Unraveling the mystery of the hypothesis through

Kouji Matsushima, Shigenori Nagai

J Clin Invest. 2012;122(3):801-804. https://doi.org/10.1172/JCI61466.

Commentary

Epidemiological studies have revealed an inverse association between Helicobacter pylori infection and the incidence of allergic . This association is consistent with the hygiene hypothesis, which posits that exposure to microbes early in life prevents the later development of allergic diseases, and has been reproduced in mouse models of asthma. In this issue of the JCI, Oertli and colleagues report that H. pylori infection in neonates elicits tolerogenic DCs that produce IL-18, which drive the generation of Tregs that subsequently protect the mice from allergic asthma. This finding strengthens the intriguing link between pathogen exposure and allergic disease.

Find the latest version: https://jci.me/61466/pdf commentaries sis and the need for continued research if 2010;40(4):923–932. and natural killer cell deficiency. J Clin Invest. 2012; 4. Orange JS. Human natural killer cell deficien- 122(3):821–832. we are to develop therapeutics that benefit cies and susceptibility to infection. Microbes Infect. 13. Hughes CR, et al. MCM4 mutation causes adrenal affected patients. 2002;4(15):1545–1558. failure, short stature, and natural killer cell deficien- 5. Orange JS. Human natural killer cell deficiencies. cy in humans. J Clin Invest. 2012;122(3):814–820. Acknowledgments Curr Opin Clin Immunol. 2006;6(6):399–409. 14. Eidenschenk C, et al. A novel primary immunode- 6. Tortorella D, Gewurz BE, Furman MH, Schust DJ, ficiency with specific natural-killer cell deficiency I thank Emily Mace for the critical review Ploegh HL. Viral subversion of the . maps to the centromeric region of chromosome 8. of this commentary, as well as the support Annu Rev Immunol. 2000;18:861–926. Am J Hum Genet. 2006;78(4):721–727. of NIH-NIAID R01067946. 7. Biron CA, Byron KS, Sullivan JL. Severe herpesvirus 15. O’Riordan SM, Lynch SA, Hindmarsh PC, Chan in an adolescent without natural killer LF, Clark AJ, Costigan C. A novel variant of famil- cells. N Engl J Med. 1989;320(26):1731–1735. ial glucocorticoid deficiency prevalent among the Address correspondence to: Jordan S. 8. Fleisher G, Starr S, Koven N, Kamiya H, Douglas Irish Traveler population. J Clin Endocrinol Metab. Orange, Children’s Hospital of Philadel- SD, Henle W. A non-x-linked syndrome with sus- 2008;93(7):2896–2899. ceptibility to severe Epstein-Barr infections. 16. Bochman ML, Schwacha A. The Mcm complex: phia Research Institute, 3615 Civic Center J Pediatr. 1982;100(5):727–730. unwinding the mechanism of a replicative helicase. Blvd., ARC 907A, Philadelphia, Pennsylva- 9. Jawahar S, Moody C, Chan M, Finberg R, Geha R, Microbiol Mol Biol Rev. 2009;73(4):652–683. nia 19104, USA. Phone 267.426.5622, Fax Chatila T. Natural Killer (NK) cell deficiency associ- 17. Shima N, et al. A viable allele of Mcm4 causes chro- ated with an epitope-deficient Fc receptor type IIIA mosome instability and mammary adenocarcino- 267.426.0947; E-mail: [email protected]. (CD16-II). Clin Exp Immunol. 1996;103(3):408–413. mas in mice. Nat Genet. 2007;39(1):93–98. 10. Dickinson RE, et al. Exome sequencing identifies 18. Freud AG, Caligiuri MA. Human natural killer cell 1. Sun JC, Lanier LL. NK cell development, homeosta- GATA-2 mutation as the cause of dendritic cell, development. Immunol Rev. 2006;214:56–72. sis and function: parallels with CD8 T cells. Nat Rev monocyte, B and NK lymphoid deficiency. Blood. 19. Cooley S, et al. A subpopulation of human periph- Immunol. 2011;11(10):645–657. 2011;118(10):2656–2658. eral blood NK cells that lacks inhibitory receptors 2. Orange JS. Formation and function of the lytic 11. Hsu AP, et al. Mutations in GATA2 are associated for self-MHC is developmentally immature. Blood. NK-cell immunological synapse. Nat Rev Immunol. with the autosomal dominant and sporadic mono- 2007;110(2):578–586. 2008;8(9):713–725. cytopenia and mycobacterial infection (MonoMAC) 20. Bernard F, et al. A novel developmental and immu- 3. Lee SH, Biron CA. Here today — not gone tomor- syndrome. Blood. 2011;118(10):2653–2655. nodeficiency syndrome associated with intrauter- row: roles for activating receptors in sustaining 12. Gineau L, et al. Partial MCM4 deficiency in patients ine growth retardation and a lack of natural killer NK cells during viral infections. Eur J Immunol. with growth retardation, adrenal insufficiency, cells. Pediatrics. 2004;113(1 pt 1):136–141.

