Background
Environmental agriculture organic dust exposures are rich in Toll-like receptor (TLR) agonists that are associated with regulating airway allergic and non-allergic inflammatory diseases [
1]. Early life exposures to these TLR-enriched environments appears to be protective against the development of IgE-mediated diseases, including eosinophilic asthma [
2‐
4]. However, workplace exposure to these organic dusts is associated with increased occupational asthma, workplace exacerbated asthma, neutrophil-predominant pulmonary inflammation, chronic bronchitis, and chronic obstructive pulmonary disease (COPD) [
2,
5‐
7]. This dichotomy implies that persistent exposures to these environmental inflammatory-inducing agent(s) modulate the lung inflammatory response with potential long-term consequences.
The complexity and biodiversity of agriculture dust exposures is increasingly recognized, and animal and human studies have defined roles for TLR2/TLR4/TLR9-signaling pathways [
8‐
12]. The TLR/IL-1R/IL-18R adaptor protein myeloid differentiation factor 88 (MyD88) that is used by all TLRs except for TLR3 [
13] has been shown to have a fundamental role in the acute inflammatory response to organic dust [
10,
14,
15] as well as other inflammatory exposures [
16,
17]. MyD88 signaling mediates bleomycin-induced IL-17B expression in alveolar macrophages to promote pulmonary fibrosis [
16]. In silica-induced fibrosis, silica dust increases MyD88 expression in macrophages [
17]. In experimental asthma, MyD88 expression in epithelial cells mediates eosinophilia whereas MyD88 expression in conventional dendritic cells controls the neutrophilic response [
18]. In response to agriculture organic dust extract (ODE), MyD88 knockout (KO) mice demonstrate reduced airway hyper-responsiveness and near absence of neutrophil influx and inflammatory cytokine release following acute ODE exposure [
10]. However, these animals demonstrate an elevated, as opposed to reduced, mucus metaplasia response to acute ODE challenges [
14]. Despite the well-characterized role for MyD88 in mediating acute responses to ODE, its impact on airway inflammatory responses to repetitive, prolonged exposures is unknown. This information could be important in future targeted approaches.
Repetitive exposures with ODE from large animal farming facilities induces perivascular lymphocytic aggregates that slowly, but not completely, resolve in size and number by 4 weeks after the inflammatory insult is removed [
19]. It is not known if there is a heightened or dampened airway inflammatory response upon re-exposure. In other settings, endotoxin exposure can induce a refractory state of hypo-responsiveness for several days/weeks [
20]. Alternatively, the lung microenvironment could be poised to be hyper-responsive to rechallenge, which collectively would have implications for workers returning to the job site after prolonged absences. It is unclear whether MyD88 signaling is involved in rechallenge responses.
In this study, we sought to investigate the lung cellular and microenvironment response to repeated ODE exposure for 3 weeks and to determine whether prolonged recovery (i.e. 4 weeks) following repetitive exposures would result in a heightened or refractory response to subsequent ODE rechallenge. Moreover, we hypothesized that the MyD88 signaling pathway would be central to governing lung responses to ODE. Collectively, our findings demonstrate a prominent role for MyD88 in mediating repetitive ODE-induced airway inflammatory responses, including an adaptive role for regulating the expression of epithelial barrier integral proteins. Moreover, our results also suggest that repetitive ODE treatment heightens neutrophil influx but decreases inflammatory cytokine release, while simultaneously upregulating an anti-inflammatory and macrophage response following a later ODE insult. These studies highlight a contextual role of MyD88-dependent signaling in orchestrating the pulmonary inflammatory response to acute and repetitive organic dust exposures.
