- Email: [email protected]
IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes
Accepted Manuscript Title: IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes Author: Ran Yu Yumei Ding Lijuan Zhu Yinying Qu Chenguang Zhang Lin Liu Lili Chen PII: DOI: Reference:
S0003-9969(16)30193-5 http://dx.doi.org/doi:10.1016/j.archoralbio.2016.08.001 AOB 3656
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
3-5-2015 14-7-2016 2-8-2016
Please cite this article as: Yu Ran, Ding Yumei, Zhu Lijuan, Qu Yinying, Zhang Chenguang, Liu Lin, Chen Lili.IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.08.001 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 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.
TITLE PAGE
IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes
Ran Yu1*, Yumei Ding1*, Lijuan Zhu1, Yinying Qu2, Chenguang Zhang1, Lin Liu2# and Lili Chen1#
1.Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China 2. Dalian Stomatological Hospital of Dalian Medical University, 116021Dalian, China
*, equal contribution: The first two author contributed equally to this work.
#
, corresponding author: The last two authors are the corresponding authors.
Highlights
IL-22 and STAT3 were involved in oral mucosal wound healing, and the value change tendency of Il-22 and STAT3 during the wound healing was similar.
STAT3 was activated after the keratinocytes or the tongue tissue were stimulated by IL-22,
IL-22 could obviously promote the proliferation of keratinocytes, but not fibroblasts.
IL-22 could mediate gene expression in keratinocytes, such as type I collagen and MMP-1 which may contribute to re-epithelialization and scarless healing in oral wound healing.
Abstract Objective Wounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity.
Design The function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22，collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes.
Results IL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1，which may contribute to scarless healing.
Conclusion Our study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing.
Key words: Wound healing, IL-22, STAT3
ARTICLE PROPER 1. Introduction Repair following injury involves several processes, such as inflammation, re–epithelialization, scar formation. Interleukin-22 (IL-22), which is released by leukocytes, such as Th1, Th17, Th-22, and NK cells (Aujla & Kolls, 2009; Liang et al., 2010; Wolk & Sabat, 2006; Wolk et al., 2011), is an IL-10 cytokine family member, such as IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28a/b, IL-29, and the type I IFN (Donnelly, Sheikh, Kotenko, & Dickensheets, 2004; Pestka et al., 2004). It has been shown that the IL-22R1 subunit expression decides whether a cell is a target of IL-22 or not (Sabat, Witte, Witte, & Wolk, 2014). In resting and activated immune cells including monocytes, B cells, T cells, NK cells, macrophages, and DC populations, as well as bone marrow, blood mononuclear cells, thymus, and spleen, there is a lack of IL-22R1 expression(Kunz et al., 2006; Wolk et al., 2004; Wolk et al., 2005; Wolk et al., 2008), and signal transduction induced by IL-22 does not occur in any of these cells. Therefore, IL-22 does not have any influences on these cells in vitro and in vivo (Wolk et al., 2004). In contract, IL-22R1 is expressed in keratinocytes, bronchial and intestinal epithelial cells, intestinal subepithelial myofibroblasts, hepatocytes, pancreas acinar cells, and a range of respective tissue cell-derived cell lines (Wolk & Sabat, 2006; Wolk, Witte, Witte, Warszawska, & Sabat, 2010). IL-22, a critical mediator in mucosal host defense can promote keratinocyte migration, enhance the reconstitution of human epidermis and restrain a set of differentiation-related Ags (Aujla & Kolls, 2009; Boniface et al., 2005; Pickert et al., 2009). Through enhanced regulation of the expression of numerous antimicrobial molecules such as S100A7, S100A8, S100A9 and Matrix metalloproteinase-1 (MMP-1), and the b-defensin 2 (BD 2) and BD3, IL-22 increases the innate immunity of keratinocytes (Wolk et al., 2004; Wolk et al., 2011; Wolk et al., 2006). MMP-1 is the proteolytic enzyme of type I collagen, which plays a central role in extracellular matrix (ECM) remodeling and cell movement during the wound healing process. Many autoimmune disorders, such as psoriasis, rheumatoid arthritis, inflammatory bowel disease, or mucosal-associated infections have an association with IL-22 (Costa et al., 2013; Ouyang, 2010). High IL-22 levels have been found in tissue-repair processes, indicating that IL-22 plays a protective role in diseases such as myocarditis or tuberculosis (Behm, Babilas, Landthaler, & Schreml, 2012; Chang et al., 2006; Chen et al., 2013; McGee et al., 2013; Ye et al., 2012) STAT3 is the major and essential mediator of IL-22 signaling (Chang et al., 2006). STAT3 phosphorylation in Tyr705 and Ser727 residues is required when STAT3 is activated (Wen, Zhong, &
Darnell, J. E. Jr, 1995). Current studies have revealed that STAT3 is involved in psoriasis and colonic inflammation by IL-22-dependent mucosal wound healing (Aujla et al., 2008; Miyoshi et al., 2011). However, few studies are available on oral mucosal disease. This study will focus on the function of IL-22 in oral mucosal wound healing. We suggest that IL-22 regulate the oral mucosal wound healing via STAT3 in keratinocytes.
2. Materials and Methods
2.1 Mice All experiments involving male C57BL/6 mice were approved by the Wuhan University and maintained under specific pathogen-free conditions.
2.2 Experimental mouse models Wounds (length 5mm, depth 1mm) were made in the middle of the mouse tongues with a surgical knife blade. The depth was controlled by making a mark on the knife, 1mm from the knifepoint.. And mice were killed at 2h, 24h, 3d, 5d, or 7d after the treatment (n=5). Tongue samples were harvested and frozen for Western blot analysis, immunohistochemistry and real-time PCR.
2.3 In vitro keratinocytes culture Keratinocytes in primary culture were prepared from plastic surgery skin obtained from healthy volunteers, as previously described(Albanesi et al., 2000). Cells were cultured in six-well plates in the serum-free medium (KGM) for at least 3-5 days at 70-90% confluence before conducting experiments. In all experiments, third-passage keratinocytes were used. Stimulation with recombinant human or mouse IL-22 was performed in keratinocyte basal medium or fibroblast. Mouse fibroblast cell line NIH-3T3 was purchased from Wuhan University.
2.4 Scratch and proliferation assays Keratinocytes and fibroblasts were cultured in wells until they reached 100% confluence. A uniform cell-free area was created by scraping the cell monolayer with the tip of a p-200 pipette. Cell migration to the cell-free zone was measured at different time points of culture in the presence or absence of IL-22 (20 ng/ml), using an inverted phase contrast microscope. The effects of the IL-22 on fibroblast and keratinocyte proliferation were determined utilizing CCK-8 assay. Briefly, 5000 cells were seeded into the 96-well plates for 24 hours to adhere, and then stimulated with different concentrations of IL-22. We evaluated the cell proliferation in its logarithmic growth phases.
2.5 Western blot analysis Total proteins were extracted from tongue tissues and keratinocytes as previously reported 31, and the concentration of protein was determined using a BCA protein assay kit. After being subjected to SDS PAGE, proteins were transferred to PVDF membranes and probed with primary Abs diluted in TBST containing 5% nonfat dried milk. Western blot filters were developed using the ECL-plus detection system. The Abs employed for the study were as follows: anti-IL-22 (antgene), anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology), MMP-1, type I collagen (abclonal). Samples were normalized to GAPDH.
