|Home | About | Journals | Submit | Contact Us | Français|
Atopic dermatitis (AD) is characterized by scratching and by Th2-dominated immune response to cutaneously introduced antigens. Antigen application to skin mechanically injured by tape stripping results in Th2-dominated skin inflammation.
To examine the effect of tape stripping on the capacity of skin dendritic cells (DCs) to polarize T cells towards a Th2 phenotype.
CD11c+ DCs were isolated from skin of BALB/c or C57BL/6 mice. FITC+ and FITC− DCs were isolated from draining lymph nodes (DLN) 24 hrs after painting the skin with FITC. DCs were assessed for their ability to induce cytokine secretion by OVA stimulated naïve CD4+ T cells from TCR-OVA transgenic DO11.10 mice. Cytokine mRNA levels were examined by quantitative PCR.
DCs isolated from the skin of wild-type (WT), but not TSLPR−/− or IL-10−/−, mice 6 hrs after tape stripping elicited significantly more IL-4 and IL-13, and significantly less interferon-γ production by CD4+ cells, than DCs isolated from unmanipulated skin, and expressed significantly more mRNA for the Th2 skewing molecules IL-10, Jagged1, and Jagged2, but significantly less mRNA for the Th1 skewing cytokine IL-12. CD11c+FITC+ cells isolated from DLN of shaved and tape stripped skin of WT, but not TSLPR−/− or IL-10−/−, mice polarized T cells significantly more towards Th2 and expressed significantly more IL-10, Jagged1 and Jagged2 mRNA than CD11c+FITC+ cells isolated from DLN of shaved skin. Tape stripping significantly increased TSLP levels in the skin, and TSLP was shown to play an essential role in the Th2 polarization of skin DCs by tape stripping.
Tape stripping upregulates TSLP levels in the skin, which polarizes skin DCs to elicit a Th2 response via the induction of IL-10.
Mechanical injury inflicted by scratching in patients with AD may cause skin DCs to polarize T cells to secrete Th2 cytokines.
Mechanical injury to the skin, such as that caused by scratching in AD, may cause skin DCs to polarize T cells to secrete more allergy-mediating Th2 cytokines.
Atopic dermatitis (AD) is characterized by skin inflammation with dermal infiltration by T cells and eosinophils 1. Acute AD skin lesions express the Th2 cytokines IL-4, IL-5, and IL-13, while chronic lesions also express the Th1 cytokine interferon (IFN)-γ 2. The majority of AD patients exhibit systemic Th2 dominated immune responses with elevated serum IgE and evidence of antigen specific IgE 3.
The hallmark of AD is dry itchy skin due to a disrupted skin barrier with increased transepidermal water loss 4, 5. This results in intense scratching, which inflicts mechanical injury to the skin, and entry of antigens otherwise excluded by a normal skin barrier. Epicutaneous (EC) sensitization with allergens plays an important role in the pathogenesis of AD, illustrated by the observation that application of allergen to the abraded uninvolved skin of patients with AD provokes an eczematous rash with eosinophilic infiltration 6.
Dendritic cells (DCs) are essential for the generation of an immune response. Langerhans cells form a DC network in the epidermis where they sample antigens that get through the skin barrier. Antigen that reaches the interstitial spaces is taken by dermal DCs. Following antigen uptake, and in the presence of danger signals generated by microbial antigens from the skin flora, and/or mechanical injury, inflammatory mediators such as IL-1 and TNF-α are released 7, 8. These cause the conversion of the immature skin DCs into mature professional antigen-presenting cells by upregulating the expression of MHC class II molecules and key co-stimulatory molecules, such as CD80 and CD86. DCs that capture antigen in the skin express CCR7, and migrate to draining lymph nodes (DLN), which express the chemokines CCL19 and CCL21 9, where they present antigenic peptides to naïve recirculating T cells, to cause T cell proliferation and differentiation. In the case of CD4+ T cells, differentiation leads to the generation of Th1 cells that secrete IFN-γ, Th2 cells that secrete IL-4, IL-5, and IL-13 and other Th subsets, which include Th17, Th22 and Th9 10–12.
