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The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA), and acts selectively on keratinocytes triggered through Toll-like receptor (TLR) 3. The significance of this is seen by observations that TLR3 activation is required for normal inflammation after injury, and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. These findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.
All complex metazoans are colonized with a myriad of microbial organisms, a group that has been referred to as the “microbiome”. On the skin, bacterial colonization is abundant, diverse and constant, but inflammation is undesirable and an indication of disease. The two principal normal stimuli of inflammation are injury and infection. In infections, the detection of microbes is accomplished in part by Toll-like receptors (TLRs) that are best known as stimuli of inflammation. Although the role of TLRs in response to infection is well defined, the mechanisms involving TLRs that regulate inflammation in the skin injury are poorly understood.
Epithelial surfaces in the gut regulate the magnitude and duration of TLR signaling, thus shaping and maintaining normal mucosal immunity1-3 via the induction of proinflammatory and anti-inflammatory cytokine synthesis 4,5. Moreover, commensal bacteria Lactococcus lactis and Bacteroides vulgatus can prime the host to produce IL-10, trefoil factors and TGF-β suggested to modulate inflammation in the intestine 6-8. However, TLRs that have been generally thought to act as proinflammatory signals in response to microbial products also may recognize self-epitopes that are released from damaged cells or are present at the surface of apoptotic cells in autoimmune diseases9,10. These observations have raised the possibility that TLRs may play a role in initiating inflammation in response to injury11,12.
In skin injury, tissue damage results in necrosis and apoptosis, but it is not known if this event is involved in the initiation of the inflammatory response that is critical to normal wound repair. In the absence of infection, uncontrolled inflammation during wound repair is undesirable and may cause dysfunction during healing. Uncontrolled inflammation after minor trauma is also well known to exacerbate several human skin diseases such as psoriasis. Therefore, normal immune defense requires that a balance is maintained to minimize unnecessary inflammation yet rapidly respond to infection and injury. This balance is particularly difficult to maintain at epithelial surfaces that are in contact with the external environment and has frequent trauma and exposure to the products of the microbiome. The innate systems that enable the control of this inflammatory response are generally unknown.
Given the hypothesis that epithelial flora may serve to protect the host from unintended inflammatory diseases, and the importance of TLRs in the recognition of microbial products and potential role after tissue damage, we set out to study how these systems may be involved in homeostatic control of skin inflammation. Staphylococcal species are the most frequently cultured normal inhabitant of the healthy human skin 13 and have been hypothesized to serve a role in human health14. It has been entirely unknown how these bacteria could affect cutaneous homeostasis. In this study we describe a mechanism by which a product of staphylococci, LTA, suppresses skin inflammation during wound repair.
Recent observations in several systems have suggested that activation of TLRs on keratinocytes will trigger inappropriate production of proinflammatory cytokines in the epidermis as an element in the pathogenesis of several skin diseases 15-17. However, despite production of TLR ligands, several commensal bacterial species including Staphylococcus epidermidis (S. epidermidis) normally reside in contact with keratinocytes in the epidermis and do not initiate inflammation. We therefore hypothesized that S. epidermidis could influence the inflammatory response of keratinocytes through negative regulation of TLR signaling. Primary human keratinocytes treated with a panel of TLR ligands showed that polyriboinosinic polyribocytidylic acid [poly(I:C)], a stimulus for TLR318,19, was the most potent stimulus for expression of TNF-α by keratinocytes and that prior exposure to a <10 kDa product of S. epidermidis (SE) suppressed poly(I:C)-induced IL-6 and TNF-α expression (Fig. 1a and Supplementary Fig. 1a). This suppression persisted over time and affected both TNF-α mRNA and protein (Supplementary Fig. 1b).