Unraveling the mystery of the hygiene hypothesis through Helicobacter pylori infection Kouji Matsushima1 and Shigenori Nagai2

1Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 2Department of Microbiology and , Keio University School of Medicine, Tokyo, Japan.

Epidemiological studies have revealed an inverse association between Heli- sue lymphoma, gastroesophageal reflux cobacter pylori infection and the incidence of allergic asthma. This associa- disease, and iron deficiency anemia (4). tion is consistent with the hygiene hypothesis, which posits that exposure The flip side of the equation is that, in to microbes early in life prevents the later development of allergic diseases, an epidemiological study, Chen and Blaser and has been reproduced in mouse models of asthma. In this issue of the reported an inverse association between JCI, Oertli and colleagues report that H. pylori infection in neonates elicits infection with H. pylori expressing the viru- tolerogenic DCs that produce IL-18, which drive the generation of Tregs that lence factor cytotoxin-associated protein A subsequently protect the mice from allergic asthma. This finding strength- (CagA) and the incidence of asthma and ens the intriguing link between pathogen exposure and allergic disease. allergy (5). This observation is consistent with the hygiene hypothesis, which states Nearly half of the world’s population is with age. German scientists first observed that exposure to microbes (both pathogen- infected with the Gram-negative bacterium the spiral-shaped bacterium in the lining of ic and commensal) early in life prevents the Helicobacter pylori. Infection rates are higher the human stomach in 1875 (2). However, later development of allergic diseases (6). in developing countries than in developed Australian scientists, Marshall and Warren, The basis for this phenomenon is unclear, countries; in fact, the bacterium is gradu- were the first to culture this bacterium in but, recently, Müller and colleagues dem- ally disappearing from many populations 1984, and they went on to link the presence onstrated that H. pylori infection prevents in developed countries (1). Infection seems of H. pylori in the gut to in allergic asthma in mouse models of the con- to occur in early childhood in most cases, the stomach (gastritis) and ulceration of dition through the induction of Tregs (7). with the incidence of infection increasing the stomach or duodenum (peptic ulcer The logical question raised by this dis- disease) (3). Marshall and Warren were covery was what is the mechanism underly- awarded the 2005 Nobel Prize in Physiol- ing the induction of Tregs in mice infected Conflict of interest: The authors have declared that no conflict of interest exists. ogy or Medicine for this finding. H. pylori with H. pylori? In this issue of the JCI, Oertli, Citation for this article: J Clin Invest. 2012; infection has been subsequently associated Müller, and colleagues provide insight into 122(3):801–804. doi:10.1172/JCI61466. with gastric cancer, mucosa-associated tis- this process (8). Specifically, they show that

The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 3 March 2012 801 commentaries

Figure 1 H. pylori–exposed tolerogenic DCs drive naive CD4+ T cells to become Tregs. DCs in the airways capture allergens, process , and migrate into pulmonary lymph nodes, in which they initiate -specific Th2-skewed immune responses, which ultimately cause asthma. When H. pylori infection occurs in neonates, H. pylori–exposed DCs in the lamina propria of the gut display impaired maturation, resulting in tolerogenic DCs. IL-18 derived from these DCs acts directly on naive CD4+ T cells in gut regional lymph nodes (mesenteric lymph nodes), caus- ing them to become Tregs that subsequently suppress asthmatic immune responses in the airways.

