Background
Airway epithelial cells (AECs) preserve a near-sterile microenvironment via mucociliary clearance mechanisms and more importantly, by an adaptive mucosal immune response [
1‐
4]. The conducting airway epithelium consists of basal, ciliated, club (or Clara) and mucous (or goblet) cells [
4‐
6]. In smaller airways, primarily club cells differentiate into mucous cells as an innate immune response to airway injury also referred to as mucous cell metaplasia (MCM) [
4,
5,
7‐
9]; however other epithelial cells can also differentiate into mucous cells [
6,
10] . While mucous cell differentiation is vital to pulmonary health, dysregulation can lead to aberrant mucin secretion and obstruction of airways as observed in chronic respiratory diseases [
4,
11]. The most abundant mucins secreted by airway epithelial cells (AECs) are MUC5AC and MUC5B, which in combination with other proteins, lipids and glycosylated factors form a mucous layer [
12‐
14]. The airway surface mucous layer not only serves as a barrier but also traps inhaled particles for mucociliary clearance [
4,
11,
14]. Several inflammatory factors and toxicants activate the EGFR/ERK pathway and induce MUC5AC and MUC5B expression to alter innate immune responses [
4,
11,
13,
15,
16].
The ambient air is contaminated with various organic or inorganic compounds including lipopolysaccharide (LPS), a component of gram-negative bacteria. Our previous studies showed that intratracheal instillation of 1000 μg LPS in rats causes extensive neutrophilic inflammation and epithelial cell hyperplasia accompanied by MCM [
7,
17]. The resolution process for this extensive inflammation required a period of 40 d for the inflammation to subside and the lung epithelia to resemble the non-exposed condition [
17].
The airway epithelium has evolved several regulatory mechanisms to control hyperreactive inflammatory responses to minimize deleterious effects. Lymphocytes, monocytes, macrophages, and dendritic cells possess ‘dynamic cellular programing’ or ‘memory’ to adjust the immune response to secondary challenges [
18‐
22]. Various animal studies have highlighted the important role of adaptive T cells, both CD4 and CD8 T lymphocytes, and macrophages in regulating immune responses to previously encountered insults [
23‐
26]. However, only few reports have reported on epithelial cell ‘innate-programming’ or ‘memory’ responses [
27,
28]. Therefore, the present study was designed to investigate the response of airway epithelium to a secondary LPS challenge. Possible interaction of T cells and AECs in regulating memory responses was investigated by using T cell-sufficient and -deficient mice.
Discussion
The present study shows that airway epithelial cells that have been previously primed with LPS respond to subsequent LPS challenge with a rapid increase in MCM and epithelial cell hyperplasia (ECH). This memory response may be due to a sustained spatiotemporal localization of TLR4 and EGFR that initiate a rapid mucus expression via ERK1/2 activation.
Innate memory-based responses have been described in several immune cell populations including monocytes, macrophages and NK cells [
39‐
41]. Recent seminal studies have highlighted the role of memory-based or ‘trained’ immune responses as summarized recently by Netea et al. [
42]. These trained responses are adaptive and beneficial because mice primed with microbial ligands or PRRs are protected against subsequent lethal infection [
39,
43,
44]. These trained responses have been mechanistically attributed to the transcriptional and epigenetic reprogramming that involved histone modifications but DNA methylation, microRNA and/or long noncoding RNA could also be implicated [
39,
45]. Future studies will investigate the molecular basis of reprogramming in AECs that resulted in the observed augmented memory-based or trained responses.
Airway epithelial responses including MCM, ECH, and nuclear localization of pERK were elevated in L/0 rats compared with L/0 wild-type mice. This disparity could be due to differences in epithelial cell types present in the conducting airway epithelium. At airway generation 5, the site for all quantifications in the current study, serous cells are present in rats but absent in mice [
7]. Further, mice in general respond with minimal mucous cell metaplasia in response to many insults possibly due to a more resistant genotype that results in less sustained activation of the signaling pathways and transcription factors compared to rats [
31]. This was also evident by the dose of LPS used for challenge because 1 μg LPS caused MCM in LPS-pretreated rats (L/1) but had no discernible airway epithelial changes in mice irrespective of the LPS priming (personal observation). As was previously reported [
8,
9], Muc5ac-positive mucous cells differentiate from the Scgb1a1-positive club cells. Consistent with these observations, a marked suppression of Scgb1a1 expression was observed by us following the LPS challenge in all rodents.