Cutaneous inflammation induced by poly(I:C) in mice were next examined to determine the in vivo relevance of the response observed to SE. Ears pretreated with SE prior to poly(I:C) showed less inflammation compared to those treated only with poly(I:C) (Fig. 1b,c). The decrease in inflammation corresponded with a decrease in the expression of IL-6 and TNF-α mRNA ( Fig. 1d). However, the inhibitory effects of SE in whole skin were not observed when inflammation was induced by lipopolysaccharide (LPS) (Supplementary Fig. 1c), or phorbol 12-myristate 13-acetate (PMA) (Supplementary Fig. 1d). Thus, these data show SE functions as a selective suppressor of poly(I:C)-mediated inflammation in the skin.
Analysis of similar soluble <10 kDa products of other staphylococcal strains demonstrated that the capacity to inhibit poly(I:C)-induced TNF-α expression was present in multiple staphylococcal strains, except one hospital-isolated strain of Staphylococcus aureus (S. aureus) Rosenbach (Supplementary Fig. 2). SE also suppressed the expression of IL-8, but was not able to suppress IFN-β or IL-1β (data not shown). Moreover, SE inhibited the capacity of mouse epidermal sheets to express TNF-α and IL-6 in response to poly(I:C) (Supplementary Fig. 3a). However, in contrast to keratinocytes and epidermal sheets, other cell types representative of those present in skin but below the epidermis were not inhibited by SE and had increased expression of IL-6 and TNF-α in response to poly(I:C) and SE (Supplementary Fig. 3 b–d). Taken together, these results show poly(I:C) is a potent stimulus to release proinflammatory cytokines in the skin, but a soluble low molecular weight product from some bacteria will inhibit this response selectively in keratinocytes.
To understand the immunological relevance of suppression of TLR3-induced cytokine release by keratinocytes we next sought to investigate conditions in which TLR3 might be activated in skin. In gastrointestinal epithelium TLR3 has been implicated as a mechanism for detection of cell death 20. Skin injury results in the generation of necrotic and apoptotic cells 21,22. Therefore, we examined if TLR3 might be a mechanism for detection of skin injury and initiation of inflammation. To test this hypothesis, aseptic, full-thickness incisions were performed on the back of Tlr3-deficent mice and matched wild-type controls. Tlr3-deficent mice showed significantly less production of IL-6 and TNF-α at the wound edge compared to wild-type controls (Fig. 2a). This correlated with a decrease in inflammation and leukocyte recruitment (Fig. 2b and Supplementary Fig.4). Furthermore, consistent with the inhibitory effects of SE on poly(I:C)-induced skin inflammation, application of SE also inhibited wound-induced IL-6, TNF-α, and inflammation in wild-type mice, but had no significant effect on the injury response in Tlr3−/− mice (Fig. 2a,b and Supplementary Fig.4). Thus, these findings establish that Tlr3 is required for part of the normal inflammatory response following injury, and that SE acts on this pathway to decrease the magnitude of inflammation.
Wounding results in the rapid generation of abundant amounts of damaged cells, including necrotic and apoptotic keratinocytes. To test if a product of damaged keratinocytes can stimulate TLR3-dependent inflammation in adjacent normal cells, cultured keratinocytes were treated with UVB radiation to induce apoptosis and cell death. These cells (UVR-cells) were then collected and added to separate cultures of normal human keratinocytes. TNF-α release significantly increased when normal keratinocytes were exposed to UVR-cells but not when exposed to equal amounts of dead, non-irradiated cells (Fig. 2c and Supplementary Fig. 5a). This response was dependent on TLR3 since targeted knock down of TLR3 by siRNA abrogated the response to UVR cells (Fig. 2c and Supplementary Fig. 6). Consistent with response to poly(I;C) or wounding, SE also inhibited TNF-α release induced by UVR-cells (Fig. 2d). The product of UVR-cells that activated TLR3 in normal keratinocytes was RNA as RNase abrogated the capacity of UVR-cells to stimulate the production of TNF-α, but treatment with DNase did not (Fig. 2e). UVR-cells were also sorted into intact, Annexin V+ cells that excluded propidium iodide (PI), and dead cells whose membrane was permeable after UVB irradiation and thus stained with PI. Non-irradiated cells (normal cells) disrupted by sonication, or early apoptotic cells that were annexin V+ but excluded PI (annexin V+/ PI− cells), could not induce TNF-α release or TLR3 expression (Supplementary Fig.5a, b). However, UVR-cells staining with PI (PI+ cells) significantly increased TNF-α release and TLR3 expression (Supplementary Fig. 5a, b). Thus, these results show that RNA released from necrotic cells can induce TNF-α and TLR3 expression by keratinocytes.
