Doina Diaconu

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.

Keratinocyte but Not Endothelial Cell-Specific Overexpression of Tie2 Leads to the Development of Psoriasis

Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.

Chronic Skin-Specific Inflammation Promotes Vascular Inflammation and Thrombosis

Patients with psoriasis have systemic and vascular inflammation and are at increased risk for myocardial infarction, stroke, and cardiovascular death. However, the underlying mechanism(s) mediating the link between psoriasis and vascular disease is incompletely defined. This study sought to determine whether chronic skin-specific inflammation has the capacity to promote vascular inflammation and thrombosis. Using the KC-Tie2 doxycycline-repressible (Dox-off) murine model of psoriasiform skin disease, spontaneous aortic root inflammation was observed in 33% of KC-Tie2 compared to 0% of control mice by 12 months of age (P=0.04) and was characterized by the accumulation of macrophages, T-lymphocytes and B-lymphocytes and reduced collagen content and increased elastin breaks. Importantly, aortic inflammation was preceded by increases in serum TNF-α, IL-17A, VEGF, IL-12, MCP-1 and S100A8/A9 as well as splenic and circulating CD11b+Ly-6Chi pro-inflammatory monocytes. Doxycycline treatment of old mice with severe skin disease eliminated skin inflammation and aortic root lesion presence in 1 year old KC-Tie2 animals. Given the bi-directional link between inflammation and thrombosis, arterial thrombosis was assessed in KC-Tie2 and control mice; mean time to occlusive thrombus formation was shortened by 64% (P=0.002) in KC-Tie2 animals; doxycycline treatment returned thrombosis clotting times to control mouse levels (P=0.69). These findings demonstrate that sustained skin-specific inflammation promotes aortic root inflammation and thrombosis and suggest that aggressive treatment of skin inflammation may attenuate pro-inflammatory and prothrombotic pathways that produce cardiovascular disease in psoriasis patients.

Cutaneous denervation of psoriasiform mouse skin improves acanthosis and inflammation in a sensory neuropeptide-dependent manner.

Nervous system involvement in psoriasis pathogenesis is supported by increases in nerve fiber numbers and neuropeptides in psoriatic skin and by reports detailing spontaneous plaque remission following nerve injury. Using the KC-Tie2 psoriasisform mouse model, we investigated the mechanisms by which nerve injury leads to inflammatory skin disease remission. Cutaneous nerves innervating dorsal skin of KC-Tie2 animals were surgically axotomized and beginning 1d following denervation, CD11c+ cell numbers decreased by 40% followed by a 30% improvement in acanthosis at 7d and a 30% decrease in CD4+ T cell numbers by 10d. Restoration of SP signaling in denervated KC-Tie2 skin prevented decreases in CD11c+ and CD4+ cells but had no affect on acanthosis; restoration of CGRP signaling reversed the improvement in acanthosis and prevented denervated-mediated decreases in CD4+ cells. Under innervated conditions, small molecule inhibition of SP in KC-Tie2 animals resulted in similar decreases to those observed following surgical denervation for cutaneous CD11c+ and CD4+ cell numbers; whereas small molecule inhibition of CGRP resulted in significant reductions in CD4+ cell numbers and acanthosis. These data demonstrate that sensory nerve-derived peptides mediate psoriasiform dendritic cell and T cell infiltration and acanthosis and introduce targeting nerve-immunocyte/keratinocyte interactions as potential psoriasis therapeutic treatment strategies.

Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice.

Background  There is ongoing debate regarding the initiation of psoriatic plaque as primarily arising from an anomaly in epidermal keratinocytes (KCs) or from abnormalities in immunocytes that secondarily activate otherwise normal KCs. In mice engineered to overexpress the angiopoietin receptor Tie2 in KCs, skin spontaneously develops the characteristic clinical, histological and immune cell phenotypes of psoriasis which can be reversed with either transgene repression or ciclosporin administration, suggesting key roles for both KCs and T cells in mediating the skin disease in this murine model.

Botulinum neurotoxin A decreases infiltrating cutaneous lymphocytes and improves acanthosis in the KC-Tie2 mouse model

