1. Poly(I:C) Drives Type I IFN- and TGFβ-Mediated Inflammation and Dermal Fibrosis Simulating Altered Gene Expression in Systemic Sclerosis 
Giuseppina A. Farina, Michael R. York, Michael Di Marzio, Cindy A. Collins, Stephan Meller, Bernhard Homey, Ian R. Rifkin, Ann Marshak-Rothstein, Timothy R.D.J. Radstake, Robert Lafyatis 
Journal of Investigative Dermatology 
Volume 130, Issue 11, Pages (November 2010)
DOI: /jid
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Type I IFN-responsive genes: expression in the skin of patients with diffuse cutaneous systemic sclerosis (dcSSc) and dermal fibroblast induction by specific Toll-like receptor (TLR) ligands. (a, b) mRNA expression of Siglec1 (n=25) and OAS2 in lesional (n=36, SSc L) and non-lesional (n=15, SSc N/L) dcSSc and control skin (n=6). The average fold change in SSc L skin was increased for Siglec1 (5.75-fold increase) and OAS2 (3.17-fold increase). The fold change of OAS2 in SSc N/L skin (n=15) was also increased (2.8-fold increase), *P<0.05; **P<0.01. (c) mRNA expression of OAS2 (n=6) in SSc (n=6), lupus erythematosus (LE; n=12), and control skin (n=6). OAS2 in LE skin was higher than that in control skin (43.1-fold increase) and also higher than that in SSc lesional skin (7.6-fold increase, **P<0.01). (d) mRNA induction of OAS2 in normal (▵) and in SSc () dermal fibroblasts by TLR ligands: Pam3CSK4 (1μgml−1), Poly(I:C) (2.5μgml−1), lipopolysaccharide (LPS, 10μgml−1), imiquimod (5μgml−1); sspolyU/lyovec (100μgml−1), or CpGA (5mM); IFNα, IFNβ, and IFNγ (each at 250Uml−1); or transforming growth factor-β (TGFβ, 5ngml−1). TLR2 (P<0.01) and TLR3 ligands (P<0.0001), and IFNα/β (P<0.001) and IFNγ (P<0.01) significantly increased the expression of OAS2 in SSc and normal fibroblasts.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Type II IFN-responsive genes: expression in skin from patients with diffuse cutaneous systemic sclerosis (dcSSc) and dermal fibroblast induction by specific Toll-like receptor (TLR) ligands. (a, b) Increased mRNA expression of type II IFN-regulated genes, CCL2 (3.5-fold increase, **P<0.01) and CXCL9 (7.03-fold increase, *P<0.05), in SSc lesional (L) and in SSc non-lesional (N/L; and 5.8-fold increase, *P<0.05; **P<0.01, respectively) compared with control skin. (c) Increased expression of CXCL9 in dcSSc skin compared to lupus erythmatosus (LE) skin. (d) mRNA induction of CXCL9 in normal (▵) and SSc () dermal fibroblasts by TLR ligands (as in Figure 1), IFNα/β, IFNγ, and transforming growth factor-β (TGFβ). TLR2 (P<0.01) and TLR3 ligand (P<0.001), and IFNα/β (P<0.01) and IFNγ (P<0.001) induced CXCL9 in SSc and N/L fibroblasts compared with unstimulated cell lines. (e, f) mRNA (e) and protein (f) induction of CXCL10 in normal (▵) and in SSc () dermal fibroblasts by polyinosinic/polycytidylic acid (Poly(I:C)) (P<0.001), IFNα (P<0.01), IFNβ (P<0.01), IFNγ (P<0.001), or the combination of IFNβ and IFNγ (P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Induction of transforming growth factor-β (TGFβ)-responsive genes by Toll-like receptor (TLR) ligands in systemic sclerosis (SSc) and normal dermal fibroblasts. (a) Increased COMP mRNA expression in diffuse cutaneous SSc (dcSSc) and lupus erythematosus (LE) skin compared with control skin. (b, c) COMP and PAI-1 mRNA expression by dermal fibroblasts from dcSSc () and control (▵) subjects treated with TLR agonists (as in Figure 1), IFNα/β, IFNγ, or TGFβ. COMP and PAI-1 expression show significant induction with agonists of TLR2 (P<0.05), TLR3/Poly(I:C) (P<0.05), and TGFβ (P<0.001). (d) mRNA expression of TLR3 mRNA expression in lesional (SSc L; 1.5-fold increase, P=NS), in non-lesional skin (SSc N/L; 0.92-fold increase, P=NS) from dcSSc patients compared with control skin (n=6). (e) Increased TLR3 mRNA expression by dermal fibroblasts from control (▵) and dcSSc () subjects cultured as in Figure 2: TLR2 agonist (17.1-fold increase, P<0.05); TLR3 agonist (86.9-fold increase, P< ); IFNα (56.1-fold increase, P< ); IFNβ (39.5-fold increase, P< ); and IFNγ (13.8-fold increase, P<0.01). (f) Immunohistochemical staining with anti-TLR3 (left panel) and control immunoglobulin (right panel) in dcSSc skin. Bar=50μm. *P<0.05; **P<0.01.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Toll-like receptor 3 (TLR3) ligand, polyinosinic/polycytidylic acid (Poly(I:C)) causes inflammation and fibrosis in mouse skin. (a) Hematoxylin and eosin-stained cross section of skin from a C57BI6 mouse 1 week after subcutaneous pump injection of Poly(I:C) (B) or phosphate-buffered saline (A). Arrows indicate inflammatory infiltrates (heavy arrow) or dermal fibrosis (light arrow). Original magnification × 40. (b) Expression of the IFN-responsive genes MX2 (A) and CXCL9 (B), or transforming growth factor-β (TGFβ)-responsive genes COMP (C) and PAI-1 (D) by reverse transcriptase-PCR of skin RNA from control or polyinosinic/polycytidylic acid (Poly(I:C))-treated mice compared with mice injected by using a subcutaneous pump with increasing doses of TGFβ. Fold change shown in the graph is normalized to mRNA expression by control phosphate-buffered saline-treated mouse. Bar=200μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Effect of IFN or TRIF/TICAM deletion, or transforming growth factor-β (TGFβ) inhibition on polyinosinic/polycytidylic acid (Poly(I:C)) induction of IFN- and TGFβ-responsive genes. Expression of IFN-responsive genes MX2 (a) and CXCL9 (b), and TGFβ-responsive genes PAI-1 (c) and TSP1 (d) by reverse transcriptase-PCR analysis of skin mRNA from C57BI/6 wild type (WT) (n=10), C57BI/6/IFNAR-/- (n=8), and C57BI/6 TICAM-/- (n=10) mice 1 week after Poly(I:C) pump insertion; *P<0.05; **P<0.01. Expression of IFN-responsive genes MX2 (e) and CXCL9 (f), and TGFβ-responsive genes PAI-1 (g) and TSP1 (h) mRNA from untreated C57BI/6 WT (n=3) mice 1 week after phosphate-buffered saline pump insertion, or mice treated with TGFβ-neutralizing antibody (n=3) 1 week after Poly(I:C) pump insertion. Fold changes shown on the graph are normalized to mRNA expression by control mice. Results presented are means±SE and are representative of three independent experiments. *P<0.05; **P<0.01.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions