Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes

This information is current as Sreya Das, Srisathya Srinivasan, Ankita Srivastava, Sushant of September 27, 2021. Kumar, Gargi Das, Suman Das, Alka Dwivedi, Atharva Karulkar, Khushi Makkad, Richa Bilala, Ankit Gupta, Abhijeet Sawant, Chitra Nayak, Prakriti Tayalia and Rahul Purwar

J Immunol published online 13 February 2019 Downloaded from http://www.jimmunol.org/content/early/2019/02/12/jimmun ol.1800823

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 13, 2019, doi:10.4049/jimmunol.1800823 The Journal of Immunology

Differential Influence of IL-9 and IL-17 on Actin Cytoskeleton Regulates the Migration Potential of Human Keratinocytes

Sreya Das,*,1 Srisathya Srinivasan,*,1 Ankita Srivastava,*,1 Sushant Kumar,* Gargi Das,* Suman Das,* Alka Dwivedi,* Atharva Karulkar,* Khushi Makkad,* Richa Bilala,† Ankit Gupta,† Abhijeet Sawant,‡ Chitra Nayak,† Prakriti Tayalia,* and Rahul Purwar*

T cells mediate skin immune surveillance by secreting specific and regulate numerous functions of keratinocytes, including migration during homeostasis and disease pathogenesis. Keratinocyte migration is mediated mainly by proteolytic cleavage of the extracellular matrix and/or by cytoskeleton reorganization. However, the cross-talk between cytokines and actomyosin ma-

chinery of human primary keratinocytes (HPKs), which is required for cytoskeleton reorganization and subsequent migration, Downloaded from remains poorly examined. In this study, we describe that IL-9 profoundly reduced the actin stress fibers, inhibited contractility, and reduced the cortical stiffness of HPKs, which resulted in inhibition of the migration potential of HPKs in an adhesion- and MMP-independent manner. Similarly, IL-9 inhibited the IFN-g–induced migration of HPKs by inhibiting the actomyosin ma- chinery (actin stress fibers, contractility, and stiffness). IL-17A increased the actin stress fibers, promoted cellular contractility, and increased proteolytic collagen degradation, resulting in increased migration potential of HPKs. However, IL-9 inhibited the

IL-17A–mediated HPKs migration. Mechanistically, IL-9 inhibited the IFN-g– and IL-17A–induced phosphorylation of myosin http://www.jimmunol.org/ L chain in HPKs, which is a major regulator of the actomyosin cytoskeleton. Finally, in addition to HPKs, IL-9 inhibited the migration of A-431 cells (epidermoid carcinoma cells) induced either by IFN-g or IL-17A. In conclusion, our data demonstrate the influence of T cell cytokines in differentially regulating the actomyosin cytoskeleton and migration potential of human keratino- cytes, which may have critical roles in skin homeostasis and pathogenesis of inflammatory diseases as well as skin malignancies. The Journal of Immunology, 2019, 202: 000–000.

ealthy human adult skin contains twice as many effector Very recently, IL-9–secreting Th cells (Th9 cells) were dis- memory T cells compared with blood (1, 2). Skin resi- covered and reported to be present in healthy and lesional skin of by guest on September 27, 2021 dent effector memory T cells are the major mediators of skin inflammatory disorders, such as psoriasis and atopic derma- H + immune surveillance (1, 2). These CD4 T cells mediate effector titis (AD) (8). The effector functions of IL-9 are mediated by functions by secreting specific cytokines that are categorized as IL-9R, composed of a-chain and g-chain (9). IL-9R is expressed Th1 (IFN-g), Th2 (IL-4/IL13), Th17 (IL-17), and Th9 (IL-9), on numerous cell types, including mast cells, CD4+ T cells, based on their profile (3, 4). Although numerous stud- B cells, airway epithelial cells, and keratinocytes (10–13). Re- ies report the presence and functions of various Th cell subsets in cently, we and others have demonstrated that treatment with IL-9 multiple skin inflammatory diseases (5, 6) and malignancies (7), and Th9 cells leads to tumor regression in murine models of the roles of Th9 cells on human primary keratinocyte (HPK) (2, 14). Increased levels of IL-9 in serum and in skin functions have not been reported yet. More importantly, no study lesions are reported to have a positive correlation with the disease so far has probed the cross-talk between T cell cytokines (IL-9, progression of AD (15). In psoriatic lesional skin, higher numbers IFN-g, and IL-17) as well as actomyosin machinery of HPKs of IL-9 producing T cells were found compared with AD and and their subsequent impact on keratinocyte functions, including healthy skin (8). Recently, IL-9 has been reported to increase migration potential and proliferation. CXCL8 secretion in keratinocytes isolated from adult foreskin (13).

*Department of Biosciences and Bioengineering, Indian Institute of Technology zymography experiments, and the analysis. A.D. and A.K. contributed in FACS Bombay, Mumbai, Maharashtra 400076, India; †Department of Skin and Venereal experiments. K.M. and P.T. contributed in optimizing the three-dimensional migra- Diseases, Topiwala National Medical College and BYL Nair Charitable Hospital, tion assay and editing the manuscript. A.G., R.B., and A. Sawant contributed in the Mumbai, Maharashtra 400008, India; and ‡Department of Plastic Surgery, Topiwala skin sample procurement. C.N. contributed to the design of the study and skin sample National Medical College and BYL Nair Charitable Hospital, Mumbai, Maharashtra procurement. R.P. contributed to the design of the study, interpretation of the results, 400008, India and writing of the manuscript. All authors read and approved the final manuscript. 1Sreya Das, S.S., and A. Srivastava contributed equally to this work. Address correspondence and reprint requests to Dr. Rahul Purwar, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, ORCIDs: 0000-0003-1933-7749 (Suman Das); 0000-0002-4056-2628 (P.T.). Maharashtra 400076, India. E-mail address: [email protected] Received for publication June 13, 2018. Accepted for publication January 18, 2019. The online version of this article contains supplemental material. This work was supported by funds from an intramural seed grant from the Indian Abbreviations used in this article: AD, atopic dermatitis; AFM, atomic force micros- Institute of Technology Bombay, the Council for Scientific and Industrial Research, copy; 3D, three-dimensional; DPBS, Dulbecco’s PBS; ECM, extracellular matrix; the Tata Education and Development Trust, Bristol-Myers Squibb, and the Wadhwani FA, focal adhesion; HPK, human primary keratinocyte; Mlc, myosin L chain; MMP, Foundation (to R.P.). matrix metalloproteinase; NMMIIA, nonmuscle myosin IIA; t, de-adhesion time Sreya Das, S.S., and A. Srivastava contributed in the design of the study, performed constant; ttotal, total de-adhesion time. the experiments, analyzed the data, and drafted the manuscript. S.K. contributed in the establishment of keratinocyte isolation and culture methods, and performed the Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 ELISA. G.D. and Suman Das contributed in performing the proliferation assay,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800823 2 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION

