Candida esophagitis represents a severe threat to an immunocompromised body and the course of the infection is determined by both pathogen- and host-dependent factors [
15,
24,
38,
39]. It is well established that epithelial cells of the esophagus are the central target of an oro-esophageal invasive
Candida infection but there are only very limited data on the host response preventing a
Candida esophagitis. In the present study we identified the NF-κB and MAPK/AP-1 pathways as central regulators of epithelial hBD-2 and hBD-3 expression during
C. albicans infection. Furthermore we were able to show a crucial role of the interaction of PMNs with the epithelial cell compartment for the induction of hBD-2 and hBD-3 expression during
Candida infection of esophageal epithelial cells. Finally hBD-3 expression is dependent on transactivation of EGFR by TGF-α. This is the first report delineating molecular mechanisms leading to upregulation of hBD-2 and hBD-3 in esophageal
Candida infection suggesting differential regulation of these two central epithelial antibiotic peptides in response to a
C. albicans infection. We were able to show a concentration dependency of hBD-2 and hBD-3 induction upon stimulation with
C. albicans supernatants (Figure
1B). This dose dependent effect could be at least in part be mediated through stronger induction of NF-κB (Figure
4) and AP-1 (Figure
6) through higher concentrations of
C. albicans. A comparable concentration-dependent effect of supernatants of environmental airborne fungi on cytokine release of eosinophils [
40] and of
C. albicans on the activation of NF-κB [
9] was recently reported.
There is some controversy about the relevance of the NF-κB and AP-1 binding sites in the promotor of hBD-2 for full induction of hBD-2 expression after treatment with IL-1β or infection with microbiota. Wehkamp and collegues showed that the parallel activation of NF-κB and AP-1 is needed for full transcriptional activation of the hBD-2-promotor after IL-1β stimulation, treatment of keratinocytes with supernatants of
Pseudomonas aeruginosa [
22], or
E. coli mediated hBD-2 induction [
41]. On the other hand another study demonstrated induction of hBD-2 expression through
Fusobacterium nucleatum in human gingival epithelial cells independent of NF-κB activation [
42] and there is also evidence for induction of hBD-2 through Salmonella enteritidis [
43] or H. pylori [
44] in the absence of functional AP-1. In the present work we were able to show that both NF-κB and AP-1 activation are required for full upregulation of the hBD-2 mRNA after treatment with either supernatants of
C. albicans or the coculture of
C. albicans with PMNs. Inhibiton of the NF-κB or of the MAPK/AP-1 pathway significantly reduced the induction of hBD-2 expression confirming the central role of both transcription factors. These observations are in line with recent results for H. pylori infection [
20] and the effects of lactobacilli and the VSL#3 bacterial mixture on enterocytes [
45]. The role of NF-κB is less clear in the regulation of hBD-3 expression. In contrast to a recently reported role of NF-κB in the regulation of the hBD-3 gene in keratinocytes [
31] the majority of data failed to show a functional relevance for NF-κB in controlling hBD-3 expression [
33,
46]. By using siRNA targeting the RelA/p65 subunit of NF-κB we could demonstrate that
C. albicans mediates hBD-3 upregulation through a MAPK/AP-1 pathway independently of the observed NF-κB acitvation. To investigate the proposed role of PMNs in the immune response the
C. albicans infection [
25,
26] we established an in vitro model (Figure
2). PMNs alone induced NF-κB and AP-1 leading to hBD-2 and hBD-3 expression. Coincubation of PMNs with
C. albicans lead to a significant upregulation of hBD-2 and hBD-3 expression compared to effects of PMNs or
C. albicans alone. Inhibition of the MAPK/AP-1 pathway reduced the expression of both hBDs under this condition. The fact that PMNs alone induced hBD expression indicated that PMNs could contribute to the induction of antimicrobial peptides in epithelia during inflammation. There is some evidence that a TLR-4 mediated interplay between PMNs and epithelial cells is important for the protective response against
Candida infections [
5] but the exact mechanisms remained elusive. One the one hand PMNs induced expression and release of IL-6 and IL-8 in O21 cells (Additional file
1) which might in turn activate NF-κB and AP-1 leading to hBD-2 and hBD-3 expression. On the other hand the induction of a TH1 response, i.e. TNF-α and interferon-γ might directly induce or enhance the expression of hBDs as reported recently [
47]. On the other hand PMNs are able to establish an anti-fungal response by upregulate the expression of TLR4 in epithelial cells [
5]. The observed more pronounced effect on hBD-3 expression are in line with the theory that hBD-3 might be clinically more relevant than hBD-2 since hBD-2 and hBD-3 have potent fungicidal activity against
C. albicans at micromolar concentrations, with hBD-3 being about 10 times more fungicidal than hBD-2 [
16,
17,
19]. Finally we demonstrated that hBD-3 expression was induced by transactivation of the EGFR independent of EGF (Figure
8 and
9). Many signals besides EGF converge and result in EGFR-dependent signaling which is important for various biological processes including normal growth, development and as shown recently for inflammation or innate immune response [
48,
49]. Using blocking antibodies for the EGFR-ligands EGF, HB-EGF, amphiregulin and TGF-α we were able to show that most likely TGF-α is involved in the EGFR mediated upregulation of hBD-3 expression (Figure
9). Since we are also able to inhibit the induction of hBD3 by the supernatants of 1 × 10
5 C. albicans cells/ml by EGFR and/or TGF-α blocking antibodies we speculate that the EGFR-ligand is secreted by OE21 cells. Ligands of the EGFR are expressed as transmembrane precursors. These are released from the cell surface following shedding of the extracellular domain by a family of metalloproteinases (a disintegrin and metalloprotease (ADAM)). ADAM10, -12 and -17 are the sheddases of the EGFR ligands in response to various stimuli. Since human cathelicidin cationic antimicrobial protein (hCAP)-18 and its active peptide LL-37 have been shown to be involved in the transactivation of the EGFR at the airway epithelial surfaces [
50,
51] it is tempting to speculate that the activation of PMNs by
C. albicans leads to secretion of proinflammatory mediators including leukotriene B4 (LTB4) and LL-37 [
52,
53] which in turn amplifies the inflammatory response [
54,
55] and leads to the shedding of EGFR-ligands.