Yi Fritz

Keratinocyte Overexpression of IL-17C Promotes Psoriasiform Skin Inflammation

IL-17C is a functionally distinct member of the IL-17 family that binds IL-17 receptor E/A to promote innate defense in epithelial cells and regulate Th17 cell differentiation. We demonstrate that IL-17C (not IL-17A) is the most abundant IL-17 isoform in lesional psoriasis skin (1058 versus 8 pg/ml; p < 0.006) and localizes to keratinocytes (KCs), endothelial cells (ECs), and leukocytes. ECs stimulated with IL-17C produce increased TNF-α and KCs stimulated with IL-17C/TNF-α produce similar inflammatory gene response patterns as those elicited by IL-17A/TNF-α, including increases in IL-17C, TNF-α, IL-8, IL-1α/β, IL-1F5, IL-1F9, IL-6, IL-19, CCL20, S100A7/A8/A9, DEFB4, lipocalin 2, and peptidase inhibitor 3 (p < 0.05), indicating a positive proinflammatory feedback loop between the epidermis and ECs. Psoriasis patients treated with etanercept rapidly decrease cutaneous IL-17C levels, suggesting IL-17C/TNF-α–mediated inflammatory signaling is critical for psoriasis pathogenesis. Mice genetically engineered to overexpress IL-17C in KCs develop well-demarcated areas of erythematous, flakey involved skin adjacent to areas of normal-appearing uninvolved skin despite increased IL-17C expression in both areas (p < 0.05). Uninvolved skin displays increased angiogenesis and elevated S100A8/A9 expression (p < 0.05) but no epidermal hyperplasia, whereas involved skin exhibits robust epidermal hyperplasia, increased angiogenesis and leukocyte infiltration, and upregulated TNF-α, IL-1α/β, IL-17A/F, IL-23p19, vascular endothelial growth factor, IL-6, and CCL20 (p < 0.05), suggesting that IL-17C, when coupled with other proinflammatory signals, initiates the development of psoriasiform dermatitis. This skin phenotype was significantly improved following 8 wk of TNF-α inhibition. These findings identify a role for IL-17C in skin inflammation and suggest a pathogenic function for the elevated IL-17C observed in lesional psoriasis skin.

Proteomics of Skin Proteins in Psoriasis: From Discovery and Verification in a Mouse Model to Confirmation in Humans

Herein, we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n = 3) were compared with littermate controls (n = 3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average p = 0.0082; cystatin A, human ortholog); slc25a5 (average fold change of 46.2 and an average p = 0.0318); serpinb3b (average fold change of 35.6 and an average p = 0.0345; serpinB1, human ortholog); and kallikrein related peptidase 6 (average fold change of 4.7 and an average p = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, p < 0.0001), KLK6 (9-fold, p = 0.002), stefin A1 (7.3-fold; p < 0.001), and slc25A5 (1.5-fold; p = 0.05) using qRT-PCR on a second cohort of animals (n = 8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; p < 0.05), 29,000-fold (stefinA1; p < 0.01), 322-fold (KLK6; p < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes versus healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3-fold), KLK6 (5.8-fold), and serpinB1 (76-fold; all p < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover, slc25a5, cystatin A, KLK6, and serpinB1 protein were all increased in lesional psoriasis skin compared with normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.

Imiquimod has strain-dependent effects in mice and does not uniquely model human psoriasis.