2.6 RNA isolation and real-time PCR Total RNA was extracted from tongue tissue using the TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions, and RNA was converted to cDNA using a reverse transcription kit (Thermo). Samples were heated at 42˚C for 60 min and 70˚C for 5min. Real-time PCR was performed on cDNA using SYBR green chemistry. GAPDH was used as an internal control. The sequences of primers and probes were as follows (table1):
2.7 Immunohistochemistry Firstly, the tissue section was dewaxed and rehydrated, After washing, the sections were incubated overnight at 4ºC with the MMP-1, collagen1, p-stat3 antibody. And then the slides were incubated with biotinylated secondary antibody for 30 minutes. After colourated with 3,3-diaminobenzidin (DAB), the
slides were counterstained for 1 minute with hematoxylin. At last, the slides were dehydrated with sequential ethanol washing 1 minute with 75%, followed by 80% and finishing with a 100% ethanol..
2.8 Histopathological analysis Tongue tissue were first fixed in 4% paraformaldehyde and then embedded in paraffin. Thin sections were obtained and stained with hematoxylin and eosin for routine histology.
2.9 Statistical analysis Data were presented as mean ± SEM from at least three independent experiments. Comparisons were evaluated using ANOVA, and differences were considered significant at the values of *, P < 0.05; **, P < 0.01; ***, P < 0.001.
2. Result
3.1 STAT3 was specifically induced by IL-22 in oral mucosa The wound healing process is always started by some cytokines via chemotactic signals to inflammatory and resident cells (Larjava, 2011). IL-22 can induce changes in systemic physiology and lead to an acute-phase response (Liang et al., 2010). To identify whether IL-22 and p-stat3 were involved in oral diseases, C57BL/6 mice were chosen and a wound model was made (Figure 1B). Firstly, H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues were made (Figure1A). The process of wound healing and the development of the horny layer could be observed. Then the expression of IL-22 and p-stat3 was confirmed by Western blot and real-time PCR at oh, 2h, 24h, 3d, 5d, 7d after wound being made (Figure2A and B). To study the connection between IL-22 and oral epithelial STAT3 activity, the tongue pieces from C57BL/6 mice were cultured in absence or presence of IL-22. Subsequently, p-stat3 was detected by immunostaining and Western blot analysis. As a result, treated tongue tissues showed higher expression levels of p-STAT3 compared with the untreated group (Figure2 D and E). A
further study for the effect of IL-22 on p-stat3 in
keratinocytes had been made,，and as we expected the p-STAT3 upregulated within 30min after IL-22 treatment (Figure2 C).
3.2 IL-22 could promote the proliferation and migration of keratinocytes in vitro
Depending on the target cells, STAT3 regulates signal transduction of several cytokines and proinflammatory mediators, such as IL-22 and IL-6 (Chapman et al., 2000; Levy & Darnell, 2002). We investigated the effect of IL-22 (20 ng/ml) on keratinocytes and fibroblasts by CCK-8 (Figure3 A and B) and scratch assays at different time point (Figure3 C). While IL-22 could obviously promote the proliferation of keratinocytes, it had little effect on fibroblasts.
3.3 Cytokine expression in keratinocytes during the wound healing in vivo. The wound healing process is a complicated process, which involves a range of processes, such as inflammation, formation of granulation tissue, re-epithelialization, and remodeling. The success of wound healing depends on chemokines, cytokines, and growth factors involved in a complex integration of signal networks. MMP-1 and type I collagen play an important role in wound healing. We confirmed the expression of IL-22 by Western blot analysis, Immunohistochemistry and real-time PCR (Figure4A, E and F). Western blot analysis and immunostaining were used to examine the activation of the STAT3 (Figure4 D and E). We have also examined the expression of type I collagen and MMP-1 (Figure4 B, C, E and F). The results showed that IL-22, MMP-1, collagen1 and STAT3 were involved in oral mucosal wound healing.
3.4 The effect of IL-22 on the expression of MMP-1 and collagen I Repair following injury involves several processes, such as inflammation, re–epithelialization, scar formation. The process of re–epithelialization occurred at the early stage of wound healing. The movement of epithelial in a wound setting is different from the migration of fibroblasts, angiogenic endothelial cells. It is a commonly thought that cell-matrix need to be broken to allow keratinocytes the freedom to move forward. MMP-1 is required for migration of keratinocytes on native type I collagen. And MMP-1 can contribute to resolution of scarring. To investigate how IL-22 could promote the migration of keratinocytes and the formation of scar, we stimulated the keratinocytes with IL-22, and then the expression of collagen I and MMP-1 were examined (Figure5 A and B). The results showed that IL-22 could induce the expression of MMP-1 and decrease the expression of type I collagen. The results suggested that IL-22 could promote the migration of keratinocytes at the early stage of
wound healing and contribute to scarless healing at the late stage of wound healing by induce the expression of MMP-1 via STAT3.
4. Discussion Th22, which is characterized by the production of IL-22, but not of IL-17 or IFN-γ, is the novel subpopulation of the effector CD4+ T cells (Duhen, Geiger, Jarrossay, Lanzavecchia, & Sallusto, 2009; Trifari, Kaplan, Tran, Crellin, & Spits, 2009). Th22 cells are the important source of IL-22 during later stages of inflammation. Being released as a signature cytokine in innate immunity, regeneration, and protection from damage, IL-22 plays either a pathogenic or a protective role in different situations. IL-22 is upregulated in a number of chronic inflammatory and autoimmune diseases, and the exact role of IL-22 appears to depend on the specific inflammatory microenvironment. The protective role of IL-22 has been found in a myocarditis model, whereas the inflammatory role of IL-22 has been demonstrated in rheumatoid arthritis. Through activation of STAT3-signaling cascades, the cytokine induces proliferative and anti-apoptotic pathways, as well as anti-microbial molecules, which help prevent tissue damage and aid in its repair (Lejeune et al., 2002; Wolk et al., 2010) In our previous studies, we have elucidated that the adipose stem cell-conditioned medium (ASC-CM) was of benefit to the migration and proliferation of fibroblasts, keratinocytes, and endothelial cells (Hu, Zhao, Liu, Gong, & Chen, 2013) In addition, IL-22 probably acts as an important role in ASC-CM because we found that the signal of IL-22 in ASC-CM exceeded 700-fold (Zhao, Hu, Liu, Gong, & Chen, 2013) IL-22 plays an important role in skin inflammatory processes and wound healing. However, oral mucosa and skin differ in keratinization patterns, resistance to external pressure, and so on. Oral mucosa, exposed to large amounts of antigens, is mostly nonkeratinized and moist with saliva at all times (Liu, Mao, & Chen, 2011). Until now, there has been little research about the effects and mechanism of IL-22 on oral mucosal diseases. Here, we determined the expression of IL-22 in the normal and wound mucosa. We found that there were two peaks of IL-22 expression after injury: an early peak with a maximum at 2 hour after wounding, and a second peak at about day 7 after wounding IL-22 was almost undetectable in tongue tissues on days 0, 1, and 3 after suffering injuries.
To explore the function of IL-22 on re-epithelialization in wound healing, we further stimulated both the fibroblasts and keratinocytes with IL-22. The results indicated that IL-22 promoted the migration and proliferation of keratinocytes while having little effect on fibroblasts. Taken together, these data indicate that IL-22 induces the re-epithelialization by promoting the migration of keratinocytes at the early stage of wound healing. Our experiment also focused on the connection between IL-22 and STAT3, MMP-1, type I collagen during wound healing. The results showed that the p-stat3 had the same regularity as the expression of IL-22 during wound healing. Both the expression of IL-22 and p-stat3 were obviously higher at 2h and 7d than on days 1 and 3. In addition, we stimulated the tongue mucosa and keratinocytes with IL-22 and detected the activation of p-stat3. We also discovered that IL-22 could affect the keratinocytes secreting type I collagen and MMP-1 to mediate wound healing. Gene expression of type I collagen is increased in tongue wound model.