However, the role of IL-9 in the cellular responses of human ker- with 1% BSA for 15 min and washed thrice with 13 Dulbecco’s PBS atinocytes, such as proliferation and migration, remains unexplored. (DPBS). Cells were seeded on the collagen-coated coverslips for various Apart from IL-9 producing Th9 cells, the presence of Th17 cells experiments. have been reported in lesional skin and blood of patients with skin Pan cytokeratin staining for assessment of culture purity inflammatory diseases and malignancies as well (16, 17). Specific Isolated HPKs were stained for cytokeratin to ensure the purity of the cells. targeting of IL-17A with humanized mAbs, such as , has HPKs were seeded on collagen-coated 18-mm glass coverslips. The cells were been seen to improve clinical symptoms of psoriasis (18). IL-17 has washed thrice with warm 13 DPBS, fixed with 4% paraformaldehyde, and been observed to play a critical role in psoriatic inflammation (19), permeabilized using 0.1% Triton X-100. The cells were blocked with 1% proliferation (20), and tumor progression (21). Interestingly, the role BSA and incubated overnight with pan cytokeratin primary Ab (Invitrogen Carlsbad, CA) at 4˚C followed by Alexa Fluor 488 Goat anti-mouse secondary of IL-17A in promoting matrix metalloproteinase (MMP) secretion Ab (1:1000) at room temperature for 2 h. The images were acquired using a in keratinocytes and other cell types is well described (22–24). DMi8 microscope (Leica, Wetzlar, Germany) under 403 magnification. Despite numerous studies highlighting the roles of actin cyto- Stimulation of HPKs and epidermoid carcinoma cells (A-431) skeleton on keratinocyte functions (migration and proliferation) in with cytokines homeostasis, inflammatory skin diseases, such as psoriasis (25) and in skin malignancies (26), no study has probed the effects of To examine the role of IL-9, HPKs were pretreated overnight with IL-4 IL-17 or IL-9 on the actomyosin machinery of keratinocytes and (20 ng/ml) plus TGF-b (10 ng/ml) in complete media for upregulation their subsequent impact on keratinocyte functions. of IL-9R surface expression. The next day, the culture media was replaced with minimal media (without bovine pituitary extract and without epi- In the current study, we have determined the role of IL-9 in actin dermal ), and HPKs were treated with either IL-9 (50 ng/ml) cytoskeleton remodeling of HPKs and its subsequent impact on or left untreated (control). Downloaded from cellular functions, with focus on migration potential using physio- To examine the functions of IL-17A and IFN-g, HPKs were treated with logically relevant models. IL-9 significantly reduced the actin stress either IL-17A (50 ng/ml) or IFN-g (10 ng/ml) in minimal media (-bovine pituitary extract, -epidermal growth factor) or left untreated (control) fibers and abrogated the actomyosin contractility in a highly repro- without pretreatment. A-431 cells were treated with IL-9 (50 ng/ml), ducible manner, resulting in profound suppression of the migration IL-17A (50 ng/ml), and IFN-g (10 ng/ml) in complete DMEM with high potential of HPKs. Mechanistically, IL-9 reduced the phosphorylated glucose media without pretreatment. myosin L chain (Mlc) levels in HPKs, indicating a role in regulating Flow cytometry http://www.jimmunol.org/ nonmuscle myosin IIA (NMMIIA) activity. IL-9 also had a profound inhibitory effect on the IFN-g– and IL-17A–induced migration of After cytokine treatment, the HPKs were harvested by the addition of 0.25% HPKs as well as the malignant epidermoid carcinoma cell line A-431. trypsin-EDTA for 3–4 min at 37˚C. After trypsinization, the cells were washed with staining buffer (13 DPBS with 2% FBS) followed by incu- Interestingly, IL-9–mediated inhibition of keratinocyte migration was bation with anti-human CD129 Ab conjugated with PE (BioLegend, San independent of MMPs and focal adhesions (FAs). Pharmaceutical Diego, CA) for 30 min on ice. The cells were washed thrice, and the cell inhibition of NMMIIA using blebbistatin disrupted the actomyosin pellet was resuspended in 250 ml of staining buffer and acquired using BD contractility and resulted in inhibition of HPKs migration, similar to FACSVerse flow cytometer. The live population was gated based on side scatter and the forward scatter, and IL-9R expression was analyzed using IL-9. To our knowledge, this is the first report demonstrating the BD FACSuite software as described earlier (28). The data represent mean effects of IL-9 on the migration potential of healthy human ker- fluorescence intensity compared with controls. by guest on September 27, 2021 atinocytes and malignant epidermoid carcinoma cells through Quantification of actin stress fibers regulation of actin cytoskeleton remodeling. Cells were seeded on collagen-coated coverslips, and appropriate treatments Materials and Methods were given. The cells were fixed with 4% paraformaldehyde for 20 min at 3 Sample collection and keratinocyte isolation room temperature. Following washes with 1 DPBS, the cells were per- meabilized using 0.1% Triton X-100 for 5 min, blocked with 1% BSA for Human skin specimens were obtained after cosmetic surgery according to 30 min, and incubated with Alexa Fluor 647–conjugated phalloidin for 2 h at the Declaration of Helsinki Principles and upon approval of ethical com- room temperature. Images were acquired using LSM 780 microscope (Zeiss, mittee of the regional hospital (BYL Nair Charitable Hospital, Mumbai, Oberkochen, Germany) under 633 magnification and ZEN Black software. India). HPK cultures were established from discarded samples of cosmetic The images were processed using the Filament Sensor software, as previ- surgery as described previously (27). The skin sample was cut into pieces ously described (29). The images were first processed using Gaussian and and incubated overnight at 4˚C in 2.4 U of Dispase II (Roche, Mannheim, Laplacian filters. The line sensor filter was used to detect the stress fibers, Germany). Next, the epidermis was separated from the dermis and placed and filaments were then traced. The quantified stress fibers were represented in trypsin-EDTA solution (0.25%) (HiMedia, Mumbai, India) for 20 min at as a ratio between the cytokine-treated cells and their respective controls. 37˚C. After stopping the trypsin reaction by addition of neutralizing media (media with 10% FBS), the cell suspension was filtered through sterile Trypsin de-adhesion assay gauze (40 mm) and washed twice in neutralizing media. Cells were seeded at a density of 30,000 cells on collagen-coated 18-mm 2 3 coverslips (5 mg/cm ). The cells were washed with warm 1 PBS (37˚C), and prewarmed trypsin (0.25%, 500 ml) was then added to the well. Live The single cell suspension of keratinocytes was incubated in serum-free cell images were captured every 3 s until the cells rounded up but remained attached to the substrate. For quantifying de-adhesion dynamics, the nor- EpiLife Keratinocyte Growth Medium (0.06 mM Ca++) (Life Technolo-  gies) and supplemented with EpiLife Defined Human Keratinocyte Growth malized change in area (A) was calculated using the following formula: Supplements (Life Technologies, Carlsbad, CA) at 37˚C in a humidified   atmosphere containing 5% CO . For experiments, keratinocytes in pas- Ai2A 2 A ¼ t ; sages 2–5 cultured in hydrocortisone-free medium were used. A i2Af Human epidermoid carcinoma A-431 cell line was obtained from Na- tional Centre for Cell Sciences cell repository, Pune, India. Cells were where Ai represents the cell area at time t = 0, Atrepresents the area at cultured in DMEM with high glucose (Sigma-Aldrich, St. Louis, MO) time t, and Af represents the area at the final time point. The experimental supplemented with 1 mM sodium pyruvate, 10% FBS, and 1% penicillin– de-adhesion curves were fitted with the Boltzmann equation, streptomycin antibiotic solution (Life Technologies).   Preparation of collagen-coated coverslips A ¼ 1 1 þ eðt2t1Þ=t2 Circular glass coverslips (12 and 18 mm) were sterilized using 70% ethanol and UV treatment and incubated overnight with rat tail collagen type I to obtain the de-adhesion time constants (t)1 and t2, respectively. Total de- 2 (5 mg/cm ) (Life Technologies) at 4˚C. The coated coverslips were blocked adhesion time (ttotal) was calculated as the sum of t1 and t2. The analysis The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. IL-9 disrupts the actomyosin contractile dynamics (contractility and stiffness), resulting in inhibition of the HPKs migration. HPKs were pretreated with IL-4 plus TGF-b overnight for upregulation of IL-9R. The next day, HPKs were washed and further stimulated with IL-9 or left untreated (control) for an additional 24 h. (A and B) The HPKs were stained with fluorescently tagged phalloidin to visualize actin fibers, and the images were processed as described in Materials and Methods.(A) The representative images of actin stress fibers are shown at original magnification 363. Scale bar, 10 mm. (B) The cumulative analysis of actin stress fibers is depicted as bar graphs (n = 4 independent experiments, (Figure legend continues) 4 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION of the images was done using ImageJ software and the de-adhesion curves with Alexa Fluor 488 Goat anti-mouse secondary Ab (Invitrogen) for 2 h at were fitted using GraphPad Prism 7 software. room temperature. HPKs treated with the appropriate cytokines were then seeded at a density of 5000 cells per well on these cover slips and incu- Atomic force microscopy bated for 6 h. The cells were then fixed with 4% paraformaldehyde, and actin was stained with Alexa Fluor 647–conjugated phalloidin. DAPI was HPKs were seeded on 18-mm coverslips coated with collagen, and treat- ments were given. Atomic force microscopy (AFM) measurements were used to visualize the nucleus. Images of tagged collagen, actin, and the nucleus of individual cells were taken in a DMi8 fluorescence microscope performed with an Asylum MFP-3D AFM (Asylum Research, Santa 3 Barbara, CA) coupled to a Nikon TE2000E2 epifluorescence microscope. (Leica) under 20 magnification. The area of the degraded collagen, Individual cells were indented using a pyramid-tipped probe with a nominal seen as a dark background because of the loss of fluorescence signal, was measured by autothreshold-based particle analysis as described in spring constant of 30 pN/nm. The first 1 mm of force-indentation curves for Supplemental Fig. 2A. This area was normalized to the cell area from their individual cells was fitted with the Hertzian model for a pyramidal tip to obtain estimates of cortical stiffness. Experimental and theoretical curves corresponding actin images and the normalized degradation per condition were extracted and analyzed to estimate the cortical stiffness of the cells. was quantified as described in Supplemental Fig. 2B and 2C. At least 30 cells in each experiment were analyzed per condition. The mRNA isolation, cDNA synthesis, and quantitative PCR analysis of the indentation curves to obtain the elastic modulus of the cells was performed using MATLAB software. RNA isolation from cytokine-treated HPKs was performed using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). First strand cDNA synthesis Three-dimensional migration assay was performed using QuantiTect Reverse Transcription Kit (QIAGEN), A 24-well plate was treated with 3% glutaraldehyde for 20 min, washed and quantitative PCR was performed using a SYBR Premix Ex Taq II thrice with sterile distilled water, and sterilized by UV treatment. Col- Kit (TlI Plus) (Takara, Kusatsu, Japan) by StepOnePlus Real-Time PCR System (Applied Biosystems). Target were amplified (cycle condi- lagengelswerepreparedataworkingconcentration1mg/ml,and1N tions as per manufacturer’s protocol) using human MMP-2, MMP-9, NaOH was used to adjust the pH. Cells were treated with cytokines, Downloaded from added to the collagen, mixed well, and gently layered in the wells. The MMP-13, and GAPDH predesigned primers obtained from QuantiTect gels and were allowed to solidify for 1 h at 37˚C. Cells embedded in (QIAGEN). Quantification of the relative expression was performed collagen were further treated with appropriate cytokines or drugs by using StepOne Software v2.3 (Applied Biosystems, Foster City, CA). layering the media with the treatments on top of the gels. The HPKs and Gelatin zymography A-431 cells were imaged by time-lapse microscopy using a spinning disc confocal microscope (Zeiss) under 103 magnification for 24 and 12 h, Cell-free supernatants at 24, 48, and 72 h were collected from cytokine- respectively. Acquisition was done using ZEN Blue software, and the treated–cultured HPKs. Equal volumes of these supernatants were loaded migration of the cells was tracked using the Manual Tracking plugin of on 8% SDS–PAGE gel with 2 mg/ml gelatin and run at 125 V for 120 min http://www.jimmunol.org/ ImageJ software. on ice. The gel was then incubated in 13 Renaturation buffer (2.5% Triton X-100) for 30 min at room temperature and transferred to 13 Developing p-Mlc quantification by immunofluorescence buffer (0.5 M Tris base, 2 M NaCl, and 0.05 M CaCl2) at room temperature 3 To analyze the phosphorylation of a conserved Ser19 residue on the Mlc, for 30 min. This was followed by 20 h incubation in fresh 1 developing HPKs were seeded on collagen-coated coverslips. The cells were washed buffer at 37˚C. Staining was done for 1–2 h by QC Colloidal Coomassie with 13 DPBS and then fixed with ice-cold 4% paraformaldehyde so- Stain (Bio-Rad Laboratories, Hercules, CA). Destaining was carried out in lution for 20 min at room temperature. After three washes with cold 13 water till the zone of clearance was clearly visible. DPBS, the cells were permeabilized with 0.1% Triton X-100 for 4 min at Quantification of FA room temperature. Next, cells were washed thrice, and blocking was done with 2% BSA for 30 min at room temperature. Incubation with the For the quantifying the FA, HPKs were seeded on collagen-coated cov- by guest on September 27, 2021 primary Ab (Phospho-MYL9 Ser19 mAb, 1:100 dilution in 0.1% BSA; erslips, and appropriate treatments were given. The cells were washed with Thermo Fisher Scientific) was done overnight at 4˚C. After incubation 13 DPBS and then permeabilized with an ice-cold 1:1 solution of 4% and washes, Alexa Fluor 594–conjugated Goat anti-rabbit secondary Ab paraformaldehyde and 0.5% Triton X-100 for 1 min on ice. The cells were (1:1000) and Alexa Fluor 647–conjugated phalloidin (1:200) were added washed with cold 13 DPBS and incubated for 5 min with cold 4% para- and incubated for 2 h at room temperature to visualize p-Mlc and actin, formaldehyde on ice. After three washes with cold 13 DPBS and incubating respectively. The nucleus was visualized using DAPI (1:1000). The with blocking buffer (1.5% BSA in 0.5% Triton X-100) for 30 min on ice, images were acquired using an LSM 780 microscope (Zeiss) under 633 the samples were incubated at 4˚C overnight in anti-vinculin primary Ab magnification and ZEN Black software. The analysis of the fluorescence (Invitrogen) at 4˚C. Next, secondary Ab (Alexa Fluor 488 Goat anti-mouse) intensity was performed using ImageJ software. The cell area and the and Alexa Fluor 647–conjugated phalloidin were added and incubated for integrated density were measured after selecting the cell of interest. 2 h at room temperature to visualize vinculin and actin, respectively. Background fluorescence was measured in an area adjacent to the cells. The images were acquired using an LSM 780 microscope (Zeiss) under 633 The mean of the corrected total cell fluorescence obtained from the magnification and ZEN Black software. The total area of the FAs was quan- formula: CTCF = Integrated cell density 2 (Area of selected cell 3 Mean tified as previously described (30). The raw fluorescence images were pro- fluorescence of the background). cessed using the threshold analysis method after subtraction of background fluorescence and enhancement of local contrast (CLAHE). The total area of the Collagen degradation assay FAs was quantified by particle analyzer function using predefined parameters 2 (size and circularity). The analysis was performed using ImageJ software. The coverslips (12 mm) were coated with 10 mg/cm collagen. After overnight incubation, coverslips were washed followed by blocking with ELISA 0.1% BSA for 20 min. Further collagen-coated coverslips were incubated with anti-collagen mouse primary Ab (1:200 dilution; Invitrogen) over- Cell-free supernatant was collected from cytokine-treated HPKs for 24, 48, night at 4˚C. After incubation, the coverslips were washed and incubated and 72 h. One hundred microliters of supernatant was used in duplicate to