T cells affect the generation of mucous cell metaplasia in response to various challenges [
23,
46]. Based on the threshold and type of insult, the immunologic tolerance dominates the primary response and is generally driven in part by T cells, specifically by resident memory (T
m) and regulatory (T
reg) T cells [
23,
47,
48]. Therefore, to isolate the epithelial memory responses from that of T cells we analyzed the secondary response in the absence of T cells. Moreover the memory-based immune responses have also been previously demonstrated as lymphocyte-independent mechanism using athymic and
Rag1-deficient mice [
39,
49]. We found that athymic Foxn1
nu mice responded with enhanced MCM in response to LPS or saline challenge compared to Foxn1
WT mice. Higher apical expression of TLR4 and EGFR in Foxn1
nu mice that rapidly activates ERK1/2 phosphorylation and nuclear localization may be involved in this process. Validation of these findings by in situ hybridization or q-PCR from microdissected airway cells would require more comprehensive kinetic (several time-point) analyses to adequately match the observed changes at protein levels.
The L/0 Foxn1
nu mice showed higher number of mucous cells even when instilled with saline as a secondary challenge (Fig.
4b) with a corresponding increased TLR4 expression compared to L/0 Foxn1
WT mice (Fig.
5a). We believe that these mice are more responsive to saline challenge and, therefore, showed increased mucous cell numbers and TLR4 expression. Form our previous studies, we have not observed differences in mucous cells at baseline, and we assume that 40 d after LPS challenge all mucous cells were resolved also in Foxn1
nu mice as in wild-type mice. Other studies have also documented that following a primary LPS exposure there were no discernible differences reported in athymic mice lacking T lymphocytes compared to euthymic wild-type mice [
50]. Our previous studies were focused on the kinetics of LPS response in rats, and the T cells along with other immune cells returned back to normal levels by day 40 [
17].
T
reg or T
m cells may suppress TLR4 and EGFR expression in AECs to establish a tolerogenic homeostasis in airway mucosa [
23,
47,
48]. Future studies will investigate which T cell sub-population contributes to the suppression of apical TLR4 and EGFR expression on AECs. In vitro studies using air-liquid interface cultures of AECs will also help determine if this memory-response in AECs requires a signal from T cells to suppress LPS-induced TLR4 expression.
The number of AECs was increased in LPS-pretreated rats one day after saline challenge. Because all inflammation was resolved by day 40 with epithelial cell numbers returning the levels found in naïve rats by day 30 [
17], this rapid increase in ECH by saline instillation suggests that LPS-primed airway epithelial cells can proliferate rapidly. Whether the observed ‘innate memory’ that was established in response to LPS instillation may also respond to a secondary challenge by insults other than LPS will be investigated in the future. TLR4 is one of the pattern-recognition receptors (PRRs) that regulate maladaptive immune response to LPS [
51]. LPS responsiveness is fine-tuned by the levels of TLR4 present on the cell surface which in turn is determined by the amount of TLR4 trafficking between the Golgi and the plasma membrane, and the TLR4 internalized into endosomes [
52]. Dysregulation of TLR4 expression or localization at the epithelial interface results in impaired host response to LPS as observed in cystic fibrosis [
51]. TLR4 integrates its signaling with EGFR and helps regulate the proliferative responses [
53‐
55]. Interestingly, in athymic Foxn1
nu mice, the levels of TLR4 and EGFR were augmented with some AECs showing nuclear pERK in LPS-pretreated mice even in the absence of LPS challenge. Therefore, EGFR and ERK1/2, the known inducers of MCM [
35‐
38] could be responsible for the ‘memory’ in airway epithelial cells.
Mucous cell metaplasia is a reversible adaptive response that increases mucous secretions to help clear the airways through mucociliary clearance. However, whether these metaplastic cells retain a memory to a prior exposure has not been previously investigated. We and others have reported that the hyperplastic epithelial cells undergo apoptosis during the resolution process [
17,
56‐
58]. Whether some of the hyperplastic cells remain following the resolution process and serve as ‘memory’ cells remains to be investigated. In addition, AECs secrete mucous and inflammatory factors, and interact with other epithelial and mucosal immune cells in an autocrine and a paracrine manner. The mucus layer helps trap inhaled toxicants and dilutes local inflammatory factors or chemoattractants to suppress the effect on other epithelial and immune cells. Thus, the intricate and tightly regulated crosstalk between immune and epithelial cells may be required to adjust a tolerant versus a hyperreactive epithelium. The reported suppressive role of T cells on the hyperactive mucous response could also help understand the pulmonary responses to inhaled toxicants that result in chronic mucous hypersecretion in certain susceptible and immunocompromised population.