Introduction
Obesity is increasingly recognized as an important risk factor for asthma. Obesity increases both the prevalence and incidence of asthma [
1]. Obesity also increases the severity of asthma and reduces the efficacy of standard asthma control medications [
2,
3]. Furthermore, in obese asthmatics, weight loss improves asthma symptoms and reduces airway hyperresponsiveness (AHR), a canonical feature of asthma [
4,
5]. These effects of obesity are particularly prominent in non-atopic asthmatics [
5].
Ozone (O
3), a common air pollutant generated from automobile exhaust in the presence of sunlight, is a non-atopic asthma trigger. Exposure to O
3 reduces lung function, causes AHR, and induces symptoms of asthma [
6‐
8]. Hospital admissions and emergency room visits for asthma are higher after days when environmental O
3 concentrations are elevated [
6,
7]. Both overweight and obesity exacerbate O
3-induced decrements in lung function [
9,
10] and the effects of obesity on O
3-induced changes in lung function are magnified in subjects with pre-existing AHR [
9]. Pulmonary effects of acute O
3 exposure, including airway obstruction and AHR, are also greater in obese than lean mice [
11,
12]. These observations suggest a link between obesity, responses to air pollution, and asthma. Greater understanding of this link could lead to improved therapeutic options for the obese asthmatic population.
We have reported that IL-33, a member of the IL-1 cytokine family, contributes to the pulmonary responses to acute O
3 exposure in obese
db/db mice [
12]. IL-33 and its receptor, ST2, are both also genetically linked to asthma [
13]. IL-33 is expressed in airway epithelial cells and is released upon cell necrosis [
14], including after O
3-induced injury [
15]. Indeed, bronchoalveolar lavage (BAL) fluid concentrations of IL-33 increase following acute O
3 exposure and these increases are greater in
db/db than lean mice [
12]. Furthermore, in
db/db mice, treatment with a blocking antibody to ST2 attenuates O
3-induced airway obstruction, O
3-induced AHR, and also attenuates O
3-induced neutrophil recruitment to the lungs [
12].
While the study described above indicates an important role for IL-33 in the ability of obesity to augment responses to O
3, the study was limited to obese
db/db mice. These mice are obese because of a genetic deficiency in the receptor for leptin, a satiety hormone. The purpose of the study described herein was to examine the hypothesis that IL-33 also contributes to the effects of O
3 in mice with diet-induced obesity (DIO) caused by high fat diet (HFD) feeding. Mice with DIO have intact leptin receptors and increased circulating leptin [
16], a situation similar to that observed in human obesity. Therefore, we placed weanling wildtype (WT) mice and mice with a genetic deficiency in ST2 (ST2
−/− mice) either on regular chow diets or on diets in which 60% of the calories derived from fat in the form of lard. Diets were maintained for 12–14 weeks. Mice were then exposed to O
3 (2 ppm) or room air for 3 h. Our results indicate reductions in O
3-induced AHR and neutrophil recruitment in ST2 deficient versus WT chow-fed but not HFD-fed mice.
We have recently reported a role for the microbiome in the effects of ST2 deficiency on O
3-induced AHR in lean male mice: compared to WT mice, ST2 deficient mice housed with other ST2-deficient mice (same housed mice) have reduced O
3-induced AHR, but housing ST2 deficient mice with WT mice (cohousing) reverts the magnitude of their O
3-induced to that observed in WT mice [
17]. Because mice ingest some of the fecal microbiota of their cagemates either during grooming or as a result of coprophagy, cohousing transfers gut microbiota from one mouse to its cagemates and rapidly normalizes differences in their gut microbiota [
18,
19]. Indeed, we observed effects of both ST2 deficiency and effects of cohousing on the gut microbial community structures of these lean chow-fed mice [
17]. Consistent with these observations, others have reported differences in the gut microbiomes of IL-33 deficient and WT mice [
18]. The data suggest a role for the microbiome in mediating effects of ST2 deficiency on pulmonary responses to O
3 in lean male mice. HFD feeding causes marked changes in the gut microbiome [
20,
21]. Moreover, the gut microbiome contributes to obesity-related increases in pulmonary responses to O
3 [
22]. To determine whether the microbiome might also contribute to differences in the impact of ST2 deficiency on pulmonary responses to O
3 in chow-fed versus HFD-fed mice, effects of ST2 deficiency on O
3-induced AHR and inflammation were examined in both same housed and cohoused mice fed HFD. Our data indicate that whereas cohousing attenuates effects of ST2 deficiency on O
3- induced AHR and neutrophil recruitment in chow-fed mice, cohousing has no effect on responses to O
3 in HFD-fed mice, likely because HFD-feeding overrides ST2- and cohousing-related changes in the gut microbiome.
