LETTERS https://doi.org/10.1038/s41591-018-0324-z Healthy infants harbor intestinal bacteria that protect against food allergy Taylor Feehley1,9, Catherine H. Plunkett1,9, Riyue Bao 2,3,9, Sung Min Choi Hong1, Elliot Culleen1, Pedro Belda-Ferre 1, Evelyn Campbell1, Rosita Aitoro4, Rita Nocerino4, Lorella Paparo4, Jorge Andrade 2,3, Dionysios A. Antonopoulos5,6, Roberto Berni Canani4,7,8 and Cathryn R. Nagler 1* There has been a striking generational increase in life-threat- formula to manage ongoing allergic symptoms, whereas the healthy ening food allergies in Westernized societies1,2. One hypoth- donors received a standard cow’s milk-based formula5. Initial trans- esis to explain this rising prevalence is that twenty-first fer recipients were used as living repositories for subsequent experi- century lifestyle practices, including misuse of antibiotics, ments (see Online Methods). dietary changes, and higher rates of Caesarean birth and for- Groups of germ-free mice and mice colonized with either the mula feeding have altered intestinal bacterial communities; healthy or CMA infant microbiota were sensitized with BLG and early-life alterations may be particularly detrimental3,4. the mucosal adjuvant cholera toxin. Consistent with previous To better understand how commensal bacteria regulate food reports7,11, germ-free mice, devoid of any bacterial colonization, allergy in humans, we colonized germ-free mice with feces were highly susceptible to anaphylactic responses to food, as evi- from healthy or cow’s milk allergic (CMA) infants5. We found denced by a drop in core body temperature (Fig. 1a) and production that germ-free mice colonized with bacteria from healthy, of BLG-specific IgE and IgG1 (Fig. 1b,c). We also measured a sub- but not CMA, infants were protected against anaphylactic stantial reduction in core body temperature in mice colonized with responses to a cow’s milk allergen. Differences in bacterial fecal samples from each of the four CMA donors in response to BLG composition separated the healthy and CMA populations in challenge (Fig. 1a). Sensitized CMA-colonized mice produced sig- both the human donors and the colonized mice. Healthy and nificantly higher serum concentrations of BLG-specific IgE (Fig. 1b), CMA colonized mice also exhibited unique transciptome sig- IgG1 (Fig. 1c) and mouse mast cell protease-1 (mMCPT-1) (Fig. 1d) natures in the ileal epithelium. Correlation of ileal bacteria compared with healthy-colonized mice. Notably, all of the mice that with genes upregulated in the ileum of healthy or CMA colo- received the four healthy infant microbiotas were protected from an nized mice identified a clostridial species, Anaerostipes caccae, anaphylactic response to BLG challenge; their core body tempera- that protected against an allergic response to food. Our find- ture post-challenge was significantly different from that measured ings demonstrate that intestinal bacteria are critical for regu- in germ-free or CMA-colonized mice (Fig. 1a). Histological analysis lating allergic responses to dietary antigens and suggest that did not reveal any evidence of pathology or inflammation in ileal or interventions that modulate bacterial communities may be colonic tissue samples taken post-challenge (Extended Data Fig. 1) therapeutically relevant for food allergy. or after long-term colonization (Extended Data Fig. 2). Microbial Work from our laboratory and others has demonstrated that analysis revealed that community diversity and evenness were simi- the fecal microbial communities of infants with CMA are mark- lar between healthy- and CMA-colonized mouse groups (Extended edly different from those of their healthy counterparts5,6. Based on Data Fig. 3). To examine whether the cow’s-milk-containing formula these results, as well as evidence that members of the microbiota contributed to microbiota-independent protection against anaphy- can be allergy protective7, we used a gnotobiotic mouse model laxis in the healthy-colonized mice, we performed additional fecal to investigate whether commensal bacteria have a causal role in transfers from breast-fed healthy and CMA donors (Supplementary protection against an allergic response to the cow’s milk allergen Table 2). Recipient mice received only plant-based mouse chow. β -lactoglobulin (BLG). Germ-free mice were colonized with human Mice colonized with feces from a breast-fed healthy donor were pro- feces from four healthy and four immunoglobulin E (IgE)-mediated tected from an anaphylactic response to BLG sensitization and chal- CMA infant donors who were matched for age, gender, and mode lenge. However, mice colonized with feces from a breast-fed CMA of birth8,9 (Supplementary Table 1). It has previously