Highlights d Controlled release of lab mice into the wild alters the state of the immune system d Rewilded mice harbor an altered microbiota including increases in intestinal fungi

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32The immune systems of free-living mammals such as humans and wild mice display a 33 heightened degree of activation compared with laboratory mice maintained under artificial 34 conditions. Here, we demonstrate that releasing inbred laboratory mice into an outdoor enclosure 35to mimic life in a natural environment alters the state of immunity. In addition to enhancing the 36 differentiation of T cell populations previously associated with pathogen exposure, we found that 37 outdoor release of mice led to an increase in circulating granulocytes. However, rewilded mice 38 were not infected by pathogens previously implicated in immune activation. Rather, changes to 39 the immune system were associated with an altered composition of the microbiota, and fungi 40 isolated from rewilded mice were sufficient to increase circulating granulocytes. These findings 41 establish an experimental procedure to investigate the impact of the natural environment on 42 immune development and identify a role for sustained fungal exposure in determining 43 granulocyte numbers. 44 45 46 48 research and has enabled fundamental advances in basic immunology. Yet, this ubiquitous model 49 fails to recreate certain aspects of human immunity. Inbred laboratory mice and adult humans 50 differ in the proportion of leukocyte subsets, transcriptional responses to microbial challenges, 51 and other immune parameters (Masopust et al., 2017; Tao and Reese, 2017). Such differences 52 may limit the predictive value of experiments with mice when studying complex inflammatory 53 and infectious diseases, resulting in significant shortcomings in translating laboratory 54 observations to humans. 55Recent findings suggest that this shortcoming of the rodent model may be due to the 56 specific pathogen free (SPF) environment in which they are maintained. Wild mice and pet store 57 mice, both of which are exposed to a litany of pathogens that are typically excluded from SPF 58 facilities, display an abundance of differentiated memory T cells that more closely resembles the 59 state of immunity in adult humans (Abolins et al., 2017; Beura et al., 2016; Choi et al., 2019). 60Similarly, transferring embryos from lab mice into wild mice generates commensal-and 61 pathogen-exposed offspring (wildlings) that more faithfully recreate human immunity than 62 standard SPF mice, including the unresponsiveness to immunotherapies that failed in clinical 63 trials (Rosshart et al., 2019). Sequentially infecting SPF mice with 3 viruses and a helminth shifts 64 the gene expression profile of peripheral blood mononuclear cells (PBMCs) towards that of pet 65 store mice and adult humans (Reese et al., 2016), further highlighting the role for pathogen 66 experience in normalizing the immune system. SPF mice are also distinguished from free-living 67 mammals by the lack of exposure to potentially immuno-stimulatory members of the microbiota 68 that are absent in a laboratory animal facility. For example, the offspring of germ-free mice 69 inoculated with ileocecal contents from ...

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.). ...