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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: J Invest Dermatol. 2019 Feb 16;139(8):1753–1761.e4. doi: 10.1016/j.jid.2019.02.006

Staphylococcus aureus Lipoteichoic Acid Damages the Skin Barrier through an IL-1 mediated Pathway

Anne M Brauweiler 1, Elena Goleva 1, Donald YM Leung 1,2,3
PMCID: PMC6650368  NIHMSID: NIHMS1521954  PMID: 30779913

Abstract

Staphylococcus aureus is a significant bacterial pathogen that may penetrate through the barrier into the epidermis and dermis of the skin. We hypothesized that the S. aureus cell wall product, lipoteichoic acid (LTA), may contribute to the development of inflammation and skin barrier defects; however, the effects of LTA in vivo are not well understood. In this study, we examined the effects induced by intradermal S. aureus LTA. We found that keratinocytes in LTA treated skin were highly proliferative, expressing 10-fold increased levels of Ki67. Furthermore, we observed that LTA caused damage to the skin barrier with substantial loss of filaggrin and loricrin expression. In addition, levels of the IL-1 family of inflammatory cytokines as well as the neutrophil attracting chemokines, Cxcl1 and Cxcl2, were increased. Concomitantly, we observed significant numbers of neutrophils infiltrating into the epidermis. Finally, we determined that LTA induced signals were mediated in part through IL-1, as an IL-1R1 antagonist ameliorated the effects of LTA, blocking neutrophil recruitment and increasing expression of skin barrier proteins. In summary, we demonstrate that S. aureus LTA alone is sufficient to promote keratinocyte proliferation, inhibit expression of epidermal barrier proteins, induce IL-1 signaling and recruit cells involved in skin inflammation.

INTRODUCTION

Staphylococcus aureus is a gram-positive extracellular bacterium that causes a wide spectrum of human diseases and nearly 500,000 hospitalizations per year in the United States alone (Hersh et al., 2008, McCaig et al., 2006). Infection usually begins in the skin and epidermal keratinocytes, as part of the innate immune response, play a significant role as the first line of defense against bacterial challenge. Previously, we tested the keratinocyte response to a panel of S. aureus products and found that cell wall lipoteichoic acid (LTA) induced major changes in patterns of gene expression (Brauweiler et al., 2015). Furthermore, a gene array analysis of primary keratinocytes showed that more than 300 genes were up or down regulated by LTA. In particular, we found that LTA repressed a number of genes involved in keratinocyte differentiation. This process was dependent on p63 (Brauweiler et al., 2017), a transcription factor required for keratinocyte proliferation and skin development (Koster et al., 2007, Yang et al., 1999).

In contrast, the effects of LTA in the skin in vivo are relatively unexplored. S. aureus LTA is not present on normal skin, however, physiological levels are detected on the lesional skin of a majority of patients with atopic dermatitis (AD) (Travers et al., 2010), an inflammatory skin disease associated with a skin barrier defect and loss of expression of the crucial barrier proteins filaggrin and loricrin (Boguniewicz and Leung, 2011, McAleer and Irvine, 2013). Here, we examined the effects of LTA using in vitro and in vivo models of S. aureus LTA exposure. We determined the effects of intradermal LTA on skin maturation, expression of epidermal barrier proteins, inflammatory cytokine signaling and recruitment of cells involved in the innate immune response.

