AllergoOncology: Microbiota in allergy and cancer—A European Academy for Allergy and Clinical Immunology position paper

Abstract The microbiota can play important roles in the development of human immunity and the establishment of immune homeostasis. Lifestyle factors including diet, hygiene, and exposure to viruses or bacteria, and medical interventions with antibiotics or anti‐ulcer medications, regulate phylogenetic variability and the quality of cross talk between innate and adaptive immune cells via mucosal and skin epithelia. More recently, microbiota and their composition have been linked to protective effects for health. Imbalance, however, has been linked to immune‐related diseases such as allergy and cancer, characterized by impaired, or exaggerated immune tolerance, respectively. In this AllergoOncology position paper, we focus on the increasing evidence defining the microbiota composition as a key determinant of immunity and immune tolerance, linked to the risk for the development of allergic and malignant diseases. We discuss novel insights into the role of microbiota in disease and patient responses to treatments in cancer and in allergy. These may highlight opportunities to improve patient outcomes with medical interventions supported through a restored microbiome.

vice versa. Thereis growing evidence that microbiota play a paramount role in the control of immune-mediated diseases such as allergy and cancer, the two complementary diseases in the frame of AllergoOncology. 2

| Hygiene hypothesis in allergy: from epidemiology to mechanisms
The allergy epidemics correlate with improved hygiene practices associated with urban lifestyles (Table S1). Milestone studies revealed that this epidemic can be counter-regulated in part by exposure to a traditional farming environment. The cornerstones of the allergy protective farm effect are (a) farm activities during pregnancy, (b) early life farm exposure, and (c) raw milk consumption, but farms are also protective against viral infections. Protection depends on the farm dust levels in an area and depends on the type of farming. To give an example, children from Amish traditional preindustrial farming communities were less prone to allergy development than Hutterites who use modern industrial farming methods. Amish farm dust initiated innate immune pathways associated with protection from allergy development in a mouse model. Additionally, rich microbial exposure by cohabitation with wild mice improves the immune response in laboratory mice. In line with this, dog keeping is reported to protect children from asthma. Also helminth infections can support the establishment of protective microbiota (for summary and references, see Table S1).
Molecular mechanisms are increasingly understood. Viral antigens due to molecular mimicry counteract specific allergen sensitization and induce specific T effector memory responses. Exposure to N-glycolylneuraminic acid (Neu5Gc), a sialic acid compound in farm dust, induces regulatory pathways. Endotoxin exposure of bronchial epithelial cells counteracts allergen-induced Th2 responses. Prenatal farm exposure supports Treg and Th17 cell differentiation. The

G R A P H I C A L A B S T R A C T
Increasing scientific evidence indicates the influence of microbiota on the immune response contributing to the prevention or progression of immune-related diseases such as allergy and cancer. A variety of factors have been identified influencing microbiota composition. This opens new avenues to beneficially modulate patients' responses to cancer or allergy treatments.

Term Definition
Microbiota Microorganisms (bacteria, viruses, fungi, protozoa, and archaea) populating the inner and outer surfaces of the human body Microbiome Collective genome of all microorganisms Mutualism A relationship between two organisms of different species resulting in benefits for both organisms from the interaction Symbionts Two different organisms of the same or of different species with a close and persistent biological interaction UNTERSMAYR ET AL.
| 1039 protective immunity associated with the consumption of raw milk induces FOXP3 demethylation and T regulatory cells (Tregs). Exposure of bronchial epithelia to farm dust, for instance containing CpG-DNA, or of gut epithelia to farm milk enhances epithelial barrier integrity, resulting in protective innate immunity to allergens and viruses (Table S1).
In accordance, immature gut microbial composition at age 1 year was positively associated with asthma risk at 5 years in children with asthmatic mothers. 3