DCs exposed to H. pylori are programmed and responding to danger signals, such as to promote self tolerance (the lack of to become tolerogenic, driving Treg dif- those on invading pathogens. DCs respond immune responsiveness to self antigens). ferentiation and thereby protection from to danger signals by endocytosing local Interestingly, certain helminth and bacte- asthma, through the production of IL-18. antigens, which they then degrade, gen- rial pathogens, including Fasciola hepatica, This study suggests novel cellular and erating antigenic peptides that form com- Candida albicans, Schistosoma japonicum, molecular pathways regulating allergic dis- plexes with MHC molecules that are then Schistosoma mansoni, Bordetella pertussis, and eases in individuals infected with H. pylori. expressed on the DC surface in order to Vibrio cholerae, are known to promote DC However, there remain many unanswered initiate antigen-specific T cell responses. tolerogenicity by producing TGF-β and questions regarding the underlying mech- DCs also respond to danger signals by IL-10 mimetics or by inducing host DCs to anisms of H. pylori infection–associated undergoing a process known as matura- produce these cytokines and consequently asthma protection. tion, during which they begin to express drive Treg induction to modulate the host the chemokine receptor CCR7, which immune response (10). H. pylori–conditioned tolerogenic DCs enables them to migrate, in a chemokine- The effects of H. pylori infection described DCs, originally discovered by Stein- dependent manner, to the lymph nodes by Oertli et al. (8) are mediated by patho- man and Cohn in 1973 (9), are now well draining the site of antigen encounter. gen-induced tolerogenic DCs. In vitro expo- established to be the critical antigen-pre- In addition to initiating the adaptive sure of mouse bone marrow–derived DCs senting cells responsible for activating immune response to invading pathogens, to H. pylori induced a population of DCs naive T cells. DCs link the innate and DCs also continuously present self anti- that were unable to activate T cells. Com- acquired immune systems by detecting gens, in the context of MHC molecules, parable populations of DCs were present