TLR-TLR cross-talk can suppress inflammatory responses 23,24, and LTA is a known molecular signal for recognition of staphylococci, thus we sought to determine whether this molecule could be responsible for the observed effects. LTA from S. epidermidis has two forms, cellular and exocellular LTA 25. Exocellular LTA can be recovered from liquid growth medium, and is the form present in SE, whereas commercial LTA is derived from the cell wall and membrane. Commercially available LTA from S. aureus (LTA-SA), but not LPS or Flagellin, suppressed the production of TNF-α induced by poly(I:C) (Fig.3a). LTA directly purified from SE (LTA-SE) or commercial LTAs from some other staphylococcal species suppressed poly(I:C)-induced TNF-α, whereas TLR2/1, TLR2/6 ligands (Pam3CSK4, FSL-1 and PGN-SA) either synergistically induced TNF-α, or failed to suppress TNF-α (Malp-2, PGN-EK and Zymosan) when combined with poly(I:C) (Fig. 3b). Interestingly, LTAs from Streptococcus faecalis and Bacillus subtilis (LTA-SF and LTA-BS) were not active (Fig. 3b).
The identity of LTA as the molecule in SE responsible for suppression of keratinocyte TNF-α production was confirmed by use of a specific LTA-neutralizing antibody25 (Fig. 3c). Furthermore, synthetic LTAs containing two or three D-alanine suppressed poly(I:C)-induced TNF-α, whereas synthetic LTAs only containing an anchor or containing two or five N-acetylglucosamine (NacGlc) failed to suppress TNF-α (Fig. 3d). The necessity of D-alanine modification was then confirmed by use of LTA from a S.aureus dltA mutant that lacks D-alanine modifications 26. Preparations from this mutant bacterial culture supernatant (SA dltA) partially lost the capacity to suppress poly(I:C)-induced TNF-α compared to its parental strain (SA wt) (Fig. 3e).
SE was observed to block nuclear translocation of NF-kB1/p50, but not the translocation of IRF3, both stimulated by poly(I:C) in keratinocytes (Supplementary Fig. 7a,b). Thus, we next investigated negative regulators involved in TLR-mediated NF-kB signaling including TNF receptor-associated factor 1 (TRAF1), TNF alpha-induced protein 3 (TNFAIP3, A20) and interleukin-1 receptor-associated kinase M (IRAK-M)27. LTA and SE significantly induced TRAF1 in keratinocytes within one hour and maximally between 6 and 9 hrs (Fig. 4a and Supplementary Fig. 8a). The expression of other negative regulatory genes (NLRX1, A20 or IRAK-M) involved in both the NF-kB and RIG-like helicase pathways, were not induced (Supplementary Fig. 8a). The role of TRAF1 as a mediator of the LTA inhibitory effect was confirmed by experiments in Traf1−/− mice where LTA was unable to suppress the production of IL-6 and TNF-α at wound edge compared to wild-type controls (Fig. 4b).
It has been hypothesized that TRAF1 is processed by caspase 8 to an active N-terminal fragment (N-TRAF1) followed by binding to TRIF in order to serve as a negative regulator of TLR3. The cleavage of TRAF1 to N-TRAF1 was observed in the presence of SE and poly(I:C) (Fig. 4c) and occurred in a caspase 8-dependent manner since treatment with a caspase 8 inhibitor blocked the generation of N-TRAF1 (Fig. 4d). Association of TRAF1 with TRIF was observed by immunoprecipitation of SE-treated keratinocytes with TRAF1-specific antibody and detected with antibody to TRIF (Supplementary Fig. 8b). Furthermore, addition of a caspase 8 inhibitor increased TNF-α and completely abrogated the inhibitory effects of SE and LTA (Supplementary Fig. 8c,d).