Psoriatic skin contains more nerve fibers (Naukkarinen et al., 1989), has increased levels of sensory nerve-derived calcitonin gene related peptide (CGRP) and substance P (SP) (Chan et al., 1997; Jiang et al., 1998), and psoriasis disease severity can be exacerbated by stress (Griffiths and Richards, 2001) possibly by increasing cutaneous nerve numbers and SP and CGRP expression (Joachim et al., 2007; Remrod et al., 2007). The clinical observation that psoriasis undergoes remission following loss of innervation, nerve function or nervous system injury (Dewing, 1971; Farber et al., 1990; Joseph et al., 2005; Perlman, 1972; Stratigos et al., 1998) further supports a role for the nervous system in psoriasis pathogenesis; however, the neural-mediated mechanisms underlying disease resolution in these cases have not been explored. Recent work by our group identified increases in cutaneous nerve fibers and nerve-derived SP and CGRP in the KC-Tie2 murine model of psoriasiform dermatitis, providing an experimental paradigm to explore neural contributions to psoriasis pathogenesis. Surgical elimination of the cutaneous nerves in KC-Tie2 mouse dorsal skin resulted in a 30% improvement in acanthosis, a 40% decrease in CD11c+ dendritic cells (DCs) and a 30% decrease in CD4+ T cells. These outcomes were SP and CGRP dependent; as reconstitution of SP and CGRP in denervated KC-Tie2 skin prevented improvement in the phenotype and inhibition of SP and CGRP in innervated KC-Tie2 skin recapitulated the findings elicited by experimental denervation in a sensory neuropeptide specific manner (Ostrowski et al., 2011). These findings provide insight into potential mechanisms underlying clinical reports of disease improvement following nervous system injury and identify key roles for nerve derived SP and CGRP in sustaining chronic psoriasiform skin inflammation. Botulinum neurotoxins of various serotypes act by inhibiting the exocytosis of neurotransmitters from nerve endings. Botulinum neurotoxin A (BoNT-A, known commonly by the tradenames BOTOX®, Dysport® and Xeomin®) does so by cleaving the SNAP25 protein, and is best known for its capacity to inhibit acetylcholine release in neuromuscular junctions and at the neurovascular interface and therefore is often used to treat glabellar lines, hemifacial spasm, cervical dystonia, blepharospasm, hyperhidrosis and Raynauds disease. However BoNT-A also inhibits nerve-derived release of CGRP and SP and can be used for treating pain syndromes and potentially neurogenic inflammation (Carmichael et al., 2010; Meng et al., 2007). This is particularly significant and provides mechanistic insight into the recent unpublished observation of psoriatic plaque remission in a patient injected with BoNT-A for the treatment of upper limb spasticity related to stroke (Dr. Jim Andrews, personal communication) and the subjective clinical observation of disease improvement in inverse psoriasis following BoNT-A administration (Zanchi et al., 2008). This study sought to investigate whether one intradermal injection of BoNT-A into KC-Tie2 mouse skin would provide similar levels of improvement in skin disease as surgical denervation and/or chemical inhibition of SP and CGRP. Adult KC-Tie2 mouse dorsal skin was intradermally injected with BoNT-A (Dysport®; 9units/kg/100ul) and saline (100ul volume) in anatomically separate locations, the regions were marked and images were taken to ensure that the same locations were harvested for analyses. Two (n=5) or six weeks (n=6) later, mice were sacrificed, and BoNT-A and saline injected skin was harvested and processed for histological and immunostaining analyses as described previously (Ostrowski et al., 2011). All animal protocols were approved by the Case Western Reserve University institutional animal care and use committee. Dorsal skin injected once with BoNT-A showed significant improvement in acanthosis compared to saline injected skin at 2 weeks (~17% decrease; P=0.031) and at 6 weeks (~25% decrease; P=0.011; Figure 1a–b; Figure 2). Dermal DC infiltration was also significantly reduced, evidenced by a significant decrease in the number of dermal CD11c+ DCs in BoNT-A injected skin compared to saline injected skin (Figure 1c–d; Figure 2) at 2 weeks (29% decrease; P=0.002) and at 6 weeks (38% decrease; P<0.0001). CD4+ T cell numbers also decreased significantly in BoNT-A injected skin compared to saline injected skin (Figure 1e–f; Figure 2) at 2 weeks (24% decrease; P=0.017) and at 6 weeks (34% decrease; P<0.002). The number of F4/80+ macrophages and CD8+ T cells did not differ between BoNT-A and saline injected skin, nor were any changes observed for dermal angiogenesis (blood vessel number or size) at either of the time points examined (not shown). Figure 1 Botulinum neurotoxin A (BoNT-A) improves skin disease severity in KC-Tie2 mice Figure 2 Acanthosis, CD11c+ DC and CD4+ T cell numbers decrease following BoNT-A injection These findings demonstrate a significant improvement in psoriasiform skin inflammation and epidermal hyperplasia following one intradermal injection of BoNT-A, and identify decreases in infiltrating CD4+ T cells and CD11c+ DCs that occur concomitant with the improvement in acanthosis. These findings are similar to those we previously reported, and the percent improvement for each outcome was comparable to that observed following both surgical elimination of the cutaneous nerves and following 4 weeks of CGRP and SP inhibition (Ostrowski et al., 2011) and suggest that BoNT-A might serve as a useful psoriasis therapeutic. However, the use of BoNT-A as monotherapy for patients with high PASI scores (>50) is not a practical approach. Nonetheless, in some psoriasis patients, recalcitrant plaques remain, even following treatment with strong systemic and biological therapies such as TNFα inhibitors. The persistence of some plaques may be explained by the local microenvironment within the tissue combined with signals yet unaccounted for that have strong proinflammatory effects, such as nerve-derived SP and CGRP. Our previous results (Ostrowski et al., 2011) identified these neuropeptides as necessary to sustain and exacerbate tissue hyperproliferation and suggest that the presence of nerves and their derived proteins play critical roles in the cascade of events that sustain inflammation and acanthosis. The ability to interfere with cutaneous nerve signaling presents an opportunity to address recalcitrant plaque psoriasis, thus BoNT-A may serve as a supplemental therapeutic approach to otherwise successful topical or biologic regimens. The current data validates this idea and demonstrates that a single intradermal injection of BoNT-A, a known inhibitor of CGRP/SP release, leads to significant improvement in disease severity as early as two weeks post treatment and provides new insight into the cellular mechanisms underlying disease improvement following BoNT-A administration in psoriasis patients.

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.

Using optical coherence tomography for the longitudinal non-invasive evaluation of epidermal thickness in a murine model of chronic skin inflammation

Non‐invasive methods are desirable for longitudinal studies examining drug efficacy and disease resolution defined as decreases in epidermal thickness in mouse models of psoriasiform skin disease. This would eliminate the need for either sacrificing animals or collecting serial skin biopsies to evaluate changes in disease progression during an individual study. The quantitation of epidermal thickness using optical coherence tomography (OCT) provides an alternative to traditional histology techniques.

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.