However, the expression in human keratinocyte is decreased by
treatment with IL-22. Kubo’s (Kubo et al., 2014) study showed that expression of IL-10 and type I collagen increased significantly from 3 days to 7 days in skin burn injury. Shi JH’s (Shi et al., 2013) study proved that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col I, Col3, and up-regulate the mRNA expression levels of MMP1 and MMP8. The expression of type I collagen in human keratinocyte is decreased by treatment with both IL-22 and IL-10, while the expression of IL-10, IL-22 and type I collagen are increased in wound model, .Wound healing is a complicated process. Numbers of cytokines are released during the wound healing. The reason of the phenomenon may be that other cytokines contributed to the expressions of type I collagen in vivo. These findings suggested that IL-22 affect the expression of MMP-1, type I collagen and the phosphorylation of STAT3 to participate in wound healing. Gene knock-out mice and transfection techniques are necessary for further experiments. Based on our study, Particular attention should be paid on oral mucosal diseases such as RAU and OLP. Gaining a better understanding of the role of IL-22 in diseases of the oral mucosa is important for development of IL-22 as a potential drug target.
5. Conclusions This study shows that IL-22 can induce the proliferation and migration of keratinocytes at the early stage of wound healing and contribute to scarless healing at the late stage of wound healing by promoting the expression of MMP-1. And it can also promote oral mucosal wound healing by activating STAT3. . All the data imply that IL-22 promotes the oral mucosal wound healing via STAT3 in keratinocytes
. Funding This work was supported by the Major Project of National Natural Science Foundation of China (Grant No. 31110103905), Natural Science Foundation of China (Grant No. 81100738) and Fund of Hubei Province (Grant No. 2014BHE0014)
Competing interests No declare
Acknowledgements We thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Reference Albanesi, C., Scarponi, C., Sebastiani, S., Cavani, A., Federici, M., De Pita, O., . . . Girolomoni, G. (2000). IL-4 Enhances Keratinocyte Expression of CXCR3 Agonistic Chemokines. The Journal of Immunology, 165(3), 1395-1402. doi: 10.4049/jimmunol.165.3.1395 Aujla, S. J., Chan, Y. R., Zheng, M., Fei, M., Askew, D. J., Pociask, D. A., . . . Kolls, J. K. (2008). IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med, 14(3), 275-281. doi: 10.1038/nm1710 Aujla, S. J., & Kolls, J. K. (2009). IL-22: a critical mediator in mucosal host defense. J Mol Med (Berl), 87(5), 451-454. doi: 10.1007/s00109-009-0448-1 Behm, B., Babilas, P., Landthaler, M., & Schreml, S. (2012). Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol, 26(7), 812-820. doi: 10.1111/j.1468-3083.2011.04415.x Boniface, K., Bernard, F. X., Garcia, M., Gurney, A. L., Lecron, J. C., & Morel, F. (2005). IL-22 Inhibits Epidermal Differentiation and Induces Proinflammatory Gene Expression and Migration of Human Keratinocytes. The Journal of Immunology, 174(6), 3695-3702. doi: 10.4049/jimmunol.174.6.3695 Chang, H., Hanawa, H., Liu, H., Yoshida, T., Hayashi, M., Watanabe, R., . . . Aizawa, Y. (2006). Hydrodynamic-Based
Delivery
of
an
Interleukin-22-Ig
Fusion
Gene
Ameliorates
Experimental Autoimmune Myocarditis in Rats. The Journal of Immunology, 177(6), 3635-3643. doi: 10.4049/jimmunol.177.6.3635 Chapman, R. S., Lourenco, P., Tonner, E., Flint, D., Selbert, S., Takeda, K., et al. (2000). The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3. Adv. Exp. Med. Biol . 480, 129–138. Chen, J., Feng, J., Chen, X., Xu, H., Zhou, Z., Shen, X., . . . Shen, Z. (2013). Immunoexpression of interleukin-22 and interleukin-23 in oral and cutaneous lichen planus lesions: a preliminary study. Mediators Inflamm, 2013, 801974. doi: 10.1155/2013/801974 Costa, M. M., Saraceni, P. R., Forn-Cuni, G., Dios, S., Romero, A., Figueras, A., & Novoa, B. (2013). IL-22 is a key player in the regulation of inflammation in fish and involves innate immune cells and PI3K signaling. Dev Comp Immunol, 41(4), 746-755. doi: 10.1016/j.dci.2013.08.021 Donnelly, R. P., Sheikh, F., Kotenko, S. V., & Dickensheets, H. (2004). The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J Leukoc Biol, 76(2), 314-321. doi: 10.1189/jlb.0204117 Duhen, T., Geiger, R., Jarrossay, D., Lanzavecchia, A., & Sallusto, F. (2009). Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol, 10(8), 857-863. doi: 10.1038/ni.1767 Hu, L., Zhao, J., Liu, J., Gong, N., & Chen, L. (2013). Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocytes. Exp Ther Med, 5(3), 701-706. doi: 10.3892/etm.2013.887 Kubo, H., Hayashi, T., Ago, K., Ago, M., Kanekura, T., & Ogata, M. (2014). Temporal expression of wound healing-related genes in skin burn injury. Leg Med (Tokyo), 16(1), 8-13. doi: 10.1016/j.legalmed.2013.10.002 Kunz, S., Wolk, K., Witte, E., Witte, K., Doecke, W. D., Volk, H. D., . . . Sabat, R. (2006). Interleukin (IL)-19, IL-20 and IL-24 are produced by and act on keratinocytes and are distinct from classical ILs. Exp Dermatol, 15(12), 991-1004. doi: 10.1111/j.1600-0625.2006.00516.x
Larjava H. (2011). Oral Wound Healing: An Overview. Endodontic Topics, 24(1), 1-3. Lejeune, D., Dumoutier, L., Constantinescu, S., Kruijer, W., Schuringa, J. J., & Renauld, J. C. (2002). Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL-10. J Biol Chem, 277(37), 33676-33682. doi: 10.1074/jbc.M204204200 Liang, S. C., Nickerson-Nutter, C., Pittman, D. D., Carrier, Y., Goodwin, D. G., Shields, K. M., . . . Fouser, L. A. (2010). IL-22 induces an acute-phase response. J Immunol, 185(9), 5531-5538. doi: 10.4049/jimmunol.0904091 Levy, D. E., & Darnell, J. E. Jr. (2002). Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol, 3(9), 651–662. Liu, J., Mao, J. J., & Chen, L. (2011). Epithelial-mesenchymal interactions as a working concept for oral
mucosa
regeneration.
Tissue
Eng
Part
B
Rev,
17(1),
25-31.
doi:
10.1089/ten.TEB.2010.0489 McGee, H. M., Schmidt, B. A., Booth, C. J., Yancopoulos, G. D., Valenzuela, D. M., Murphy, A. J., . . . Horsley, V. (2013). IL-22 promotes fibroblast-mediated wound repair in the skin. J Invest Dermatol, 133(5), 1321-1329. doi: 10.1038/jid.2012.463 Miyoshi, K., Takaishi, M., Nakajima, K., Ikeda, M., Kanda, T., Tarutani, M., . . . Sano, S. (2011). Stat3 as a therapeutic target for the treatment of psoriasis: a clinical feasibility study with STA-21, a Stat3 inhibitor. J Invest Dermatol, 131(1), 108-117. doi: 10.1038/jid.2010.255 Ouyang, W. (2010). Distinct roles of IL-22 in human psoriasis and inflammatory bowel disease. Cytokine Growth Factor Rev, 21(6), 435-441. doi: 10.1016/j.cytogfr.2010.10.007 Pestka, S., Krause, C. D., Sarkar, D., Walter, M. R., Shi, Y., & Fisher, P. B. (2004). Interleukin-10 and related
cytokines
and
receptors.
