A novel immune modulator IM33 mediates a glia-gut-neuronal axis that controls lifespan

Abstract

Aging is a complex process involving various systems and behavioral changes. Altered immune regulation, dysbiosis, oxidative stress, and sleep decline are common features of aging, but their interconnection is poorly understood. Using Drosophila, we discover that IM33, a novel immune modulator, and its mammalian homolog, secretory leukocyte protease inhibitor (SLPI), are upregulated in old flies and old mice, respectively. Knockdown of IM33 in glia elevates the gut reactive oxygen species (ROS) level and alters gut microbiota composition, including increased Lactiplantibacillus plantarum abundance, leading to a shortened lifespan. Additionally, dysbiosis induces sleep fragmentation through the activation of insulin-producing cells in the brain, which is mediated by the binding of Lactiplantibacillus plantarum-produced DAP-type peptidoglycan to the peptidoglycan recognition protein LE (PGRP-LE) receptor. Therefore, IM33 plays a role in the glia-microbiota-neuronal axis, connecting neuroinflammation, dysbiosis, and sleep decline during aging. Identifying molecular mediators of these processes could lead to the development of innovative strategies for extending lifespan.

Keywords: Drosophila; IM33; brain-gut axis; glia-gut axis; longevity; neuroimmunology.

Figure 1.. Glia-derived IM33 is required to maintain the lifespan.

(A) Relative mRNA level of head IM33 of young and old wild-type flies. Mean ± S.E.M.. Two-tailed unpaired t-test. Each dot represents a pool of 15 fly heads. (B and C) The lifespan of flies with neuronal (B) or glial (C) knockdown of IM33. Log-rank test. (D) Knockdown of IM33 in glia from the adulthood stage shortens the lifespan. Log-rank test. (E) The lifespan of control (Repo/+, IM33/+) and IM33-overexpressing flies (Repo>IM33). Log-rank test. (F) Climbing assay of young (7 d) and old (30 d) flies with indicated genotype. Mean ± S.E.M.. Two-way ANOVA with Sidak’s multiple-comparisons test. Each dot represents one testing vial that contains 10 flies. (G) Representative images showing the staining of RFP and Dapi of the fly brain. Arrows indicate the areas enriched with RFP-positive cells at the brain border. Genotype: IM33-Gal4>UAS-RedStinger. Scale bar: 50 μm. (H) Double staining of RFP and glia marker Repo of the brain area highlighted in (G). Arrowheads indicate the RFP+ cells colocalized with Repo. Scale bar: 5 μm.

Figure 2.. Glia-derived IM33 shapes the gut microbiota via immune modulation.

(A) The number of colony-forming units (CFU) in wild-type and IM33 deficient flies 3 h post-E. coli infection. Mean ± S.E.M.. Two-tailed unpaired t-test. Each dot represents one fly. (B) The relative mRNA abundance of anti-Gram negative antimicrobial peptides in wild-type and IM33 knockout flies with or without E. coli infection. Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents a pool of 4 flies. (C) The relative mRNA level of gut AMPs of control (Repo/+, IM33 RNAi/+) or glial IM33-deficient flies (Repo>IM33 RNAi). Mean ± S.E.M.. Two-way ANOVA with Sidak’s multiple-comparisons test. Each dot represents a pool of 15 fly guts. (D) PCR of Acetobacter and Lactobacillus from control or IM33 RNAi fly gut. (E) The quantification of (D). Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents a pool of 15 fly guts. (F) qPCR of L. plantarum abundance in flies with glial IM33 knockdown. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents a pool of 15 fly guts. (G) The lifespan of flies with intact microbiota (untreated), microbiota depletion (ABX), L. brevis (LB) or L. plantarum (LP). Log-rank test. (H) Representative images of DHE staining of the gut from flies with indicated genotype and treatment. Scale bar: 200 μm. (I) Quantification of the gut DHE fluorescence of flies with indicated genotype and treatment. Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents one fly gut. (J) Lipoic acid (LA) treatment extends the lifespan of the flies with glial IM33 knockdown. Log-rank test.

Figure 3.. Glia-derived IM33 modulates sleep in a microbiota-dependent manner.

(A) Sleep profiles of flies with indicated genotype and treatment. (B-D) Quantification of total (B), daytime (C) and nighttime (D) sleep bout length, sleep bout number and sleep time in (A). Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents one fly. (E) Survival curves of flies with indicated genotype and treatment. Log-rank test. (F) Quantification of daytime sleep bout length and number of flies with indicated genotype and treatment. Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents one fly.

Figure 4.. The daytime sleep regulation by glial IM33 is mediated by L. plantarum.

(A) Sleep profiles of untreated flies, flies with ABX treatment, and flies transferred with L. brevis (LB) or L. plantarum (LP). (B) Quantification of daytime sleep quality in (A). Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents one fly. (C) The abundance of L. plantarum from ZT0-ZT24 at 4-hour intervals. Mean ± S.E.M.. N=4 for each time point, each N is a pool of 15 fly guts.

Figure 5.. The activation of insulin-producing cells by L. plantarum causes daytime sleep fragmentation.

(A) tSNE plot of all the samples with annotated clusters. (B) tSNE plot of the three samples with indicated color. (C) Heatmap showing the average scaled expression of sleep-related genes in the three groups. (D) The natural CaLexA signal in IPCs after ABX treatment or L. plantarum transfer. Arrowheads indicate the IPCs. Scale bar: 20 μm. (E) Quantification of the CaLexA intensity in the cell bodies of IPCs from flies with indicated treatment. Mean ± S.E.M.. One-way ANOVA with Tukey’s multiple-comparisons test. Each dot represents one fly brain. (F) Quantification of the daytime sleep bout length and number under indicated conditions. Genotype: ILP2>Shibire^ts. Mean ± S.E.M.. Two-tailed unpaired t-test. Each dot represents one fly. LP: L. plantarum; LB: L. brevis.

Figure 6.. L. plantarum-derived peptidoglycan signals on PGRP-LE to activate IPCs.

(A) Quantification of the daytime sleep bout length and number of flies fed with water or DAP-type peptidoglycan (10 ug/ml). Mean ± S.E.M.. Two-tailed unpaired t-test. Each dot represents one fly. (B) Representative images showing the CaLexA signal in IPCs from water-treated or PG-treated flies. Scale bar: 20 μm. (C) Quantification of the CaLexA intensity in the cell bodies of IPCs from flies with or without PG. Mean ± S.E.M.. Two-tailed unpaired t-test. Each dot represents one fly brain. (D) Representative images of GCaMP7c signal in IPCs in the absence or presence of peptidoglycan. Scale bar: 10 μm. (E) Traces of GCaMP7c signal in IPCs upon peptidoglycan treatment. (F) Quantification of the normalized GCaMP7c signal in IPCs before and after PG treatment. Paired t-test. Each dot pair represents one fly brain. (G) L. plantarum transfer-induced daytime sleep fragmentation is prevented by PGRP-LE knockdown in IPCs. Mean ± S.E.M.. Two-way ANOVA with Sidak’s multiple-comparisons test. Each dot represents one fly. (H) Knockdown of PGRP-LE in IPCs blocks the PG treatment-induced daytime sleep fragmentation. Mean ± S.E.M.. Two-way ANOVA with Sidak’s multiple-comparisons test. Each dot represents one fly. (I) Knockdown of PGRP-LE in IPCs rescues the L. plantarum transfer-caused shortened lifespan. Log-rank test. (J) Survival curve of flies with indicated genotype. Log-rank test. LP: L. plantarum.