802 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 3 March 2012 commentaries in the mesenteric lymph nodes and lamina classes of Tregs, just as they have been found genic and provide protection from allergic propria (the thin layer of loose connective to guide the generation of different subsets disease. However, important questions tissue that lies beneath mucosal epithe- of helper T cells. For example, under Th1 remain about how events in the gut affect lia, which contains lymphoid tissues and inflammatory conditions, IFN-γ induces the lungs. As illustrated in Figure 1, we subsets of leukocytes) of H. pylori–infected the expression of the transcription factor believe that antigen-independent systemic mice and patients, respectively. T-bet in FoxP3+ Tregs, optimizing these cells surveillance by Tregs generated in the mes- Oertli et al. went on to show that direct for the suppression of Th1 responses (14). enteric lymph nodes draining the H. pylori– contact between H. pylori and DCs was On the other hand, Tregs in the periphery infected gut represents the most plausible essential for the induction of tolerogenic express high levels of IFN regulatory factor-4 mechanism for how this could occur. DCs (8). In investigating the molecular (IRF4), which is required for these cells to basis for these events, they determined be able to suppress Th2 responses (15). It is Why is neonatal infection so critical? that neither the host molecules TLR2 unclear at this stage whether IL-18 induces As demonstrated in a paper from Müller and and MyD88 (a key intracellular signal general Treg populations or specifically colleagues (7), asthma is strongly reduced in transduction molecule for TLRs) nor the induces Tregs optimized for suppressing mice infected with H. pylori neonatally but not H. pylori type IV secretion system (which Th2 responses, which are key to the patho- in mice infected during adulthood. In neona- delivers CagA into epithelium) were genesis of asthma. Therefore, it will be very tally infected mice, the reduction in asthma involved. As no evidence of a role for the important to define how IL-18–mediated was accompanied by an increase in Tregs; in TLR family of pathogen-sensing receptors signals regulate the activation of FoxP3, mice infected during adulthood, numbers of in DC recognition of H. pylori was found by the master regulator of Treg induction Tregs remained unchanged. These observa- Oertli et al., it is possible that one or more (16), and to define the effect of IL-18 on tions raise the question, why do Tregs domi- members of the nod-like receptor (NLR) the signaling pathways that determine Treg nate in H. pylori infection in neonates? Given family of pathogen-sensing receptors is heterogeneity. the data from Oertli et al. (8), this phenome- involved. In this context, Watanabe et al. non could be due to a difference in IL-18 pro- have shown that γ-d-glutamyl-meso-diami- Tolerogenic signals from the gut duction between H. pylori–programmed DCs nopimelic acid (iE-DAP; a cell wall peptido- to the airways in neonates and adults. However, it could glycan derivative from H. pylori) activates One of the biggest puzzles raised by the also be due to a barrier difference between DCs in a manner dependent on nucleotide- work of Oertli et al. is how H. pylori in the the guts of neonates and adults. Although binding oligomerization–domain contain- mucus of the stomach influences DCs in the finding that asthma is strongly reduced ing 1 (Nod1, also known as NLRC1) when the lamina propria. It has been reported in mice infected with H. pylori neonatally but added to culture medium and also induces that H. pylori is able to sense the pH gradi- not in those infected during adulthood is type I IFN and, eventually, IL-10 through ent within the mucus layer, moving away consistent with the epidemiologic data indi- the Nod1-TBK1-IRF7 pathway in human from the acidic lumen toward the more cating that H. pylori infection occurs early in epithelial cell lines (11). Future studies neutral pH environment of the epithe- life, its mechanistic basis remains unclear. should examine whether this pathway lial cell surface, even invading the gastric is also involved in H. pylori induction of mucosa and translocating to the gastric DC-derived IL-18 as a potential IL-18–producing tolerogenic DCs. lymph nodes (17, 18). However, it remains therapeutic target unclear how efficiently live H. pylori migrate In summary, the study by Oertli, Müller, and Treg induction by DC-derived IL-18 to the lamina propria and the draining colleagues (8) has extended our understand- The role of IL-18 in allergic diseases is com- lymph nodes, in which they may regulate ing of the molecular and cellular basis of the plicated. IL-18 is produced mainly by mac- the function of DCs. Ito et al. have shown hygiene hypothesis in the context of H. pylori rophages and epithelial cells in a caspase-1– that H. pylori are mainly phagocytosed in infection. They have revealed that DCs exposed dependent manner (12). IL-18 was first the lamina propria and translocated into to H. pylori are destined to become tolerogen- identified based on its ability, in combina- the gastric lymph nodes by macrophages, ic, driving Treg differentiation through IL-18 tion with IL-12, to induce the production of but not by DCs, in the induction of gas- production and thereby inducing protection IFN-γ by T cells and other leukocytes. There- tritis (19). At the same time, there are a from allergic asthma. If DC-derived IL-18 is fore, coadministration of IL-18 and IL-12 number of reports suggesting that Peyer’s found to be generally involved in Treg induc- induces potent antiallergic activity. On the patches (aggregations of lymphoid tis- tion in vivo, targeting IL-18 as a means of Treg other hand, IL-18 in combination with IL-2 sue in the small intestine) are the sites of regulation could present an attractive strategy increases the expression of CD40L on CD4+ T cell priming and effector T cell genera- for the prevention and treatment of the vari- T cells and, in the absence of antigen, can also tion in H. pylori infection (20, 21). In a clini- ous human diseases in which Treg function induce the production of Th2 cytokines such cal setting, H. pylori infection only occurs in is dysregulated, which include autoimmune as IL-4 and IL-13, which are key contributors the stomach. It seems unlikely that DCs in diseases and cancer as well as asthma. to the pathogenesis of asthma (13). the lamina propria of the stomach egress to One of the most important observations the airways via the lymphatics and circula- Acknowledgments made in the study by Oertli et al. (8) is that tion. Although this fact highlights a short- We are very grateful to Francis H.W. Shand DC-derived IL-18 plays a central role in the coming of the model used by Oertli et al. for reviewing the manuscript and provid- conversion of naive T cells to Tregs that (8), in which H. pylori–conditioned DCs ing helpful comments. possess potent inhibitory activity against were administered intranasally, their study Th2 responses. Cytokine signals have been represents important proof of the concept Address correspondence to: Kouji Matsu- implicated in the generation of different that H. pylori–exposed DCs become tolero- shima, Department of Molecular Preventive