BackgroundImiquimod (IMQ) produces a cutaneous phenotype in mice frequently studied as an acute model of human psoriasis. Whether this phenotype depends on strain or sex has never been systematically investigated on a large scale. Such effects, however, could lead to conflicts among studies, while further impacting study outcomes and efforts to translate research findings.MethodsRNA-seq was used to evaluate the psoriasiform phenotype elicited by 6 days of Aldara (5% IMQ) treatment in both sexes of seven mouse strains (C57BL/6 J (B6), BALB/cJ, CD1, DBA/1 J, FVB/NJ, 129X1/SvJ, and MOLF/EiJ).ResultsIn most strains, IMQ altered gene expression in a manner consistent with human psoriasis, partly due to innate immune activation and decreased homeostatic gene expression. The response of MOLF males was aberrant, however, with decreased expression of differentiation-associated genes (elevated in other strains). Key aspects of the IMQ response differed between the two most commonly studied strains (BALB/c and B6). Compared with BALB/c, the B6 phenotype showed increased expression of genes associated with DNA replication, IL-17A stimulation, and activated CD8+ T cells, but decreased expression of genes associated with interferon signaling and CD4+ T cells. Although IMQ-induced expression shifts mirrored psoriasis, responses in BALB/c, 129/SvJ, DBA, and MOLF mice were more consistent with other human skin conditions (e.g., wounds or infections). IMQ responses in B6 mice were most consistent with human psoriasis and best replicated expression patterns specific to psoriasis lesions.ConclusionsThese findings demonstrate strain-dependent aspects of IMQ dermatitis in mice. We have shown that IMQ does not uniquely model psoriasis but in fact triggers a core set of pathways active in diverse skin diseases. Nonetheless, our findings suggest that B6 mice provide a better background than other strains for modeling psoriasis disease mechanisms.

Induction of Alternative Proinflammatory Cytokines Accounts for Sustained Psoriasiform Skin Inflammation in IL-17C+IL-6KO Mice

IL-6 inhibition has been unsuccessful in treating psoriasis, despite high levels of tissue and serum IL-6 in patients. In addition, de novo psoriasis onset has been reported after IL-6 blockade in patients with rheumatoid arthritis. To explore mechanisms underlying these clinical observations, we backcrossed an established psoriasiform mouse model (IL-17C+ mice) with IL-6-deficient mice (IL-17C+KO) and examined the cutaneous phenotype. IL-17C+KO mice initially exhibited decreased skin inflammation; however, this decrease was transient and reversed rapidly, concomitant with increases in skin Tnf, Il36α/β/γ, Il24, Epgn, and S100a8/a9 to levels higher than those found in IL-17C+ mice. A comparison of IL-17C+ and IL-17C+KO mouse skin transcriptomes with that of human psoriasis skin revealed significant correlation among transcripts of skin of patients with psoriasis and IL-17C+KO mouse skin, and confirmed an exacerbation of the inflammatory signature in IL-17C+KO mice that aligns closely with human psoriasis. Transcriptional analyses of IL-17C+ and IL-17C+KO primary keratinocytes confirmed increased expression of proinflammatory molecules, suggesting that in the absence of IL-6, keratinocytes increase production of numerous additional proinflammatory cytokines. These preclinical findings may provide insight into why patients with arthritis being treated with IL-6 inhibitors develop new onset psoriasis and why IL-6 blockade for the treatment of psoriasis has not been clinically effective.

Transgenic expression of human amphiregulin in mouse skin: inflammatory epidermal hyperplasia and enlarged sebaceous glands.

To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5′ and 3′ untranslated region sequences (AREG‐UTR) led to a >10‐fold increase in hAREG expression compared to an otherwise‐identical construct containing only the coding region (AREG‐CDR). AREG‐UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG‐CDR mice. Histologically, AREG‐UTR mice presented with marked epidermal hyperplasia of tail skin (2.1‐fold increase in epidermal thickness with a 9.5‐fold increase in Ki‐67+ cells) accompanied by significantly increased CD4+ T‐cell infiltration. Dorsal skin of AREG‐UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG‐UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki‐67+ cells. To determine the response of AREG‐UTR animals to a pro‐inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG‐UTR and wild type mice (1.7‐ and 2.2‐fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen.

Interleukin 6 regulates psoriasiform inflammation–associated thrombosis

Psoriasis patients are at increased risk of heart attack and stroke and have elevated MRP8/14 levels that predict heart attack. The KC-Tie2 psoriasiform mouse model exhibits elevated MRP8/14 and is prothrombotic. Mrp14-/- mice, in contrast, are protected from thrombosis, but, surprisingly, KC-Tie2xMrp14-/- mice remain prothrombotic. Treating KC-Tie2xMrp14-/- mice with anti-IL-23p19 antibodies reversed the skin inflammation, improved thrombosis, and decreased IL-6. In comparison, IL-6 deletion from KC-Tie2 animals improved thrombosis despite sustained skin inflammation, suggesting that thrombosis improvements following IL-23 inhibition occur secondary to IL-6 decreases. Psoriasis patient skin has elevated IL-6 and IL-6 receptor is present in human coronary atheroma, supporting a link between skin and distant vessel disease in patient tissue. Together, these results identify a critical role for skin-derived IL-6 linking skin inflammation with thrombosis, and shows that in the absence of IL-6 the connection between skin inflammation and thrombosis comorbidities is severed.

Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis.

Psoriasis is a chronic inflammatory skin disease affecting 2.5–6 million patients in the United States. The cause of psoriasis remains unknown. Previous human and animal studies suggest that patients with a susceptible genetic background and some stimulus, such as barrier disruption, leads to a coordinated signaling events involving cytokines between keratinocytes, endothelial cells, T cells, macrophages and dendritic cells. Ceramides are endogenous skin lipids essential for maintaining skin barrier function and loss of ceramides may underlie inflammatory and premalignant skin. Ceramides act as a double-edged sword, promoting normal skin homeostasis in the native state, but can be metabolized to sphingosine-1-phosphate (S1P), linked to inflammation and tumorigenesis. To overcome this difficulty, we synthesized solenopsin analogs which biochemically act as ceramides, but cannot be metabolized to S1P. We assess their in vivo bioactivity in a well-established mouse model of psoriasis, the KC-Tie2 mouse. Topical solenopsin derivatives normalized cutaneous hyperplasia in this model, decreased T cell infiltration, interleukin (IL)-22 transcription, and reversed the upregulation of calprotectin and Toll-like receptor (TLR) 4 in inflamed skin. Finally, they stimulated interleukin (IL)-12 production in skin dendritic cells. Thus suggesting barrier restoration has both a biochemical and physical component, and both are necessary for optimal barrier restoration.

VEGF-Trap decreases CD4+ T cells, Th17 cytokines improving psoriasis-like skin inflammation in KC-Tie2 mice.