Tape stripping inflicts mechanical injury and disrupts the skin barrier, features shared with AD skin. We, and others, have shown that application of protein antigen to tape stripped skin results in Th2 dominated skin inflammation13–15. Furthermore, application of the hapten picryl chloride to tape stripped skin skews the Th2:Th1 ratio towards Th2, compared to application to intact skin 14. In this study we examined the effect of tape stripping on the polarization of skin DCs to elicit a Th2 response in antigen specific T cells.
BALB/c were from Charles River Laboratory (Wilmington, MA). DO11.10 TCR transgenic mice on BALB/c background and TSLPR−/− mice on BALB/c background were bred in our facility. IL-10−/− mice on BALB/c background were a gift from Dr. Dale Umetsu (Children’s Hospital, Boston). All procedures performed were in accordance with the Animal Care and Use Committee of the Children’s Hospital Boston.
A Tegaderm patch (Westnet Inc.) was applied to shaved skin, then immediately ripped off. The procedure was repeated for 6 times on dorsal skin and 12 times on ear skin.
Preparation of ear cells was performed as described previously16. Single cell suspensions were incubated with Fc block (2.4G2), stained with mAbs for B220, CD11b, CD11c, CD19, CD40, CD80, CD86, Langerin, and MHC class II (eBioscience), then ran on FACSCanto (Becton Dickinson) and results were analyzed using FlowJo software (Tree Star Inc.).
Dorsal skin of mice was tape-stripped followed by application of 100 μL of 10 mg/ml FITC dissolved in 1:1 acetone/dibutylphthalate (DBP). Inguinal and axillary lymph nodes were removed 2, 6, 24, and 48 hrs after FITC application, and incubated with 300 U/ml type I collagenase (Worthington Biochemical) and 100 U/ml DNase I (Sigma) in PBS at 37 °C for 60 min. After filtration through 70 μm cell strainer, single cell suspensions were stained with mAbs and analyzed as described above.
CD11c+ cells were isolated to >95% purity by CD11c+ Microbeads (Miltenyi Biotec). Purified cells were stained with PE-conjugated CD11c antibody (eBioscience) and CD11c+FITC+ or CD11c+FITC− cells were sorted on FACS Vantage SE (BD). Splenic CD4+ T cells from DO11.10 mice were purified to > 98% purity by CD4+ Microbeads using AutoMacs (Miltenyi Biotec). 1 × 105 CD4+ T cells and 1 × 104 DCs were cultured with 2 μM of OVA peptide323–339 (OVAp) for 96 hrs in RPMI 1640 (Invitrogen)/10% FCS. Proliferation was assessed by the incorporation of 1 μCi/well [3H]-thymidine during the last 8 hrs of 96 hrs cultures. Cytokines were measured in supernatants. by ELISA kits (IL-4 and IFN-γ eBioscience; and IL-13 R&D Systems).
RNA extraction, reverse transcription, PCR reactions, and calculation of relative gene expression to that of the housekeeping gene GAPDH, were performed as previously described 17.
Skin was homogenized using Polytoron RT-3000 (Kinematica: AG) in T-PER tissue protein extraction reagent (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). Protein concentration was quantified with Nanodrop (Thermo Scientific). TSLP was measured by ELISA (R&D systems).
Statistical significance was determined by using GraphPad Prism, version 4.0a (Graphpad Software). Statistical differences were determined by student’s t test (between 2 groups) and one-way analysis of variance (ANOVA) (between multiple groups). A p value of < 0.05 was considered to indicate statistical significance.
DCs were isolated from ear skin of BALB/c mice 6 hrs after tape stripping and compared to DCs isolated from unmanipulated ear skin. We used the 6 hrs time point in order to capture DCs from injured skin prior to their migration to LN, and at a time when expression of inflammatory cytokines (IL-1, IL-6 and TNF-α) in tape stripped skin has peaked 7, 8, 18, 19. The percentages of CD11c+ DCs in tape stripped and unmanipulated skin were comparable (Fig. 1A). The two DC populations were comparable in subsets composition including myeloid DCs (CD11chiCD11b+B220−CD19−Langerin−), plasmacytoid DCs (CD11cloCD11b−B220+CD19−Langerin−), and Langerhans cells (CD11c+CD11b+B220−CD19−Langerin+) (Fig. 1B). DCs from unmanipulated and tape stripped ear skin had comparable surface expression of CD40, CD80, CD86, MHC class II, and OX40L (Fig. S1A). Tape stripping was previously shown to induce CCR7 expression by skin DCs 19.