five to six cells were analyzed in each experiment per condition). (C–E) Contractility of HPKs (retraction dynamics) was examined by trypsin de-adhesion assay. (C) Representative time-lapse images of the retraction dynamics of HPKs. Scale bar, 25 mm. (D) The de-adhesion curves obtained were fitted using the Boltzmann sigmoidal equation to obtain the time constants t1 and t2.(E) ttotal is calculated as the sum of t1 and t2 (n = 5 independent experiments, 10–12 cells were analyzed in each experiment per condition). (F and G) AFM was performed to analyze the cortical stiffness of cytokine- and blebbistatin- treated HPKs (as indicated). Representative indentation curves (F) and quantification of the cortical stiffness of HPKs treated with IL-9 and blebbistatin are shown (G)(n = 5 independent experiments, 25–30 cells were probed in each experiment per condition). (H) Effect of IL-9 on migration of HPKs was examined by embedding the HPK in a collagen matrix, and migration of HPKs in the x–y-axis was quantified. Representative track plots of control, IL-9–, and blebbistatin-treated HPKs are shown (left). Cumulative data (right) are depicted as bar graph (n = 5 independent experiments, 10–12 cells were analyzed in each experiment per condition). The control in Fig. 1 represents the cells pretreated with IL-4 plus TGF-b. Data are represented as mean 6 SEM for the line graph and mean + SEM for bar graph. AFM data are represented as minimum-to-maximum box plot with median. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. IL-9 inhibits IFN-g–induced increase in actomyosin contractility and subsequently inhibits migration potential of HPKs. HPKs were treated with the indicated cytokines or left untreated (control) for 24 h. (A and B) Actin stress fiber formation was analyzed by phalloidin staining. (A) The representative images of actin stress fibers under original magnification 363 are shown. Scale bar, 10 mm. (B) Actin stress fibers were quantified, and the bar graph represents the cumulative data (n = 5 independent experiments, five to six cells were analyzed in each experiment per condition). (C–E) Contractility of HPKs was analyzed in cytokine-treated and control (untreated) cells using trypsin de-adhesion assay. (Figure legend continues) 6 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION quantify the levels of CXCL8 and CXCL10 as per manufacturer’s independent experiments, p , 0.05). To confirm if IL-9 exerts its instructions. effects on the actomyosin contractility of HPKs, blebbistatin, an Proliferation assay NMMIIA inhibitor, was used, which also significantly reduced the stress fibers, similar to IL-9 (Fig. 1A, 1B; n = 4 independent ex- HPKs were seeded at a density of 5000–7000 cells, and imaging was done periments, p , 0.01). at 0 and 48 h under 103 magnification. Cytokine treatment was given every 24 h in minimal media. The cells were manually counted using the The actomyosin cytoskeleton machinery controls the contrac- cell counter function in ImageJ software. tility (retraction dynamics) and stiffness of the cell, thereby reg- ulating cellular functions, including cell migration. The effect of Statistical analysis IL-9 on the cellular contractility was also probed using a trypsin Statistical analysis for all the experiments performed was done using the de-adhesion assay, which quantifies the ability of a cell to retract standard Student t test (two-tailed). Statistical analysis for the IL-9R ex- from the substrate in the presence of trypsin. Temporal curves of pression was performed using a paired t test. A p value ,0.05 was con- sidered to be statistically significant. The extent of statistical significance normalized change in area were fitted with the Boltzmann equation was designated as *p , 0.05, **p , 0.01, and ***p , 0.001, based on the to obtain the time constants t1 and t2. ttotal was calculated as the p value obtained. sum of time constants, t1 and t2. As depicted in Fig. 1C, IL-9 significantly delayed the retraction of HPKs compared with con- Results trol (pretreatment with IL-4 plus TGF-b) in a highly reproducible Regulation of IL-9R expression on HPKs manner (Fig. 1C, 1D; p , 0.001) and significantly increased the HPKs express IL-9R at a basal level (13). However, the regulation ttotal (Fig. 1E; n = 5 independent experiments). Similarly, blebbis- of IL-9R under various inflammatory conditions has not been tatin treatment of HPKs increased the ttotal by reducing the acto- Downloaded from examined yet. HPKs were isolated from normal-appearing skin of myosin contractility (Fig. 1D, 1E; n = 7 independent experiments, adult individuals undergoing cosmetic surgery as described in p , 0.001). These data indicate that IL-9, similar to blebbistatin, Materials and Methods. All cells were positive for pan cytokeratin regulates cell contractility via the actomyosin pathway. staining, indicating 100% purity of keratinocytes (Supplemental Because contractility (cell elasticity) is closely associated with Fig. 1A). Next, HPKs were treated with Th1 (IFN-g, TNF-a), Th2 cortical stiffness of the cell, the role of IL-9 in regulating the

(IL-4), and other proinflammatory cytokines, such as TGF-b alone cortical stiffness of HPKs was examined by AFM. HPKs were http://www.jimmunol.org/ or in various combinations (Supplemental Fig. 1C–H). Stimulation indented using a pyramid-tipped probe, and the elastic modulus of of HPKs with IL-4 plus TGF-b significantly increased the IL-9R each cell was computed from individual force curves obtained from surface expression reproducibly in multiple experiments (Supplemental the indentation (Fig. 1F). It was observed that IL-9 treated cells Fig. 1B, 1C). Neither IL-4 (Supplemental Fig. 1D) nor TGF-b were significantly softer compared with control cells, similar to (Supplemental Fig. 1E) alone had a significant effect on IL-9R blebbistatin-treated cells (Fig. 1G; n = 5 independent experiments, expression in HPKs isolated from three different individuals. In p , 0.05). addition, there was no significant difference in IL-9R in all other Next, we examined the functional significance of IL-9–mediated conditions tested (Supplemental Fig. 1F–H). disruption of actomyosin contractility. Mechanical properties, such as cellular contractility and stiffness, are critical for the by guest on September 27, 2021 IL-9 disrupts the actomyosin contractile dynamics migration of cells. Therefore, the role of IL-9 in the migration (contractility and stiffness), which results in potential of HPKs was examined in a three-dimensional (3D) inhibition of the migration potential of HPKs in a confined microenvironment, a physiologically relevant model to three-dimensional environment study cell migration. HPKs were embedded in collagen matrix, Because we observed that IL-4 plus TGF-b upregulates the ex- and their migration was examined for 24 h using time-lapse mi- pression of IL-9R on HPKs (Supplemental Fig. 1B, 1C), we croscopy. The migration on x–y-axis was quantified as described attempted to examine the impact of IL-9 on the mechanical in the Materials and Methods section. IL-9, similar to blebbistatin, properties and migration of HPKs upon upregulation of IL-9R. inhibited the migration potential of HPKs in a highly reproducible HPKs were pretreated with IL-4 plus TGF-b to upregulate manner as compared with the control cells (Fig. 1H; n = 5 inde- IL-9R expression. The next day, HPKs were washed and treated pendent experiments, p , 0.05). with IL-9 for 24 h or left untreated (control). The contractility of To examine the role of IL-9 in cytoskeleton remodeling and HPKs was examined by quantifying the actin stress fibers, which migration without upregulating the IL-9R by prestimulation of IL-4 were stained using fluorescently tagged phalloidin and quantified plus TGF-b, HPKs were treated with IL-9 alone or left untreated in multiple cells as described in the Materials and Methods sec- (control). The effect of IL-9 on contractility (stress fiber quanti- tion (Fig. 1A, 1B). The images were processed using Gaussian and fication: Supplemental Fig. 1I; trypsin de-adhesion assay: Laplacian filters by Filament Sensor software. The line sensor Supplemental Fig. 1J; cortical stiffness by AFM: Supplemental filter was then used to detect the stress fibers, and filaments were Fig. 1K) and migration of HPKs (Supplemental Fig. 1L) was then traced and quantified. IL-9 reduced the numbers of actin examined. IL-9 neither significantly altered any of the tested stress fibers significantly compared with the control (pretreatment mechanical properties nor had any impact on migration of HPKs with IL-4 plus TGF-b) in multiple experiments (Fig. 1A, 1B; n =4 compared with control (untreated cells).

(C) Figure depicts the representative time-lapse images of trypsin de-adhesion assay. Scale bar, 25 mm. (D) The de-adhesion curves plotted were fitted using the Boltzmann sigmoidal equation to obtain the time constants (t1 and t2). (E) Data shows ttotal obtained from five independent experiments (n =5 independent experiments, 10–12 cells were analyzed in each experiment per condition). (F and G) Representative indentation curves (F) and quantification of the cortical stiffness of HPKs stimulated with cytokine are shown (G)(n = 5 independent experiments, 25–30 cells were probed in each experiment per condition). (H) Effect of IL-9 on IFN-g–mediated migration of HPKs was examined by embedding the HPKs in a collagen matrix and its migration in the x–y-axis was quantified. Representative track plots (H, left) and cumulative data (H, right) are depicted (n = 5 independent experiments, 10–12 cells were analyzed in each experiment per condition). Data are represented as mean 6 SEM for the line graph and mean + SEM for bar graph. AFM data are represented as minimum-to-maximum box plot with median. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. IL-9 reduces the IL-17A–induced increase in actomyosin contractility and inhibits migration of HPKs. HPKs were treated with IL-17A or both IL-17A plus IL-9 or left untreated (control) for 24 h. (A and B) Actin stress fiber formation was analyzed by phalloidin staining. (A) The repre- sentative images of actin stress fibers under original magnification 363 are shown. Scale bar, 10 mm. (B) The quantification of the actin stress fibers is performed as described in Materials and Methods and the bar graph represents the cumulative data (n = 3–5 independent experiments, five to six cells were analyzed in each experiment per condition.). (C–E) Contractility of HPKs was analyzed using trypsin de-adhesion assay. (C) (Figure legend continues) 8 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION

IL-9 inhibits IFN-g–induced increase in actomyosin carcinoma cell line, A-431, were treated with IFN-g or IL-17A contractility and subsequently inhibits migration with or without IL-9 (no pretreatment). Although there was no potential of HPKs statistically significant effect of the tested cytokines (IFN-g and IFN-g is a pathogenic T cell–derived cytokine observed in skin IL-17A) on the actin stress fiber formation (Fig. 4A, 4B; n =3 . lesions of various skin inflammatory disorders (5) and is known to independent experiments, p 0.05) and cellular contractility . affect keratinocyte migration, as shown previously in a murine (Fig. 4C, 4D; n = 4 independent experiments, p 0.05), IL-9 model of wound healing (31). To strengthen our observation that significantly reduced the IFN-g–mediated cellular contractility IL-9 inhibits contractility and subsequently inhibits migration as examined by quantifying the ttotal (Fig. 4D; n = 4 independent , potential of HPKs, we examined if IL-9 was able to inhibit the experiments, p 0.05). Similar to healthy HPKs, both IL-17A effects of IFN-g–mediated migration of HPKs. IL-9 inhibited the and IFN-g promoted the migration of A-431 cells compared with IFN-g–induced actin stress fibers (Fig. 2A, 2B; n = 4 independent control (untreated cells) (Fig. 4E, 4F; n = 3 independent experi- , experiments, p , 0.01) as well as significantly decreased the ments, p 0.001). Interestingly, IL-9 inhibited the IFN-g– as well IFN-g–induced contractility (Fig. 2C–E; n = 5 independent ex- as IL-17A–mediated migration of A-431 cells (Fig. 4E, 4F; n =3 , , periments, p , 0.001) and stiffness (Fig. 2F, 2G; n = 5 indepen- independent experiments, p 0.05 and p 0.001). However, dent experiments, p , 0.001) in a highly reproducible manner. IL-9 alone had no effect on cytoskeletal remodeling and migration Interestingly, the disruption of contractility by IL-9 resulted in in- of A-431 cells (data not shown). These data demonstrate that IL-9 hibition of IFN-g–induced migration potential of HPKs (Fig. 2H; inhibits IL-17A– and IFN-g–induced migration of malignant n = 5 independent experiments, p , 0.001). These data collectively epidermoid carcinoma cells (A-431). However, these effects suggest that, although IFN-g significantly increases the actomyosin seem to be partially regulated by the actomyosin cytoskeleton Downloaded from contractility and migration potential of HPKs, IL-9 is able to inhibit remodeling. this increase in the actomyosin contractility and the migration IL-9 downregulates the IFN-g– and IL-17A–induced p-Mlc potential of HPKs. To delineate the underlying molecular mechanism by which T cell IL-9 inhibits IL-17–induced increase in actomyosin cytokines (IL-9, IFN-g, and IL-17A) impact migration of HPKs, contractility and subsequently inhibits migration we studied the levels of Mlc phosphorylation, a major regulator of http://www.jimmunol.org/ potential of HPKs actomyosin cytoskeleton. NMMIIA, in particular the phosphory- IL-17A is known to promote the secretion of proteolytic enzymes lation of Mlc, and actin work in tandem to generate the contractile that degrade the extracellular matrix (ECM). However, the roles of forces necessary for the cell to migrate (32). Because NMMIIA is IL-17A on the actin cytoskeleton remodeling and their subsequent an actin-dependent molecular motor, we analyzed the role of IL-9 in effects on migration of HPKs has not been examined yet. We ob- activating NMMIIA by immunofluorescence of the phosphorylated served that IL-17A increased the actin stress fibers (Fig. 3A, 3B; Ser19 conserved residue on the Mlc. IL-9 did not significantly alter n = 5 independent experiments, p , 0.001), increased the con- the p-Mlc levels in HPKs, irrespective of whether prestimulation tractility by significantly decreasing ttotal (Fig. 3C–E; n =5inde- with IL-4 plus TGF-b was performed or not. (Supplemental Fig. pendent experiments, p , 0.001) and increased the migration of 1M) (Fig. 5A; n = 3 independent experiments, p . 0.05). by guest on September 27, 2021 HPKs in a confined environment compared with the control (un- Because IFN-g and IL-17A significantly increased the con- treated cells) (Fig. 3H; n = 5 independent experiments, p , 0.001). tractile dynamics of HPKs, we then analyzed if these cytokines However, IL-17A treatment did not significantly alter the cortical had an effect on the p-Mlc levels. IFN-g (Fig. 5B; n = 3 inde- stiffness of the HPKs (Fig. 3F, 3G; n = 5 independent experiments, pendent experiments, p , 0.001) as well as IL-17A (Fig. 5C; p . 0.05). Interestingly, IL-9 reversed the IL-17A–mediated acto- n = 5 independent experiments, p , 0.001) significantly in- myosin contractility by reducing the stress fibers (Fig. 3A, 3B; n =3 creased the phosphorylation by almost 2.5-fold each as com- independent experiments, p , 0.05), increasing the retraction time pared with the control (untreated cells). Importantly, IL-9 (Fig. 3C–E; n = 4 independent experiments, p , 0.001) and re- significantly reduced the IFN-g– as well as IL-17A–induced ducing the stiffness (Fig. 3F, 3G; n = 3 independent experiments, p-Mlc levels (Fig. 5B, 5C; n = 3 independent experiments, p , 0.05). Subsequently IL-9 significantly inhibited the IL-17A– p , 0.05). Collectively, these findings suggest that IL-9 down- induced migration of HPKs (Fig. 3H; minimum n = 2 independent regulates the IFN-g– as well as IL-17A–induced phosphorylated/ experiments, p , 0.05). These data indicate that, although IL-17A active state of NMMIIA. promotes the migration of HPKs by regulating the actomyosin IL-9 and IL-17A differentially regulate ECM remodeling cytoskeleton, IL-9 significantly inhibits the IL-17A–mediated con- ability of HPKs tractility and migration. In addition to cell contractility (retraction dynamics), proteolytic IL-9 abrogates IFN-g– and IL-17A–induced enhanced activity of cells is another major contributing factor toward mi- migration of epidermoid carcinoma cells (A-431) gration in a confined 3D microenvironment. We analyzed the roles To study the effect of IL-9 on IL-17A– and IFN-g–induced mi- of IL-9 and IL-17A on the ECM remodeling ability using gration in malignant epithelial cells, cells of an epidermoid the collagen degradation assay, which quantifies the proteolytic