RESULTS

S. aureus LTA induces inflammatory cytokine expression in primary keratinocytes in a p63 dependent manner.

In our previous array analysis, we determined that keratinocytes respond robustly to staphylococcal LTA (Brauweiler et al., 2017). Following LTA treatment, the pattern of gene expression was changed, with significant inhibition of early keratinocyte differentiation (Brauweiler et al., 2017). In the array, we also observed increased expression of the inflammatory cytokine, IL-1β. However, this observation was not pursued further. Here, we examined inflammatory cytokines induced by 10 μg/ml LTA treatment, an amount found on the skin of AD patients (Travers et al., 2010). We found that keratinocytes exposed to LTA for 24 hours up-regulated mRNA expression of a number of cytokines in the IL-1 family. Notably, IL-1β, was induced 10-fold (Fig. 1). IL-36α and IL-1α were also induced by LTA treatment. A dose response (Sup. Fig. 1), shows that 10 μg/ml LTA gives a near maximal response. Expression of the IL-1 family cytokine IL-33, however, was unaffected by LTA (Fig. 1). Expression of other IL-1 family members was marginally regulated by LTA (Sup. Fig. 1). In addition, we found that a number of neutrophil chemotactic cytokines were also induced by LTA. These included IL-8 as well as CXCL2 (Fig. 1). Small changes in CXCL1 expression were observed. Finally, LTA induced expression of the matrix metalloproteinase, MMP-1. We conclude that LTA stimulates essential components of the innate immune response in keratinocytes, causing cytokine and chemokine induction and matrix metalloproteinase expression that, in turn, could contribute to infiltration of immune cells.

Fig 1. S.aureus LTA induced inflammatory cytokine expression is mediated by p63 in primary keratinocytes.

Fig 1.

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Primary human keratinocytes transfected with control or p63 siRNA were treated +/− LTA for 24 hrs and analyzed by RT-PCR for the expression of cytokine and neutrophil chemotactic genes. Notably, LTA induced cytokine expression is absent in p63 siRNA silenced cells. Data are mean ± SEM, n = 3. *P<0.05; **P<0.01; ***P<0.001.

We were interested in exploring the mechanism for LTA induced changes in gene expression. Previously, we determined that LTA inhibited early keratinocyte differentiation through the master regulatory transcription factor, p63 (Brauweiler et al., 2017). We therefore determined the role of p63 in the induction of inflammatory cytokine expression. Keratinocytes were transfected with control or p63 siRNA. During the transfection process, cells were allowed to remain adherent, as this prevented the keratinocyte death associated with p63 silencing (Brauweiler et al., 2017). Adherent p63 silenced cells are viable and visually indistinguishable from control cells (Brauweiler 2017). Supplemental Fig. 1 shows by RT-PCR that expression of several housekeeping genes are unchanged by p63 knockdown.

Remarkably, LTA mediated induction of cytokines and chemokines was completely blocked in p63 siRNA silenced cells (Fig. 1). Therefore LTA mediated signals require p63 to regulate target cytokine gene expression. We therefore conclude that in addition to its established role in regulating epidermal proliferation and development, p63 also plays a significant role in the keratinocyte inflammatory response.

Intradermal injection of S. aureus LTA induces epidermal thickening and hyperproliferation.

We were interested in determining the biological effects of LTA on the skin in vivo. Previously published studies have shown that 50 μg of LTA injected intradermally (ID) into the back of mice can modulate cytokine induction (Lai et al., 2009). To this end, mice were injected intradermally (ID) with PBS or LTA. Skin samples at the site of injection were harvested 48 hours later. Figure 2a shows a representative H&E staining of skin showing significant epidermal thickening induced by LTA treatment. A dose response and time course of LTA mediated epidermal thickening are shown in Supplemental Fig. 2. The epidermal thickening was associated with increased proliferation as significantly increased numbers of Ki67+ cells were present in the epidermis following LTA treatment (Fig. 2b). Interestingly, most of the cells in the basal layer were positive for Ki67 after LTA treatment, while control basal cells were only occasionally positive. In addition, suprabasal Ki67+ cells were also observed in LTA treated skin. Furthermore, quantitative analysis of Ki67+ cells in the epidermis revealed more than a 10-fold increase in proliferation (Fig. 2d). Previous studies have shown that skin cell development and proliferation requires the activity of p63 (Yang et al., 1999). Although we find that proliferation was rapidly induced in vivo upon exposure to LTA, we were unable to explore the effects of p63 further as mice deficient in this gene do not have skin and die shortly after birth (Yang et al., 1999). The rapid induction of keratinocyte proliferation by LTA, however, is highly suggestive of regulation of p63 activity by LTA.

Fig 2. Intradermal injection of S. aureus LTA induces epidermal thickening and hyperproliferation.

Fig 2.

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Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) Representative H&E staining of skin showing epidermal thickening upon LTA treatment. Bar = 100 μM. (b) Increased numbers of Ki67+ cells in the epidermis following LTA treatment. Bar = 40 μM. Dashed line represents epidermal-dermal junction. (c) Increased numbers of Keratin 5 (K5) positive cells after LTA treatment indicates keratinocyte involvement in epidermal thickening. (d) Quantitative analysis of Ki67+ cells in the epidermis. Data are mean ± SEM, n = 6, ***P<0.001.