DCs from tape stripped skin induced CD4+DO11.10 T cells, stimulated with OVAp, to secrete significantly more IL-4 and IL-13 compared to DCs from unmanipulated skin (Fig. 1C). Induction of IFN-γ secretion, proliferation, and kinetics of 3H-thymidine incorporation into DNA by the two DC populations were comparable (Fig. 1C, 1D, and S1B).
DC-derived IL-10 and the Notch ligands Jagged1 and Jagged2 have been implicated in Th2 cell differentiation 20, 21, 22. DCs from tape stripped ear skin of mice expressed significantly higher levels of IL-10, Jagged1, and Jagged2 mRNA than DCs from unmanipulated ear skin (Fig. 2). In contrast, they expressed significantly lower amounts of mRNA for IL-12, which is important for Th1 cell differentiation 23, 24.
As previously reported 25, 26, FITC painting of shaved back skin of BALB/c mice caused the appearance of CD11c+FITC+ DCs in DLN starting at 6 hrs and peaking at 24 hrs (Fig. 3A). The percentage of CD11c+FITC+ DCs in DLN of shaved and tape stripped (STS) skin peaked earlier (6 hrs) and at a higher level, but by 24 hrs, the percentages and subset composition of CD11c+FITC+ DCs in DLN of shaved and STS were comparable (Fig. 3B and C). The two DC populations also expressed comparable levels of CD40, CD80, CD86, and OX40L, but CD11c+FITC+ DCs from DLN of STS skin expressed higher levels of MHC class II and CCR7, consistent with more vigorous mobilization from the skin (Fig. 3D). CD11c+FITC+ DCs from DLN of both STS skin and shaved skin expressed higher levels of CD40, CD80, CD86, and MHC class II than their CD11c+FITC− counterparts (Fig. S2).
CD11c+FITC+ cells purified from DLNs of STS skin induced significantly higher secretion of IL-4 and IL-13, and lower secretion of IFN-γ than CD11c+FITC+ cells from DLNs of shaved skin (Fig. 3E). CD11c+FITC+ cells from DLNs of both STS and shaved skin induced significantly higher secretion of Th2 cytokines and lower secretion of IFN-γ, compared to their CD11c+FITC− counterparts. CD11c+FITC− cells from DLN of STS and shaved skin were comparable in their ability to induce the secretion of Th2 and Th1 cytokines. There were no significant differences between any of the CD11c+ subpopulations in their ability to support T cell proliferation (Fig. 3F).
The transcription factors GATA-3 and T-bet play critical roles in driving the differentiation of naïve CD4+ T cells into Th2 and Th1 cells respectively 27, 28. CD11c+FITC+ DCs from DLNs of STS skin induced significantly higher expression of GATA-3 mRNA and lower expression of T-bet mRNA in T cells, compared to CD11c+FITC+ DCs from DLNs of shaved skin (Fig. 3G). CD11c+FITC+ cells from DLNs of both STS and shaved skin induced significantly higher GATA-3 mRNA and lower T-bet mRNA than their CD11c+FITC−counterparts (Fig. 3G). Expression of GATA-3 and T-bet mRNA in T cells cultured with CD11c+FITC− DCs from STS and shaved skin were comparable.
CD11c+FITC+ DCs from DLN of STS skin of BALB/c mice expressed significantly higher levels of IL-10, Jagged1, and Jagged2 mRNA, and lower levels of IL-12 mRNA compared to CD11c+FITC+ DCs from DLN of shaved skin (Fig. 4). The same differences were seen when CD11c+FITC+ DCs were compared to their CD11c+FITC−counterparts in DLN of STS skin. Expression of IL-10, Jagged1, and Jagged2 mRNA by CD11c+FITC+ DCs and CD11c+FITC− DCs from DLN of shaved skin were comparable. CD11c+FITC+ DCs from shaved skin expressed lower levels IL-12 mRNA compared to their CD11c+FITC− counterparts.