Representative time-lapse images of trypsin de-adhesion assay are shown. Scale bar, 25 mm. (D) The de-adhesion curves plotted were fitted using the

Boltzmann sigmoidal equation to obtain the time constants (t1 and t2). (E) Data shows ttotal obtained from five independent experiments (n = 4–5 in- dependent experiments, 10–12 cells were analyzed in each experiment per condition). (F and G) Representative indentation curves (F) and quantification of the cortical stiffness of HPKs (G) are shown (n = 3–5 independent experiments, 25–30 cells were probed in each experiment per condition). (H) Effect of IL-17A and IL-17A plus IL-9 on migration of HPKs was examined by embedding the HPKs in a collagen matrix. The migration of HPKs in x–y-axis was quantified. Representative track plots (H, left) and cumulative data (H, right) are depicted (minimum n = 2 independent experiments, 20–25 cells were analyzed in each experiment). Data are represented mean 6 SEM for the line graph and mean + SEM for bar graph. AFM data are represented as minimum- to-maximum box plot with median. *p , 0.05, ***p , 0.001. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. IL-9 inhibits migration of malignant A-431 cells. A-431 were treated with indicated cytokines for 24 h or left untreated (control). (A and B) Actin stress fiber formation was analyzed by phalloidin staining. (A) The representative images of actin stress fibers under original magnification 363 are shown. Scale bar, 10 mm. (B) Actin stress fibers were quantified, and the bar graph represents the cumulative data (n = 3 independent experiments, seven to eight cells were analyzed in each experiment per condition). (C and D) Contractility of A-431 was analyzed using trypsin de-adhesion assay. (C) The de- adhesion curves plotted were fitted using the Boltzmann sigmoidal equation to obtain the time constants (t1 and t2). (D) Data shows ttotal obtained from four independent experiments (10–12 cells were analyzed in each experiment per condition). (E and F) Effect of cytokines on migration of A-431 cells was examined by embedding the cells in a collagen matrix and its migration in the x–y-axis was quantified. Representative track plots (E) and cumulative data (F) are depicted (n = 3 independent experiments, 10–12 cells were analyzed in each experiment per condition). Data are represented as mean 6 SEM for the line graph and mean + SEM for bar graph. *p , 0.05, **p , 0.01, ***p , 0.001. degradation ability of cells by secretion of collagenases, including cells as well as control cells were seeded on fluorescently tagged MMPs. collagen for 6 h. The representative schematic of the quantification HPKs were prestimulated with IL-4 plus TGF-b to upregulate IL-9R of normalized degradation is depicted in Supplemental Fig. 2A. As on day 0. The next day, HPKs were further stimulated with IL-9 or left shown in Fig. 6, IL-9 had no effect on collagen degradation ability untreated (control) for an additional 24 h. On day 2, IL-9–stimulated of HPKs (Fig. 6A; n = 3 independent experiments, p . 0.05). 10 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION

FIGURE 5. IL-9 downregulates the IFN-g– and IL-17–induced phosphor- ylation of p-Mlc levels in HPKs. Levels of p-Mlc were quantified by staining p-Mlc (Ser19). (A) HPKs were pretreated with IL-4 plus TGF-b overnight for up- regulation of IL-9R. The next day, HPKs were washed and further treated with IL-9 or left untreated (control) for an additional 24 h. (B and C)HPKswere 6 B treated either with IFN-g IL-9 ( ), Downloaded from IL-17A 6 IL-9 (C), or left untreated (control) for 24 h. Representative images depict actin (red), p-Mlc (green), and DAPI-visualized nucleus (blue) under original magnification 363. Scale bar, 10 mm. The fold change in the fluores- cence intensity was quantified as a mea- http://www.jimmunol.org/ sure of the change in p-Mlc levels (n =3 independent experiments, 10–12 cells were analyzed in each experiment per condition). Data are represented as and mean + SEM for bar graph. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 27, 2021

Next, we examined if IFN-g or IL-17 had an impact on The effects of IL-9, IFN-g, and IL-17A on migration of HPKs collagen degradation ability of HPKs. IFN-g had no effect on are independent of adhesion collagen degradation (Fig. 6B; n = 3 independent experiments, As FA dynamics are critical regulators of cell migration, we . p 0.05). On the contrary, IL-17A increased the collagen examined if IL-9 or IL-17A had an effect on the total size of FA matrix degradation by almost 50% compared with the corre- formed in HPKs. There was no significant change in the formation sponding control (untreated cells) (Fig. 6B; n = 3 independent of FAs in HPKs by all the cytokines tested (Supplemental Fig. 3A, , experiments, p 0.05), indicating that IL-17A but not IL-9 3B; n = 5 independent experiments, p . 0.05). These data or IFN-g promotes the ability of keratinocytes to degrade suggest that the effects of IL-9, IFN-g, and IL-17A on HPK collagen, which is the major component of the basement migration potential observed in this study are independent of FA membrane. dynamics. Next, the roles of IL-9 and IL-17A were examined on mRNA expression and activity of various MMPs by quantitative PCR IL-9 has no effect on HPK proliferation and (Supplemental Fig. 2D) and by gelatin zymography, respectively production (Supplemental Fig. 2E). Neither IL-9 nor IL-17A influenced mRNA IL-9 belongs to IL-2Rg receptor family (9) and is known to promote expression of MMP-2, MMP-9, and MMP-13 (Supplemental Fig. 2D; cell growth and development in several cell types (10, 33). In ad- n = 4–6 independent experiments). In addition, there was no statisti- dition, actin cytoskeleton remodeling also regulates proliferation of cally significant effect of IL-9 or IL-17A on MMP-9 (92 kDa) cells (34). Therefore, we examined if IL-9–mediated inhibition of and MMP-2 (67 kDa) (Supplemental Fig. 2E; n = 4–5 independent actomyosin contractility has roles in proliferation of HPKs. After experiments) activity in the gelatin zymography. These data indicate IL-9R upregulation, HPKs were treated with IL-9 or left untreated that reduced IL-9–mediated migration of the HPKs is independent of (control) for 48 h. There was no significant effect of IL-9 on pro- the collagenases, including MMPs. However, IL-17A–mediated col- liferation of HPKs (Supplemental Fig. 4A; n =2independentex- lagen degradation observed in Fig. 6C might be because some other periments) suggesting that IL-9–mediated inhibition of actomyosin collagenase activity. contractility is dispensable for HPKs proliferation. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. IL-9, IFN-g, and IL-17A play differential role in ECM degradation. Collagen degradation assay was performed to analyze the degradation ability of HPKs. The collagen (green), F-actin (red), nucleus of the cell (blue), and merge image, respectively, are shown (A and B left panel). Degraded collagen is indicated with white arrows. The area degraded by cytokine-treated cells was quantified. (A) HPKs were pretreated with IL-4 plus TGF-b overnight for upregulation of IL-9R. The next day, HPKs were washed and further treated with IL-9 or left untreated (control) for an additional 24 h. The representative images (left) and quantification of degraded area (right) is shown. (B) HPKs were treated either with IFN-g or IL-17A or left untreated (control) for 24 h. Imaging was performed under original magnification 320. Scale bar, 50 mm. The representative images (left) and quantification of degraded area (right) are shown (n = 3 independent experiments with at least 30 cells in each experiment were analyzed per condition). Bar graphs are represented as mean + SEM. *p , 0.05.