Highlights
• Microbiota composition has been linked to health protective effects representing a key determinant of immunity and immune tolerance.
• Microbiota imbalance is increasingly recognized to be associated with an enhanced risk for immune-related diseases such as allergy and cancer.
• Novel insights into the role of microbiota in disease and patient responses to cancer or allergy treatments highlight opportunities to improve patient outcomes through a restored microbiome.
prevalence of allergies and asthma. 7 Also, the pharmacologic impairment of gastric digestion is associated with gut dysbiosis and has been correlated with allergy in mice and humans. 8,9 However, study results of probiotic supplementation in childhood asthma or wheeze are inconclusive. 10 1.3 | Hygiene hypothesis in oncology: from epidemiology to mechanisms Increased incidence of certain cancers in Westernized countries 11 may be associated with under-exposure to certain microbial species, modern lifestyle, and consumption of sterilized food. 12 Higher socioeconomic status is associated with increased incidence of Hodgkin lymphoma, 13 while daycare attendance and higher number of childhood infections are linked with lower risk of acute lymphoblastic leukemia 14 and adult chronic lymphoid leukemia. 15 Increased cancer risk is observed in patients with autoimmune diseases and chronic allergic disorders. 16 Mice with rapid melanoma growth and poor immunosurveillance, exhibited relatively low levels of Bifidobacterium species which, when restored by oral feeding and co-housing, resulted in enhanced tumor antigen presentation and reduced malignant growth. 17 Epidemiological studies have also examined specific bacteria, viruses, periodontal disease, and circulating antibodies to selected pathogens in relation to cancer risk. 18 Carriage of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in prediagnostic oral wash samples was positively associated with pancreatic cancer risk, and Fusobacteria and its genus Leptotrichia were inversely associated. 19 Tannerella forsythia was positively associated with esophageal adenocarcinoma and Porphyromonas gingivalis with squamous cell carcinoma. 20 Greater abundance of genera Corynebacterium and Kingella was inversely associated with head and neck squamous cell cancer. 21 An omnivore, but not a vegetarian, diet promotes butyrate-  24 It has also been postulated that the use of antibiotics may support cancer progression through subversion of immunosurveillance. In a transgenic mouse model of spontaneous mammary carcinoma development, treatment with metronidazole and ciprofloxacin resulted in accelerated development of these tumors. 25 Repeated antibiotic use may also increase risk of certain human malignancies. 26 2 | METABOLITES CRITICALLY SHAPE THE MICROBIOME

| Dietary micronutrients
Micronutrients being essential food for microbes substantially influence microbiota composition. Most bacteria require iron and have evolved multiple strategies for sequestration including the production of hemophores and low molecular iron chelators named siderophores.
Iron supplementation promotes the establishment of bacteria relying on this metal (Table 3). In atopy, a poor iron status is associated with allergy, whereas an improved status abrogates or seems to prevent the onset of allergy. In cancer, iron contributes to tumor growth with high serum iron increasing the risk of several cancers (Table 4). 27 Vitamins A and D (Tables 3 and 4) are important for mucosal immunity. 28 Retinol supplementation does not change the risk of allergy 29 or cancer, 30 though increased serum retinol levels are associated with prostate cancer. 31 Vitamin D supplementation slightly decreases respiratory infections and the incidence of asthma in offspring, 32 prevents hematologic malignancies in elderly women and advanced colorectal adenomas, 33 but seem not to impact allergy risk. 34 Vitamin D supplementation decreases the abundance of Bifidobacterium and Coprococcus. 35 Serum folate levels are associated with serum IgE, 36 high plasma folate is associated with decreased risk of wheezing, 37 and folic acid supplementation has been linked with an increased risk for lung cancer. 38 Folate can be produced by most Bacteroidetes, Fusobacteria, and Proteobacteria, but rarely from Actinobacteria and Firmicutes. 39 In summary, dietary micronutrients shape the microbiome which contributes products essential for an effective immune defense.

| Nondigestible Oligosaccharides
Nondigestible oligosaccharides (NDO) are potential substrates for bacterial metabolism in the colon, and a declining microbial diversity in the gut is implicated in the rising incidence of allergic disorders in early life. 40 Manipulation of the gut microbiota with NDO from natural sources or supplemented galacto-oligosaccharides (GOS), lactose, and fructooligosaccharides (FOS) holds great promise for the treatment of inflammatory and allergic diseases (Table 4). NDO feeding reduced the development of atopic dermatitis (AD) in infants at risk of allergy which was associated with increased Bifidobacterium and Lactobacilli, 41,42 re-balancing the immune response from a predominant Th2-type allergic profile at birth to a more Th1-type and Treg profile. NDO protect against allergic manifestations in experimental models of food allergy and allergic asthma 43,44 and reshape the gut microbiota with increased levels of short-chain fatty acids (SCFAs). 45 Less is known concerning the anti-cancer effects of NDO. 46 Experimental models show encouraging effects of GOS, FOS, agaro-oligosaccharides, 47 and butyrate 46 in influencing microbiota diversity (eg, increased Fusobacterium) and the prevention and progression of colorectal cancer. 48 NDO showed potential in the protection against the development of cancer in healthy subjects. 49 Since Western UNTERSMAYR ET AL.
| 1041 diets are typically low on NDO, increasing the intake of NDO may benefit human health via an improved microbiome and/or via direct effects on the immune system.