Shaving and tape stripping of back skin caused a significant increase in TSLP levels after 6 hrs, with a return to baseline by 24 hrs (Fig. 5A). Shaving alone caused no detectable increase in skin TSLP levels either at 6 hrs or 24 hrs. We used TSLPR−/− mice to examine the role of TSLP in Th2 polarization of skin DCs by tape stripping. DCs from unmanipulated and tape stripped ear skin of TSLPR−/− mice expressed comparable levels of CD40, CD80, CD86, MHC class II and OX40L to their counterparts isolated from WT controls (Fig. S3). DCs isolated from tape stripped skin of TSLPR−/− mice did not promote IL-4 and IL-13 production by OVAp stimulated CD4+DO11.10 cells, compared to DCs isolated from unmanipulated skin (Fig. 5B). Lack of TSLPR had no detectable effect on the capacity of DCs from unmanipulated or tape stripped skin to drive IFN-γ production.
DCs from tape stripped skin of TSLPR−/− mice failed to upregulate expression of IL-10, Jagged1, and Jagged2 mRNA compared to DCs from unmanipulated ear skin (Fig. 5C). Addition of recombinant TSLP failed to upregulate IL-10, Jagged1, and Jagged2 mRNA expression in DCs isolated from unmanipulated skin of WT mice (Fig. S4), suggesting that TSLP synergizes with other factors to cause upregulation of these molecules.
CD11c+FITC+ DCs from DLN of FITC painted STS skin of TSLPR−/− mice did not promote IL-4 or IL-13 production by OVAp stimulated CD4+DO11.10 cells (Fig. 5D), and did not exhibit a diminished capacity to drive IFN-γ production. There was no detectable difference between CD11c+FITC− DCs from DLN of FITC painted shaved skin of TSLPR−/− and WT mice in supporting cytokine production by T cells (data not shown).
IL-10 expression by antigen pulsed splenic DCs is important for their ability to cause recipient mice to mount a Th2 response to EC challenge with antigen 20. DCs from tape stripped skin of IL-10−/− mice did not promote the production of IL-4 and IL-13 by OVAp stimulated CD4+DO11.10 cells, compared to DCs from unmanipulated skin (Fig. 6A). DCs from tape stripped and unmanipulated skin of IL-10−/− mice induced T cells to secrete higher amounts of IFN-γ than their WT counterparts. There was no difference between DCs isolated from unmanipulated skin of IL-10−/− and WT mice in supporting cytokine production by T cells (data not shown).
IL-10 mRNA expression was upregulated in DCs from tape stripped skin of WT mice but, as expected, was undetectable in IL-10−/− mice (Fig. 6B). DCs from tape stripped skin of IL-10−/− mice failed to upregulate the expression of Jagged1 and Jagged2 mRNA (Fig. 6B).
CD11c+FITC+ DCs from DLN of STS skin of IL-10−/− mice failed to enhance IL-4 and IL-13 production and to inhibit IFN-γ production compared to CD11c+FITC+ DCs from DLN of shaved skin of these mice (Fig. 6C). The latter elicited comparable secretion of IL-4 and IL-13, but significantly higher IFN-γ production, by T cells compared to their WT counterparts (Fig. 6C). There was no difference in the polarizing ability of CD11c+FITC−DCs from DLNs of IL-10−/− mice and WT controls (data not shown), and no significant difference between CD11c+FITC+ DCs isolated from DLN of STS skin of IL-10−/− and WT mice in supporting T cell proliferation (data not shown).
We have demonstrated that mechanical injury inflicted by tape stripping polarizes skin DCs to elicit a Th2 response and that TSLP is essential for this polarizing effect.
Tape stripping, which disrupts the skin barrier, upregulates mRNA expression of several cytokines including IL-10 and IL-21 18, 19, the levels of which are also upregulated in AD skin lesions. Furthermore, application of OVA patches to tape-stripped skin of WT mice mimics many features of AD 15, 19. These observations suggest that tape stripping of mouse skin might mimic the trauma induced by scratching in AD.
We demonstrate that DCs from tape stripped skin and FITC+ DCs from DLN of tape stripped skin are polarized to drive a Th2 response, compared to their counterparts from non-injured skin (Fig. 1, ,3).3). Since FITC+ DCs in DLN of FITC-painted skin likely represent skin DCs emigrants, the data indicates that mechanical injury exerts a Th2 skewing effect on skin DCs, including those that migrate to DLN, where they present antigen to re-circulating T cells. In addition, FITC+ DCs from DLN of injured skin inhibit Th1 skewing. Inhibition of Th1 polarization was not evident in DCs isolated from skin 6 hrs after tape stripping, possibly because it might have required a longer time to be manifest.
