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

Open ArchivePublished:September 23, 2017DOI:https://doi.org/10.1016/j.jid.2017.09.021
      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.

      Abbreviations:

      CVD (cardiovascular disease), FDG-PET-CT (18-fluorodeoxyglucose positron emission tomography-computed -tomography), NET (neutrophil extracellular trap), TNF (tumor necrosis factor)

      Introduction

      Chronic autoimmune diseases can affect specific organs including the skin (e.g., psoriasis) and joints (e.g., rheumatoid arthritis and spondyloarthritis). Epidemiological and clinical evidence suggests that patients with poorly controlled inflammatory disease within peripheral tissues develop persistent systemic inflammation and have an increased risk of developing and dying from cardiovascular disease (CVD). This increased risk cannot be fully explained by traditional cardiovascular risk factors (for reviews
      • Szekanecz Z.
      • Kerekes G.
      • Vegh E.
      • Kardos Z.
      • Barath Z.
      • Tamasi L.
      • et al.
      Autoimmune atherosclerosis in 3D: How it develops, how to diagnose and what to do.
      ,
      • Yim K.M.
      • Armstrong A.W.
      Updates on cardiovascular comorbidities associated with psoriatic diseases: epidemiology and mechanisms.
      ), and the underlying mechanisms remain unclear. It is likely that tissue-derived soluble factors drive systemic inflammation and directly affect distant vessels and the development of atherothrombosis. Treatment of the primary disease (i.e., psoriasis or rheumatoid arthritis) may improve CVD risk and reduce cardiovascular events.
      Clinical studies examining a reduction in the number of cardiovascular events and/or increased lifespan after treatment of the primary disease are ongoing but currently limited because of the time required to observe a longitudinal result. Attempts to circumvent this limitation include shorter duration studies that examine surrogate measures of CVD. These include coronary artery calcium scores (
      • Santilli S.
      • Kast D.R.
      • Grozdev I.
      • Cao L.
      • Feig R.L.
      • Golden J.B.
      • et al.
      Visualization of atherosclerosis as detected by coronary artery calcium and carotid intima-media thickness reveals significant atherosclerosis in a cross-sectional study of psoriasis patients in a tertiary care center.
      ), vascular inflammation (18-fluorodeoxyglucose positron emission tomography-computed tomography [FDG-PET-CT]) (
      • Naik H.B.
      • Natarajan B.
      • Stansky E.
      • Ahlman M.A.
      • Teague H.
      • Salahuddin T.
      • et al.
      Severity of Psoriasis Associates With Aortic Vascular Inflammation Detected by FDG PET/CT and Neutrophil Activation in a Prospective Observational Study.
      ), flow-mediated dilation, intima media thickness measures (
      • Di Minno M.N.
      • Ambrosino P.
      • Lupoli R.
      • Di Minno A.
      • Tasso M.
      • Peluso R.
      • et al.
      Cardiovascular risk markers in patients with psoriatic arthritis: A meta-analysis of literature studies.
      ,
      • Fang N.
      • Jiang M.
      • Fan Y.
      Association between psoriasis and subclinical atherosclerosis: a meta-analysis.
      ), and traditional serum risk factors (i.e., plasma lipids, C-reactive protein, IL-6, resistin, myeloperoxidase) (
      • Cao L.Y.
      • Soler D.C.
      • Debanne S.M.
      • Grozdev I.
      • Rodriguez M.E.
      • Feig R.L.
      • et al.
      Psoriasis and cardiovascular risk factors: increased serum myeloperoxidase and corresponding immunocellular overexpression by Cd11b(+) CD68(+) macrophages in skin lesions.