Annu
Rev
Immunol,
22,
929-979.
doi:
10.1146/annurev.immunol.22.012703.104622 Pickert, G., Neufert, C., Leppkes, M., Zheng, Y., Wittkopf, N., Warntjen, M., . . . Becker, C. (2009). STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J Exp Med, 206(7), 1465-1472. doi: 10.1084/jem.20082683 Sabat, R. E., Witte, K., Witte, E., & Wolk. K. (2014). IL-22 and IL-17: An Overview. Blood. Shi, J. H., Guan, H., Shi, S., Cai, W. X., Bai, X. Z., Hu, X. L., . . . Hu, D. H. (2013). Protection against TGF-beta1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring. Arch Dermatol Res, 305(4), 341-352. doi: 10.1007/s00403-013-1314-0 Trifari, S., Kaplan, C. D., Tran, E. H., Crellin, N. K., & Spits, H. (2009). Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol, 10(8), 864-871. doi: 10.1038/ni.1770 Wen, Z., Zhong, Z., & Darnell, J. E. Jr. (1995). Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell, 82(2):241–250. Wolk, K., Kunz, S., Witte, E., Friedrich, M., Asadullah, K., & Sabat, R. (2004). IL-22 increases the innate immunity of tissues. Immunity, 21(2), 241-254. doi: 10.1016/j.immuni.2004.07.007 Wolk, K., & Sabat, R. (2006). Interleukin-22: a novel T- and NK-cell derived cytokine that regulates the biology of tissue cells. Cytokine Growth Factor Rev, 17(5), 367-380. doi: 10.1016/j.cytogfr.2006.09.001
Wolk, K., Warszawska, K., Hoeflich, C., Witte, E., Schneider-Burrus, S., Witte, K., . . . Sabat, R. (2011). Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa. J Immunol, 186(2), 1228-1239. doi: 10.4049/jimmunol.0903907 Wolk, K., Witte, E., Reineke, U., Witte, K., Friedrich, M., Sterry, W., . . . Sabat, R. (2005). Is there an interaction between interleukin-10 and interleukin-22? Genes Immun, 6(1), 8-18. doi: 10.1038/sj.gene.6364144 Wolk, K., Witte, E., Wallace, E., Docke, W. D., Kunz, S., Asadullah, K., . . . Sabat, R. (2006). IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis. Eur J Immunol, 36(5), 1309-1323. doi: 10.1002/eji.200535503 Wolk, K., Witte, E., Witte, K., Warszawska, K., & Sabat, R. (2010). Biology of interleukin-22. Semin Immunopathol, 32(1), 17-31. doi: 10.1007/s00281-009-0188-x Wolk, K., Witte, K., Witte, E., Proesch, S., Schulze-Tanzil, G., Nasilowska, K., . . . Sabat, R. (2008). Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes. J Leukoc Biol, 83(5), 1181-1193. doi: 10.1189/jlb.0807525 Ye, Z. J., Zhou, Q., Yuan, M. L., Du, R. H., Yang, W. B., Xiong, X. Z., . . . Shi, H. Z. (2012). Differentiation and recruitment of IL-22-producing helper T cells stimulated by pleural mesothelial cells in tuberculous pleurisy. Am J Respir Crit Care Med, 185(6), 660-669. doi: 10.1164/rccm.201107-1198OC Zhao, J., Hu, L., Liu, J., Gong, N., & Chen, L. (2013). The effects of cytokines in adipose stem cell-conditioned medium on the migration and proliferation of skin fibroblasts in vitro. Biomed Res Int, 2013, 578479. doi: 10.1155/2013/578479
Figures legends Fig 1. H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues (A) H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues. (B) The tongue piece of different day during the wound healing.（n=5） Fig 2. STAT3 was specifically induced by IL-22 in oral mucosal (A) Western blot analysis of the expression of p-stat3 activity and IL-22 after the wound made. (B) Real-time PCR analysis for the expression of IL-22 after the wound made. (C) Western blot analysis of p-stat3 of keratinocytes for 0 min, 30 min, and 60 min in presence or absence of recombinant IL-22 (20 ng/ml). (D,E) p-stat3 immunostaining and Western blot of the tongue tissues after the tongue pieces were cultured in medium for 0 hour, 1 hour and 2 hour in presence or absence of recombinant IL-22 (20 ng/ml)..Data from five mice per time point are provided as mean±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 3. IL-22 could promote the proliferation and migration of keratinocytes in vitro (A) The effect of IL-22 of different concentrations on the proliferation of keratinocytes.. (B) The effect of IL-22 of different concentrations on the proliferation of fibroblasts (C) The effect of IL-22 (20 ng/ml) on fibroblast and keratinocyte migration utilizing scrape-wound healing assay. Results showed that IL-22 had little effect on fibroblasts migration, but it could promote keratinocytes migration (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 4. Cytokine expression in keratinocytes during the wound healing in vivo. (A) IL-22 immunostaining of the tongue tissues during the wound healing. (B) MMP-1 immunostaining of the tongue tissues during the wound healing. (C) Type I collagen immunostaining of the tongue tissues during the wound healing. (D)
p-stat3 immunostaining of the tongue tissues during the wound healing. (E)Western blot
analysis of p-stat3 activity, MMP1, type I collagen and the expression of IL-22 in the mice tongue tissues. (F) Real-time PCR analysis for MMP-1, type I collagen from the tongue tissues (n=5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 5. The effect of IL-22 (20 ng/ml) on the expression of type I collagen and MMP-1 in keratinocytes. (A) Real-time PCR analysis of effect of IL-22 on the expression of type I collagen and MMP-1 in keratinocytes. (B) Western blot analysis of effect of IL-22 on the expression of type I collagen and MMP-1 in keratinocytes. Data from three independent experiments are listed as the mean±SEM. Beta-actin served as the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Table 1. Primer sequences for Real-time PCR Mouse
Forward: 5’-ACCTTTCCTGACCAAACTCA-3’
IL-22 Reverse: 5’-AGCTTCTTCTCGCTCAGACG-3’ Mouse Collagen1 Mouse
Forward : 5’-GACATGTTCAGCTTTGTGGACCTC-3’ Reverse: 5’-GGGACCCTTAGGCCATTGTGTA-3’ Forward : 5’-TCCCTGGAATTGGCAACAAAG-3’
MMP-1 Reverse: 5’-GCATGACTCTCACAATGCGATTAC-3’ Human
Forward : 5’-ACTCCCTCCATCCCAACCT-3’
Collagen1 Reverse: 5’ -ACCAAGCTTCCTTTTTTAAAAAC-3’ Human
Forward : 5’-AAGCGTGTGACAGTAAGCTAALL-3’
MMP-1 Reverse: 5’-AACCGGACTTCATCTCTG-3’
S0003-9969(16)30193-5 http://dx.doi.org/doi:10.1016/j.archoralbio.2016.08.001 AOB 3656
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
3-5-2015 14-7-2016 2-8-2016
Please cite this article as: Yu Ran, Ding Yumei, Zhu Lijuan, Qu Yinying, Zhang Chenguang, Liu Lin, Chen Lili.IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.08.001 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 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.