Increased CD86 expression by DCs may favor Th2 polarization, while increased expression of CD80 and CD40 may enhance Th1 polarization 29, 30. DCs isolated directly from tape stripped skin versus unmanipulated skin, and FITC+ DCs isolated from DLN of FITC-painted STS skin versus shaved skin had comparable expression of CD40, CD80 and CD86 costimulatory molecules, DC subset composition, and ability to support T cell proliferation to antigen (Fig. 1, ,3,3, S1). Thus, differences in the expression of costimulatory molecules, DC subsets distribution, or ability to present antigen could not have accounted for the Th2 polarizing effect of mechanical injury on skin DCs. The failure to detect upregulation of costimulatory molecules on DCs isolated from skin 6 hrs after tape stripping, suggests that this time period may not have been sufficient to detect upregulation of these molecules following mechanical injury. FITC+DCs from shaved skin showed higher expression of maturation markers than FITC− DCs (Fig. S2). This is possibly mediated by TSLP, because TSLP skin levels increase following painting with FITC in acetone:DBP solvent 31–34, and TSLP upregulates CD40, CD80, CD86, and MHC class II expression on human DCs35. Comparable upregulation of costimulatory molecules on FITC+ DCs from DLN of FITC-painted STS and shaved skin suggests that additional induction of TSLP by tape stripping could not further upregulate the expression of these markers induced by FITC painting alone. An additional effect of tape stripping was vigorous mobilization of skin DCs to DLN, possibly because it induces expression of IL-1β and TNF-α which promote DC migration to DLN 7, 8, 38.
DCs isolated directly from injured skin and skin-derived FITC+ DC emigrants isolated from DLN of FITC-painted tape stripped skin upregulated the expression of mRNA for the Th2 polarizing molecules IL-10, Jagged1, and Jagged2 (Fig. 2, ,4).4). Conversely, they downregulated the expression of mRNA for the Th1 polarizing cytokine IL-12. A number of studies, but not all, have implicated Jagged1 and Jagged2 expression by DCs in Th2 polarization 21, 22, 39–42. Further experiments are needed to elucidate the role of these Notch ligands in the Th2 polarization of skin DCs by mechanical injury. Given the pivotal role of IL-12 in driving Th1 polarization 23, 24, decreased expression of IL-12 mRNA by CD11c+FITC+ DCs in DLN of STS skin likely underlies their impaired ability to drive Th1 cells and may have contributed to their Th2 skewing capacity 43.
Tape stripping induced skin DC and skin DC emigrants in DLN to induce T cells to secrete Th2 cytokines in the Th1 biased C57BL/6 background (Fig. S5 and Supplemental text). The only difference in the response of skin DCs compared to BALB/c mice was that FITC+DCs from both STS and shaved skin induced higher amount of IFN-γ secretion in T cells than their FITC− counterparts. This is consistent with the observation that IFN-γ mRNA expression is upregulated in skin of C57BL/6, but not BALB/c mice, following EC sensitization with OVA 15, 44.
Shaving followed by tape stripping, but not shaving alone, caused a significant increased in skin TSLP levels within 6 hrs, consistent with upregulation of TSLP expression in the skin by tissue damage 45. Mechanical injury induces the expression of inflammatory cytokines in the skin such as IL-1, TNF-α and IL-33 (7, 8, 18, 19, 46 and data not shown) that induce the expression of TSLP. DCs isolated directly from injured skin of TSLPR−/− mice and as FITC+ skin DC emigrants from DLN of FITC-painted injured skin of these mice failed to cause Th2 polarization compared to control DCs derived from uninjured skin of TSLPR−/− mice (Fig. 5). These results indicate that TSLP released in the skin following mechanical injury is essential for the Th2 polarizing effect of mechanical injury on skin DCs. DCs from tape stripped skin of TSLPR−/− mice failed to upregulate mRNA expression of the putative Th2 skewing molecules IL-10, Jagged1, and Jagged2, suggesting that failure to upregulate these molecules contributed to the failure of these DCs to cause Th2 skewing. We did not detect a difference in OX40L expression between DCs isolated from tape stripped skin of TSLPR−/− mice and WT controls, and in a previous study found no differences in OX40L expression by FITC+ DCs isolated from DLN of FITC painted shaved skin of TSLPR−/− mice and WT controls 17.
