Housing laboratory mice deficient for Nod2 and Atg16l1 in a natural environment uncovers genetic and environmental contributions to immune variation

Abstract

29The relative contributions of genetic and environmental factors to variation in immune 30 responses are still poorly understood. Here, we performed a deep phenotypic analysis of 31 immunological parameters of laboratory mice released into an outdoor enclosure, carrying 32 susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory 33 bowel diseases. Variations of immune cell populations were largely driven by environment, 34whereas cytokine production in response to stimulation was affected more by genetic 35 mutations. Multi-omic models identified transcriptional signatures associated with differences 36 in T cell populations. Subnetworks associated with responses against Clostridium perfringens, 37 Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, 38 exposing laboratory mice carrying different genetic mutations to a natural environment 39 uncovered important contributors to immune variation.40 41 One sentence summary 42 Natural environment exposure in laboratory mice primarily promotes variation in population 43 frequencies of immune cells, whereas cytokine responses to stimulation are affected more by 44 genetic susceptibility to inflammatory bowel disease. 45 46 47 93 in wild type animals, we reasoned that they could be an interesting model system to 94 investigate gene-environment interactions in the outdoor enclosure.95We hypothesized that by introducing laboratory mice carrying IBD susceptibility 96 mutations into the outdoor enclosure, the drastic change in environment may trigger 97 alterations in the immune response and the microbiota, which would have greater adverse 98 effects on mutant mice than wildtype mice. Hence, to study the immunological consequences 99 of rewilding, we released Atg16l1 T316A/T316A , Atg16l1 T316A/+ , Nod2 -/mice in addition to 100 C57BL/6J wild type (WT) mice to determine whether mutations in these genes alter the 101 immune response to microbial exposure in a natural environment. Here we present a detailed 102 systems immunology profile of both rewilded and laboratory mice to further investigate 103 variation in immune response and the relationship with environment and genetic mutations. 105 RESULTS 106Study design for immune profiling of rewilded and laboratory mice 107 From 116 mice released into the outdoor enclosure for 6-7 weeks we recovered 104 mice 108 (25 WT, 28 Nod2 -/-, 27 Atg16l1 T316A/+ , and 24 Atg16l1 T316A/T316A ) in time for analysis and 109 compared them to 80 matched controls (19 WT, 19 Nod2 -/-, 20 Atg16l1 T316A/+ , 22 110 Atg16l1 T316A/T316A ) maintained under specific pathogen free (SPF) conditions (herein referred 111 to as lab mice) ( Figure 1A). Blood samples were collected at the time of sacrifice and 112 analyzed by flow cytometry with a lymphocyte panel (Table S1). Additionally, cytokine 113 production in the plasma was assayed ( Figure 1A). Fecal samples and cecal contents were 114 collected for microbial profiling, and for reconstitution experiments (Companion study, 115 Yeung et. al.). ...

Altered Immunity of Laboratory Mice in the Natural Environment Is Associated with Fungal Colonization

“…Additional C57BL/6J mice were purchased from Jackson Laboratory and bred onsite to supplement WT controls for experiments. 16S microbial diversity at the conclusion of the experiment did not show appreciable differences in microbial composition within the lab populations (see companion manuscript Lin et al, 2020;Figure S5). Outdoor enclosures were previously described (Budischak et al, 2018;Leung et al, 2018) and the protocols for releasing the laboratory mice into the outdoor enclosure facility were approved by Princeton IACUC (#1982-17).…”

“…To determine the consequences of microbial colonization and exposure to the natural environment on the steady-state immune system, we applied multicolor flow cytometry to analyze the immune cell composition of blood and mesenteric lymph nodes (MLNs) from SPF mice aged 6-8 weeks old at the time of release into the enclosure and captured 6-7 weeks later. Details of the cohort of rewilded mice are described in the STAR Methods and includes male and female wild-type (WT) C57BL6/J mice as well as an additional cohort of mice harboring mutations in inflammatory bowel disease (IBD) genes (Atg16L1 and Nod2) described in the companion study (Lin et al, 2020). Matched control mice were maintained under SPF conditions in the institutional vivarium, herein referred to as lab mice.…”

“…Matched control mice were maintained under SPF conditions in the institutional vivarium, herein referred to as lab mice. The initial flow cytometry analyses were performed on all rewilded and lab mice because genotype did not affect the outcome (Lin et al, 2020). We provide the detailed breakdown by Atg16L1 and Nod2 mutation status in subsequent analyses.…”

“…In a companion manuscript (Lin et al, 2020), we describe deep immune profiling of rewilded mice to identify a series of changes that occur to the immune system upon exposure to the natural environment and apply computational analyses to infer the impact of genetic and environmental variables. Here, we tested the hypothesis that colonization by microorganisms present in the natural environment contributes to the maturation of the immune system that occurs in free-living mammals.…”

Integrating microbiome science and evolutionary medicine into animal health and conservation

“…Nevertheless, the Microbiome Rewilding Hypothesis has direct applicability to captive animals. In laboratory mice, rewilding via exposure to outdoor enclosures influenced immune cell frequencies and increased colonisation by symbiotic, intestinal fungi and bacteria, some of which were causally linked to increased circulation of immune-boosting white blood cells (Lin et al, 2020;Yeung et al, 2020). In captive-born harlequin frogs (Atelopus varius), a 'softrelease' to outdoor mesocosms resulted in rapid changes to the structure and anti-fungal properties of the skin microbiomes, more similar to communities seen in in-situ frogs (Kueneman et al, 2022).…”