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Medicine, The University of Tokyo, 7-3-1 2011;121(8):3088–3093. JR, Urdahl KB, Campbell DJ. The transcription 8. Oertli M, et al. DC-derived IL-18 drives Treg dif- factor T-bet controls homeosta- Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. ferentiation, murine Helicobacter pylori–specific sis and function during type 1 inflammation. Nat Phone: 81.3.5841.3431; Fax: 81.3.5841.3393; , and asthma protection. J Clin Immunol. 2009;10(6):595–602. E-mail: [email protected]. Invest. 2012;122(3):1082–1096. 15. Zheng Y, et al. Regulatory T-cell suppressor pro- 9. Steinman RM, Cohn ZA. Identification of a novel gram co-opts transcription factor IRF-4 to control cell type in peripheral lymphoid organs of mice. Th2 responses. Nature. 2009;458(7236):351–356. 1. Covacci A, Telford JL, Del Giudice G, Parsonnet J, I. Morphology, quantitation, tissue distribution. 16. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Reg- Rappuoli R. Helicobacter pylori virulence and genet- J Exp Med. 1973;137(5):1142–1162. ulatory T cells and immune tolerance. Cell. 2008; ic geography. Science. 1999;284(5418):1328–1333. 10. Maldonado RA, von Andrian UH. How tolerogenic 133(5):775–787. 2. Blaser MJ. An endangered species in the stomach. dendritic cells induce regulatory T cells. Adv Immu- 17. Schreiber S, et al. The spatial orientation of Helico- Sci Am. 2005;292(2):38–45. nol. 2010;108:111–165. bacter pylori in the gastric mucus. Proc Natl Acad Sci 3. Marshall BJ, Warren JR. Unidentified curved bacilli 11. Watanabe T, et al. NOD1 contributes to mouse host U S A. 2004;101(14):5024–5029. in the stomach of patients with gastritis and peptic defense against Helicobacter pylori via induction 18. Petersen AM, Krogfelt KA. Helicobacter pylori: an ulceration. Lancet. 1984;1(8390):1311–1315. of type I IFN and activation of the ISGF3 signaling invading microorganism? A review. FEMS Immunol 4. Superbaum S, Michetti P. Helicobacter pylori infec- pathway. J Clin Invest. 2010;120(5):1645–1662. Med Microbiol. 2003;36(3):117–126. tion. N Engl J Med. 2002;347(15):1175–1186. 12. Gu Y, et al. Activation of Interferon-gamma induc- 19. Ito T, et al. Helicobacter pylori invades the gas- 5. Chen Y, Blaser MJ. Inverse associations of Helico- ing factor mediated by Interleukin-1beta convert- tric mucosa and translocates to the gastric lymph bacter pylori with asthma and allergy. Arch Intern ing enzyme. Science. 1997;275(5297):206–209. nodes. Lab Invest. 2008;88(6):664–681. Med. 2007;167(8):821–827. 13. Yoshimoto T, et al. Nonredundant roles for CD1d- 20. Nagai S, et al. Role of Peyer’s patches in the induc- 6. Strachan DP. Hay fever, hygiene, and household restricted natural killer T cells and conventional tion of Helicobacter pylori-induced gastritis. Proc size. BMJ. 1989;299(6710):1259–1260. CD4+ T cells in the induction of immunoglobulin Natl Acad Sci U S A. 2007;104(21):8971–8976. 7. Arnold IC, et al. Helicobacter pylori infection pre- E antibodies in response to interleukin 18 treat- 21. Kiriya K, et al. Essential role of Peyer’s patches in vents allergic asthma in mouse models through ment of mice. J Exp Med. 2003;197(8):997–1005. the development of Helicobacter-induced gastritis. the induction of regulatory T cells. J Clin Invest. 14. Koch MA, Tucker-Heard G, Perdue NR, Killebrew Int Immunol. 2007;19(4):435–446.