TITLE PAGE
IL-22 mediates the oral mucosal wound healing via STAT3 in keratinocytes
Ran Yu1*, Yumei Ding1*, Lijuan Zhu1, Yinying Qu2, Chenguang Zhang1, Lin Liu2# and Lili Chen1#
1.Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430022 Wuhan, China 2. Dalian Stomatological Hospital of Dalian Medical University, 116021Dalian, China
*, equal contribution: The first two author contributed equally to this work.
#
, corresponding author: The last two authors are the corresponding authors.
Highlights
IL-22 and STAT3 were involved in oral mucosal wound healing, and the value change tendency of Il-22 and STAT3 during the wound healing was similar.
STAT3 was activated after the keratinocytes or the tongue tissue were stimulated by IL-22,
IL-22 could obviously promote the proliferation of keratinocytes, but not fibroblasts.
IL-22 could mediate gene expression in keratinocytes, such as type I collagen and MMP-1 which may contribute to re-epithelialization and scarless healing in oral wound healing.
Abstract Objective Wounds are common in the oral cavity. During wound healing, several cytokines are released, which are probably helpful in providing wound debridement, removal of damaged tissues and microbes. Most of the target cells of IL-22 are epithelial cells, which play an important role in mucosa immunity.
Design The function of IL-22 in oral diseases is not well understood. We investigated the expression level of IL-22，collagen I and p-stat3 (Tyr705) via a mice tongue wound model in vivo and detected the effect of IL-22 on the expression of MMP-1, type I collagen and p-stat3 in keratinocytes.
Results IL-22 and p-stat3 were associated with wound healing, and STAT3 was activated when the keratinocytes or the tongue tissue were stimulated by IL-22. In addition, IL-22 could mediate gene expression involved in wounds involving keratinocytes, such as type I collagen and MMP-1，which may contribute to scarless healing.
Conclusion Our study suggests that IL-22 mediates wound healing via STAT3 in keratinocytes. This study reveals a new role for IL-22 in mediating wound healing.
Key words: Wound healing, IL-22, STAT3
ARTICLE PROPER 1. Introduction Repair following injury involves several processes, such as inflammation, re–epithelialization, scar formation. Interleukin-22 (IL-22), which is released by leukocytes, such as Th1, Th17, Th-22, and NK cells (Aujla & Kolls, 2009; Liang et al., 2010; Wolk & Sabat, 2006; Wolk et al., 2011), is an IL-10 cytokine family member, such as IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28a/b, IL-29, and the type I IFN (Donnelly, Sheikh, Kotenko, & Dickensheets, 2004; Pestka et al., 2004). It has been shown that the IL-22R1 subunit expression decides whether a cell is a target of IL-22 or not (Sabat, Witte, Witte, & Wolk, 2014). In resting and activated immune cells including monocytes, B cells, T cells, NK cells, macrophages, and DC populations, as well as bone marrow, blood mononuclear cells, thymus, and spleen, there is a lack of IL-22R1 expression(Kunz et al., 2006; Wolk et al., 2004; Wolk et al., 2005; Wolk et al., 2008), and signal transduction induced by IL-22 does not occur in any of these cells. Therefore, IL-22 does not have any influences on these cells in vitro and in vivo (Wolk et al., 2004). In contract, IL-22R1 is expressed in keratinocytes, bronchial and intestinal epithelial cells, intestinal subepithelial myofibroblasts, hepatocytes, pancreas acinar cells, and a range of respective tissue cell-derived cell lines (Wolk & Sabat, 2006; Wolk, Witte, Witte, Warszawska, & Sabat, 2010). IL-22, a critical mediator in mucosal host defense can promote keratinocyte migration, enhance the reconstitution of human epidermis and restrain a set of differentiation-related Ags (Aujla & Kolls, 2009; Boniface et al., 2005; Pickert et al., 2009). Through enhanced regulation of the expression of numerous antimicrobial molecules such as S100A7, S100A8, S100A9 and Matrix metalloproteinase-1 (MMP-1), and the b-defensin 2 (BD 2) and BD3, IL-22 increases the innate immunity of keratinocytes (Wolk et al., 2004; Wolk et al., 2011; Wolk et al., 2006). MMP-1 is the proteolytic enzyme of type I collagen, which plays a central role in extracellular matrix (ECM) remodeling and cell movement during the wound healing process. Many autoimmune disorders, such as psoriasis, rheumatoid arthritis, inflammatory bowel disease, or mucosal-associated infections have an association with IL-22 (Costa et al., 2013; Ouyang, 2010). High IL-22 levels have been found in tissue-repair processes, indicating that IL-22 plays a protective role in diseases such as myocarditis or tuberculosis (Behm, Babilas, Landthaler, & Schreml, 2012; Chang et al., 2006; Chen et al., 2013; McGee et al., 2013; Ye et al., 2012) STAT3 is the major and essential mediator of IL-22 signaling (Chang et al., 2006). STAT3 phosphorylation in Tyr705 and Ser727 residues is required when STAT3 is activated (Wen, Zhong, &
Darnell, J. E. Jr, 1995). Current studies have revealed that STAT3 is involved in psoriasis and colonic inflammation by IL-22-dependent mucosal wound healing (Aujla et al., 2008; Miyoshi et al., 2011). However, few studies are available on oral mucosal disease. This study will focus on the function of IL-22 in oral mucosal wound healing. We suggest that IL-22 regulate the oral mucosal wound healing via STAT3 in keratinocytes.
2. Materials and Methods
2.1 Mice All experiments involving male C57BL/6 mice were approved by the Wuhan University and maintained under specific pathogen-free conditions.
2.2 Experimental mouse models Wounds (length 5mm, depth 1mm) were made in the middle of the mouse tongues with a surgical knife blade. The depth was controlled by making a mark on the knife, 1mm from the knifepoint.. And mice were killed at 2h, 24h, 3d, 5d, or 7d after the treatment (n=5). Tongue samples were harvested and frozen for Western blot analysis, immunohistochemistry and real-time PCR.
2.3 In vitro keratinocytes culture Keratinocytes in primary culture were prepared from plastic surgery skin obtained from healthy volunteers, as previously described(Albanesi et al., 2000). Cells were cultured in six-well plates in the serum-free medium (KGM) for at least 3-5 days at 70-90% confluence before conducting experiments. In all experiments, third-passage keratinocytes were used. Stimulation with recombinant human or mouse IL-22 was performed in keratinocyte basal medium or fibroblast. Mouse fibroblast cell line NIH-3T3 was purchased from Wuhan University.
2.4 Scratch and proliferation assays Keratinocytes and fibroblasts were cultured in wells until they reached 100% confluence. A uniform cell-free area was created by scraping the cell monolayer with the tip of a p-200 pipette. Cell migration to the cell-free zone was measured at different time points of culture in the presence or absence of IL-22 (20 ng/ml), using an inverted phase contrast microscope. The effects of the IL-22 on fibroblast and keratinocyte proliferation were determined utilizing CCK-8 assay. Briefly, 5000 cells were seeded into the 96-well plates for 24 hours to adhere, and then stimulated with different concentrations of IL-22. We evaluated the cell proliferation in its logarithmic growth phases.
2.5 Western blot analysis Total proteins were extracted from tongue tissues and keratinocytes as previously reported 31, and the concentration of protein was determined using a BCA protein assay kit. After being subjected to SDS PAGE, proteins were transferred to PVDF membranes and probed with primary Abs diluted in TBST containing 5% nonfat dried milk. Western blot filters were developed using the ECL-plus detection system. The Abs employed for the study were as follows: anti-IL-22 (antgene), anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology), MMP-1, type I collagen (abclonal). Samples were normalized to GAPDH.
