Commensal microorganisms have been proposed to influence host immune responses and host-pathogen interactions in the gut28
. In the current study we hypothesized that specific elements of the resident microbiota of normal human skin can modulate cutaneous immune responses triggered by TLR ligands. Our results confirmed this hypothesis while also revealing for the first time that TLR3 is a critical element in the induction of inflammation after skin injury. Inhibition of this inflammatory event is accomplished by specific staphylococcal LTAs, and mediated by TLR2 on keratinocytes. The mechanism for LTA-TLR2 mediated suppression of TLR3 signaling is by induction of the negative regulatory factor TRAF1, an event we show is important to limit the extent of cutaneous inflammation. Thus, we show for the first time that a sensitive balance exists between stimulating and inhibitory mediators during wound healing, and that epithelial cells uniquely detect these signals to achieve tissue homeostasis following injury.
It was previously unclear why keratinocytes are highly sensitive to TLR3 ligands since the classical ligand for TLR3, viral dsRNA, is not a frequent initiator of major inflammatory responses in the skin. The epidermis can be exposed to dsRNA from viral infections, but this is a relatively uncommon event compared to other skin pathogens. We show here that the significance of the high sensitivity to TLR3 ligands is likely because the epidermis uses TLR3 for recognition of injury to self. Our data show RNA from necrotic cells trigger TLR3 on undamaged keratinocytes, leading to local release of proinflammatory cytokines. This is probably a frequent mechanism for detection of injury and maintenance of homeostasis as necrotic cells are abundant at the wound edge. Therefore, it is reasonable to speculate that TLR3 in the normal epidermis is an important sensor of injury, and that systems must exist to modulate this response to prevent excessive or unwanted inflammation. Furthermore, since SE or LTA not only inhibited proinflammatory cytokine production by isolated keratinocytes in culture, but also inhibited inflammation in vivo, this argues that the production of proinflammatory cytokines by keratinocytes is a major contributor to some forms of skin inflammation.
Our findings demonstrate that LTA produced by staphylococcal species have a uniqe anti-inflammatory action on keratinocytes. In contrast, LTA initiates the opposite response when exposed to other immune cells. It is logical that LTA would have distinct effects on cytokine release depending on the cell type exposed. LTA acts as a proinflammatory factor for cells that normally exist in a sterile environment, such as macrophages (Supplementary Fig.3b
), monocytes and mast cells29,30
. These cells are not normally exposed to the surface microbiome and appropriately recognize LTA as foreign. However, keratinocytes are unique in that they are frequently exposed to LTA. Furthermore, the structure of LTA appears to be important for the nature of the keratinocyte response. Addition of D-alanine to the LTA core is an important factor to dictate activity but further analysis is necessary to understand these LTA structure-function relationships. Moreover, other TLR2 ligands that depend on formation of a heterodimer with TLR1 or TLR6 do not inhibit TLR3 in keratinocytes but rather have a proinflammatory effect. Thus, the specificity of the response is dictated by cell type and specific structure of the TLR2 ligand produced by the microbe. In particular, S. epidermidis
, a normal inhabitant of the skin, may have a uniquely structured TLR2 ligand that maximizes anti-inflammatory action yet minimizes the capacity to initiate inflammation.
To limit TLR-induced inflammation several negative regulatory systems exist including sequestration of signaling molecules, blockade of their recruitment, degradation of target proteins, or inhibition of transcription27,31
. These negative regulators can be part of microbial virulence. Examples of this include decoy receptors in some bacterial infections that prevent a direct interaction between TLRs and their microbial ligands, and vaccinia virus production of several proteins that interfere with viral recognition through both TLR3 and helicases32-34
. Therefore, there is potential for pathogenic staphylococci such as S. aureus
to exploit suppression of keratinocyte activation as a mechanism of virulence, while S. epidermidis
may benefit the host by dampening unwanted inflammation. This hypothesis requires further testing but is supported by data obtained from germ-free mice that shows these animals lack normal expression of Traf1
in the skin. These observations support additional findings that the ability of TLR2 to recognize commensal bacteria is not irrelevant under normal conditions. Rather, the activation of TLR2 in keratinocytes has its own beneficial effect in maintaining homeostasis.
Taken together, these findings are best appreciated when one recognizes that inflammation is an undesirable condition on skin but is fundamentally a necessary protective response after injury. Prolonged and dysregulated production of inflammatory cytokines leads to excessive neutrophil influx, resulting in sustained inflammatory responses and poor healing, subsequently causing extensive tissue damage35,36
. On the other hand, without an appropriate inflammatory response wound healing is also delayed and the host is more susceptible to microbial invasion. Local modulation of the inflammatory response by products of bacterial commensals at the site of such an injury might be a beneficial therapeutic strategy for management of wound healing complicated by excessive inflammation, or control of other inflammatory skin disorders. The trick will be to evoke a reduction in the detrimental aspects of inflammation without increasing the risk of wound infection. Our findings emphasize the potential benefit of the resident bacteria on skin, and the potential negative consequences of complete depletion of microflora from skin by indiscriminate use of topical and systemic antibiotics.