Our studies present a system “perturbation” that is caused by an interrelated, multifactoral process. We have presented a model of a clinically feasible, common scenario that occurs for a number of humans: antibiotic treatment followed by a non-life-threatening low-grade increase in fungal microbiota accompanied by an increase in the enteric microbiota. These are interrelated, dependent processes. In our studies, we have viewed this as one “change,” a perturbation in the system. Our data demonstrate that the perturbation itself does not elicit an allergic response. Airway antigen exposure alone also does not elicit an allergic response. However, if the system is perturbed and then antigen exposure occurs, a vigorous airway allergic response develops. This is a cause-and-effect equation with two variables, (i) antibiotic-induced change with fungal microbiota increase and (ii) airway exposure to antigen. The conclusion of these studies is that inhalation of antigen induces an allergic response only when the physiologic perturbation has occurred. This study may raise more questions than it answers. However, it is important to realize that this study definitively points out one very important point: we have demonstrated in an animal model a type of system perturbation that allows airway allergic responses to be generated where they would not normally occur. This perturbation is a clinically relevant model. We do not know which aspect of the perturbation is most important; however, the perturbation is the cause of the immune deviation.
These studies address a major concept of the hygiene hypothesis and experimentally test in mice whether the correlation between antibiotics or microbiota changes and allergies in humans could be a cause-and-effect relationship. Repeated Aspergillus
spore exposure of untreated mice did not produce any indices of an allergic response. In sharp contrast, repeated Aspergillus
spore exposure of Anb/Ca (microbiota-disrupted) mice produced a strong CD4 T-cell-mediated allergic response as indicated by increased levels of eosinophils, mast cells, IL-5, IL-13, IFN-γ, IgE, and mucus-secreting cells in the airways. The presence of IFN-γ along with Th2 cytokines in the lungs is consistent with an allergic airway response (19
Is this response restricted to mold spores and C57BL/6 mice? We have performed additional studies similar to those described in this paper using BALB/c mice for mold spore challenge. An allergic response also developed in the lungs of Anb/Ca but not non-antibiotic-treated BALB/c mice (our unpublished data). We also used a similar multiple intranasal challenge protocol with ovalbumin (OVA) in Anb/Ca and non-antibiotic-treated BALB/c mice (which did not include any systemic priming to OVA). The allergic response in the airways of untreated mice exposed intranasally to OVA was low, while Anb/Ca mice produced a significant allergic response in the airways (our unpublished data). Thus, our additional studies indicate that the pulmonary allergic response in mice with altered microbiota can occur in other inbred genetic backgrounds of mice and in response to nonfungal antigens.
In this study, we have demonstrated that the physiologic perturbation of cefoperazone and increased yeast microbiota can allow or promote the development of an allergic airway response upon subsequent exposure to Aspergillus
spores. This is a complex physiologic perturbation but one that is clinically common. While our studies demonstrate a cause-and-effect relationship between this perturbation and the subsequent response, at this point we cannot identify which factors are most important. Cephalosporins and other antibiotics have been shown to have direct effects on leukocytes (for examples, see references 34
, and 63
). This is not likely the major mechanism of immune deviation in our mice because exposure of mice treated with antibiotics but not inoculated with C. albicans
conidia did not induce the fulminant allergic response seen in Anb/Ca mice (data not shown), indicating that both antibiotic-induced and fungal microbiota changes appear to be necessary for promoting an allergic response in the airways. At this point, we cannot rule out the possibility that the growth of the fungal microbiota alone is all that is necessary. However, it must be kept in mind that growth of the fungal microbiota is largely influenced by the bacterial microbiota, which in turn will be significantly influenced by antibiotics. Our data demonstrate that this complex physiologic perturbation can alter immune regulation in the lungs, leading to allergic airway disease upon mold spore exposure.