Neutrophils are recruited to the site of LTA injection.

48 hours following LTA treatment, H&E staining revealed the presence of a massive cell infiltrate in regions of the stratum corneum of LTA treated mice (Fig. 3a). This cell infiltrate was not present in PBS treated mice. Immunofluorescence staining shows that the infiltrate was positive for Myeloperoxidase, a neutrophil specific marker (Sup. Fig. 4). Further analysis by quantitative RT-PCR using the neutrophil specific marker, Ly-6G, reveals that levels of this marker were increased 500-fold by LTA injection (Fig. 3b and Sup Fig, 3). We also show that the neutrophil attracting chemokines Cxcl1 and Cxcl2 were also highly induced in response to LTA (Fig. 3c). These observations establish that LTA treatment in vivo is able to induce expression of chemokines resulting in neutrophil accumulation in the skin.

Fig 3. Neutrophils are recruited to the site of LTA injection.

Fig 3.

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Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) Representative H&E staining of skin showing neutrophil infiltration into the epidermis following LTA treatment (arrow). Bar = 100 μM. (b) Quantitation by RT-PCR for the neutrophil marker Ly6G, showing a 500-fold increase in neutrophils following LTA treatment. (c) RT-PCR analysis showing that neutrophil attracting chemokines are potently induced by LTA. Data are mean ± SEM, n = 6. **P<0.01; ***P<0.001.

Intradermal LTA injection causes reductions in filaggrin and loricrin expression.

In addition to being a crucial part of the innate immune response, keratinocytes also generate proteins crucial for skin barrier function. Expression of the barrier proteins, loricrin and filaggrin are essential for a healthy epidermis. However, the high levels of neutrophils present in the stratum corneum may cause the epidermal barrier to be damaged by LTA. We stained for filaggrin and loricrin in the skin of mice 48 hours after LTA treatment. Notably, in regions of neutrophil recruitment, we find that filaggrin and loricrin staining is completely absent (Fig. 4). A dose response and time course of loss of filaggrin and loricrin expression are shown in Supplemental Fig. 5. We consider it likely that the neutrophil recruitment causes proteolysis resulting in damage to the barrier, as neutrophils are known to secrete a large variety of proteases (Pham, 2008). It is also possible that LTA triggers a wounding response resulting in increased protease expression. In summary, our results suggest that the innate immune response triggered by LTA also causes significant damage to the skin barrier resulting in disrupted expression of filaggrin and loricrin.

Fig 4. Neutrophil infiltration is associated with loss of filaggrin and loricrin expression.

Fig 4.

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Mice were injected ID with PBS or LTA. Skin samples at the site of injection were harvested 48 hours later. Neutrophil infiltration is observed with LTA treatment (H&E). Loss of filaggrin and loricrin staining (red stain at top of epidermis) is associated with neutrophil infiltration. Bar = 200 μM. Dashed line represents epidermal-dermal junction.

Inflammatory cytokine expression is induced by intradermal LTA.

We next examined the effect of S. aureus LTA on inflammatory cytokine expression in the skin. We focused on IL-1 family members as these cytokines are known to be essential for recruitment of neutrophils. Furthermore, we find they are induced by keratinocytes in vitro. Mice were treated with an ID injection of LTA and 48 hours later, biopsies were taken at the site of injection. mRNA was isolated and cytokine gene expression was evaluated by RT-PCR. We found that LTA caused a significant up-regulation of a number of inflammatory cytokines (Fig. 5a and Sup. Fig. 6). LTA induced Il-1β 30-fold and Il-36α 20-fold. In Fig. 5b, we show by immunofluorescence that IL-1β expression is increased in the epidermis after LTA treatment. However, IL-1α and IL-36α protein expression as detected by immunostaining, do not appear to be significantly affected (Sup. Fig. 7).

Fig 5. IL-1 cytokines are induced by intradermal LTA.

Fig 5.