2.6 RNA isolation and real-time PCR Total RNA was extracted from tongue tissue using the TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions, and RNA was converted to cDNA using a reverse transcription kit (Thermo). Samples were heated at 42˚C for 60 min and 70˚C for 5min. Real-time PCR was performed on cDNA using SYBR green chemistry. GAPDH was used as an internal control. The sequences of primers and probes were as follows (table1):
2.7 Immunohistochemistry Firstly, the tissue section was dewaxed and rehydrated, After washing, the sections were incubated overnight at 4ºC with the MMP-1, collagen1, p-stat3 antibody. And then the slides were incubated with biotinylated secondary antibody for 30 minutes. After colourated with 3,3-diaminobenzidin (DAB), the
slides were counterstained for 1 minute with hematoxylin. At last, the slides were dehydrated with sequential ethanol washing 1 minute with 75%, followed by 80% and finishing with a 100% ethanol..
2.8 Histopathological analysis Tongue tissue were first fixed in 4% paraformaldehyde and then embedded in paraffin. Thin sections were obtained and stained with hematoxylin and eosin for routine histology.
2.9 Statistical analysis Data were presented as mean ± SEM from at least three independent experiments. Comparisons were evaluated using ANOVA, and differences were considered significant at the values of *, P < 0.05; **, P < 0.01; ***, P < 0.001.
2. Result
3.1 STAT3 was specifically induced by IL-22 in oral mucosa The wound healing process is always started by some cytokines via chemotactic signals to inflammatory and resident cells (Larjava, 2011). IL-22 can induce changes in systemic physiology and lead to an acute-phase response (Liang et al., 2010). To identify whether IL-22 and p-stat3 were involved in oral diseases, C57BL/6 mice were chosen and a wound model was made (Figure 1B). Firstly, H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues were made (Figure1A). The process of wound healing and the development of the horny layer could be observed. Then the expression of IL-22 and p-stat3 was confirmed by Western blot and real-time PCR at oh, 2h, 24h, 3d, 5d, 7d after wound being made (Figure2A and B). To study the connection between IL-22 and oral epithelial STAT3 activity, the tongue pieces from C57BL/6 mice were cultured in absence or presence of IL-22. Subsequently, p-stat3 was detected by immunostaining and Western blot analysis. As a result, treated tongue tissues showed higher expression levels of p-STAT3 compared with the untreated group (Figure2 D and E). A
further study for the effect of IL-22 on p-stat3 in
keratinocytes had been made,，and as we expected the p-STAT3 upregulated within 30min after IL-22 treatment (Figure2 C).
3.2 IL-22 could promote the proliferation and migration of keratinocytes in vitro
Depending on the target cells, STAT3 regulates signal transduction of several cytokines and proinflammatory mediators, such as IL-22 and IL-6 (Chapman et al., 2000; Levy & Darnell, 2002). We investigated the effect of IL-22 (20 ng/ml) on keratinocytes and fibroblasts by CCK-8 (Figure3 A and B) and scratch assays at different time point (Figure3 C). While IL-22 could obviously promote the proliferation of keratinocytes, it had little effect on fibroblasts.
3.3 Cytokine expression in keratinocytes during the wound healing in vivo. The wound healing process is a complicated process, which involves a range of processes, such as inflammation, formation of granulation tissue, re-epithelialization, and remodeling. The success of wound healing depends on chemokines, cytokines, and growth factors involved in a complex integration of signal networks. MMP-1 and type I collagen play an important role in wound healing. We confirmed the expression of IL-22 by Western blot analysis, Immunohistochemistry and real-time PCR (Figure4A, E and F). Western blot analysis and immunostaining were used to examine the activation of the STAT3 (Figure4 D and E). We have also examined the expression of type I collagen and MMP-1 (Figure4 B, C, E and F). The results showed that IL-22, MMP-1, collagen1 and STAT3 were involved in oral mucosal wound healing.
3.4 The effect of IL-22 on the expression of MMP-1 and collagen I Repair following injury involves several processes, such as inflammation, re–epithelialization, scar formation. The process of re–epithelialization occurred at the early stage of wound healing. The movement of epithelial in a wound setting is different from the migration of fibroblasts, angiogenic endothelial cells. It is a commonly thought that cell-matrix need to be broken to allow keratinocytes the freedom to move forward. MMP-1 is required for migration of keratinocytes on native type I collagen. And MMP-1 can contribute to resolution of scarring. To investigate how IL-22 could promote the migration of keratinocytes and the formation of scar, we stimulated the keratinocytes with IL-22, and then the expression of collagen I and MMP-1 were examined (Figure5 A and B). The results showed that IL-22 could induce the expression of MMP-1 and decrease the expression of type I collagen. The results suggested that IL-22 could promote the migration of keratinocytes at the early stage of
wound healing and contribute to scarless healing at the late stage of wound healing by induce the expression of MMP-1 via STAT3.
4. Discussion Th22, which is characterized by the production of IL-22, but not of IL-17 or IFN-γ, is the novel subpopulation of the effector CD4+ T cells (Duhen, Geiger, Jarrossay, Lanzavecchia, & Sallusto, 2009; Trifari, Kaplan, Tran, Crellin, & Spits, 2009). Th22 cells are the important source of IL-22 during later stages of inflammation. Being released as a signature cytokine in innate immunity, regeneration, and protection from damage, IL-22 plays either a pathogenic or a protective role in different situations. IL-22 is upregulated in a number of chronic inflammatory and autoimmune diseases, and the exact role of IL-22 appears to depend on the specific inflammatory microenvironment. The protective role of IL-22 has been found in a myocarditis model, whereas the inflammatory role of IL-22 has been demonstrated in rheumatoid arthritis. Through activation of STAT3-signaling cascades, the cytokine induces proliferative and anti-apoptotic pathways, as well as anti-microbial molecules, which help prevent tissue damage and aid in its repair (Lejeune et al., 2002; Wolk et al., 2010) In our previous studies, we have elucidated that the adipose stem cell-conditioned medium (ASC-CM) was of benefit to the migration and proliferation of fibroblasts, keratinocytes, and endothelial cells (Hu, Zhao, Liu, Gong, & Chen, 2013) In addition, IL-22 probably acts as an important role in ASC-CM because we found that the signal of IL-22 in ASC-CM exceeded 700-fold (Zhao, Hu, Liu, Gong, & Chen, 2013) IL-22 plays an important role in skin inflammatory processes and wound healing. However, oral mucosa and skin differ in keratinization patterns, resistance to external pressure, and so on. Oral mucosa, exposed to large amounts of antigens, is mostly nonkeratinized and moist with saliva at all times (Liu, Mao, & Chen, 2011). Until now, there has been little research about the effects and mechanism of IL-22 on oral mucosal diseases. Here, we determined the expression of IL-22 in the normal and wound mucosa. We found that there were two peaks of IL-22 expression after injury: an early peak with a maximum at 2 hour after wounding, and a second peak at about day 7 after wounding IL-22 was almost undetectable in tongue tissues on days 0, 1, and 3 after suffering injuries.
To explore the function of IL-22 on re-epithelialization in wound healing, we further stimulated both the fibroblasts and keratinocytes with IL-22. The results indicated that IL-22 promoted the migration and proliferation of keratinocytes while having little effect on fibroblasts. Taken together, these data indicate that IL-22 induces the re-epithelialization by promoting the migration of keratinocytes at the early stage of wound healing. Our experiment also focused on the connection between IL-22 and STAT3, MMP-1, type I collagen during wound healing. The results showed that the p-stat3 had the same regularity as the expression of IL-22 during wound healing. Both the expression of IL-22 and p-stat3 were obviously higher at 2h and 7d than on days 1 and 3. In addition, we stimulated the tongue mucosa and keratinocytes with IL-22 and detected the activation of p-stat3. We also discovered that IL-22 could affect the keratinocytes secreting type I collagen and MMP-1 to mediate wound healing. Gene expression of type I collagen is increased in tongue wound model.
