Abnormally differentiating keratinocytes in the epidermis of systemic sclerosis patients show enhanced secretion of CCN2 and S100A9

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1 Abnormally differentiating keratinocytes in the epidermis of systemic sclerosis patients show enhanced secretion of CCN2 and S1A9 Joanna Nikitorowicz-Buniak UCL Division of Medicine

Systemic sclerosis () A heterogeneous, severe rheumatic connective tissue disease 1 in 1, affected, Female predominance - 4:1 Complex pathogenesis: infection agents, environmental triggers and

2 Systemic sclerosis () A heterogeneous, severe rheumatic connective tissue disease 1 in 1, affected, Female predominance - 4:1 Complex pathogenesis: infection agents, environmental triggers and genetic factors Progressing autoimmunity, inflammation, and vasculopathy leading to fibrosis Most patients develop skin fibrosis patients classified into subsets depending on the pattern of skin involvement

Skin a complex tissue compartment Studies on skin have focused on the dermis and dermal fibroblasts Epidermis-dermis cross-talk important in homeostasis, wound healing and

3 Skin a complex tissue compartment Studies on skin have focused on the dermis and dermal fibroblasts Epidermis-dermis cross-talk important in homeostasis, wound healing and cancer (Warner et al., 27) epidermis dermis In hypertrophic scaring and keloids epidermis can promote dermal fibrosis (Bellemere et al., 25; Simon et al., 211; Funayama et al., 23)

4 Epidermis in Increased ECM and Ca2+ binding proteins, and those associated with oxidative stress, protein folding, contractility and mobility Skin pigmentation changes epidermis Promotes fibroblast activation and fibroblast CCN2 release via IL-1α Activated keratinocytes express K6 and K16 Active MAPK and c-met signalling

5 Hypothesis and objectives epidermal keratinocytes have an important role in the pathogenesis of skin disease. 1. Explore keratinocyte differentiation in 2. Profile the epidermal and dermal secretomes 3. Investigate epidermal-fibroblast interactions

6 Increased epidermal thickness in Control Thickness Thickness ** median ± SEM HC n=6, n=6 **p<.5

7 Hypertrophy of epidermal keratinocytes HC n=7, n=1; Mann-Whitney U test *p<.5 Measurements based on the average of 6 fields of view for each section

8 Altered terminal differentiation markers loricrin Control involucrin filaggrin HC n= 6, n= 9, *p<.5 **p<.5

DMEM overnight epidermis Serum free KM Controls

9 Profiling the secretome - multiplex 4mm forearm biopsy Luminex Trypsin/ EDTA dermis Serum free DMEM overnight epidermis Serum free KM Controls n = 8 or 12 n = 8 or 12 ELISA (Reiss et al., 29),

10 CCN2 marker and mediator of fibrosis in Involved in proliferation, migration, adhesion, matrix production, and wound healing. Upregulated in various fibrotic diseases including (Igarashi 1996). Elevated levels of serum CCN2 in diffuse linked to pulmonary complications (Sato 2). Plasma and dermal interstitial fluid levels for N-CCN2 elevated and correlate with severity of skin disease (Dziadzio 25). fibroblasts constitutively overexpress CCN2 despite the absence of TGF-β (Denton 21).

11 Increased CCN2 levels released by epidermis HC n=8-12 n=8-12 *p<.5, CCN2 mrna in epidermal blister sheets HC n=9 and n=32; * p<.5.

12 CCN2 expression pattern in skin Control Early Established

13 S1A9 in the skin Control

Relative CTGF mrna levels CTGF protein (OD) Change in proliferation (%) The scratch clousure compared to unstimulated (%) Effect of S1A9 on fibroblast production of CCN2 15 1 5-5 -1 Control.

14 Relative CTGF mrna levels CTGF protein (OD) Change in proliferation (%) The scratch clousure compared to unstimulated (%) Effect of S1A9 on fibroblast production of CCN Control * * * ** Control.5ug/ml 1ug/ml 2ug/mml -15 S1A9 (ug/ml) CTGF 1 8 * GAPDH None S1A9 S1A9 TAK-242 +TAK-242 * 2 None S1A9 S1A9+TAK-242 TAK HC n=3 n=3 None S1A9 S1A9+TAK-242 TAK-242

15 Number of cells Number of cells Number of cells Number of cells Control TGFβ signaling in skin Merged Phospho-Smad2/3 Cytokeratin 14 p-smad2/3 p-smad2/3 positive positive fibroblasts fibroblasts p-smad2/3 p-smad2/3 positive positive keratinocytes * 2 2 ** Control Control Control Control

16 Copy number Copy number Epithelial and mesenchymal markers Control Control Merge Merge Merge merge Mer ge Merge Merge Col IV Col IV E-cadherin E-cadherin FSP-1 FSP-1 Vimentin Vimentin 8 SNAI1 * 6 SNAI HC dc HC dc

17 Summary 1. patients have a thickened epidermis and hypertrophic keratinocytes. 2. keratinocytes exhibit altered expression of differentiation markers. 3. Activated keratinocytes in the skin of patients produce elevated levels of CCN2 and S1A9. 4. Stimulation of dermal fibroblasts with S1A9 induce CCN2 production via TLR4. 5. Changes consistent with partial EMT are present in the epidermis.