NPs — heart hormones that regulate brown fat? Andrew J. Whittle and Antonio Vidal-Puig

University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Cambridge, United Kingdom.

Thermogenesis in brown adipose tissue (BAT) is well characterized as being induction is fundamental to not just ther- under the control of the sympathetic nervous system. The energy-burning mogenic activity but also the differentia- capacity of BAT makes it an attractive target for anti-obesity therapies. How- tion of adipocytes toward a more “brown” ever, previous attempts to manipulate BAT’s sympathetic activation have cell fate (5). In this issue of the JCI, Bordic- lacked specificity. In this issue of the JCI, Bordicchia et al. provide new data chia, Collins, and colleagues demonstrate a indicating that cardiac natriuretic peptides (NPs) are also able to activate novel mechanism for activating this genetic thermogenic machinery in adipose tissue. Their findings suggest a novel program in adipose tissue (6). strategy to increase energy dissipation in adipose tissue, independent of Adrenergic signaling is the classical adrenergic receptors. activator of the thermogenic machin- ery. Catecholamines drive increased Regulation of the thermogenic an essential role in this process, and there is intracellular cAMP levels, directly acti- program via p38 MAPK now substantial evidence that BAT depots vating cAMP-dependant protein kinase Obesity is a growing epidemic that places exist and are active in adult humans (1–3). (PKA), which subsequently phosphory- an impossible burden on the health care Following exposure to cold temperatures, lates hormone-sensitive lipase (HSL). systems of both developed and develop- the brain coordinates increased activation But as shown previously by the Collins ing countries across the globe, due to the of BAT via the sympathetic nervous sys- team, PKA can also phosphorylate kinas- negative effects of excess fat accumulation tem (SNS). Brown adipocytes in BAT and es in the p38 MAPK pathway (7, 8). The on multiple organs. Fat is stored mainly populations of “brown-like” adipocytes in UCP1 promoter contains motifs bound in the well-described white adipose tissue WAT respond by increasing transcription by multiple transcription factors, such as (WAT), but there exists a second fat reser- of genes important for mobilizing and oxi- activating transcription factor–2/cAMP voir known as brown adipose tissue (BAT). dizing lipids to generate heat. This genetic response element–binding (ATF2/CREB) Maintenance of core body temperature is response is known as the thermogenic pro- and PPARγ, the transcriptional effects essential to all mammals, and brown fat gram, the key components of which are of which are all enhanced by increased is highly specialized to generate heat. In lipases and the specialized mitochondrial p38 MAPK activation. Pharmacological smaller organisms such as rodents, it is well transporter uncoupling protein 1 (UCP1). inhibition of p38 MAPK is sufficient to established that brown adipocytes perform UCP1 enables the separation of lipid oxi- negate the effects of adrenergic stimula- dation from ATP production, allowing a tion on UCP1 transcription (7). higher metabolic rate and the conversion Previous strategies aimed at increas- Conflict of interest: The authors have declared that no conflict of interest exists. of nutritional energy to heat (4). Under- ing energy dissipation in BAT through Citation for this article: J Clin Invest. 2012; standing the regulation of the thermogenic enhanced activation of the adrenergic 122(3):804–807. doi:10.1172/JCI62595. program is of particular importance, as its system have proved successful at reduc-

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