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Mice were injected ID with PBS or LTA. Skin samples at site of injection were harvested 48 hours later. (a) mRNA was analyzed by rt-PCR for expression of cytokine genes. Data are mean, n = 6. **P<0.01. (b) Staining of skin showing epidermal thickening and increased IL-1b expression in the epidermis upon LTA treatment. Bar = 40 μM. Dashed line represents epidermal-dermal junction.

IL-1R1 receptor antagonist treatment ameliorates the loss of filaggrin and loricrin expression associated with LTA injection.

Our in vivo studies show that a single ID injection of LTA increases expression of the IL-1 family of inflammatory cytokines, concomitant with neutrophil recruitment. We hypothesized that damage to the barrier was induced by neutrophil accumulation. Previous studies have shown that blockade of IL-1R1 signaling inhibits neutrophil recruitment (Miller et al., 2007). We therefore tested whether the IL-1R1 antagonist protein, Anakinra, a known inhibitor of IL-1R1 activity, could alleviate damage induced by LTA. Supplemental Fig. 8 shows that the LTA induced increase in epidermal thickening is a result of keratinocyte proliferation combined with cell infiltration. In the IL-1R1 antagonist/LTA treated samples, the epidermis still shows thickening with significant increases in keratinocyte K5 staining, but there is a strong reduction in the cell infiltrate. The reduction in neutrophil involvement correlates with a reduction in epidermal damage.

Following treatment with Anakinra, neutrophil infiltration induced by LTA was reduced by 80% as indicated by the marker Ly6G (Fig. 6a). RT-PCR analysis showed that LTA induced expression of the neutrophil attracting chemokines, Cxcl1 and Cxcl2, was reduced by the IL-1R1 antagonist as well (Fig. 6b and 6c). Consistent with a reduction in neutrophil recruitment, we also find the LTA mediated inhibition of Flg and Lor expression was also ameliorated by the IL-1R1 antagonist (Fig. 6d and 6e). In contrast, the increase in epidermal thickness induced by LTA was unaltered by the IL-1R1 antagonist (Fig. 6f). In conclusion, we show that neutrophil recruitment and subsequent damage of the epidermal barrier induced by LTA can be alleviated by IL-1R1 blockade, however, not all processes were reversed by Anakinra, as epidermal proliferation was still apparent.

Fig 6. The IL-1R1 antagonist blocks LTA induced neutrophil recruitment and increases Flg and Lor expression.

Fig 6.

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Mice were injected ID with PBS, the IL-1 receptor 1 antagonist (IL-1Ra), LTA, or LTA combined with IL-1Ra. Skin samples at site of injection were harvested 48 hours later. (a) Quantitation by RT-PCR for the neutrophil marker Ly6G, shows that IL-1Ra causes an 80% reduction in neutrophils following LTA treatment. (b and c) RT-PCR analysis shows that LTA induced expression of neutrophil attracting chemokines is reduced by IL-1Ra. The LTA induced reduction of Flg (d) and Lor (e) expression is ameliorated by IL-1Ra. (f) The LTA induced increase in epidermal thickness is unchanged by IL-1Ra. Data are mean ± SEM, n = 6. *P<0.05; **P<0.01.

DISCUSSION

S. aureus is a significant bacterial pathogen that is able to penetrate through the barrier into the epidermis and dermis of the skin (Liu et al., 2017, Nakatsuji et al., 2016). Our previous in vitro gene array analysis shows that S. aureus LTA triggers an innate response in epidermal keratinocytes that induces expression of genes involved in proliferation and inhibits expression of genes involved in differentiation (Brauweiler et al., 2017). However, the effects of LTA in vivo are not well understood. In this study, we show that LTA alone has a significant impact on the skin by promoting skin proliferation, inhibiting expression of epidermal barrier proteins, inducing IL-1 signaling, and recruiting cells involved in the innate immune response that break down the epidermal barrier.

