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Physiological microbial exposure transiently inhibits mouse lung ILC2 responses to allergens

Abstract

Lung group 2 innate lymphoid cells (ILC2s) control the nature of immune responses to airway allergens. Some microbial products, including those that stimulate interferons, block ILC2 activation, but whether this occurs after natural infections or causes durable ILC2 inhibition is unclear. In the present study, we cohoused laboratory and pet store mice as a model of physiological microbial exposure. Laboratory mice cohoused for 2 weeks had impaired ILC2 responses and reduced lung eosinophilia to intranasal allergens, whereas these responses were restored in mice cohoused for ≥2 months. ILC2 inhibition at 2 weeks correlated with increased interferon receptor signaling, which waned by 2 months of cohousing. Reinduction of interferons in 2-month cohoused mice blocked ILC2 activation. These findings suggest that ILC2s respond dynamically to environmental cues and that microbial exposures do not control long-term desensitization of innate type 2 responses to allergens.

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Fig. 1: Mouse lung immune cell populations are altered by cohousing.
Fig. 2: Short-term cohousing impairs eosinophil and ILC2 responses to intranasal allergens.
Fig. 3: Acute IL-33, IL-5 and IL-13 responses after Alt exposure are reduced in 2-month cohoused mice.
Fig. 4: Comparable immune infiltration in SPF and 2-month cohoused mice after repeated Alt exposure.
Fig. 5: Cohousing with pet store mice alters the fecal microbiota.
Fig. 6: Cohousing with pet store mice induces transient systemic and lung inflammation.
Fig. 7: Poly(I:C) suppresses ILC2 and eosinophil responses to Alt in SPF and 2-month cohoused mice.

Data availability

The 16S rRNA amplicon sequence data are deposited in the SRA under the primary accession no. SRP371485. The SILVA rRNA database can be found at https://www.arb-silva.de/ (PMID: 1794732). THe UCHIME database can be found at https://drive5.com/uchime/uchime_download.html (PMID: 21700674). The Ribosomal Database Project can be found at http://rdp.cme.msu.edu/ (PMID: 19004872). Source data are provided with this paper.

Code availability

Code for 16S rRNA-seq-based microbiome analysis is available in PMID: 30227891 (ref. 69).

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Acknowledgements

We thank UMN Flow Cytometry Resource Facility, CFI Dirty Mouse Colony, UMN BSL-3 Program, Minnesota Supercomputing Institute, Cytokine Reference Laboratory and UMN Research Animal Resources for support. We thank Jamequist and Kita lab members for thoughtful discussion. This work was supported by the NIH (grant nos. R01HL117823 to H.K., T32HL007741 to K.E.B. and R35GM140881 to T.S.G.), Minnesota Partnership for Biotechnology and Medical Genomics (grant no. 16.48 to S.C.J. and H.K.) and UMN Medical School (grant no. AIRP-CP-21 to S.C.J.).

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Authors and Affiliations

Authors

Contributions

K.E.B. designed and performed experiments and analyzed and interpreted the data. K.I., M.J.P., D.A.W., R.T., T.A.K., J.X. and T.S.G. performed experiments. M.H.K. and C.S. carried out 16S rRNA-seq-based microbiome analysis. H.J.M. provided the HpARI reagent. S.C.J. and H.K. supervised the project. K.E.B. and S.C.J. wrote the manuscript. H.K., T.S.G. and H.J.M. edited the manuscript.

Corresponding authors

Correspondence to Hirohito Kita or Stephen C. Jameson.

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The authors declare no competing interests.

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Nature Immunology thanks Donata Vercelli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Serology screening for SPF pathogens in cohoused mice.

