample altered gut resistome profile in NSG-CD34+ mice. (A) Box plots showing α-diversities (Chao 1) of gut resistome in NSG_Hu-FMT (mice administered with human fecal microbiota transfer after gut bacteriome depletion with antibiotic cocktail) and NSG_GWIV (mice administered with human fecal microbiota transfer from GWI Veteran’s stool sample after gut bacteriome depletion with antibiotic cocktail. (B) β-Diversity analysis (Bray–Curtis) of NSG_Hu-FMT and NSG_GWIV groups. (C) Bar graph representation of relative abundances of altered antibiotic resistance genes in NSG_Hu-FMT and NSG_GWIV groups; p < 0.05 was considered as statistically significant.

Figure 11

Altered expressions of systemic proinflammation biomarkers, after human fecal microbiota transfer from GWI Veteran’s stool sample, in NSG-CD34+ mice. Bar graph representation of systemic cytokines (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TNF R-1 in NSG_Hu-FMT and NSG_GWIV groups. (E) Bar graph representation of serum endotoxemia, measured by LAL assay in NSG_Hu-FMT and NSG_GWIV groups; p < 0.05 was considered as statistically significant.