We also examined if IFN-g–mediated cytoskeleton remodeling 3) IL-9 inhibits the IFN-g– and IL-17A–induced migration of affected the proliferation of HPKs. HPKs were treated with HPKs as well as malignant epidermoid carcinoma cells. IFN-g 6 IL-9 for 48 h or left untreated (control). Neither IFN-g In this study, we demonstrate that IL-9 inhibits migration of HPKs nor IFN-g plus IL-9 had any significant effect on the proliferation and A-431 epidermoid carcinoma cells in a confined 3D ECM using of HPKs as compared with control (Supplemental Fig. 4B; n =2 a physiologically relevant model. Cell migration is a multistep independent experiments). process and is dependent upon protrusive (forward pulling) forces Next, we quantified the effect of IL-9 on chemokine (CXCL8 and that drive cell motility and frictional forces that resist cell motility CXCL10) production. HPKs were pretreated with IL-4 plus TGF-b (35, 36). During migration through a confined 3D microenviron- on day 0, followed by IL-9 treatment on day 1. Cell-free super- ment of ECM, cells sense frictional force and steric hindrance natants were collected after 24, 48, and 72 h of treatment. As shown imposed by the 3D ECM because of smaller pore size compared in Supplemental Fig. 4, there was marginal and inconsistent increase in with the cell (35). To migrate through this 3D ECM, cells must CXCL8 (Supplemental Fig. 4C) and CXCL10 (Supplemental Fig. 4D) either degrade the ECM by proteolytic enzymes, including MMPs levels in multiple donors (n = 10 independent experiments). (37), and/or physically deform themselves. A balanced interplay of the actomyosin machinery that encompasses actin stress fibers Discussion turnover, myosin, and contractility (retraction dynamics) is essen- Our data demonstrate, to our knowledge, a few novel observations: tial for migration in multiple cell types (35). One of the major 1) profound suppression of migration potential of keratinocytes by regulators of cellular contractility in cells is myosin. Myosin IIA, IL-9 depends on actomyosin contractility (retraction dynamics) an isoform of myosin, has been known to influence the formation and is independent of MMP pathway and cell adhesion to the of actin stress fibers, thereby regulating cellular contractility (32). matrix; 2) IL-17A promotes migration potential of HPKs by both Previous studies have demonstrated that a combination of con- actomyosin contractile mechanisms and proteolytic pathways; and tractility and stiffness controls the deformability of many cell types 12 ROLES OF IL-9 AND IL-17 IN KERATINOCYTE MIGRATION

(38, 39). To our knowledge, this is the first report describing References actomyosin contractility as a key mechanism controlling cytokine- 1. Purwar, R., J. Campbell, G. Murphy, W. G. Richards, R. A. Clark, and mediated keratinocyte migration. IL-9 reduces the contractile T. S. Kupper. 2011. Resident memory T cells (T(RM)) are abundant in human lung: diversity, function, and antigen specificity. PLoS One 6: e16245. dynamics and the migration of cytokine-treated keratinocytes in a 2. Purwar, R., C. Schlapbach, S. Xiao, H. S. Kang, W. Elyaman, X. Jiang, highly reproducible manner. Further, pharmaceutical abrogation A. M. Jetten, S. J. Khoury, R. C. Fuhlbrigge, V. K. Kuchroo, et al. 2012. Robust of NMMIIA by blebbistatin abolished both contractility and tumor immunity to melanoma mediated by -9-producing T cells. Nat. Med. 18: 1248–1253. stiffness in a manner very similar to IL-9 and resulted in inhibition 3. Elyaman, W., E. M. Bradshaw, C. Uyttenhove, V. Dardalhon, A. Awasthi, of keratinocyte migration potential. In line with our observa- J. Imitola, E. Bettelli, M. Oukka, J. van Snick, J. C. Renauld, et al. 2009. IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural tions, previous studies have also shown that contractility is in- regulatory T cells. Proc. Natl. Acad. Sci. USA 106: 12885–12890. dispensable for 3D migration, and disruption of contractility by 4. Raphael, I., S. Nalawade, T. N. Eagar, and T. G. Forsthuber. 2015. T cell subsets blebbistatin (40) leads to the reduction of migration in a 3D and their signature cytokines in autoimmune and inflammatory diseases. Cyto- kine 74: 5–17. microenvironment. We also observed that IL-9 neither increased 5. Hijnen, D., E. F. Knol, Y. Y. Gent, B. Giovannone, S. J. Beijn, T. S. Kupper, the MMP expression levels nor its activity, indicating that IL- C. A. Bruijnzeel-Koomen, and R. A. Clark. 2013. CD8(+) T cells in the lesional 9–mediated keratinocyte migration is independent of MMP- skin of atopic dermatitis and psoriasis patients are an important source of IFN-g, IL-13, IL-17, and IL-22. J. Invest. Dermatol. 133: 973–979. mediated mechanisms. 6. Thepen, T., E. G. Langeveld-Wildschut, I. C. Bihari, D. F. van Wichen, F. C. van Next, IL-9 suppressed both IL-17A– and IFN-g–induced mi- Reijsen, G. C. Mudde, and C. A. Bruijnzeel-Koomen. 1996. Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial TH2 gration of human keratinocytes. Mechanistically, IL-9 regulated response to a TH1 response in situ: an immunocytochemical study. J. Allergy the IFN-g– and IL-17A–mediated physical properties (stress fibers Clin. Immunol. 97: 828–837. formation, contractility, and stiffness) of human keratinocytes. 7. Murugaiyan, G., and B. Saha. 2009. Protumor vs antitumor functions of IL-17. Downloaded from J. Immunol. 183: 4169–4175. Similar to our study, previous studies have shown the effect of 8. Schlapbach, C., A. Gehad, C. Yang, R. Watanabe, E. Guenova, J. E. Teague, IFN-g on actin cytoskeleton reorganization in various cell types L. Campbell, N. Yawalkar, T. S. Kupper, and R. A. Clark. 2014. Human TH9 (41). Similar to human keratinocytes, IL-9 suppressed the IFN- cells are skin-tropic and have autocrine and paracrine proinflammatory capacity. Sci. Transl. Med. 6: 219ra8. g– and IL-17A–induced migration of epidermoid carcinoma cells 9. Bauer, J. H., K. D. Liu, Y. You, S. Y. Lai, and M. A. Goldsmith. 1998. Hetero- (A-431). Unlike healthy human keratinocytes, surprisingly, IFN-g, merization of the gammac chain with the interleukin-9 receptor alpha subunit leads to STAT activation and prevention of . J. Biol. Chem. 273: 9255–9260. IL-17A, and IL-9 alone did not significantly alter the stress fibers 10. Renauld, J. C., A. Goethals, F. Houssiau, E. Van Roost, and J. Van Snick. 1990. http://www.jimmunol.org/ formation and stiffness (data not shown) of A-431 cells. However, and expression of a cDNA for the human homolog of mouse T cell and IL-9 altered the contractility of IFN-g– and IL-17A–treated cells, growth factor P40. Cytokine 2: 9–12. 11. Uyttenhove, C., R. J. Simpson, and J. Van Snick. 1988. Functional and structural indicating that IL-9–mediated inhibition of A-431 migration could characterization of P40, a mouse glycoprotein with T-cell growth factor activity. be partially mediated by actomyosin-dependent pathways. The Proc. Natl. Acad. Sci. USA 85: 6934–6938. opposing effect of IL-9 on contractility observed between HPKs 12. Goswami, R., and M. H. Kaplan. 2011. A brief history of IL-9. J. Immunol. 186: 3283–3288. and epidermoid carcinoma cells (A-431) might be due to nature of 13. Hong, C. H., K. L. Chang, H. J. Wang, H. S. Yu, and C. H. Lee. 2015. IL-9 cells (healthy versus transformed). Further studies will be needed induces IL-8 production via STIM1 activation and ERK phosphorylation in epidermal keratinocytes: a plausible mechanism of IL-9R in atopic dermatitis. to elucidate the underlying mechanism of the observed differences J. Dermatol. Sci. 78: 206–214. of IL-9 roles in healthy and malignant cells. 14. Lu, Y., S. Hong, H. Li, J. Park, B. Hong, L. Wang, Y. Zheng, Z. Liu, J. Xu, J. He, by guest on September 27, 2021 In conclusion, our results demonstrate that disruption of acto- et al. 2012. Th9 cells promote antitumor immune responses in vivo. J. Clin. Invest. 122: 4160–4171. myosin contractility by IL-9 inhibits the migration potential of 15. Ma, L., H. B. Xue, X. H. Guan, C. M. Shu, J. H. Zhang, and J. Yu. 2014. Possible HPKs in an MMP-independent manner. In contrast, IL-17A in- pathogenic role of T helper type 9 cells and interleukin (IL)-9 in atopic der- creased the migration of HPKs in a protease-dependent manner. matitis. Clin. Exp. Immunol. 175: 25–31. 16. Kryczek, I., M. Banerjee, P. Cheng, L. Vatan, W. Szeliga, S. Wei, E. Huang, Our observations also suggest that IFN-g and IL-17A promote E. Finlayson, D. Simeone, T. H. Welling, et al. 2009. Phenotype, distribution, migration of keratinocytes by altering cell mechanics, which generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114: 1141–1149. could indicate a pathogenic role in skin inflammatory diseases and 17. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein, skin malignancies. More importantly, IL-9 inhibits the IFN-g– J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, et al. 2007. Develop- and IL-17A–induced migration of both healthy keratinocytes and ment, cytokine profile and function of human -producing helper T cells. Nat. Immunol. 8: 950–957. malignant epidermoid carcinoma cells, suggesting that IL-9 might 18. Leonardi, C., R. Matheson, C. Zachariae, G. Cameron, L. Li, E. Edson-Heredia, play a protective role in skin diseases in which keratinocyte mi- D. Braun, and S. Banerjee. 2012. Anti-interleukin-17 monoclonal antibody gration is critical for disease pathogenesis, such as inflammatory ixekizumab in chronic plaque psoriasis. N. Engl. J. Med. 366: 1190–1199. 19. Ha, H. L., H. Wang, P. Pisitkun, J. C. Kim, I. Tassi, W. Tang, M. I. Morasso, diseases and malignant carcinomas. Our data suggest that strate- M. C. Udey, and U. Siebenlist. 2014. IL-17 drives psoriatic inflammation via distinct, gies that abrogate actomyosin contractility, such as biologics, in- target cell-specific mechanisms. Proc. Natl. Acad. Sci. U S A 111: E3422–E3431. cluding rIL-9, or pharmacological inhibitors, such as blebbistatin, 20. Wu, L., X. Chen, J. Zhao, B. Martin, J. A. Zepp, J. S. Ko, C. Gu, G. Cai, W. Ouyang, G. Sen, et al. 2015. A novel IL-17 signaling pathway controlling may be useful in inhibiting keratinocyte migration in various skin keratinocyte proliferation and tumorigenesis via the TRAF4-ERK5 axis. J. Exp. inflammatory diseases as well as malignancies. Although these Med. 212: 1571–1587. 21. Nardinocchi, L., G. Sonego, F. Passarelli, S. Avitabile, C. Scarponi, C. M. Failla, data highlight that regulation of actomyosin contractility is one of S. Simoni, C. Albanesi, and A. Cavani. 2015. Interleukin-17 and interleukin-22 the key mechanisms of IL-9–, IFN-g–, and IL-17A–mediated promote tumor progression in human nonmelanoma skin cancer. Eur. J. Immu- keratinocyte migration potential, in vivo studies are required to nol. 45: 922–931. 22. Shibata, M., Y. Shintaku, K. Matsuzaki, and S. Uematsu. 2014. The effect of IL-17 further probe the role of these T cell–derived cytokines in kera- on the production of proinflammatory cytokines and matrix metalloproteinase-1 by tinocyte migration. human periodontal ligament fibroblasts. Orthod. Craniofac. Res. 17: 60–68. 23. Starodubtseva, N. L., V. V. Sobolev, A. G. Soboleva, A. A. Nikolaev, and S. A. Bruskin. 2011. [Expression of genes for metalloproteinases (MMP-1, Acknowledgments MMP-2, MMP-9, and MMP-12) associated with psoriasis]. Genetika 47: We thank the Industrial Research and Consultancy Centre, at the Indian 1254–1261. Institute of Technology Bombay for the use of the central facilities at 24. Sakurai, T., D. Yoshiga, W. Ariyoshi, T. Okinaga, H. Kiyomiya, J. Furuta, I. Yoshioka, K. Tominaga, and T. Nishihara. 2016. Essential role of - the Department of Biosciences and Bioengineering. activated kinases in IL-17A-induced MMP-3 expression in human sy- novial sarcoma cells. BMC Res. Notes 9: 68. 25. van der Kammen, R., J. Y. Song, I. de Rink, H. Janssen, S. Madonna, Disclosures C. Scarponi, C. Albanesi, W. Brugman, and M. Innocenti. 2017. Knockout The authors declare no financial conflict of interest. of the Arp2/3 complex in epidermis causes a psoriasis-like disease The Journal of Immunology 13