Blood samples were collected and tested for antibodies against common murine pathogens. Each column represents an animal. Each row indicates a pathogen. Filled boxes indicate positive results, lighter shaded boxes indicate equivocal (weak positive) results. White boxes indicate negative results. a, Serology results from representative pet store (n = 8), cohoused B6 (n = 26) and F1 IL-5v (n = 11) mice cohoused for approximately two weeks at the time of blood collection. b, Serology results from representative pet store and cohoused B6, F1 IL-5v, and BALB/c mice cohoused for at least two months at the time of blood collection (n = 26/group). CPIL = Clostridium piliforme, ECUN = Encephalitozoon cuniculi, EDIM = rotavirus, LCMV = lymphocytic choriomeningitis virus, MAV1 + 2 = mouse adenovirus 1 and 2, MCMV = murine cytomegalovirus, MHV = mouse hepatitis virus, MNV = murine norovirus, MPUL = Mycoplasma pulmonis, MPV1 + 2 = mouse parvovirus type 1 and type 2, MVM = minute virus of mice, POLY = polyoma virus, PVM = pneumonia virus of mice, REO = reovirus, SEND = murine respirovirus (Sendai virus), TCMV = GDVII Theiler’s murine encephalomyelitis virus. c. Representative flow cytometry histograms of protein expression of lung ILC2 in age-matched SPF (blue) and CoH (red) mice at 2wk and >2mo. Vertical gray lines are added to aid in comparison of expression among groups.

Extended Data Fig. 2 Example flow cytometry gating.

Representative flow plots to illustrate gating strategy of immune cells in the lungs. a, Neutrophil, eosinophil, and alveolar macrophage gating. b, γδ T cell, CD4+ T cell, CD8+ T cell, and NK cell gating. c, ILC2 and B cell gating.

Extended Data Fig. 3 A. alternata response is susceptible to inhibition by microbial factors.

Eosinophils (CD45+ SiglecF+ CD11b+ CD11c cells) were identified by being in the lung parenchyma or airways by being unlabeled by a fluorescently tagged anti-CD45 antibody injected intravenously three minutes before euthanasia. Shown are B6 mice treated 24 hours prior with intranasal PBS or A. alternata (Alt) or HpARI and Alt. Numbers in plots represent percent of cells in the gate. b, Number of eosinophils in the lungs and airways (i.v. CD45) 24 hours after indicated intranasal treatment. Mice were B6 (white circles) or B6xBALB/c IL-5WT/venus (IL-5v F1, light blue squares). Bar graph shows mean +SD of log-transformed values. c, Representative flow plots of IL-5 venus expression in IL-5v F1 lung ILC2. d, IL-5 venus gMFI of IL-5 venus+ lung ILC2. e-f, ST2 (e) and CD25 (f) of B6 and IL-5v F1 mice. gMFIs from multiple experiments normalized to PBS-treated group set to 100. b and e, f, Pooled from one B6 and one IL-5v F1 experiment (n = 5−9/group). d, Data from one experiment in IL-5v F1 mice (n = 2-3/group). Bar graphs show mean +SD. Each symbol represents a mouse. P values were determined with one-way ANOVA with Tukey’s multiple comparisons test; n.s. p ≥ 0.05. Source Data contains exact P-values and group sizes.

Source data

Extended Data Fig. 4 ILC2 numbers and CD4+ T cell data from two-week cohoused mice.

Flow analysis 24 hours after intranasal PBS or Alt treatment. Mice were B6 (white) or IL-5v F1 (light blue) and were cohoused with pet store mice for approximately two weeks (2wk) or were age-matched SPF controls. a, f, Data in Figures 2a and 2d are presented again, with samples from each experiment indicated with a different symbol/color combination so that experiment-to-experiment variation can be visualized. b, Number of lung ILC2. c, d, IL-5 venus expression within lung (i.v. CD45-) CD4+ T cells of IL-5v F1 SPF and 2wk CoH mice 24 hours after PBS or Alt treatment. c, Representative flow plots of IL-5 venus expression in lung CD4+ T cells. d, Percent IL-5 venus+ and e, normalized IL-5 venus gMFI of IL-5 venus+ lung CD4+ T cells. b, Pooled from four B6 experiments and two IL-5v F1 experiments (n = 12–14/group). c-e, Pooled from two IL-5v F1 experiments (n = 4-6/group). Numbers in plots represent percent of cells in the gate. Bar graphs show mean +SD. g, Mice were treated with intranasal PBS or papain every other day three times and analyzed 24 hours after the last treatment and are from the same experiments described in Fig. 2g-j. Number of lung i.v.- CD4+ T cells in 2wk or >2mo CoH B6 mice or age-matched SPF controls. Bar graphs show mean +SD of log-transformed values. Each symbol represents a mouse. P values were determined with a 2-way ANOVA with Tukey’s multiple comparisons test; n.s. p ≥ 0.05. Source Data contains exact P-values and group sizes.