However, the expression in human keratinocyte is decreased by
treatment with IL-22. Kubo’s (Kubo et al., 2014) study showed that expression of IL-10 and type I collagen increased significantly from 3 days to 7 days in skin burn injury. Shi JH’s (Shi et al., 2013) study proved that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col I, Col3, and up-regulate the mRNA expression levels of MMP1 and MMP8. The expression of type I collagen in human keratinocyte is decreased by treatment with both IL-22 and IL-10, while the expression of IL-10, IL-22 and type I collagen are increased in wound model, .Wound healing is a complicated process. Numbers of cytokines are released during the wound healing. The reason of the phenomenon may be that other cytokines contributed to the expressions of type I collagen in vivo. These findings suggested that IL-22 affect the expression of MMP-1, type I collagen and the phosphorylation of STAT3 to participate in wound healing. Gene knock-out mice and transfection techniques are necessary for further experiments. Based on our study, Particular attention should be paid on oral mucosal diseases such as RAU and OLP. Gaining a better understanding of the role of IL-22 in diseases of the oral mucosa is important for development of IL-22 as a potential drug target.
5. Conclusions This study shows that IL-22 can induce the proliferation and migration of keratinocytes at the early stage of wound healing and contribute to scarless healing at the late stage of wound healing by promoting the expression of MMP-1. And it can also promote oral mucosal wound healing by activating STAT3. . All the data imply that IL-22 promotes the oral mucosal wound healing via STAT3 in keratinocytes
. Funding This work was supported by the Major Project of National Natural Science Foundation of China (Grant No. 31110103905), Natural Science Foundation of China (Grant No. 81100738) and Fund of Hubei Province (Grant No. 2014BHE0014)
Competing interests No declare
Acknowledgements We thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Reference Albanesi, C., Scarponi, C., Sebastiani, S., Cavani, A., Federici, M., De Pita, O., . . . Girolomoni, G. (2000). IL-4 Enhances Keratinocyte Expression of CXCR3 Agonistic Chemokines. The Journal of Immunology, 165(3), 1395-1402. doi: 10.4049/jimmunol.165.3.1395 Aujla, S. J., Chan, Y. R., Zheng, M., Fei, M., Askew, D. J., Pociask, D. A., . . . Kolls, J. K. (2008). IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia. Nat Med, 14(3), 275-281. doi: 10.1038/nm1710 Aujla, S. J., & Kolls, J. K. (2009). IL-22: a critical mediator in mucosal host defense. J Mol Med (Berl), 87(5), 451-454. doi: 10.1007/s00109-009-0448-1 Behm, B., Babilas, P., Landthaler, M., & Schreml, S. (2012). Cytokines, chemokines and growth factors in wound healing. J Eur Acad Dermatol Venereol, 26(7), 812-820. doi: 10.1111/j.1468-3083.2011.04415.x Boniface, K., Bernard, F. X., Garcia, M., Gurney, A. L., Lecron, J. C., & Morel, F. (2005). IL-22 Inhibits Epidermal Differentiation and Induces Proinflammatory Gene Expression and Migration of Human Keratinocytes. The Journal of Immunology, 174(6), 3695-3702. doi: 10.4049/jimmunol.174.6.3695 Chang, H., Hanawa, H., Liu, H., Yoshida, T., Hayashi, M., Watanabe, R., . . . Aizawa, Y. (2006). Hydrodynamic-Based
Delivery
of
an
Interleukin-22-Ig
Fusion
Gene
Ameliorates
Experimental Autoimmune Myocarditis in Rats. The Journal of Immunology, 177(6), 3635-3643. doi: 10.4049/jimmunol.177.6.3635 Chapman, R. S., Lourenco, P., Tonner, E., Flint, D., Selbert, S., Takeda, K., et al. (2000). The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3. Adv. Exp. Med. Biol . 480, 129–138. Chen, J., Feng, J., Chen, X., Xu, H., Zhou, Z., Shen, X., . . . Shen, Z. (2013). Immunoexpression of interleukin-22 and interleukin-23 in oral and cutaneous lichen planus lesions: a preliminary study. Mediators Inflamm, 2013, 801974. doi: 10.1155/2013/801974 Costa, M. M., Saraceni, P. R., Forn-Cuni, G., Dios, S., Romero, A., Figueras, A., & Novoa, B. (2013). IL-22 is a key player in the regulation of inflammation in fish and involves innate immune cells and PI3K signaling. Dev Comp Immunol, 41(4), 746-755. doi: 10.1016/j.dci.2013.08.021 Donnelly, R. P., Sheikh, F., Kotenko, S. V., & Dickensheets, H. (2004). The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J Leukoc Biol, 76(2), 314-321. doi: 10.1189/jlb.0204117 Duhen, T., Geiger, R., Jarrossay, D., Lanzavecchia, A., & Sallusto, F. (2009). Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol, 10(8), 857-863. doi: 10.1038/ni.1767 Hu, L., Zhao, J., Liu, J., Gong, N., & Chen, L. (2013). Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocytes. Exp Ther Med, 5(3), 701-706. doi: 10.3892/etm.2013.887 Kubo, H., Hayashi, T., Ago, K., Ago, M., Kanekura, T., & Ogata, M. (2014). Temporal expression of wound healing-related genes in skin burn injury. Leg Med (Tokyo), 16(1), 8-13. doi: 10.1016/j.legalmed.2013.10.002 Kunz, S., Wolk, K., Witte, E., Witte, K., Doecke, W. D., Volk, H. D., . . . Sabat, R. (2006). Interleukin (IL)-19, IL-20 and IL-24 are produced by and act on keratinocytes and are distinct from classical ILs. Exp Dermatol, 15(12), 991-1004. doi: 10.1111/j.1600-0625.2006.00516.x
Larjava H. (2011). Oral Wound Healing: An Overview. Endodontic Topics, 24(1), 1-3. Lejeune, D., Dumoutier, L., Constantinescu, S., Kruijer, W., Schuringa, J. J., & Renauld, J. C. (2002). Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL-10. J Biol Chem, 277(37), 33676-33682. doi: 10.1074/jbc.M204204200 Liang, S. C., Nickerson-Nutter, C., Pittman, D. D., Carrier, Y., Goodwin, D. G., Shields, K. M., . . . Fouser, L. A. (2010). IL-22 induces an acute-phase response. J Immunol, 185(9), 5531-5538. doi: 10.4049/jimmunol.0904091 Levy, D. E., & Darnell, J. E. Jr. (2002). Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol, 3(9), 651–662. Liu, J., Mao, J. J., & Chen, L. (2011). Epithelial-mesenchymal interactions as a working concept for oral
mucosa
regeneration.