been reported donor exhibited a significantly greater drop in core body tempera- that diet is important for the stable colonization of germ-free mice ture compared with healthy-colonized mice (Extended Data Fig. 4a) with human feces10. To support the growth of human bacteria in and higher levels of BLG-specific IgE (Extended Data Fig. 4b). the murine hosts, mice received feces from formula-fed healthy or We also compared sensitization to BLG in germ-free mice fed water CMA infants and were fed the same formulas consumed by their or Enfamil. Both groups of mice responded robustly to sensitiza- human infant donors in addition to plant-based mouse chow. tion with BLG (Extended Data Fig. 5). There was no significant The CMA infant donors received an extensively hydrolyzed casein difference in their drop in core body temperature post-challenge 1Department of Pathology and Committee on Immunology, The University of Chicago, Chicago, IL, USA. 2Center for Research Informatics, The University of Chicago, Chicago, IL, USA. 3Department of Pediatrics, The University of Chicago, Chicago, IL, USA. 4Department of Translational Medical Science, Section of Pediatrics, University of Naples Federico II, Naples, Italy. 5Department of Medicine, The University of Chicago, Chicago, IL, USA. 6Biosciences Division, Argonne National Laboratory, Argonne, IL, USA. 7European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. 8CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy. 9These authors contributed equally: Taylor Feehley, Catherine H. Plunkett, Riyue Bao. *e-mail: [email protected] NatURE MEDICINE | www.nature.com/naturemedicine LETTERS NATURE MEDICINE abGerm-free P = 0.099 cd 1 Healthy 8,000 4,000 * 3,000 ** *** ) CMA ) 2,000 –1 –1 4,000 2,000 ) 1,000 g ml –1 μ 0 ( 2,000 500 500 (°C) ** 400 400 T ** 1,500 Δ 300 300 –1 1,000 200 200 mMCPT-1 (ng ml 500 BLG-specific IgE (ng ml 100 100 BLG-specific IgG1 –2 0 0 0 0510 15 20 25 30 35 40 45 50 55 60 65 70 CMA CMA CMA Time (min) Healthy Healthy Healthy Germ-free Germ-free Germ-free Fig. 1 | Transfer of healthy, but not CMA, infants’ microbiota protects against an allergic response to food. a, Change in core body temperature at indicated time points following first challenge with BLG in germ-free mice and in mice colonized with feces from each of eight donors (four healthy, four CMA; see Supplementary Table 1) that had been sensitized with BLG plus cholera toxin; n = 42 CMA, 31 healthy and 24 germ-free mice, with 4–12 mice for each of the eight donors, collected from two independent experiments. b–d, Serum BLG-specific IgE (b), BLG-specific IgG1 (c), and mMCPT-1 (d) from mice in a. For a, circles represent mean, and error bars represent s.e.m. For b–d, circles represent individual mice, and bars represent mean + s.e.m. Linear mixed-effect models were used to compare groups in a–d with the BH-FDR method for multiple testing correction. *P < 0.05, **P < 0.01, ***P < 0.001. a Healthy CMA b e Healthy CMA ) Protective 2,500 50 OTU abundance score per sample –1 Healthy CMA 40 OTUs 2,000 30 Unclassified_Lachnospiraceae 20 10 1,500 Unclassified_Erysipelotrichaceae 0 Unclassified_Enterobacteriaceae Non-protective 40 1,000 Streptococcus Other_Enterobacteriaceae 30 OTUs 20 500 Salmonella 10 Enterococcus 0 BLG-specific IgE (ng ml 0 Unclassified_Barnesiellaceae 4 321 8765 0 1234 Ruminococcus No. protective OTUs/ Unclassified_ruminococcaaceae DmDmDmDmDmDmDmDm Coprobacillus 1111294|Enterobacteriaceae no. non-protective OTUs 360015|Lachnospiraceae Other_Clostridiaceae New147|Clostridiaceae Unclassified_Clostridiales 628226|Clostridiaceae c 349024|Streptococcaceae ) Healthy CMA Other_Clostridiales 712677|Clostridiales_unclassified –1 500 780650|Clostridiaceae Blautia 843459|Clostridiaceae Parabacteroides 262095|Erysipelotrichaceae g ml 400 New166|Lachnospiraceae µ 579851|Erysipelotrichaceae ( 551822|Clostridiaceae Protective OTUs –4 –2 024 298247|Lachnospiraceae 300 345540|Enterobacteriaceae ( log [LDA score] 582691|Clostridiaceae n 10 259772|Lachnospiraceae = 34) 200 325419|Clostridiaceae 797229|Enterobacteriaceae 557627|Clostridiaceae 828483|Clostridiaceae 100 f 299267|Enterobacteriaceae 4448331|Enterobacteriaceae 3376513|Lachnospiraceae 0 h 813217|Enterobacteriaceae BLG-specific IgG1 01234 Healthy New56927|Lachnospiraceae g c 335701|Lachnospiraceae e d CMA 191999|Lachnospiraceae No. protective OTUs/ f b 183865|Lachnospiraceae a 231787|Enterobacteriaceae no. non-protective OTUs i 342397|Lachnospiraceae 581782|Enterobacteriaceae 304641|Enterobacteriaceae 4389289|Enterobacteriaceae d Healthy CMA 541119|Enterobacteriaceae 195258|Bacteroidaceae 800 a: Coriobacteriaceae 315846|Barnesiellaceae 551902|Ruminococcaceae ) b: Coriobacteriales 318190|Ruminococcaceae –1