The studies presented here indicate that alterations in the microbiota can significantly modify a T-cell-mediated immune response in the lungs. It has been proposed that the lung microenvironment is generally predisposed to Th2 responses (9
). However, repeated intranasal antigen exposure in the lungs leads to decreasing reactivity, a form of tolerance (18
). Emerging models of T-regulatory responses suggest that both Th1 and Th2 responses can be down-modulated by regulatory T cells (Treg cells) and/or Th3 responses (38
). Oral tolerance is an example of a Th3 response that has been studied in models of experimental allergic encephalitis (40
). Oral tolerance can suppress the Th1 response in the central nervous system during experimental allergic encephalitis (40
) and has also been reported to modulate an airway Th2 response in a model of OVA hypersensitivity (8
). The GI microbiota likely plays an important role in tolerance since oral tolerance cannot be generated in germfree mice (35
). The mechanism underlying this phenomenon remains to be determined. A number of studies have demonstrated that fluids, particles, and microbes introduced into the nasal cavity are largely found in the GI tract shortly thereafter (13
). Even volumes as small as 2.5 μl introduced intranasally into mice will largely end up being swallowed due to the mucociliary anatomy of the nasopharyngeal cavity (46
). Thus, the GI tract will be exposed to any antigens to which the respiratory tract is also exposed. Since ingestion of antigens can induce tolerance to that antigen (“oral tolerance” [71
]), the GI tract may act as a “sensor” for the development of tolerance to inhaled antigens. Oral tolerance is believed to be mediated by regulatory T-cell (Treg/Th3) responses and these Treg/Th3 cells may down-modulate Th2 responses in the airways to the same antigens (49
). Furthermore, oral tolerance is defective in germfree mice (58
), indicating a role for the microbiota in the development of the response. Thus, our data are consistent with the hypothesis that alterations of the GI bacterial and fungal microbiota may prevent the development of Treg/Th3 responses that control overexuberant mucosal Th2 responses to inhaled antigens such as mold spores.
This is also the first animal model of fungal airway allergy in which immunocompetent mice develop an allergic response to mold spores without prior systemic immunization with the fungus or fungal antigens. It has been previously demonstrated that some pure Aspergillus
proteins alone can induce an airway allergic response (17
); however, intranasal challenge with Aspergillus
spores, culture filtrate, or mycelial extracts cannot induce an allergic response without prior systemic sensitization (16
). Systemic immunization and airway challenge models have been extremely useful for the study of the manifestation and pathological process of the allergic response. However, these models cannot address potential afferent mechanisms of allergic responses in humans because humans are exposed to mold spores via inhalation, not systemic immunization. Our studies present a new model to study the afferent phase of allergic airway responses.
These studies also indicate that increased numbers of yeast cells in the microbiota can be a contributing factor in upregulating Th2 responses to antigen exposure in the lungs. In these studies, C. albicans
was never isolated from the lungs, even immediately following gavage, and >99.9% of the yeast cells were found in the GI tract when analyzed 1 day posttreatment. If oral C. albicans
was not included in the system perturbation (i.e., mice treated with antibiotic only), then a fulminant allergic response did not occur (data not shown), indicating a requirement for an alteration in the fungal microbiota in this system. C. albicans
is a normal constituent of the human microbiota (14
), and one potential mechanism for the immunomodulatory activity of C. albicans
is via the production of prostaglandin-like oxylipins (41
). C. albicans
(and many other fungi) secrete PGE2
- and PGD2
-like molecules de novo or via conversion of exogenous arachidonic acid (42
). A PGE2
-cross-reactive compound can be purified from C. albicans
and other fungi that are biologically active on mammalian cells with activity comparable to that of purified PGE2
). Prostaglandins such as PGE2
are potent immunomodulatory molecules (3
), and microbe-derived PGD2
can alter dendritic cell migration and biology (2
). Fungal cell wall glucans are also powerful inflammatory stimulants in tissues (15
) and may also play a role in the immunomodulatory activity of yeast in the GI tract. Thus, increased levels of fungal microbiota, such as often occurs during antibiotic therapy, may diminish the ability to generate Treg/Th3 responses to swallowed antigens, possibly by interfering with tolerance-inducing antigen presentation via fungal oxylipins and glucans.
The studies presented here are the first to examine whether antibiotics and fungal microbiota changes can promote the development of an airway Th2 response. The potential link between antibiotic use and allergies is one of the major observations that led to the hygiene hypothesis of allergic diseases (56
). We propose that the link between antibiotic use and dysregulated pulmonary immunity is through antibiotic-induced long-term alterations in the bacterial and fungal GI microbiota, which we predict disrupts the regulatory T-cell response. We have also demonstrated that enteric bacteria numbers can remain elevated for 2 weeks after the end of antibiotic therapy, and we have additional data that these numbers remain stably elevated for at least 3 weeks (data not shown). Numerous studies have shown a correlation between altered fecal bacterial microbiota counts (including high levels of enteric bacteria) and the development of allergies (4
). A number of other studies have suggested a correlation between antibiotic use and allergies in humans (1
). Furthermore, other factors that may affect the incidence of allergies, such as diet and probiotic therapy (24
), also affect the GI microbiota composition. So, while it remains to be tested whether human allergies result from altered microbiota, we have demonstrated in an animal model that antibiotic use leading to altered bacterial and fungal microbiota can allow the development of an airway allergic response to subsequent allergen exposure via the nose. These studies also provide a conceptual mechanistic framework for investigating whether probiotic and prebiotic regimens may be useful in preventing or reducing allergies in infants and even adults.