| Microbiota metabolites
Dairy products such as yogurt and butter contain short-chain fatty acids (SCFAs), while SCFAs are also produced by intestinal bacteria following their fermentation of fibers present in vegetables and fruits. 50 The major SCFAs are acetate, propionate, and butyrate.

| Cells of the epithelial barrier
Epithelial cells have a very close interaction with compartment-specific microbiota ( Figure 1A). These cells are able to sense conserved

| Dendritic cells
As professional antigen presenting cells (APC), DC are in contact with both invading pathogens and commensal microbiota and maintain the balance between inflammatory and tolerogenic immune responses.
Human monocyte-derived dendritic cells (moDCs), when matured in the presence of the SCFA butyrate ( Figure 1D), increase IL-10, but decrease IL-6 and IL-12 expression. 62 Similarly, treatment of mice with the SCFA propionate generates new bone marrow DC precursors with high phagocytic capacity but an impaired ability to promote T helper type 2 (Th2) responses in the lung. This effect is dependent on G protein-coupled receptor 41 (GPR41, also called free fatty acid receptor 3 or FFAR3), but not GPR43 (also called free fatty acid receptor 2 or FFAR2). 55 DCs can also exert roles in adoptive T-cell therapy (ACT) in cancer ( Figure 1E), and the composition of the gut microbiome or treatment with antibiotics could lead to an increase in CD8α + DCs, and consequent IL-12 release that sustains the anti-tumor ACT. 63

| Macrophages
The microbiota and its metabolites such as SCFAs strongly influence myelopoiesis and the tissue-resident macrophages. In the intestine, microbial SCFA butyrate activates its endogenous receptor GPR109a

| Mast cells
Mast cells (MC) are crucial effector cells in allergy and other inflammatory diseases. 66 They also play important roles in regulating the microenvironment of various tumors. 66 There is increasing evidence that MC function can be modulated by commensal, symbiotic, and pathogenic microorganisms. 67 Microorganisms may influence MC activation via direct interaction or via secreted metabolites. For example, co-culture with Lactobacillus rhamnosus ( Figure 1C)

| Eosinophils
Eosinophils are granulocytic leukocytes that exert important functions in protective immune responses against helminths and other pathogens. 73 They also serve as key effector cells in allergies and other inflammatory diseases. Recent studies demonstrated that eosinophils crucially participate in maintaining the specific tissueresident microbiome. 74 Conversely, functions of eosinophils are regulated by pathogenic as well as probiotic microorganisms; for example, human eosinophils were found to ingest Clostridium difficile ( Figure 1B), which subsequently stimulated the release of eosinophil-derived neurotoxin. 75 In contrast, ingestion of the probiotic strain Bifidobacterium bifidum resulted only in a minor neurotoxin release. 75 Comparably, mouse eosinophils were found to take up the probiotic Lactobacillus reuteri. 76

| Tregs
Tregs are potent immune-regulating cells that play a central role in controlling immune responses ( Figure 1A). Tregs can support the reduction of allergic diseases, and, on the other hand, the progression of cancer. 79

| B cells and Bregs
B cells are known to promote allergy through antigen presentation and class-switching to IgE, whereas under certain conditions, tumorinfiltrating B cells can be associated with improved patient survival in cancer. 79,84 In contrast, Breg-associated immune tolerance can lead to control of allergy or tumor progression in cancer. 79 The microbiome may have a role in mediating these multifaceted and opposing B-cell effects in allergy and cancer. Microbiota regulate activation and differentiation of B cells. 85 Gut microbiota antigens directly trigger B-cell activation by binding BCRs, and microbial products activate TLR-expressing B cells, increasing B-cell survival, antigen presentation, and antibody production. 86 Microbiota also mediate the release of epithelial cell and eosinophil-derived cytokines/chemokines that activate and recruit B cells, and may promote T follicular helper cell-mediated differentiation of plasma cells. 85 Conversely, the gut microbiome reportedly induces DCs to produce cytokines, such as IL-1β and IL-6, which promote B-cell differentiation to Bregs. 87 These findings point to a functional cross talk between humoral immunity and the microbiome.