Discussion
Our primary goal was to determine whether IL-33 contributes to pulmonary responses to O
3 in mice with obesity induced by HFD feeding. We have reported reductions in O
3-induced AHR and neutrophil recruitment in lean chow-fed ST2
−/− versus WT mice [
17]. However, in obese HFD-fed mice that were derived from the same litters, no such effect of ST2 deficiency was observed (Figs.
6b,
7a). These data indicate diet/obesity-related differences in the role of IL-33 in pulmonary responses to O
3.
Although ST2 deficiency had no effect on O
3-induced AHR (Fig.
6b) or neutrophil recruitment (Fig.
7a) in obese HFD-fed mice, treatment with anti-ST2 antibodies attenuates both the AHR and the neutrophil recruitment associated with O
3-exposure in obese
db/db mice [
12]. IL-33 is known to cause release of type 2 cytokines from ILC2 and other cells within the airways [
12], so the marked reductions in BAL IL-5 in ST2
−/− versus WT HFD-fed mice that were exposed to O
3 (Fig.
8b) indicate that the IL-33 signaling pathway was activated in response to O
3 even in these HFD-fed mice. It is possible that differences in the ability of ST2 deficiency to reduce responses to O
3 in
db/db mice [
12] but not mice with DIO (Figs.
6,
7) is due to differences in the sex of the mice: sex impacts responses to O
3 [
17] and the
db/db mice were female [
12] but we used male mice in this study because female mice are resistant to the induction of obesity by HFD feeding [
24]. However, it is also possible that the modality of obesity contributed. For example, others have reported differences in the impact of obesity on bacterial pneumonia depending on the type of obese mice used [
34].
Db/db are more hyperglycemic than mice with DIO and marked obesity develops much more rapidly in
db/db than in mice with DIO [
35].
Db/db mice are obese because they lack the longform of the receptor for leptin, a satiety hormone expressed in adipose tissue. Importantly, adipose tissue expression of both IL-33 and ST2 are observed in mice with DIO but not in
db/db mice [
36], suggesting a possible role for leptin in the regulation of the IL-33 signaling pathway. The observation that IL-33 increases the expression of the leptin receptor in some cell types [
37] also suggest that there might be differences in the role of IL-33 in
db/db mice versus mice with other forms of obesity. Such a difference could explain the impact of ST2 deficiency on responses to O
3 in
db/db mice but not mice with DIO.
Compared to chow-fed WT mice, HFD-fed WT mice had increases in serum concentrations of many pro-inflammatory cytokines and chemokines but reduced concentrations of IL-5 (Fig.
5). This imbalance between acute phase cytokines and type 2 cytokines has been reported by others studying adipose tissue in obesity [
38]. The fact that this imbalance was also observed in serum suggests that these serum cytokines may derive from adipose tissue. Importantly, in chow-fed but not HFD-fed mice, ST2 deficiency caused significant reductions in serum IL-5. The data suggest that IL-33 maintains a type 2 dominant state in lean mice, consistent with the reports of others [
38] and that these effects of IL-33 are impaired in the obese mice. However, it is unlikely that these obesity-related differences in the impact of IL-33 on systemic type 2 cytokines account for obesity-related differences in the impact of IL-33 on O
3-induced AHR. First, in same housed mice, O
3-induced AHR was greater in WT HFD-fed than WT chow-fed mice (Fig.
6b), even though serum IL-5 was lower in the HFD- than the chow-fed mice (Fig.
5a). Second, in the lung, ST2 deficiency reduced IL-5 in both the chow-fed and HFD-fed mice and IL-5 was actually greater in the HFD fed mice (Fig.
8b).
Instead, our data suggest a role for the microbiome in diet/obesity-related differences in the impact of IL-33 on pulmonary responses to O
3. In chow-fed mice, cohousing the ST2
−/− mice with WT mice abolished differences in their response to O
3 (Figs.