We show here that LTA treated skin is hyperproliferative with high expression of Ki67. In this regard, LTA treated skin can be compared to AD skin, as both show hyper-proliferation with thickening of the epidermis (Guttman-Yassky et al., 2011). It is interesting to note that AD skin is frequently colonized with S. aureus (Ong and Leung, 2016). Furthermore, recent studies have shown that S. aureus colonization precedes development of AD (Meylan et al., 2017), implicating the involvement of S. aureus products in development of disease. It is therefore plausible that hyperproliferating keratinocytes in AD skin may be responding to LTA and thereby preventing terminal differentiation. Currently, the triggers that drive keratinocyte hyper-proliferation in AD are unknown, but our data indicates that S. aureus LTA may be a contributing factor. In addition, we find that LTA treated skin, like AD skin, is associated with a substantial loss of filaggrin and loricrin expression, proteins that are required for skin barrier function (Boguniewicz and Leung, 2011). In summary, we find similarities between LTA treated skin and AD skin, as both show increased levels of proliferation and reduced levels of barrier proteins.

We have previously observed that certain aspects of the wounding response in skin keratinocytes are induced by LTA administered in vitro (Brauweiler et al., 2015). During skin wound healing, there is an essential role for the transcription factor p63 to initiate keratinocyte proliferation (Koster et al., 2007). We find here that LTA mediated inflammatory cytokine induction by keratinocytes in vitro also requires p63. Further supporting evidence for a role for p63 in inflammation is shown by in vitro studies demonstrating p63 binding sites in the IL-1α promoter (Barton et al., 2010) and p63 induced expression of IL-1α, IL-1β, and IL-8 in squamous carcinoma cells (Yang et al., 2011). However, we have not pursued the role of p63 in vivo because p63 deficient animals do not have skin and are not viable (Yang et al., 1999).

We find that the IL-1 family of cytokines, IL-1β and IL-36α as well as the neutrophil attracting chemokines, Cxcl1 and Cxcl2, are significantly induced by keratinocytes in vitro. Although another group has examined effects of LTA in inducing chemokine expression in vitro (Olaru and Jensen, 2010), to our knowledge this is the first study examining the pro-inflammatory effects of LTA in vivo. Here, we show that the skin of mice treated cutaneously with LTA had potently induced expression of genes of the IL-1 family as well as neutrophil attracting chemokines. Concomitantly, high levels of neutrophils infiltrated into the epidermis. Interestingly, IL-1β is also induced during S. aureus skin infections (Miller et al., 2007), and is crucial for neutrophil mediated bacterial clearance. For example, mice deficient in IL-1β develop elevated bacterial counts and have decreased neutrophils following S. aureus intradermal challenge.

The cytokines IL-1β and IL-36α are also increased in the skin of individuals with AD (Kezic et al., 2012, Suarez-Farinas et al., 2015). Both of these cytokines cause neutrophil recruitment. However, the role of neutrophils in AD is controversial (Choy et al., 2012, Dhingra et al., 2013). In AD skin, neutrophils and the neutrophil associated chemokines, CXCL1 and CXCL2 are elevated compared to normal skin, but are less than that of the lesional skin of psoriasis. However, neutrophil signatures are increased in lesional AD skin infected by S. aureus (Dhingra et al., 2013). These results suggest a possible contribution of the neutrophil axis to AD pathogenesis.

There are also similarities between LTA injected skin and skin treated epicutaneously with S. aureus. Both treatments induce inflammatory cytokines and epidermal thickening (Liu et al., 2017). It is currently undetermined whether epicutaneous application of S. aureus results in damage to the skin barrier as caused by LTA, although it is known that epicutaneous treatment can induce expression of Th2 cytokines (Nakatsuji et al., 2016), an event associated with development of AD. Notably, it has been shown that penetration of the skin barrier by epicutaneously applied S. aureus requires bacterial viability and protease activity (Nakatsuji et al., 2016). This paper also shows that S. aureus can penetrate the epidermis and dermis of AD lesional skin, but is not detected in normal skin. We find that epicutaneous LTA application did not induce epidermal proliferation or immune cell infiltration in normal mouse skin (data not shown). We hypothesize that epicutaneously applied LTA is unable to penetrate the skin barrier and reach epidermal keratinocytes. Therefore, in the absence of ID entry, additional factors, such as wounding or a barrier defect, are likely required for LTA mediated effects. LTA may also affect other cells of the skin, including dendritic cells, by inducing cytokine expression (Iwamoto et al., 2018, Volz et al., 2018). These cytokines may in turn, also modulate keratinocyte responses in vivo.

