Treatment with Bacterial Biologics Promotes Healthy Aging and Traumatic Brain Injury Responses in Adult Drosophila, Modeling the Gut-Brain Axis and Inflammation Responses

Abstract

Drosophila are widely used to study neural development, immunity, and inflammatory pathways and processes associated with the gut-brain axis. Here, we examine the response of adult Drosophila given an inactive bacteriologic (IAB; proprietary lysate preparation of Lactobacillus bulgaricus, ReseT^®) and a probiotic (Lactobacillus rhamnosus, LGG). In vitro, the IAB activates a subset of conserved Toll-like receptor (TLR) and nucleotide-binding, oligomerization domain-containing protein (NOD) receptors in human cells, and oral administration slowed the age-related decline of adult Drosophila locomotor behaviors. On average, IAB-treated flies lived significantly longer (+23%) and had lower neural aggregate profiles. Different IAB dosages also improved locomotor function and longevity profiles after traumatic brain injury (TBI) exposure. Mechanistically, short-term IAB and LGG treatment altered baseline nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) signaling profiles in neural and abdominal tissues. Overall, at select dosages, IAB and LGG exposure has a positive impact on Drosophila longevity, neural aging, and mild traumatic brain injury (TBI)-related responses, with IAB showing greater benefit. This includes severe TBI (sTBI) responses, where IAB treatment was protective and LGG increased acute mortality profiles. This work shows that Drosophila are an effective model for testing bacterial-based biologics, that IAB and probiotic treatments promote neuronal health and influence inflammatory pathways in neural and immune tissues. Therefore, targeted IAB treatments are a novel strategy to promote the appropriate function of the gut-brain axis.

Keywords: Toll-like receptor (TLR); anti-microbial peptide (AMP); gut–brain axis; inactive bacteriologic (IAB); neural inflammaging; nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ); nucleotide-binding oligomerization domain-containing protein (NOD2); pathogen associated molecular pattern (PAMP); probiotic; traumatic brain injury (TBI).

Figure 1

Therapeutic impact of inactive bacteriologic (IAB) dosages on Drosophila neural function and longevity. (A) Negative geotaxis responses of controls (0) and male fly cohorts exposed to IAB at 1:50, 1:100, 1:200, 1:400, or 1:800 dilutions. Starting at one week of age, climbing indexes were determined weekly (Wk). (B) Longevity profiles of fly cohorts exposed to media containing 0, 1:1200, or 1:1600 IAB dilutions. (C) Average and SEM summary profiles of two independent longevity studies (** p-values < 0.01, *** p-values < 0.001). See Supplementary Table S1A,B for fly numbers (n) and additional information.

Figure 2

Western analysis of protein aggregates following IAB treatments. Insoluble ubiquitinated proteins (IUP) and Ref(2)P aggregate marker profiles in aged neural tissues (four weeks) after one or three weeks of IAB treatment (1:800 dilution). * p < 0.05.

Figure 3

The negative geotaxis responses (NGRs) of young Drosophila before and after trauma. Geotaxis responses of flies before and after (A) two days or (B) five days of IAB pretreatment (0.0, 1:400, 1:800, 1:1200) and one week following mild traumatic brain injury or mTBI-10×. (C) The NGR profiles of flies exposed to mTBI-10× and post-treated with IAB (0.0, 1:100, 1:400, 1:800). (p values *** < 0.001). See Supplementary Table S2A–C for fly numbers (n) and additional information.

Figure 4

Impact of biological treatment on the longevity profiles of traumatized Drosophila. (A) The Kaplan–Meier Survival profiles of male flies pre-treated (two days) with different IAB dosages. (B–D) Average lifespan profiles of fly cohorts pre-treated for (B) two days or (C) five days before mTBI-10× exposure, and (D) post-treated profiles. (* p-values <0.5, *** p-values < 0.001). See Supplementary Table S3A–C for fly numbers (n) and additional information.

Figure 5

Immune and age-related responses to IAB and Lactobacillus rhamnosus (LGG) treatments. (A) Anti-microbial peptide (AMP) expression profiles in fly abdominal tissues following 24 h of IAB (1:800) or LGG (10⁵ CFU) treatment. (B) NGR of young (one-week-old) or middle-aged (three-week-old) flies after one week of control (M-9), LGG (10⁵ CFUs), IAB 1:800, or IAB 1:1200 treatment. (C) The Kaplan–Meier Survival profiles of young or middle-aged flies with control (M-9), IAB 1:800, IAB 1:1200, or LGG (10⁵ CFUs) treatment (* p-values < 0.5, ** p-values < 0.01, *** p-values < 0.001). See Supplementary Table S4A,B for fly numbers (n) and additional information.

Figure 6

Impact of biologic treatments on trauma responses. (A) Geotaxis profiles of flies pre-treated with LGG (10⁵ CFUs) or IAB (1:800), as well as one and two weeks following mTBI. See Table S5 for additional information. (B) Young flies were given different IAB dilutions two days before sTBI-1× exposure. Dead flies were counted after 24 h and 48 h, and used to calculate mortality indexes (MIs). (C) The MI profiles of control (n = 78) flies, as well the pre-treated (two days) with IAB (1:800, n = 79) or LGG (10⁵ CFUs, n = 75) fly cohorts. (* p-values < 0.5, ** p-values < 0.01, *** p-values < 0.001). See Supplementary Table S5 for additional fly numbers (n) and information.