      ) in patient populations, both before and after biologic treatment (
      • Mazzoccoli G.
      • Notarsanto I.
      • de Pinto G.D.
      • Dagostino M.P.
      • De Cata A.
      • D'Alessandro G.
      • et al.
      Anti-tumor necrosis factor-alpha therapy and changes of flow-mediated vasodilatation in psoriatic and rheumatoid arthritis patients.
      ,
      • Piaserico S.
      • Osto E.
      • Famoso G.
      • Zanetti I.
      • Gregori D.
      • Poretto A.
      • et al.
      Treatment with tumor necrosis factor inhibitors restores coronary microvascular function in young patients with severe psoriasis.
      ,
      • Pina T.
      • Corrales A.
      • Lopez-Mejias R.
      • Armesto S.
      • Gonzalez-Lopez M.A.
      • Gomez-Acebo I.
      • et al.
      Anti-tumor necrosis factor-alpha therapy improves endothelial function and arterial stiffness in patients with moderate to severe psoriasis: A 6-month prospective study.
      ,
      • Ramonda R.
      • Puato M.
      • Punzi L.
      • Rattazzi M.
      • Zanon M.
      • Balbi G.
      • et al.
      Atherosclerosis progression in psoriatic arthritis patients despite the treatment with tumor necrosis factor-alpha blockers: a two-year prospective observational study.
      ).
      A preclinical animal model that mimics human disease and develops similar CVD comorbidities could provide rapid insight into these clinically important questions. The KC-Tie2 mouse is a keratinocyte-specific, Tie2-overexpressing psoriasis model that recapitulates key aspects of human psoriasis including elevated systemic and cutaneous expression of IL-23 and IL-17A (
      • Wolfram J.A.
      • Diaconu D.
      • Hatala D.A.
      • Rastegar J.
      • Knutsen D.A.
      • Lowther A.
      • et al.
      Keratinocyte but not endothelial cell specific overexpression of Tie2 leads to the development of psoriasis.
      ). These mice respond to treatment strategies that are successful in psoriasis patients (
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ,
      • Wolfram J.A.
      • Diaconu D.
      • Hatala D.A.
      • Rastegar J.
      • Knutsen D.A.
      • Lowther A.
      • et al.
      Keratinocyte but not endothelial cell specific overexpression of Tie2 leads to the development of psoriasis.
      ) and similarly do not respond to unsuccessful human psoriasis treatment approaches (
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ,
      • Ward N.L.
      • Bhagathavula N.
      • Johnston A.
      • Dawes S.M.
      • Fu W.
      • Lambert S.
      • et al.
      Erlotinib-induced skin inflammation is IL-1 mediated in KC-Tie2 mice and human skin organ culture.
      ). KC-Tie2 mice develop CVD comorbidities subsequent to systemic inflammation, including monocytosis and neutrophilia, and are susceptible to thrombosis in an experimental occlusive thrombosis bioassay (
      • Wang Y.
      • Gao H.
      • Loyd C.M.
      • Fu W.
      • Diaconu D.
      • Liu S.
      • et al.
      Chronic skin-specific inflammation promotes vascular inflammation and thrombosis.
      ,
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ). Clinical studies have shown that targeting the critical psoriasis signature cytokines IL-23 or IL-17A using biologics leads to remarkable skin disease resolution (for review, see
      • Ritchlin C.T.
      • Krueger J.G.
      New therapies for psoriasis and psoriatic arthritis.
      ). However, it still remains unclear how the inhibition of these cytokines affects cardiovascular events and/or risk in these patients.