Painting skin with FITC in acetone:DBP upregulates TSLP expression 31–34. This may explain why FITC+DCs from DLN of FITC painted shaved skin expressed less IL-12 and were more potent in causing Th2 skewing than FITC− DCs isolated from the same LNs (Fig. 4). At the DC:T ratio of 1:10 we used in this study and in a previous study 17, we detected no difference between CD11c+FITC− DCs isolated from DLN of FITC painted shaved skin of TSLPR−/− mice and WT controls in their ability to support Th2 cytokine production by T cells. However, in a recent study, in which a lower DC:T ratio of 1:25 was used, we observed a modest (<20%), but significant decrease in the ability of CD11c+FITC+ DCs isolated from DLN of FITC painted shaved skin of TSLPR−/− mice to support Th2 cytokine production by T cells 31, suggesting a role for TSLP in the Th2 response to FITC painting of the skin in the absence of tape stripping.
An important role of TSLP in the pathogenesis of AD has been shown in several mouse models. K5-TSLP transgenic mice develop an AD-like dermatitis, and elevated serum levels of IgE 47. Selective ablation of Retinoid-X-Receptors in epidermal keratinocytes induces TSLP expression in epidermis and triggers an AD-like skin lesions in mice 48. Induced expression of TSLP by keratinocytes is essential for the pathogenesis of an AD-like dermatitis triggered by topical application of MC903, a low-calcemic analogue of vitamin D3 49. However, TSLPR−/− mice mounted a normal systemic Th2 response to EC sensitization with OVA 17, suggesting that factors other than TSLP are important for Th2 polarization in this chronic sensitization model. Nevertheless, local blockade of TSLP by a neutralizing antibody inhibited the development of allergic skin inflammation following skin challenge of EC sensitized WT mice, and cutaneous Th2 cytokine expression was severely impaired in EC sensitized TSLPR−/− mice 17, 32, suggesting that TSLP is important for Th2 cytokine secretion by skin-infiltrating T cells.
DCs isolated from injured skin of IL-10−/− mice failed to polarize a Th2 response (Fig. 6). Since tape stripping did not induce IL-10 expression in skin DCs from TSLPR−/− mice, these observations suggest that TSLP dependent IL-10 expression by DCs from mechanically injured skin may be important for their Th2 polarizing activity. However, we cannot rule out a role for keratinocyte derived IL-10. TSLP by itself did not upregulate IL-10 expression in mouse DCs, as in human DCs 50, and thus, although necessary, is not sufficient to induce IL-10 expression in mouse DCs.
Recently, it was reported that application of dust mite antigens to the skin causes accumulation of basophils in DLN, which have been proposed to play an important role in Th2 skewing 51–54. We did not detect accumulation of basophils in skin DLN 24 hrs after shaving or shaving and tape stripping, as assessed by enumerating cells that express FcεRI and CD49b (data not shown). Thus, it is unlikely that basophils contribute to the Th2 polarizing effect of mechanical injury.
Scratching of the skin elicits skin flares in patients with AD and levels of TSLP and IL-10 are increased in AD skin lesions 55, 56, 57. Our results suggest that scratching of dry skin with a disrupted barrier in AD may polarize skin DCs to secrete IL-10 and elicit a Th2 response by upregulating local expression of TSLP. Together, with the previously demonstrated role of TSLP in promoting Th2 cytokine secretion by skin infiltrating T cells 17, 20, our findings suggest that neutralization of TSLP may provide a therapeutic strategy that targets both the induction and effector arms of the immune response to cutaneously introduced antigens in AD.
Funding: This work was supported by NIH grants AR-047417, N01-AI-40030, AI068731, AR055695, and AR056113, and R.P.L. was supported by training grant CA009537, T32-GM007270, and the Howard Hughes Institute Med into Grad initiative through a grant to UW.
The authors thank Mr. Christopher Lewis for technical assistance, Dr. Seiji Kawamoto for initiating this work, and Dr. Dale T. Umetsu for his kind gift of IL-10−/− mice.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.