Source data

Extended Data Fig. 5 BALF and lung cytokine measurements.

a, b, e, f, B6 mice (white circles) or c-d, BALB/c mice (pink diamonds) were cohoused with pet store mice for at least two months (>2mo CoH) or were age-matched SPF controls. Alt (a-d), recombinant IL-33 (rIL-33, e, f) or control PBS were given intranasally to mice and lungs and bronchoalveolar lavage fluid (BALF) were collected 4.5 hours later. IL-5 and IL-13 in the lung homogenates (c, d) and BALF (a-b, e, f) were detected by ELISA and lung concentrations were normalized to the amount of total protein in the samples. a, b pooled from three experiments (n = 6–10/group). c-d pooled from four experiments (n = 6-12/group). e, f pooled from three experiments (n = 5–12/group). Bar graphs show mean +SD. Each symbol represents a mouse. P values were determined with a 2-way ANOVA with Tukey’s multiple comparisons test; n.s. p ≥ 0.05. Source Data contains exact P-values and group sizes.

Source data

Extended Data Fig. 6 Fecal microbiota analysis.

a, Principal coordinates analysis (PCoA) of Bray-Curtis distances of mouse fecal samples from cohoused Cages 2 and 3 (Cage 1 PCoA in Fig. 5b). SPF samples represented by circles are control SPF mice used in analysis of all cages and SPF samples represented by the symbols used for cohoused mice in each graph are from those same mice before cohousing. b, Distributions of abundant genera in mouse fecal samples from Cages 2 and 3. Less abundant genera accounted for less than 2.5% of the community among all samples. c, Distributions of abundant genera in mouse fecal samples from SPF control mice collected on day 0 of cohousing and 60 days later. Less abundant genera accounted for less than 2.5% of the community among all samples.

Extended Data Fig. 7 Serum cytokines and Mx1-GFP expression in cohoused mice.

a, Serum cytokine and chemokine levels of B6 mice days 0 and 60 after cohousing with a pet store mouse, (n = 8). These are from the same data in Fig. 5d. Bar graphs show mean +SD. P values were determined with a two-tailed Wilcoxon matched-pairs signed rank test; n.s. p ≥ 0.05. b, Representative flow plots of Mx1-GFP expression in blood CD8+ T cells from wild-type B6 and Mx1gfp mice either housed in SPF conditions or after seven days of cohousing. Numbers in plots represent percent of cells in the gate. c, d, Percent Mx1-GFP+ of the indicated cell populations in the spleens of (c) 2wk or (d) >2mo CoH mice or age-matched controls. e, f, Percent Mx1-GFP+ of the indicated cell populations in the lungs of (e) 2wk or (f) >2mo CoH mice or age-matched controls. c-f, Bar graphs show mean +SD. Each symbol represents a mouse. P values were determined with a Student’s t-test (two-tailed). When groups had unequal variance the t-test was conducted with Welch’s correction; n.s. p ≥ 0.05. g, Representative flow plots of Mx1-GFP expression in lung ILC2 from wild-type Mx1gfp mice 24 hours after intranasal treatment with PBS or Alt, with the indicated group treated with intranasal poly(I:C) 24 hours before Alt treatment. Numbers in plots represent percent of cells in the gate. Source Data contains exact P-values and group sizes.

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Statistical source data and exact P values.

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Statistical source data and exact P values.

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Statistical source data and exact P values.

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Source Data Extended Data Fig. 3

Statistical source data and exact P values.

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Statistical source data and exact P values.

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Statistical source data and exact P values.

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Block, K.E., Iijima, K., Pierson, M.J. et al. Physiological microbial exposure transiently inhibits mouse lung ILC2 responses to allergens. Nat Immunol 23, 1703–1713 (2022). https://doi.org/10.1038/s41590-022-01350-8

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