In contrast to the inflammatory effects shown herein, LTA can also mediate inhibitory effects, blocking TNF expression (Lai et al., 2009) and inhibiting inflammation induced by P. acnes (Xia et al., 2016). The inhibitory effects of LTA may be a consequence of TLR induced tolerance, the unresponsiveness of cells following repeated TLR stimulation (Foster et al., 2007). Alternatively, it has been shown that differing LTA structures can mediate differing inflammatory effects (Lai et al., 2009).

Finally, we observe an essential role for IL-1R1 in mediating certain LTA induced events. We find that the IL-1R1 antagonist, Anakinra, blocks neutrophil infiltration in the skin by 80%, These studies are consistent with studies showing that lesions from S. aureus infected IL-1R1 knockout mice have severely decreased recruitment of neutrophils (Miller et al., 2006). Ultimately, we note that the decreased neutrophil recruitment caused by IL-1R1 blockade correlates with recovery of filaggrin and loricrin expression. However, it is notable that IL-1R1 mediated neutrophil blockade is incomplete. Therefore, it remains possible that IL-36α, which is not blocked by IL-1R1, could also be involved in LTA mediated recruitment. Finally, IL-1R1 blockade did not affect epidermal thickness, indicating that not all effects of LTA are mediated by this receptor.

In conclusion, we identify LTA as a key molecule contributing to S, aureus induced skin inflammation. We also find that LTA treated skin shares some striking similarities with AD skin. Although inflammation is required to protect against the effects of pathogenic bacteria, it also has an injuring effect on the skin, leading to damage to the epidermal barrier and reduced differentiation of keratinocytes. Our findings demonstrate that bacterial cell wall product of S. aureus can have a significant impact in the skin and cause potentially negative effects on barrier function.

MATERIALS AND METHODS

Keratinocyte cell culture and treatments

Primary human keratinocytes (Cascade Biologics; Grand Island, NY) were grown in serum-free keratinocyte growth medium (EpiLife; Cascade Biologics), with 1% human keratinocyte growth supplement (Cascade Biologics), 0.06 mM CaCl2, and antibiotics. Keratinocytes were plated at 100,000 per well in a 24 well plate, coated with collagen matrix (Cascade Biologics) and were allowed to adhere overnight before treatment. When indicated, cells were treated with 10 μg/ml S. aureus derived LTA (InvivoGen; San Diego, CA cat #tlrl-pslta). The manufacturer states that LTA was purified such that the endotoxin level is less than 0.001 EU /μg (Morath et al., 2001).

Quantitative real-time PCR (RT-PCR)

Total RNA was isolated by RNeasy Mini Kits (Qiagen, Valencia CA) according to the manufacturer’s protocol. One microgram of RNA was reverse transcribed using the Qiagen Quantiscript kit according to manufacturers protocol. RT-PCR was performed and analyzed by the dual-labeled fluorogenic probe method by using an ABI Prism 7300 sequence detector (Applied Biosystems, Foster City CA). Probes for human IL-1a, IL-1b, IL-8, IL-33, IL-36a, CXCL1, CXCL2, MMP-1, and beta actin as well as mouse Il-1a, Il-1b, Il-36a, Cxcl1, Cxcl2, Ly-6G and beta actin were purchased from Applied Biosystems. Amplification reactions were performed in MicroAmp optical plates (Applied Biosystems) in a 25-μL volume. All reactions were normalized to beta actin.

siRNA transfection

Second-passage keratinocytes of 50–60% confluence were transfected according to the manufacturer’s instructions using Lipofectamine 2000 (Invitrogen; Grand Island, NY) with 20 μM non-targeting (microarray tested for minimal targeting of human genes), or p63, Smartpool siRNA (Dharmacon- Fisher Scientific; Pittsburgh PA). To ensure viability after p63 silencing, cells were not trypsinized after transfection.