Tissue
Eng
Part
B
Rev,
17(1),
25-31.
doi:
10.1089/ten.TEB.2010.0489 McGee, H. M., Schmidt, B. A., Booth, C. J., Yancopoulos, G. D., Valenzuela, D. M., Murphy, A. J., . . . Horsley, V. (2013). IL-22 promotes fibroblast-mediated wound repair in the skin. J Invest Dermatol, 133(5), 1321-1329. doi: 10.1038/jid.2012.463 Miyoshi, K., Takaishi, M., Nakajima, K., Ikeda, M., Kanda, T., Tarutani, M., . . . Sano, S. (2011). Stat3 as a therapeutic target for the treatment of psoriasis: a clinical feasibility study with STA-21, a Stat3 inhibitor. J Invest Dermatol, 131(1), 108-117. doi: 10.1038/jid.2010.255 Ouyang, W. (2010). Distinct roles of IL-22 in human psoriasis and inflammatory bowel disease. Cytokine Growth Factor Rev, 21(6), 435-441. doi: 10.1016/j.cytogfr.2010.10.007 Pestka, S., Krause, C. D., Sarkar, D., Walter, M. R., Shi, Y., & Fisher, P. B. (2004). Interleukin-10 and related
cytokines
and
receptors.
Annu
Rev
Immunol,
22,
929-979.
doi:
10.1146/annurev.immunol.22.012703.104622 Pickert, G., Neufert, C., Leppkes, M., Zheng, Y., Wittkopf, N., Warntjen, M., . . . Becker, C. (2009). STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J Exp Med, 206(7), 1465-1472. doi: 10.1084/jem.20082683 Sabat, R. E., Witte, K., Witte, E., & Wolk. K. (2014). IL-22 and IL-17: An Overview. Blood. Shi, J. H., Guan, H., Shi, S., Cai, W. X., Bai, X. Z., Hu, X. L., . . . Hu, D. H. (2013). Protection against TGF-beta1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring. Arch Dermatol Res, 305(4), 341-352. doi: 10.1007/s00403-013-1314-0 Trifari, S., Kaplan, C. D., Tran, E. H., Crellin, N. K., & Spits, H. (2009). Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol, 10(8), 864-871. doi: 10.1038/ni.1770 Wen, Z., Zhong, Z., & Darnell, J. E. Jr. (1995). Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell, 82(2):241–250. Wolk, K., Kunz, S., Witte, E., Friedrich, M., Asadullah, K., & Sabat, R. (2004). IL-22 increases the innate immunity of tissues. Immunity, 21(2), 241-254. doi: 10.1016/j.immuni.2004.07.007 Wolk, K., & Sabat, R. (2006). Interleukin-22: a novel T- and NK-cell derived cytokine that regulates the biology of tissue cells. Cytokine Growth Factor Rev, 17(5), 367-380. doi: 10.1016/j.cytogfr.2006.09.001
Wolk, K., Warszawska, K., Hoeflich, C., Witte, E., Schneider-Burrus, S., Witte, K., . . . Sabat, R. (2011). Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa. J Immunol, 186(2), 1228-1239. doi: 10.4049/jimmunol.0903907 Wolk, K., Witte, E., Reineke, U., Witte, K., Friedrich, M., Sterry, W., . . . Sabat, R. (2005). Is there an interaction between interleukin-10 and interleukin-22? Genes Immun, 6(1), 8-18. doi: 10.1038/sj.gene.6364144 Wolk, K., Witte, E., Wallace, E., Docke, W. D., Kunz, S., Asadullah, K., . . . Sabat, R. (2006). IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis. Eur J Immunol, 36(5), 1309-1323. doi: 10.1002/eji.200535503 Wolk, K., Witte, E., Witte, K., Warszawska, K., & Sabat, R. (2010). Biology of interleukin-22. Semin Immunopathol, 32(1), 17-31. doi: 10.1007/s00281-009-0188-x Wolk, K., Witte, K., Witte, E., Proesch, S., Schulze-Tanzil, G., Nasilowska, K., . . . Sabat, R. (2008). Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes. J Leukoc Biol, 83(5), 1181-1193. doi: 10.1189/jlb.0807525 Ye, Z. J., Zhou, Q., Yuan, M. L., Du, R. H., Yang, W. B., Xiong, X. Z., . . . Shi, H. Z. (2012). Differentiation and recruitment of IL-22-producing helper T cells stimulated by pleural mesothelial cells in tuberculous pleurisy. Am J Respir Crit Care Med, 185(6), 660-669. doi: 10.1164/rccm.201107-1198OC Zhao, J., Hu, L., Liu, J., Gong, N., & Chen, L. (2013). The effects of cytokines in adipose stem cell-conditioned medium on the migration and proliferation of skin fibroblasts in vitro. Biomed Res Int, 2013, 578479. doi: 10.1155/2013/578479
Figures legends Fig 1. H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues (A) H&E staining of paraffin-embedded sections from control tongue and wound tongue tissues. (B) The tongue piece of different day during the wound healing.（n=5） Fig 2. STAT3 was specifically induced by IL-22 in oral mucosal (A) Western blot analysis of the expression of p-stat3 activity and IL-22 after the wound made. (B) Real-time PCR analysis for the expression of IL-22 after the wound made. (C) Western blot analysis of p-stat3 of keratinocytes for 0 min, 30 min, and 60 min in presence or absence of recombinant IL-22 (20 ng/ml). (D,E) p-stat3 immunostaining and Western blot of the tongue tissues after the tongue pieces were cultured in medium for 0 hour, 1 hour and 2 hour in presence or absence of recombinant IL-22 (20 ng/ml)..Data from five mice per time point are provided as mean±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 3. IL-22 could promote the proliferation and migration of keratinocytes in vitro (A) The effect of IL-22 of different concentrations on the proliferation of keratinocytes.. (B) The effect of IL-22 of different concentrations on the proliferation of fibroblasts (C) The effect of IL-22 (20 ng/ml) on fibroblast and keratinocyte migration utilizing scrape-wound healing assay. Results showed that IL-22 had little effect on fibroblasts migration, but it could promote keratinocytes migration (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 4. Cytokine expression in keratinocytes during the wound healing in vivo. (A) IL-22 immunostaining of the tongue tissues during the wound healing. (B) MMP-1 immunostaining of the tongue tissues during the wound healing. (C) Type I collagen immunostaining of the tongue tissues during the wound healing. (D)
p-stat3 immunostaining of the tongue tissues during the wound healing. (E)Western blot
analysis of p-stat3 activity, MMP1, type I collagen and the expression of IL-22 in the mice tongue tissues. (F) Real-time PCR analysis for MMP-1, type I collagen from the tongue tissues (n=5). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Fig. 5. The effect of IL-22 (20 ng/ml) on the expression of type I collagen and MMP-1 in keratinocytes. (A) Real-time PCR analysis of effect of IL-22 on the expression of type I collagen and MMP-1 in keratinocytes. (B) Western blot analysis of effect of IL-22 on the expression of type I collagen and MMP-1 in keratinocytes. Data from three independent experiments are listed as the mean±SEM. Beta-actin served as the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Table 1. Primer sequences for Real-time PCR Mouse
Forward: 5’-ACCTTTCCTGACCAAACTCA-3’
IL-22 Reverse: 5’-AGCTTCTTCTCGCTCAGACG-3’ Mouse Collagen1 Mouse
Forward : 5’-GACATGTTCAGCTTTGTGGACCTC-3’ Reverse: 5’-GGGACCCTTAGGCCATTGTGTA-3’ Forward : 5’-TCCCTGGAATTGGCAACAAAG-3’
MMP-1 Reverse: 5’-GCATGACTCTCACAATGCGATTAC-3’ Human
Forward : 5’-ACTCCCTCCATCCCAACCT-3’
Collagen1 Reverse: 5’ -ACCAAGCTTCCTTTTTTAAAAAC-3’ Human
Forward : 5’-AAGCGTGTGACAGTAAGCTAALL-3’
MMP-1 Reverse: 5’-AACCGGACTTCATCTCTG-3’