Having established the involvement of TRAF1 in suppression of keratinocyte cytokine release, and the role of the TLR2 ligand LTA in initiating this effect, we next sought to further confirm the mechanism and physiological relevance of these observations in vivo. Analysis of the skin from germ-free mice that were never exposed to bacterial products showed that the expression of Traf1 in their skin was dramatically decreased compared to skin of normal mice housed in a pathogen-free but non-sterile conditions (Fig. 4e). In addition to skin, the expression of Traf1 in small intestine and lung, but not heart, of germ-free mice was decreased compared to those tissues from conventional mice (Supplementary Fig. 8e). Furthermore, as predicted based on the known recognition system for LTA, diminished expression of Traf1 was also observed in Tlr2−/− or Myd88−/− mice intentionally exposed to LTA (Fig. 4f). Therefore, Tlr2−/− mice could be used as another model to test the consequences of diminished Traf1 to suppress Tlr3-dependent skin inflammation following injury. As predicted, LTA failed to suppress inflammation and the production of IL-6 and TNF-α at wound edge in Tlr2−/− mice (Fig. 5a, b), and a similar loss of responsiveness to LTA was seen in Tlr2−/− mice treated with poly(I:C) (Fig. 5c).
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.
C57BL/6 wild-type, Tlr3-deficient, Tlr2-deficient and BABL/c wild-type mice were housed in VA San Diego Healthcare System Veterinary Medical Unit (VMU) and Traf1-dificient mice were purchased from the Jackson Laboratory while swiss webster conventional and germ-free mice were purchased from Taconic. All animal experiments were approved by VA San Diego Healthcare System Institutional Animal Care and Use Committee.
Bacteria were grown in Tryptic soy broth (TSB) at 37 °C for 15–16 hours. For scale-up preparation, overnight cultures were diluted 1:100 into TSB and grown for another 15–16 hours. We then collected bacterial cultures and filtered them by 0.22 μM Stericup (Millipore). We used MacroSep 10K OMEGA column (VWR) to collect the less than 10 kDa fractions from bacterial supernatants and determined concentrations of bacterial extracts by BCA™ Protein Assay Kit (Pierce). We purified LTA from S. epidermidis conditioned culture media (SE) by using the protocol of Grundling and Schneewind(2007)37.
We irradiated cultured human keratinocytes by UVB at 15 mJ/cm2 for 1 min. After 24 h, we collected the UVB-irradiated cells and sorted them into Annexin V+/ PI− cells and PI+ cells. To stimulate TNF-α we added these sorted UVR-cells to untreated normal human keratinocytes in culture, respectively. We used sonicated-non-irradiated cells (normal cells) as control. We measured TNF-α in culture media 24 h after treatment with these cells.
We used 300 μg ml−1 of S. epidermidis LTA monoclonal antibody (abcam) or monoclonal mouse IgG1 isotype control (EXBIO) to incubate with 30 μg of SE for 20 h at 4 °C before we added SE to stimulate cells. After 24 h stimulation, we collected cell supernatants for TNF-α ELISA assay.
We seeded neonatal human epidermal keratinocytes at first or second passage in 24-well plate and transfected cells with 10 nM four pairs of siRNA oligonucleotides targeted to TLR3 (Dharmacon; SMART Pool) and non-targeted control siRNA (Dharmacon) by using silentFect (BioRad) as we described before 38. We tested the efficiency of TLR3 siRNA blockage by western blot. After 24 h transfection, we added UVR-cells to stimulate cells for 24 h. We evaluated the production of TNF-α by ELISA.