hallmarked by hyperactivation of transcription factor Nrf2. Development cellular tension drives increased tissue stiffness and b-catenin activation to induce 144: 4588–4603. epidermal hyperplasia and tumor growth. Cancer Cell 19: 776–791. 26. Uzquiano, M. C., V. G. Prieto, J. W. Nash, D. S. Ivan, Y. Gong, A. J. Lazar, and 35. Lautscham, L. A., C. Ka¨mmerer, J. R. Lange, T. Kolb, C. Mark, A. H. Diwan. 2008. Metastatic basal cell carcinoma exhibits reduced actin ex- A. Schilling, P. L. Strissel, R. Strick, C. Gluth, A. C. Rowat, et al. 2015. pression. Mod. Pathol. 21: 540–543. Migration in confined 3D environments is determined by a combination of 27. Gschwandtner, M., R. Purwar, M. Wittmann, W. Ba¨umer, M. Kietzmann, adhesiveness, nuclear volume, contractility, and cell tiffness. Biophys. T. Werfel, and R. Gutzmer. 2008. Histamine upregulates keratinocyte MMP-9 J. 109: 900–913. production via the histamine H1 receptor. J. Invest. Dermatol. 128: 2783–2791. 36. Haase, I., R. Evans, R. Pofahl, and F. M. Watt. 2003. Regulation of keratinocyte 28. Purwar, R., M. Wittmann, J. Zwirner, M. Oppermann, M. Kracht, O. Dittrich- shape, migration and wound epithelialization by IGF-1- and EGF-dependent Breiholz, R. Gutzmer, and T. Werfel. 2006. Induction of C3 and CCL2 by C3a in signalling pathways. J. Cell Sci. 116: 3227–3238. keratinocytes: a novel autocrine amplification loop of inflammatory skin reac- 37. Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and tions. J. Immunol. 177: 4444–4450. M. Seiki. 1994. A matrix metalloproteinase expressed on the surface of invasive 29. Eltzner, B., C. Wollnik, C. Gottschlich, S. Huckemann, and F. Rehfeldt. 2015. tumour cells. Nature 370: 61–65. The filament sensor for near real-time detection of cytoskeletal fiber structures. 38. Ochalek, T., F. J. Nordt, K. Tullberg, and M. M. Burger. 1988. Correlation be- PLoS One 10: e0126346. tween cell deformability and metastatic potential in B16-F1 melanoma cell 30. Horzum, U., B. Ozdil, and D. Pesen-Okvur. 2014. Step-by-step quantitative variants. Cancer Res. 48: 5124–5128. analysis of focal adhesions. MethodsX 1: 56–59. 39. Yap, B., and R. D. Kamm. 2005. Cytoskeletal remodeling and cellular activation 31. Ishida, Y., T. Kondo, T. Takayasu, Y. Iwakura, and N. Mukaida. 2004. The es- during deformation of neutrophils into narrow channels. J. Appl. Physiol. 99: sential involvement of cross-talk between IFN-gamma and TGF-beta in the skin 2323–2330. wound-healing process. J. Immunol. 172: 1848–1855. 40. Srinivasan, S., V. Ashok, S. Mohanty, A. Das, S. Das, S. Kumar, S. Sen, and 32. Shutova, M., C. Yang, J. M. Vasiliev, and T. Svitkina. 2012. Functions of nonmuscle R. Purwar. 2017. Blockade of Rho-associated protein kinase (ROCK) inhibits the myosin II in assembly of the cellular contractile system. PLoS One 7: e40814. contractility and invasion potential of cancer stem like cells. Oncotarget 8: 33. Matsuzawa, S., K. Sakashita, T. Kinoshita, S. Ito, T. Yamashita, and K. Koike. 21418–21428. 2003. IL-9 enhances the growth of human mast cell progenitors under stimu- 41. Ng, C. T., L. Y. Fong, M. R. Sulaiman, M. A. Moklas, Y. K. Yong, M. N. Hakim, lation with . J. Immunol. 170: 3461–3467. and Z. Ahmad. 2015. -gamma increases endothelial permeability 34. Samuel, M. S., J. I. Lopez, E. J. McGhee, D. R. Croft, D. Strachan, P. Timpson, by causing activation of p38 MAP kinase and actin cytoskeleton alteration. Downloaded from J. Munro, E. Schro¨der, J. Zhou, V. G. Brunton, et al. 2011. Actomyosin-mediated J. Interferon Cytokine Res. 35: 513–522 http://www.jimmunol.org/ by guest on September 27, 2021 a b1000 c 800 d 1000 ns e 800 Isotype ns Control * 800 IL-4 (20ng/ml) 600 600 +TGF-β (10ng/ml) 600 500 400 400 Count 400 200 200 200 MeanIL-9R expression MeanIL-9R expression MeanIL-9R expression 0 -102 0 102 103 104 0 0 0 Control IL-4 +TGF-β Control IL-4 Control TGF-β Fluorescence intensity (IL-9R)

ns 1.5 f 800 ns g 800 h 1000 i ns ns 800 600 600 1.0 600 400 400 400 0.5 200 200 200 MeanIL-9R expression Foldchange inSF no.of MeanIL-9R expression MeanIL-9R expression 0 0 0 0.0 Control IL-9 Control TNF-α Control IFN-� Control IFN-�+TNF-α j k l m 30 ns 200 2.0 80 ns ns ns

