Candace M. Loyd

IL-1F5, -F6, -F8, and -F9: A novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression

IL-1F6, IL-1F8, and IL-1F9 and the IL-1R6(RP2) receptor antagonist IL-1F5 constitute a novel IL-1 signaling system that is poorly characterized in skin. To further characterize these cytokines in healthy and inflamed skin, we studied their expression in healthy control, uninvolved psoriasis, and psoriasis plaque skin using quantitative RT-PCR and immunohistochemistry. Expression of IL-1F5, -1F6, -1F8, and -1F9 were increased 2 to 3 orders of magnitude in psoriasis plaque versus uninvolved psoriasis skin, which was supported immunohistologically. Moreover, treatment of psoriasis with etanercept led to significantly decreased IL-1F5, -1F6, -1F8, and -1F9 mRNAs, concomitant with clinical improvement. Similarly increased expression of IL-1F5, -1F6, -1F8, and -1F9 was seen in the involved skin of two mouse models of psoriasis. Suggestive of their importance in inflamed epithelia, IL-1α and TNF-α induced IL-1F5, -1F6, -1F8, and -1F9 transcript expression by normal human keratinocytes. Microarray analysis revealed that these cytokines induce the expression of antimicrobial peptides and matrix metalloproteinases by reconstituted human epidermis. In particular, IL-1F8 increased mRNA expression of human β-defensin (HBD)-2, HBD-3, and CAMP and protein secretion of HBD-2 and HBD-3. Collectively, our data suggest important roles for these novel cytokines in inflammatory skin diseases and identify these peptides as potential targets for antipsoriatic therapies.

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.

Cutaneous manipulation of vascular growth factors leads to alterations in immunocytes, blood vessels and nerves: Evidence for a cutaneous neurovascular unit.

BACKGROUND Skin cells produce soluble factors which influence keratinocyte proliferation, angiogenesis, nerve innervation and immunocyte response. OBJECTIVE To test the hypothesis that epidermal-dermal interactions influence neural outgrowth, vascular survival, immunocyte recruitment and keratinocyte proliferation. METHODS We genetically manipulated the epidermis to express excess vascular endothelial growth factor (VEGF) and/or angiopoietin-1 (Ang1) and then examined the epidermal and dermal phenotypes. We compared these findings with those occurring following overexpression of the Ang1 receptor Tie2 in endothelial cells or keratinocytes. RESULTS Keratinocyte-overexpression of Ang1 resulted in increased epidermal thickness compared to control littermates. Keratinocyte-specific overexpression of Ang1 or VEGF increased dermal angiogenesis compared to control animals and combined Ang1-VEGF lead to further increases. Cutaneous leukocyte examination revealed increases in CD4(+) T cell infiltration in mice with keratinocyte-specific overexpression of Ang1, VEGF and Ang1-VEGF combined; in contrast only keratinocyte-specific Ang1 overexpression increased cutaneous F4/80(+) macrophage numbers. Interestingly, combined keratinocyte-derived Ang1-VEGF overexpression reduced significantly the number of F4/80(+) and Cd11c(+) cells compared to mice overexpressing epidermal Ang1 alone. Endothelial cell-specific Tie2 overexpression increased dermal angiogenesis but failed to influence the epidermal and immune cell phenotypes. Keratinocyte-specific Tie2 expressing mice had the highest levels of CD4(+), CD8(+) and CD11c(+) cell numbers and acanthosis compared to all animals. Finally, increases in the number of cutaneous nerves were found in all transgenic mice compared to littermate controls. CONCLUSION These findings demonstrate that change to one system (vascular or epidermal) results in change to other cutaneous systems and suggest that individual molecules can exert effects on multiple systems.

Mycobacterial phosphatidylinositol mannoside 6 (PIM6) up-regulates TCR-triggered HIV-1 replication in CD4+ T cells.

Tuberculosis (TB) is the leading cause of mortality among those infected with human immunodeficiency virus (HIV-1) worldwide. HIV-1 load and heterogeneity are increased both locally and systemically in active TB. Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication through dysregulation of host cytokines, chemokines, and their receptors. However the possibility that mycobacterial molecules released from MTB infected macrophages directly interact with CD4+ T cells triggering HIV-1 replication has not been fully explored. We studied the direct effect of different MTB molecules on HIV-1 replication (R5-tropic strain Bal) in anti-CD3- stimulated CD4+ T cells from healthy donors in an antigen presenting cell (APC)-free system. PIM6, a major glycolipid of the mycobacterial cell wall, induced significant increases in the percent of HIV-1 infected T cells and the viral production in culture supernatants. In spite of structural relatedness, none of the other three major MTB cell wall glycolipids had significant impact on HIV-1 replication in T cells. Increased levels of IFN-γ in culture supernatants from cells treated with PIM6 indicate that HIV-1 replication is likely dependent on enhanced T cell activation. In HEK293 cells transfected with TLR2, PIM6 was the strongest TLR2 agonist among the cell wall associated glycolipids tested. PIM6 increased the percentage of HIV infected cells and viral particles in the supernatant in a T-cell-based reporter cell line (JLTRg-R5) transfected with TLR1 and TLR2 but not in the cells transfected with the empty vector (which lack TLR2 expression) confirming that PIM6-induced HIV-1 replication depends at least partially on TLR2 signaling.

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.

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.