The reddened appearance of lesional psoriasis skin reflects increases in angiogenesis and although this increase is unlikely to be the principle cause of psoriasis it may provide new therapeutic targets. VEGF is a potent mediator of angiogenesis [1], elevated VEGF is found in lesional skin and sera of psoriasis patients and correlates with disease severity and several VEGF genetic polymorphisms have been identified associated with early onset psoriasis. VEGF contributions to psoriasis pathogenesis are multiple and include increasing cutaneous vascular permeability and inflammation, promotion of cutaneous leukocyte infiltration, eliciting pro-inflammatory T cell differentiation, and activating and inducing keratinocyte (KC) proliferation. Thus, VEGF serves autocrine and paracrine functions in psoriasis, promoting pro-inflammatory feedback loops between KCs, endothelial cells, infiltrating T cells and monocytes which together exacerbate cell-mediated immune inflammatory reactions. VEGF importance in psoriasis is further evidenced by the development of a psoriasiform phenotype in KC-VEGF overexpressing mice, where skin phenotypes similar to human psoriasis develop following oxazalone [2] or TPA [3] treatment or tape stripping in young and spontaneously in older mice [4]. In each model, KC-derived VEGF and angiogenesis increased prior to lesion development, and in lesional skin, immunocyte infiltrate included T cells and macrophages [2, 4]. Targeted VEGF-inhibition improved the skin disease, however dermal T cells remained present in one study [4], and VEGFR1 or VEGFR2-specific inhibition individually failed to modify the inflammatory response, but together resolved the psoriasiform phenotype [2]. Despite recent reports of patients experiencing psoriasis remission during bevacizumab treatment for colon cancer [5] and following sorafenib treatment for renal cell carcinoma [6], targeting VEGF in psoriasis has received minimal attention. Using the KC-Tie2 psoriasiform mouse model, we investigated the efficacy of anti-VEGF treatment in skin inflammation. KC-Tie2 mice spontaneously develop skin disease that phenocopies human psoriasis, including increases in angiogenesis, VEGF (~3-fold; P<0.02), psoriasis-related pro-inflammatory leukocytes and cytokines, and disease resolution following CsA treatment [7]. Adult KC-Tie2 mice and littermate controls were treated with subcutaneous VEGF-Trap or control FC protein (25mg/kg; n=8/group; Regeneron Pharmaceuticals, Tarrytown, NY) 2x/week for 4 weeks similar to what has previously been done [4]. VEGF-Trap is a soluble fusion protein that binds VEGF with high affinity, has an extended in vivo half-life in mice and consists of portions of human VEGFR1/R2 and the Fc domain of human IgG1. All animal protocols were approved by the Case Western Reserve University IACUC. Skin was examined histologically using H&E, immunohistochemistry and at the RNA and protein levels as previously described [7, 8]. All data are presented as mean ± SEM. Data were tested for normality and statistical significance calculated using either a Student t test or a Mann– Whitney U test, as appropriate. Significance was defined as P≤0.05. VEGF-Trap significantly reduced dermal angiogenesis to control levels in KC-Tie2 mice (P=0.04 vs KC-Tie2+FC protein) with concomitant reductions in acanthosis (47%; P=0.03 vs KC-Tie2+FC protein) and decreases in innate defense genes S100A8/A9 and DefB3 (all P<0.03; Fig. 1). Despite significant decreases in cutaneous CD11b+, CD11c+ and F4/80+ cells in VEGF-Trap treated KC-Tie2 mice (P<0.001 vs KC-Tie2+FC protein), levels remained significantly increased compared to control littermates (P<0.04). Myeloid cell-derived cytokines, TNFα, IL-1α, IL-6 and IL-12 remained elevated following VEGF-Trap treatment whereas IL-23 expression returned to control mouse levels (P=0.02 vs KC-Tie2+FC protein; Fig. 2). Interestingly, CD4+ T cells decreased (63%; P=0.001) in VEGF-Trap treated KC-Tie2 mouse skin, accompanied by significant reductions in IL-17A, IL-17F and IL-22 (all P<0.007; Fig. 2), whereas CD8+ T cells remained elevated along with IFNγ levels. Figure 1 VEGF-Trap treated KC-Tie2 mice have decreased angiogenesis, acanthosis and innate defense gene expression Figure 2 VEGF-Trap treated KC-Tie2 mice have fewer cutaneous infiltrating myeloid cells, CD4+ T cells and reduced IL-23 and Th17 cytokines Together, these data provide in vivo evidence for efficacy of VEGF-Trap treatment for psoriasis and suggest disease improvement may occur via VEGFmediated effects on the IL-23/Th17 axis of inflammation. These findings are consistent with VEGF inhibition in two prior genetic VEGF-overexpressing models of skin inflammation [2, 4], as well as recent evidence showing VEGF-inhibition (using G6-31) efficacy in the KC-specific JunB:c-Jun knockout mouse [9]. However, we provide mechanistic insight into how VEGF-mediated inhibition occurs, identifying reductions in Th17 cell presence and activation (via decreased CD4+ T cells and IL-17A/F and IL-22) in VEGF-Trap treated mice; possibly as a result of decreased levels of IL-23 but not IL-12. This observation validates and expands upon a prior report demonstrating decreased infiltrating CD3/4+ T cells and reductions in IL-22 [9]. Although significant decreases in myeloid cell infiltrates were observed in VEGF-Trap treated KC-Tie2 mouse skin, and decreased IL-23, other myeloid-cell derived cytokines, including TNFα, IL-12, IL-1α and IL-6 remained elevated, potentially explaining the significant improvement, but not complete reversal of acanthosis, Th1 presence (and increased IFNγ) and skin disease severity. This is reminiscent of prior work where significant improvement in skin disease severity, but not complete reversal, was found in KC-Tie2 mice treated with antibodies targeting TNFα [8]. Interestingly, recent attempts blocking both VEGF and TNFα, using a novel fusion protein targeting both molecules, called Valpha [3] has proven highly effective at reversing/preventing TPA-induced acanthosis and dermal lymph/angiogenesis in KC-VEGF animals at levels greater than either TNFα (Enbrel) or VEGF (VEGF-Trap) inhibition alone. VEGF-Trap also inhibits PlGF-signaling events [4], and PlGF expression levels were also elevated in KC-Tie2 mouse skin (21.66 ± 6.6pg/ml KC-Tie2 vs 8.4 ± 2.7pg/ml controls; P=0.002), thus the VEGF-Trap effects reported here likely reflect VEGF- and PlGF-inhibition. Interestingly, our observation that CD8+ T cells remain elevated along with IFNγ levels in KC-Tie2 mice treated with VEGF-Trap suggests disease severity can improve despite sustained Th1 activity if Th17 cell depletion is sufficient, and is consistent with recent clinical trials showing excellent efficacy of IL-17 inhibition in psoriasis patients. In conclusion, we demonstrate VEGF-Trap treatment improves chronic skin inflammation in KC-Tie2 mice and that disease resolution occurs in an IL-23/Th17 mediated manner. Although systemic VEGF inhibition is unlikely to become a frontline target for psoriasis drug development due in part to its potential side effects; our data provide proof of concept support for targeting this molecule and its multi-cellular functions in psoriatic skin. Targeting VEGF topically may provide an effective new approach for treating mild-to-moderate psoriasis skin lesions by inhibiting angiogenesis and reducing leukocyte infiltration and KC proliferation.