| Redox regulation in allergy and cancer
Recent reports demonstrated that certain microbes can stimulate intracellular signaling, involving PTEN, MAPK, and PTP via the generation of reactive oxygen species (ROS) in epithelia, when harboring regulatory redox-sensitive thiolates. 88 Commensal bacteria alter the epithelial redox environment by production of oxygen radicals, cause epithelial cell DNA damage, and may harbor carcinogenic properties, for example, in colon carcinoma development. 89 Furthermore, allergic asthma is associated with an increase in endogenous ROS formation, leading to oxidative stress-induced damage to the respiratory system and weakened antioxidant defenses. These may result in abnormal physiologic function of DNA, proteins, and lipids that can augment bronchial hyperresponsiveness and inflammation. 90 Overall, the microbiome is implicated in redox regulating pathways, which are relevant for both chronic inflammation and cancer.

| Lipocalins
Human lipocalins, such as tear lipocalin 1 and lipocalin 2 present in the nasal mucosa, can interfere with bacterial iron sequestration, 27 and may directly modulate site-specific microbial composition. The immunomodulatory and apoptosis-regulating properties of LCN2 have been linked to its ability to shuttle iron. 91 In line with the hygiene hypothesis, the limited "microbial exposure" of allergic individuals may also result in deficiencies of the immune regulatory machinery and this can lead to hyper-reactive responses. 92 Interestingly, nearly all major mammalian allergens belong to the lipocalin family, 93  In a recent mouse study, IL-33-deficient mice, with significantly lower levels of intestinal IgA and colon-residing IgA+ B cells, had increased DNA damage-induced tumors; observations that were ameliorated upon microbiome restoration by co-housing deficient animals with wild-type mice. 96 In humans, altered gut microbiota diversity and low total IgA levels may be associated with the development of allergies and asthma, 97 and IgA-deficient individuals have a moderately increased risk of cancer, with higher risks in gastrointestinal cancer. 98 This disease risk may be a result of impaired mucosal barrier function. However, the associations between microbiotadriven antibody production with allergies and cancer are not yet sufficiently understood.

| Oncoimmunology and allergy
Specific microbes and the microbiota in general can be considered as important drivers of immunomodulation and can contribute to establishing immune tolerance, with differential impact on the many diverse immune-mediated diseases. Due to the close interaction and the bidirectional influence of intestinal microbiota on the mucosal immune system, the gut can be considered as an essential site of immune cross talk in the human host. When it comes to allergy or cancer, however, regulation of tolerance has an opposing impact on disease development and treatment. 79 In cancer, the impact of microbiota was for a long time primarily considered in the context of dysbiosis, increased epithelial translocation and carcinogenic effector mechanisms. 99 However, in recent years, emerging knowledge points to the role of microbiota, especially in the gut, in anti-cancer immune mechanisms. 100 This is due to studies describing a reduced anti-cancer efficacy of chemotherapeutics such as cyclophosphamide and platinum salts in germ-free and antibiotic-treated mice. Moreover, the redox equilibrium of myeloid cells contained in the tumor microenvironment is influenced by intestinal microbiota and due to the importance of the gut in immune fitness, intestinal microbes are essential for the availability of immunomodulators.
In cancer immunotherapy, major advances have been made in the past years to understand the contribution of microbiota composition to successful treatment. Studies are now starting to evaluate the role of the gut microbiome in anti-PD-1 immunotherapy, 101,102 including the first clinical study focusing on PD-1 checkpoint inhibitor response in metastatic melanoma. In a recent ground-breaking study, the authors were able to demonstrate a correlation between response to PD-1 checkpoint inhibitor treatment and diversity of microbial strains in the intestine. Moreover, high abundance of Faecalibacterium and low abundance of Bacteroidales were associated with good prognosis and longer progression-free survival after checkpoint inhibitor treatment. 101 Moreover, toxicity-related side effects and a dysfunctional intestinal epithelial barrier seem to be attenuated by a beneficial microbiota composition, potentially acting not only via modulation of the host immune response but also via modulation of cancer metabolism. 103,104 Even though immunotherapy has been the treatment of choice for allergic diseases for more than a century, it seems that the cancer field is more advanced than allergy research with regard to evaluating the

| Outlook: microbiota engineering
The beneficial effect of a balanced microbiota on human health can be restored or potentiated by external/medical intervention. There are three main routes to microbiota engineering ( Figure 2 115 Cancer and allergy are widely the focus of many of these efforts (Figure 2), with a number of studies showing how microbiota composition and its engineering may ameliorate or support the appropriate immune response to contribute to clinical benefits for each condition. 116

| CONCLUSION
The biodiversity hypothesis is a cornerstone in the understanding of the allergy epidemics. Subsequently, the era of microbiota research has opened up novel perspectives on allergy pathogenesis, 117   Oligosaccharides" and to Table 4. Penichet's technology to this firm. All other authors declare no conflicts of interest in relation to this publication.