We applied 100 nM caspase 8 inhibitor (Z-IETD; R&D) 10 min before we added10 μg ml−1 of poly(I:C) with or without 36 μg ml−1 of SE or 10 μg ml−1 of LTA in cultured human keratinocytes and waited for 6 h or 48 h. We analyzed the inhibition of TRAF1 processing by western blot and the production of TNF-α by ELISA.
We stimulated cultured human keratinocytes at indicated time points. We lysed cells by using RIPA buffer (pH 7.4) containing protease inhibitor cocktail (Roche) and then sonicated them on ice-cold water. We measured protein concentrations of the extracts by BCA™ Protein Assay Kit (Pierce) and used15 μg of total protein for immunoprecipitation and 3 μg of total protein for western blot. SDS-PAGE was used to separate bands and TRAF1 or the association of TRAF1 and TRIF was detected by immunoblot with TRAF1-specific antibody (Santa Cruz) or TRIF-specific antibody (Cell signaling).
We shaved the backs of age-matched adult littermates and removed hair by using chemical depilation (Nair; Church & Dwight) as previously described39. We intradermally injected 100 μL of PBS or 100 μL of SE (24 μg) or 100 μL of LTA-SA (50 μg) 24 h and 2 h prior to wounding back skin of wild-type or Tlr3-deficient mice or Traf1-deficient mice by biopsy punches. 3 d later, we collected 2 mm of skin around wound edges either for ELISA assay or stored it in formalin (Protocol) for H&E staining.
For cutaneous inflammation in ears, we injected12 μg of SE or 10 μg of LTA-SA into the ear lobes of C57BL/6 wild-type (or BABL/c wild-type mice) and Tlr2-deficient mice 2 h before we injected 50 μg of poly(I:C) or 20 μg of LPS. After 24 h we cut mouse ears to either store them in formalin for H&E staining or homogenize them for RNA isolation. We analyzed the expression of cytokines by real-time RT-PCR.
For phorbol 12-myristate 13 –acetate (PMA)- induced skin inflammation, we injected 12 μg of SE in BABL/c wild-type mice ear lobes 2 h before we topically treated mouse ears with 20 μL of 1mg ml−1PMA (Sigma). After 6 h, we took ears for cytokines analysis.
All data are present as mean ± SEM. We used two-tailed t tests to determine significances between two groups. We did analyses of multiple groups by One-way or Two-way ANOVA with Bonferroni post test of GraphPad Prism Version 4. For all statistical tests, we considered P values <0.05 to be statistically significant.
We thank A. Peschel for advice with blocking LTA activity by the antibody; E. Raz from University of California, San Diego for providing C57BL/6 Tlr3-deficient mouse breeding pair; V. Nizet and E. Tistiskov for helpful discussion; G. Cheng, B. Beutler, R. Modlin and E. Raz for critical reading and helpful advice; D. Bird for histological sections. This work was supported by US National Institutes of Health grants R56AI083358, R01AR052728, R01 AI052453, and a US Veterans Administration Merit Award to R.L.G and US National Institutes of Health grants DC00129 and DC006279 to A.F.R.
AUTHOR CONTRIBUTIONS Y. L. and R.L.G. designed the experiments; Y. L. performed most of the experiments and analyzed data; A.D.N. helped in culture of bone-marrow-derived dendritic cells and preparation of UVB-irradiated apoptotic/necrotic cells; T.N. and A.L. helped with mouse experiments; Y.Y. and Z.-R. W. designed the LPS-induced inflammation model; A.L.C. helped to perform initial experiments; L.V.H. provided germ-free mouse ear and intestine samples; S.V.A. provided synthetic LTAs; K.A.R. showed how to make mouse wound model; C.-M. H. helped to modify the protocol for animal study; A.F.R. provided Tlr2-deficient mice; Y. L. and R.L.G. wrote and prepared the manuscript.
The authors declare no conflict of interest.