150 1.5

60 m)

20 �

( sec) ( 40 100 1.0 total �

10 Distance( 20 50 0.5 Elasticmodules (kPa) NormalizedIntensity fl.

0 0 0 0.0 Control IL-9 Control IL-9 Control IL-9 Control IL-9

Supplementary Figure 1: Regulation of lL-9 receptor by pro-inflammatory cytokines: (a) Human primary keratinocytes (HPKs) isolated from healthy skin samples and cultured HPKs at passage 2 are shown. Cytokeratin staining of the HPKs was positive in the cytoplasm (green) indicating purity of cultures (40x) (below). (b) Representative histogram showing upregulation of IL-9R expression on IL-4 (20 ng/ml) plus TGF-β (10 ng/ ml) treatment (black line) as compared to the control (blue line). The red line represents the isotype control. (c) Quantification of the IL-9 receptor expression in HPKs upon IL-4 plus TGF-β treatment by flow cytometry (n=5). (d-h) IL-9R levels upon stimulation with IL-4 (20ng/ml, n=3, d), TGF-β (10ng/ml, n=3, e), TNF-α (10 ng/ml, n=5, f), IFN-� (10 ng/ml, n=5, g) and IFN-�+TNF-α (n=5, h). (i-m) HPKs were treated with IL-9 or left untreated (control) to examine the mechanical properties of HPKs. Quantification of the actin stress fibers (i) post phalloidin staining under 63X magnification. Bar graph represents cumulative data (n=5 independent experiments). (j) Data shows �total obtained from trypsin deadhesion assay (n=4). (k) Quantification of the stiffness of HPKs treated with IL-9 are shown (n= 3 independent experiments, 25-30 cells were probed in each experiment). (l) Cumulative data of the invasion assay compared between control and IL-9 treated cells (n= 3 independent experiments, 10-12 cells were analyzed in each experiment) (m) Quantification of the fluorescence intensity upon pMLC staining (n=4 independent experiments). Control refers to untreated cells without any pretreatment. Data is represented Mean+SEM for bar graph. AFM data is represented as min max plot with median. (* p<0.05) a Thresholding Particle analysis Area of degraded collagen Area of degraded collagen Degradation = Area of the cell Area of the cell

b c Control IL-17A Normalized Normalized average of average of Area of Area of Control IL-17A degraded Cell area Degradation/ degraded Cell area Degradation/ collagen Area collagen Area Experiment 1 1 1.300345495

1 415.134 867.208 0.478701765 1 1211.835 2166.762 0.559283853 Experiment 2 1 1.609518521

2 591.613 1133.724 0.521831592 2 1440.563 1524.263 0.945088216 Experiment 3 1 1.495525617

3 720.07 1186.5 0.606885799 3 1673.512 2108.7 0.793622611 Pooled mean 1 1.468463211

4 572.403 1304.084 0.438931081 4 2179.421 2898.864 0.751818988 Pooled SD 0 0.156353031

5 949.957 1546.746 0.614164834 5 1050.761 1364.046 0.770326661 Pooled SEM 0 0.090270464 RT-PCR Zymography d e MMP-2 level MMP-9 level MMP-13 level MMP-9 level MMP-2 level ns 1.5 ns 2.0 3.0 ns ns 2.0 ns 2.0 1.0 1.5 1.5 2.0 1.5 1.0 1.0 1.0 0.5 1.0 Foldchange 0.5

0.5 0.5 Foldchange Foldchange Foldchange Foldchange 0 0 0 0 0 IL-4+TGF- + + + + + � IL-4+TGF-� + + IL-4+TGF-� + + IL-4+TGF-� IL-4+TGF-� + IL-9 - + IL-9 - + IL-9 - + IL-9 - + IL-9 - + 1.5 1.5 2.0 1.8 ns ns ns ns 1.5 1.6 1.0 1.0 1.4 1.0 0.5 0.5 1.2

Foldchange Foldchange 0.5 Foldchange 1.0 Foldchange 0 0 0 0.8 IFN- IFN- +IL9 IFN-� IFN-�+IL9 IFN-� IFN-�+IL9 IFN-� IFN-�+IL-9 � � ns ns 1.5 ns 1.5 6 1.5 2.0 ns ns 1.5 4 1.0 1.0 1.0 1.0 2 0.5 0.5 0.5

Foldchange 0.5 0 Foldchange Foldchange Foldchange Foldchange 0 0 0 0 -2 Control IL-17A Control IL-17A Control IL-17A Control IL-17A Control IL-17A

Legend in next page Supplementary Figure 2: IL-9 impacts keratinocyte migration in a MMP- independent pathway: (a) Representative schematic of the analysis performed to quantify the degradation ability of HPKs under the influence of cytokines in collagen degradation assay. The area of degraded collagen was analyzed using the auto threshold feature of ImageJ as described in the materials and methods section. The degraded area was normalized with the respective cell area. (b) Representation of the data analysis performed for the collagen degradation assay. The representative data from five cells analyzed are shown in the table and the degraded area normalized with the cell area is shown. The experiment was performed thrice and 30 cells per condition per experiment were analyzed (c) The means obtained from the 3 individual experiments performed are shown along with the pooled mean, pooled SD and pooled SEM. The data was represented in Figure 6 as Mean+SEM. (d-e): HPKs were pretreated overnight with IL-4 plus TGF-β. Next day, IL-9 was added or left untreated for 24 hours. Quantitative RT-PCR (qPCR) was performed to examine the mRNA expression of MMP-2, MMP-9 and MMP-13. The data indicates the fold change in the mRNA levels of MMP-2, MMP-9 and MMP-13 from cells treated for 8h with different cytokines. (c) Zymography was performed using cell-free supernatants of cytokine treated keratinocytes. Fold change of the degraded area was calculated to analyze the level of MMP activity. The fold change was normalized to the respective controls. Control for the IL-17A data refers to untreated cells. Actin Vinculin Merge ns a 60 ) 2

40 Control

20 Total area of FA ( µ m area FA of Total

IL-9 0 IL-4+TGF-� + + IL-9 - + b

40 ns Control ns ) 2 30 � 20 IFN-

10 Total area of FA ( µ m area FA of Total

0 Control IFN-� IL-17A IL-17A

Supplementary Figure 3: IL-9 and IL-17 treatment do not influence focal adhesion formation: HPKs were seeded on collagen coated coverslips and stained with actin (red), vinculin (green) and DAPI (blue) and total FA area was calculated as described in methods. (a): HPKs were pretreated with IL-4 plus TGF-β overnight for up- regulation of IL-9R. Next day, HPKs were washed and further treated with IL-9 or left untreated (control) for an additional 24h. (b): HPKs were treated with IFN-γ or IL-17A or left untreated (control) for an additional 24h. Representative images (left) of actin (red), vinculin (green) and DAPI (blue) and cumulative analysis (right) of multiple donors are shown (n=5 independent experiments, 5-6 cells were analyzed per condition in each experiment). a 48h b 1.5 48h 1.5 ns ns ns

1.0 1.0

0.5 0.5 Foldchange in cell number Foldchange in cell number 0.0 0.0 IFN- - + + IL-4+TGF-� + + � IL-9 - - + IL-9 - +

c 1000 24h 48h 72h d 1500 24h 48h ns ns ns 800 1000 600

400 500 Conc(pg/ml)CXCL8 of

200 Conc(pg/ml)CXCL10 of

0 0 + + IL-4+TGF-� + + + + + + IL-4+TGF-� + + IL-9 - + - + - + IL-9 - + - +

Supplementary figure 4: Role of IL-9 on proliferation and chemokine profile of HPKs: (a-b): Proliferation of HPKs under the influence of IL-9 was examined. (a) HPKs were pretreated with IL-4 plus TGF-β overnight for up-regulation of IL-9R. Next day, HPKs were washed and further stimulated with IL-9 or left untreated (control) for an additional 48h. Fold change in cell number between untreated and IL-9 treated cells at 48h is represented (b) HPKs were treated with IFN-γ ± IL-9 for 48h or left untreated (control). Proliferation was examined by cell counting using ImageJ software. Bar graph represents the fold change in the cell numbers normalized with its respective control of 48h. (n=2). (c-d): HPKs were stimulated with IL-9 after the upregulation of IL-9R or left untreated (control) and cell free supernatant was collected at different time points (24 hours, 48 hours and 72 hours) and levels of CXCL8 (c) or CXCL10 (d) were quantified in cell-free supernatant by ELISA (n=10). Bar graphs are represented as Mean + SEM.