Discussion
Organic dusts from agricultural environments contain an array of bacterial motifs recognized to play important roles in mediating airway inflammatory disease [
1], and we have previously shown that MyD88 has a fundamental role in the acute inflammatory response to organic dust [
10]. Here we demonstrate that MyD88 remains critical to mediating the airway inflammatory response following daily, repetitive exposures for 3 weeks as demonstrated by neutrophil influx and increases in a wide array of inflammatory mediators that were nearly abrogated in MyD88 KO mice. MyD88 was not responsible for lymphocyte recruitment as lymphocytes in the BALF and B cell infiltrates in the lung parenchyma were not reduced in ODE-challenged MyD88 KO mice. Mucus cell metaplasia was augmented in MyD88 KO animals, which cannot be explained by alterations in inflammatory mediators classically implicated in mucus cell biology, such as IL-6, IL-4, IL-13, and IL-33. Repetitive ODE exposure also induces a prolonged adaptation response. Despite a 4-week rest/recovery phase following repetitive ODE exposures, there was significant reduction in inflammatory mediator levels, but not neutrophil influx, in WT animals following a single rechallenge. These findings were also dependent upon MyD88. Upregulated expression of claudin-4, − 3 and occludin proteins in ODE-challenged mice further support this concept as these proteins are known to be upregulated in response to lung inflammation/injury and render adaptive roles [
37,
38]. Together, these findings illustrate the complex role of the innate immune MyD88 signaling pathway with repetitive organic dust exposures and rechallenge.
Agriculture organic dust exposures are well-established to induce a neutrophilic as opposed to an eosinophilic influx with a corresponding release of pro-inflammatory mediators including TNF-α, IL-6, and neutrophil chemoattractants CXCL1 and CXCL2 [
1]. Expanding on these findings, repetitive ODE exposures were also found to induce IL-33, the neutrophil mediator S100A8, and several factors involved with neutrophil, lymphocyte, monocyte, and mast cell recruitment (i.e. CCL2, CCL3, CCL4, CCL5, and CCL7). The classic Th2 cytokines implicated in allergic responses (i.e. IL-4, IL-5, and IL-13) were not induced. Further, tissue remodeling/pre-fibrosis/repair mediators including periostin, MMP-12, and CHI3-L1 as well as amphiregulin (AREG) were increased after repetitive ODE. Nearly all these responses were MyD88-dependent. Thus, not only is the innate MyD88 signaling pathway central to acute ODE induced inflammatory responses, these new studies suggest that there is not a compensatory response with repeated daily exposures over time regarding these multiple inflammatory attributes. Furthermore, MyD88 was central to mediating the airway inflammatory response following a prolonged rest period and subsequent rechallenge. Therefore, targeting MyD88 could represent a strategy in occupationally exposed workers to reduce airway inflammatory disease burden.
Various agriculture organic dust exposures in humans and mice also induce lymphocytic responses typified by a Th1 and/or Th17 microenvironment in the lung [
22,
39,
40]. In the current study, we focused on B cells due to our recent report demonstrating their importance in experimental ODE-induced lung disease [
22]. Current studies suggest that MyD88 may be important in resolution processes of environmentally triggered lung disease. Whereas there was no difference in B cell infiltrates with repetitive ODE exposure between WT and MyD88 KO animals, B cell infiltrates were increased in mice lacking MyD88 at 4 weeks post-recovery and a one-time rechallenge further augmented B cell infiltrates in the MyD88 KO mice. Others have also found that absence of MyD88 results in unresolved pulmonary infiltrates following long-term radiation-induced lung injury, [
41] silica-induced fibrosis [
42], and bacterial pneumonia [
43]. The explanation for the persistence of lung infiltrates has been attributed to the generation of regulatory cells triggered through TLR/MyD88 signaling [
41‐
43].
It is likely that macrophages and IL-10 contribute to the resolution process and response to ODE rechallenge. It is known that depletion of lung macrophages impairs lung recovery following repetitive ODE exposures [
44]. In addition, mice lacking scavenger receptor A (CD204), a receptor responsible for enhancing IL-10 production under inflammatory states [
45,
46], have impaired recovery [
47]. Here, we report that macrophages and IL-10 levels were significantly increased in WT mice pretreated with ODE and rechallenged with ODE, which we interpret to explain the profoundly affected (i.e. reduced) airway inflammatory cytokine response to rechallenge. This reduced response is in contrast to a hypothesis that the lung could have a heightened inflammatory response upon rechallenge as observed in Byssinosis (i.e. endotoxin-mediated disease), whereby textile workers experience worsening dyspnea on Mondays or after vacations [
48]. Agriculture organic dusts are complex and not just comprised of endotoxin, and it is likely that the wide diversity of bacterial motif exposures explain the prolonged adaptive/modulated response of the lung microenvironment. Interestingly, others have recognized the benefit of this type of immunostimulatory driver, as inhaled treatment of a synergistic combination of TLR agonists protect mice against a wide array of lethal pneumonias and are being explored for potential therapeutic applications [
49‐
51].