Protection from Psoriasis-Related Thrombosis after Inhibition of IL-23 or IL-17A

Psoriasis patients experience chronic systemic skin inflammation and develop cardiovascular comorbidities that shorten their lifespan. Whether cardiovascular disease is improved by treatment with current biologics that target disease-specific pathways is unclear. KC-Tie2 mice develop psoriasiform skin inflammation with increases in IL-23 and IL-17A and proinflammatory monocytosis and neutrophilia that precedes development of carotid artery thrombus formation. To examine whether targeted blockade of IL-23 or IL-17A in KC-Tie2 psoriasis mice improves cardiovascular outcomes, mice were treated systemically for 6 weeks with antibodies targeting IL-17A, IL-17RA, IL-12/23p40, or IL-23p19. Skin inflammation; thrombosis clotting times; and percentage of splenic monocytes, neutrophils, and CD4 T cells were examined. Skin inflammation significantly improved in KC-Tie2 mice treated with each of the antibodies targeting IL-23, IL-17A, or IL-17RA, consistent with clinical efficacy observed in psoriasis patients. The time to occlusive thrombus formation lengthened in these mice and correlated with attenuated acanthosis. This decrease in skin inflammation paralleled decreases in splenic neutrophils (CD11b+Ly6G+) but not monocytes (CD11b+Ly6Chigh) or T cells (CD4+). Our data show that targeted inhibition of IL-23 or IL-17A improves psoriasis-like skin disease and also improves cardiovascular disease in mice.

Deficiency of myeloid-related proteins 8 and 14 (Mrp8/Mrp14) does not block inflammaging but prevents steatosis

The Mrp8 and Mrp14 proteins (calprotectin) accumulate within tissues during aging and may contribute to chronic inflammation. To address this possibility, we evaluated female calprotectin-deficient Mrp14-KO and wild-type (WT) mice at 5 and 24 months of age. However, there was no evidence that age-related inflammation is blunted in KO mice. Inflammation markers were in fact elevated in livers from old KO mice, and microarray analysis revealed more consistent elevation of genes specifically expressed by B-cells and T-cells. Adipose-specific genes, however, were less consistently elevated in aged KO mice, suggesting an anti-steatosis effect of Mrp8/14 deficiency. Consistent with this, genes decreased by the anti-steatosis agent SRT1720 were decreased in old KO compared to old WT mice. Expression of lipid metabolism genes was altered in KO mice at 5 months of age, along with genes associated with development, biosynthesis and immunity. These early-age effects of Mrp8/14 deficiency, in the absence of any external stressor, were unexpected. Taken together, our findings demonstrate a pro-steatosis rather than pro-inflammatory role of calprotectin within the aging liver. This appears to reflect a developmental-metabolic phenotype of Mrp14-KO mice that is manifest at a young age in the absence of pro-inflammatory stimuli.