18 Acknowledgments Dr Richard Stratton Prof David Abraham Prof Chris Denton Dr Xu Shiwen Kristina Clark Sarah Carrar Dr Markela Ponticos Dr Sandra Guerra Dr Alan Holmes Korsa Khan

19 Control Langerin FSP-1 Merge

20 Control Control TGFβ Copy number TGFβ signaling in skin Copy number Number of cells Number of cells Number of cells Number of cells Merged Phospho-Smad2/3 Cytokeratin 14 p-smad2/3 positive fibroblasts * p-smad2/3 p p-smad2/3 positive fibroblasts C p-smad2/3 Controlpositive keratinocytes 2 15 ** Control Bright field Pan-cytokeratin Control FSP-1 Control Merged 15 1 Snail1 2ng/ml 4ng/ml 3 2 Snail2 2ng/ml 4ng/ml hrs 48hrs 72hrs 24hrs 48hrs 72hrs

21 What came first?

22 S1A9/calprotectin B/ MRP14 S1A9 in : serum and faeces (Andreasson, 211), BAL (Bargagli, 211) saliva (Giusti, 27), Key player in development of dermal fibrosis in FGF-2 KO mice (Meyer, 211). Stressed keratinocyte S1A9 Proinflammatory Pro-fibrotic Keratinocyte hyperproliferation and abnormal differentiation Leukocyte extravasation Activation of endothelial cells and phagocytes Expression of pro-inflammatory cytokines Fibroblast proliferation Inhibition of MMPs

23 S1A9 C/EBP STAT3 NF-κβ TLR4 p38 RAGE PI3K MAPK Akt JNK Erk 1/2 Expression of E-selectin, VCAM-1, ICAM-1, VEGF, CD36, TNF-α, CD4L, S1s, MCP-1, IL-1, IL-6, IL-8

25 Terminal differentiation process Basal cells Transit amplifying Terminally differentiating Change in cytokeratins expression, cross linking cellular envelop proteins, loss of nucleus, removal from the surface from Candi et al., 25

26 Epidermis in hypertrophic scaring Hypertrophic scars: - decreased production of keratinocyte derived IL-1a (which stimulate matrix degradation) and increased PDGF (which increase matrix production) leads to decreased catabolism of dermal matrix and increased formation of ECM (Niessen 21) - Co-culture of hypertrophic keratinocytes with normal wound myofibroblasts/ hypertrophic myofibroblasts/ fibroblasts induced thicker dermis due to increased synthesis of collagen and decreased MMP-1 as well as increased proliferation of dermal cells, (Bellemare 25) - Hypertrophic keratinocytes have increased secretion of TIMP-1 inhibitor of matrix degrading MMPs and correlates with dermal thickness (Simon 211)

27 Epidermis in keloids Keloid basal keratinocytes overexpress VEGF (Ong 27) Keloid keratinocytes increase proliferation and decrease apoptosis of co-cultured fibroblasts (Funayama 23) Keloid keratinocytes upregulate TGFb receptors, smad2/3 phosphorylation in co-cultured fibroblasts (Phan 25)

28 Interactions Keratinocytes co-cultured with fibroblasts induce in fibroblasts: - a-sma, CTGF, collagen I, IV, V and VI, MMP-1,MMP 3, IL-8, PAI-1, VEGF, COX 2, MCP-1, IL-6, C-SCF, ET-1,

29 Working hypothesis Injury (e.g. oxidants, solvents) activated keratinocyte thickening, abnormal differentiation including perakeratosis, keratinocyte hypertrophy,? fibroblast CTGF epidermis active TGFβ ECM overproduction and fibrosis

30 Control Smad signalling in the epidermis * P-smad2/3 positive in papillary dermis / total cells, p >.5 CK14 marker of basal cell, lost during terminal differentiation P-smad 2/3 translocated into nucleus during canonical TGFβ signalling

31 Why study the skin? Most patients develop skin fibrosis Marker of disease severity and prognosis Rapid deterioration of skin condition in the early increases the risk of renal crisis (Steen et al.,1984) Correlation between baseline skin score and disease severity and mortality (Clements et al., 2) Association between improvement in skin condition and increased survival (Steen et al., 21)

Healing & Repair. Tissue Regeneration

Healing & Repair. Tissue Regeneration Healing & Repair Dr. Srikumar Chakravarthi Repair & Healing: Are they same? Repair :Regeneration of injured cells by cells of same type, as with regeneration of skin/oral mucosa (requires basement membrane)