Mucus production and barrier control are additional innate responses to ODE that were found to be governed by MyD88. In this regard, our studies found that ODE exposure specifically increased expression of the tight junctions claudin-4, − 3 and occludin, which were lost in MyD88-deficient mice. Increased claudin-4 has been reported in the inflamed and injured lung, and has been associated with adaptive (regulatory) responses, as inhibiting claudin-4 function in mice promoted the severity of lung injury [
37,
38]. We attribute a similar adaptive role for occludin. These findings emphasize the potential communication between MyD88 signaling and barrier function in regulating the lung airway homeostasis and injury responses as these responses were absent in ODE-challenged MyD88 KO mice. Curiously, claudin-3 expression was dramatically increased in MyD88 KO mice. While we currently do not understand the implications of this finding, studies have reported independent roles for claudin-3 and claudin-4 in regulating lung epithelial barrier properties despite their high sequence similarity [
52]. The functional consequence of these changes in tight junction protein expression remains unclear. There was evidence to support that alveolar permeability was impacted with ODE exposure as protein levels were modestly increased in the lavage fluid of ODE-treated WT, which was inhibited in MyD88 KO mice. Collectively, these studies would support new directions to investigate the functional role of tight junctions through potentially utilizing genetically modified mice or through pharmacologic directed approaches.
In addition, AREG can promote the lung repair process, but prolonged overexpression can also lead to fibrotic remodeling [
27,
53,
54]. It was recently reported that ODE increases AREG levels, and importantly, a 3-day treatment period with lung-delivered recombinant AREG hastened lung resolution/recovery following dust exposure [
55]. Here, we confirm increased AREG levels with repetitive ODE exposure and demonstrate that upon re-exposure to ODE, AREG levels are dampened. These responses were inhibited in MyD88 KO animals. It is possible that the absence of an AREG response could be potentially important in explaining the persistence of the lymphoid pathology demonstrated in the MyD88 KO animals. It is also recognized that intranasal inhalation delivery method has consistently resulted in a predominance of central airway involvement as evident by pathology being focused around the bronchopulmonary compartment. However, with repeated exposures, the lung parenchyma or interstitial space is increasingly involved, but to a lesser degree as marked by increase in neutrophilic alveolitis. This neutrophilic alveolitis was dependent upon MyD88.
The exaggerated mucous cell metaplasia in mice lacking MyD88 following repetitive ODE for 3 weeks is consistent with prior work demonstrating enhanced mucus cell metaplasia after 1 week of exposure [
14]. In this previous work, there were no differences in mucous cell numbers, but negative regulation of Muc5ac transcription by MyD88 in secretory cells was demonstrated [
14]. In the current report, we demonstrated that this response remains uncoupled from classic inflammatory mucus cell metaplasia mediators, including neutrophils, IL-33, Th2 cytokines, and various pro-inflammatory cytokines. It is recognized that full-length IL-33 has modest biological activity and that its activity can be rapidly enhanced by removal of N-terminus by serine proteases released from neutrophils and mast cells as well as rapidly inactivated by disulfide bonding of critical cysteine residues. We reported on full-length IL-33, and it may be warranted to investigate processed forms of IL-33 [
56]. Increased numbers of airway mast cells are also associated with enhanced mucin [
57], and although ODE increased mast cells, there was no difference in MyD88 KO and WT animals. Interestingly, IgE levels were inherently increased in MyD88 KO mice and further augmented with ODE. Therefore, it is possible to speculate that an IgE-dependent mechanism, presumably through local mast cell activation, could be playing a potential role in ODE-induced mucus cell metaplasia. However, using tryptase as a marker of mast cell activation, we were unable to detect any differences across treatment groups in serum or BALF tryptase levels (data not shown). It might be warranted in future studies to directly target IgE in regulating mucin in this non-eosinophilic, neutrophilic-associated airway disease, as targeting IgE is a clinically available therapeutic approach.
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