Cell staining and Microscopy

Paraffin-embedded skin biosies were cut at 5 μm on frosted microscope slides. Slides were deparaffinized and then rehydrated. Skin sections were then blocked with 5% BSA in Super Block (ScyTek Laboratories, Logan, UT). Slides were then stained with antibodies directed against Loricrin (Abcam, Cambridge, MA; cat #Ab85679, 1:500); Filaggrin (Biolegend, Dedham, MA; cat #905801, 1:500); Keratin 5 (Biolegend, cat #90550, 1:2000); IL-1b (Biotechne, Minneapolis MN; cat #AF-401, 1:500); IL-1a (Biotechne, cat #AF-400, 1:1000), IL-36a (Biotechne, cat #AF-2297, 1:1000); Ki67 (EMD Millipore Corporation, Temecula CA; cat #AB9260 1:200), Myeloperoxidase (ThermoFisher, Rockford, IL; cat #PA5–16672, 1:200) or equal amounts of isotype control antibodies. Slides were incubated at 4°C overnight and then washed with PBS /Tween 0.05%. Secondary Cy-3 conjugated antibody (Jackson Labs; West Grove, PA) was added for 1 hour and cell nuclei were visualized with DAPI (Sigma). Images were taken with a Leica Microscope (Wetzlar, Germany) at 40x magnification using SlideBook software (Intelligent Imaging Innovations, Denver, CO). Mean fluorescence intensity (MFI) was determined for each exposure group using SlideBook software and was reported as mean MFI ± SE.

Mice

All procedures performed on mice were in accordance with the NIH guidelines for humane treatment of animals and were approved by the IACUC of National Jewish Health. Mice were kept under pathogen free conditions. Intradermal injections were performed on C57BL/6 x FVB mice aged 2–6 months. Mice were briefly anesthetized with Isoflurane, shaved, and injected with 200 micrograms (unless otherwise indicated) of LTA in a 100 μl volume of PBS into the back. 48 hours later, mice were euthanized by carbon dioxide inhalation, and the treated skin region was excised using a 6 mm biopsy punch (Miltex Inc.). Epicutaneous application was performed by anesthetizing with Isoflurane, shaving, followed by 5 tape strips and application of 100 micrograms LTA in a 100 μl volume of PBS in a Tagaderm dressing. Mice were harvested 48 hours later. Biopsies were then preserved in TRI reagent (MilliporeSigma) for RNA extraction, or in formalin buffered saline for paraffin-embedding for Histology. Administration of the IL-1 Receptor 1 antagonist, Anakinra (Sobi, Stockholm Sweden) was performed by 3 separate ID injections of 250 μg of active Anakinra in a 100 μl volume. The first injection was 24 hours prior to LTA treatment, the second injection was a co-treatment with LTA, and the third injection was 24 hours post LTA treatment. Mice were harvested 24 hours after the final injection.

Statistical analyses.

All statistical analysis was conducted using Graph Pad Prism. Comparisons of expression levels were performed using analysis of variance (ANOVA) techniques and Student’s t tests as appropriate.

Data availability statement.

Not applicable as there were no datasets used in this study.

Supplementary Material

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ACKNOWLEDGEMENTS

We wish to thank Desiree Garcia, Samantha Crow and Cliff Hall for expert technical support. This study was supported by NIH/NIAID grant U19 AI117673, NIH/NIAMS grant R01 AR41256, and The Edelstein Family Chair of Pediatric Allergy-Immunology at National Jewish Health.

Abbreviations

AD

Atopic Dermatitis

CXCL

Chemokine (C-X-C motif) ligand

Flg

Filaggrin

ID

Intradermal

IL

Interleukin

IL-1R1

Interleukin 1 receptor type 1

LTA

Lipoteichoic Acid

Lor

Loricrin

MMP-1

Matrix metalloproteinase-1

RT-PCR

Real Time PCR

Th2

T helper type 2

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1521954-supplement-1.tif (38.7MB, tif)
2
NIHMS1521954-supplement-2.tif (38.6MB, tif)
3
NIHMS1521954-supplement-3.tif (38.7MB, tif)
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NIHMS1521954-supplement-4.tif (38.6MB, tif)
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NIHMS1521954-supplement-5.tif (38.6MB, tif)
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NIHMS1521954-supplement-6.tif (38.6MB, tif)
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NIHMS1521954-supplement-7.tif (19.3MB, tif)
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NIHMS1521954-supplement-8.tif (38.7MB, tif)

Data Availability Statement

Not applicable as there were no datasets used in this study.

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