In this report, we dissect a mechanism by which mucosal epithelial cells use IL-1α as a signaling molecule to respond and protect themselves against infection by the pathogenic bacterium, L. monocytogenes
. We also show that the protection against invasion by L. monocytogenes
is cell autonomous, whereby epithelial cells confer resistance to the pathogen independently of dedicated innate and adaptive immune cells. In response to L. monocytogenes
, epithelial cells release IL-1α and, in an IL-1RI-dependent manner, modulate autonomous resistance to these invasive enteric pathogens. Other invasive mucosal pathogens including C. albicans
and Porphyromas gingivalis25,28,29
also induce the release of IL-1α from oral keratinocytes over time, suggesting that the epithelial response to pathogens could be generalized.
Upon invasion into keratinocytes, L. monocytogenes and P. gingivalis
induce calcium release from intracellular stores,30,31
which can activate the calcium-dependant protease, calpain. Activated calpain cleaves the 32 kDa proIL-1α to produce mature 17 kDa IL-1α, a critical step during Golgi-independent release of IL-1α. Release of the IL-1α pro-peptide also occurs apparently through non-canonical plasma membrane translocation.32,33
Both the pro-peptide and mature IL-1α are recognized by the IL-1RI,34
which is generally expressed on the surface of mucosal epithelial cells.35
Spent media from Listeria
-infected oral keratinocytes increased S100A8/A9 expression in an IL-1RI dependent manner, suggesting that released IL-1α was biologically active. Hence, IL-1α can alert or signal neighboring epithelial cells to nearby stresses in a paracrine manner.
We had hypothesized that mucosal epithelial cells use IL-1α as a signaling molecule to increase S100A8/A9-dependent resistance to Listeria
invasion in oral keratinocytes. We examined whether exogenous IL-1α induces expression of S100A8/A9 and other AMP genes in an IL-1RI dependent manner. In TR146 cells, exogenous IL-1α induces dose-dependent up-regulation of representative AMPs, including both S100A8/A9 subunits and h-BD2, consistent with our previous studies in HaCaT epithelial cells.24,36
The increased concentration of IL-1α required to maximally express S100A8/A9 in HaCaT as compared to TR146 cells appears to be a result of greater IL-1RI expression, suggesting that keratinocytes could act as sensitive sentinels in the oral mucosa. Expression of S100A8/A9 and h-BD2 by TR146 cells was maximal in response to 1 ng/ml IL-1α, which appears to be biologically relevant since L. monocytogenes
induced TR146 cells to release approximately 1.4 ng/ml IL-1α into the media. Hence, IL-1α released from epithelial cells (endogenous) or added to the cells (exogenous) can signal naïve keratinocytes through IL-1RI to induce expression of S100A8/A9 and other AMPs. Although also biologically active within the cell,37,38
intracellular IL-1α is insensitive to IL-1Ra,39
which effectively blocks extracellular IL-1α from the IL-1 type I receptor.40–42
Since either spent culture media containing IL-1α or added IL-1α appears to signal epithelial cells similarly, we concluded that exogenous IL-1Ra targets surface IL-1RI; increased AMP gene expression is dependent on surface IL-1α/IL-RI interactions.
IL-1R-dependent responses have been implicated in resistance to bacterial41,43,44
and fungal infections45,46
and in animal models. To determine whether IL-1RI contributes to resistance of oral keratinocytes to infection by L. monocytogenes
, cells were incubated for 24 h with IL-1α in the presence or absence of IL-Ra. After infection with Listeria
, an antibiotic protection assay was performed to discriminate extracellular and invaded bacteria. Exogenous IL-1α was shown to increase production of S100A8/A9 about 2-fold (), which, for the first time, was shown to be sufficient to augment keratinocyte resistance to invasive Listeria
(). Moreover, since S100A8 and S100A9 do not form homodimers within cells, the increase in S100A8/A9 protein expression is expected to reflect functional S100A8/A9 complex.47
In the presence or absence of exogenous IL-1α, IL-1Ra reduced S100A8/A9 expression, increasing the number of recoverable invaded Listeria
and reducing resistance to invasion. Since endogenous IL-1α is released by infected oral keratinocytes (), blocking IL-1R1 with IL-1Ra appeared to reduce protection of epithelial cells mediated by S100A8/A9.