7,
9), suggesting a role for the microbiome in the effects of ST2 deficiency on pulmonary responses to O
3 in these lean mice. The gut microbiome can affect pulmonary responses to O
3 [
33] and both ST2 deficiency and cohousing caused changes in the gut microbiome in chow-fed mice [
17]. HFD-feeding caused marked changes in the gut microbiome and abolished both ST2-related and cohousing-related differences in the gut microbiome that were observed in chow-fed mice (Figs.
10,
11, Fig.
S5, Tables
S1,
S2). Moreover, whereas cohousing augmented responses to O
3 in chow-fed ST2
−/− mice, no effect of cohousing was observed in HFD-fed ST2
−/− mice (Fig.
9). The ability of HFD feeding to override genetic impacts on the gut microbiome has been reported by others [
39].
Although there was no effect of ST2 deficiency on O
3-induced AHR in HFD-fed mice, ST2 deficiency did affect innate airway responsiveness in HFD-fed mice. Compared to air-exposed HFD-fed WT mice, airway responsiveness was significantly
increased in air-exposed HFD-fed ST2
−/− mice, at least at the lowest concentrations of methacholine (Fig.
6a). In contrast, no such effect of ST2 deficiency was observed in the chow-fed mice (Fig.
6a). The data suggest that IL-33 protects against the development of innate AHR in these obese mice. Similarly, IL-33 is proposed to protect against the adipose tissue inflammation of obesity [
40,
41]. Increases in IL-1β and TNFα, have been proposed to contribute to the innate AHR of obesity [
42‐
44]. Hence, we considered the possibility that ST2 deficiency augmented innate AHR in HFD-fed mice (Fig.
6a) by augmenting these cytokines. As discussed above, we did observe increases in circulating IL-1β and TNFα, as well as increases in several other pro-inflammatory cytokines in WT HFD-fed versus chow-fed mice, but ST2 deficiency ablated rather augmented HFD-related increases in serum IL-1β and TNFα (Fig.
5a, b).
There were both strengths and weaknesses to this study. An important strength was the breeding and housing strategy. Both the chow-fed and the HFD-fed mice derived from the same litters. Most of the WT and ST2−/− were also derived from the same litters because we predominantly bred ST2+/− mice. Thus, mice were inoculated with the same microbiome at birth and the environmental conditions extant in the cages of the various groups of mice from birth to weaning were matched. Our data emphasize the need for attention to mouse housing conditions and the microbiome in any study of the impact of genetic deficiencies in the setting of HFD feeding.
Regarding weaknesses, it is important to note that we examined only the gut microbiome. We cannot rule out the possibility that changes in either the lung or oral microbiome also impacted responses to O
3. We examined the gut microbiome because we have previously reported that it is the gut rather than the lung microbiome that accounts for effects of antibiotics and germ free conditions on responses to O
3 [
33]: in lean male mice, oral vancomycin reduces O
3-induced AHR [
33] but does not affect the lung microbiome [
45], even though it does affect the gut microbiome [
33].
Another potential weakness relates to the time point after initiation of HFD feeding at which the mice were examined. Although we observed greater effects of O
3 on airway responsiveness, and on BAL IL-5 and IL-1α in HFD- versus chow-fed mice (Figs.
6b,
8b, c), there was no effect of HFD feeding on BAL neutrophils (Fig.
7a) and most other BAL cytokines and chemokines were actually lower in HFD- versus chow-fed mice exposed to O
3 (Fig.
8). In contrast, we have previously reported that compared to mice fed low fat diets, O
3-induced increases in BAL concentrations of most acute phase cytokines and chemokines as well as total BAL neutrophils are greater in mice fed HFD for 30–35 weeks [
16]. The difference is likely related to the time point after the onset of HFD feeding at which the mice were evaluated (12 weeks in this study), since mice evaluated 20 weeks after the onset of HFD feeding also failed to demonstrate obesity-related increases in O
3-induced neutrophil recruitment and cytokine release [
16]. Likely the duration as well as the extent of obesity is important in determining the response to O
3. Hence it is possible that the impact of ST2 deficiency could be different in mice with DIO of more prolonged duration.
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