To show more definitively that resistance to invasion was mediated by S100A8/A9, we knocked down S100A8 and S100A9 subunit proteins using shRNA. S100A8/A9 was reduced nearly 85% in clone A8A9c10 when compared to the sham silenced Neg3 cells (). Whereas expression of h-BD2 in response to IL-1α was not affected, h-BD2 expression in response to increasing doses of IL-1α was similar in TR146 cells () and A8A9c10 (). In response to IL-1α, expression of h-BD2 by Neg3 cells was anomalously high and Neg3 resistance to invasion in some experiments appeared to be unaffected by IL-1RI stimulation. The inability of IL-1α to induce resistance in Neg3 cells may be the result of shRNA transfection, which can induce nonspecific responses in mammalian cells as previously reported.48,49
While S100A8/A9-mediated resistance to invasion by Listeria
appeared lower in A8A9c10 cells than Neg3 cells (), a second silenced clone, A8A9c27 showed significantly less resistance to invasion that Neg3 cells (). The increased h-BD2 in Neg3 cells could be argued to have increased the resistance of these cells to invasion in the presence of IL-1α, but this does not appear to be the case. Neg3 cells () and wild-type TR146 cells () showed similar reductions in invaded Listeria
in the presence of IL-1α. We conclude therefore that IL-1α increases expression of S100A8/A9, which specifically protects the epithelial cell against invasion by Listeria
, supporting our hypothesis that IL-1α-mediated resistance to Listeria
invasion is S100A8/A9-dependent.
We are currently pursuing the question of how S100A8/A9 controls intracellular resistance to invasion. Within the cell, S100A8/A9 has other activities that could contribute mechanistically to resistance to invasion. In HaCaT cells, overexpression of S100A8/A9 appeared to increase NADPH activity when incubated with phorbol 12-myristate 13-acetate50
, perhaps enhancing formation of reactive oxygen species and microbial resistance to infection.51,52
The S100A9 C-terminal extended peptide domain may play a regulatory role in resistance to Listeria
infection since truncated S100A9 enhances keratinocyte resistance to Listeria
This finding does not appear to support a NADPH/S100A8/A9-dependent mechanism of antimicrobial resistance since a similar mutation in S100A9 by Benedyk et al. showed decreased NADPH activity when compared to full-length S100A9. Other structural motifs within S100A9 also appear to play a role in resistance to invasion. We have reported that S100A8/A9 requires intact EF-hands to coordinate calcium binding, which could contribute to control of invasion by Listeria
in oral epithelial cells.3
In addition to direct antimicrobial activity, S100A8/A9 is a putative inhibitor of casein kinase I and II,53
which mediate IL-1RI signaling in intestinal epithelial cells.54
By regulating activity of casein kinase I and II, S100A8/A9 could modulate IL-1RI signal transduction and down-stream effectors of resistance to invasion.
Over 700 species of microorganisms colonize the oral cavity55
and nearly all respiratory and enteric pathogens also must pass through the oral cavity and do not cause infection. Although transient enteric pathogens such as L. monocytogenes
and Salmonella enterica serovar typhimurium
invade oral epithelial cells in vitro
these bacteria do not cause persistent oral infections in people. Unlike unilayered cuboidal epithelial cells of the intestinal mucosa, oral keratinocytes form a stratified epithelium, which as we show can respond and resist infection by bacterial pathogens autonomously, independent of infiltrating immune cells. Since myeloid cells can also release IL-1α during inflammation, exogenous IL-1α and the autonomous responses of the epithelium may generally increase production of AMPs, including S100A8/A9, to augment the innate protective barrier to infection. We speculate that this mechanism antagonizes colonization and infection by enteric intracellular pathogens in the human oral cavity.
Periodontal disease is an irreversible loss of the connective and hard tissues underlying the oral mucosa. Initiated by dental plaque colonizing the epithelial interface approximating the tooth, pathology generally reflects propagation of infiltrating inflammatory cells in response to invasive microorganisms. Neutrophils and macrophages induce resorption of the alveolar bone and loss of connective tissue attachment to the tooth to create the characteristic space or pocket between the proximal infected epithelium and the tooth, and loss of alveolar bone. Understanding how oral keratinocytes regulate S100A8/A9 and other AMPs to increase resistance to microbial infection would appear to advance our understanding of how the host resists loss of periodontal tissues and other mucosal infections.