      Results and Discussion

      KC-Tie2 animals with established skin disease were treated weekly (for 6 weeks) with antibodies targeting IL-12/23p40, IL-23p19, IL-17A, or IL-17RA (or isotype IgG) to determine whether targeted blockade of IL-23 or IL-17A improved skin phenotype and lengthened thrombosis clotting times. Acanthosis (epidermal thickness) and cutaneous CD4+ T cell numbers significantly decreased in KC-Tie2 mice treated with antibodies versus isotype IgG (acanthosis: F(5) = 32.99, P < 0.0001; CD4+ T cell number: F(5) = 9.588, P < 0.0001). Post hoc analyses showed that treatment with antibodies targeting IL-12/23p40, IL-23p19, and IL-17RA resulted in the greatest improvement in acanthosis (P < 0.0001 vs. isotype IgG and P < 0.002 vs. anti-IL-17A), followed by IL-17A inhibition (P < 0.01 vs. isotype IgG) (Figure 1a and b ). Similarly, cutaneous infiltration of CD4+ T cells decreased across all treatment groups (Figure 1c) (all P < 0.01 vs. isotype IgG), whereas infiltration of CD8+ T cells decreased in all groups except IL-17RA (Figure 1c) (P < 0.05). F4/80+ macrophages did not decrease significantly in any group relative to isotype IgG-treated mice (Figure 1c). The decrease in acanthosis with concomitant improvement in skin inflammation mirrors psoriasis patient clinical responsiveness to current biologics, including ustekinumab (IL-12/23p40 [
      • Lebwohl M.
      • Papp K.
      • Han C.
      • Schenkel B.
      • Yeilding N.
      • Wang Y.
      • et al.
      Ustekinumab improves health-related quality of life in patients with moderate-to-severe psoriasis: results from the PHOENIX 1 trial.
      ]); guselkumab, tildrakizumab, and risankizumab (IL-23p19 [
      • Amin M.
      • Darji K.
      • No D.J.
      • Wu J.J.
      Review of phase III trial data on IL-23 Inhibitors Tildrakizumab and Guselkumab for psoriasis [e-pub ahead of print].
      ,
      • Blauvelt A.
      • Papp K.A.
      • Griffiths C.E.
      • Randazzo B.
      • Wasfi Y.
      • Shen Y.K.
      • et al.
      Efficacy and safety of guselkumab, an anti-interleukin-23 monoclonal antibody, compared with adalimumab for the continuous treatment of patients with moderate to severe psoriasis: results from the phase III, double-blinded, placebo- and active comparator-controlled VOYAGE 1 trial.
      ,
      • Papp K.A.
      • Blauvelt A.
      • Bukhalo M.
      • Gooderham M.
      • Krueger J.G.
      • Lacour J.P.
      • et al.
      Risankizumab versus ustekinumab for moderate-to-severe plaque psoriasis.
      ,
      • Reich K.
      • Papp K.A.
      • Blauvelt A.
      • Tyring S.K.
      • Sinclair R.
      • Thaci D.
      • et al.
      Tildrakizumab versus placebo or etanercept for chronic plaque psoriasis (reSURFACE 1 and reSURFACE 2): results from two randomised controlled, phase 3 trials.
      ]); ixekizumab and secukinumab (IL-17A [
      • Gordon K.B.
      • Blauvelt A.
      • Papp K.A.
      • Langley R.G.
      • Luger T.
      • Ohtsuki M.
      • et al.
      Phase 3 trials of ixekizumab in moderate-to-severe plaque psoriasis.
      ,
      • Langley R.G.
      • Elewski B.E.
      • Lebwohl M.
      • Reich K.
      • Griffiths C.E.
      • Papp K.
      • et al.
      Secukinumab in plaque psoriasis—results of two phase 3 trials.
      ]); and brodalumab (IL-17RA [
      • Blauvelt A.
      • Papp K.A.
      • Lebwohl M.G.
      • Green L.J.
      • Hsu S.C.
      • Bhatt V.
      • et al.
      Rapid onset of action in patients with moderate-to-severe psoriasis treated with brodalumab: a pooled analysis of data from two phase 3 randomized clinical trials (AMAGINE-2 and AMAGINE-3).
      ]), and validates the KC-Tie2 mouse as a predictive preclinical model that mimics clinical patient response to psoriasis treatment.
      Figure 1
      Figure 1Skin inflammation and thrombosis clotting times improve in KC-Tie2 mice treated with antibodies targeting IL-12, IL-23, IL-17A, and IL-17RA. (a) Quantification of epidermal thickness (μm) of hematoxylin and eosin-stained dorsal skin sections of KC-Tie2 mice treated with isotype IgG (n = 10, mouse; n = 9, mouse/rat) or antibodies targeting IL-12p40 (n = 12), IL-23p19 (n = 14), IL-17A (n = 9), or IL-17RA (n = 11) and control littermates (WT, n = 17). Representative images of (b) dorsal skin from each group stained with hematoxylin and eosin and (c) CD4 (T cell), CD8 (T cell), and F4/80 (macrophage). (d) Occlusion times (minutes) after rose bengal induced photochemical injury of the carotid artery in KC-Tie2 mice treated with isotype IgG (n = 10, mouse; n = 8, mouse/rat) or antibodies targeting IL-12p40 (n = 12), IL-23p19 (n = 14), IL-17A (n = 7), or IL-17RA (n = 11) and control littermates (WT, n = 50). (e) Correlation between acanthosis and clotting times for all animals. Each symbol represents an individual mouse. Solid straight lines indicate the mean for each group. Data were analyzed using a Fisher least significant difference test. P < 0.05 versus isotype IgG in a, +P < 0.05 versus control littermates (WT) in d. Scale bars in b and c = 100 μm. WT, wild type.
      We previously showed that repression of skin inflammation in the KC-Tie2 model using a tetracycline-repressible genetic approach resulted in decreases in acanthosis, IL-12, IL-23, and IL-17A (
      • Wolfram J.A.
      • Diaconu D.
      • Hatala D.A.
      • Rastegar J.
      • Knutsen D.A.
      • Lowther A.
      • et al.
      Keratinocyte but not endothelial cell specific overexpression of Tie2 leads to the development of psoriasis.
      ) and lengthened thrombosis clotting times (
      • Wang Y.
      • Gao H.
      • Loyd C.M.
      • Fu W.
      • Diaconu D.
      • Liu S.
      • et al.
      Chronic skin-specific inflammation promotes vascular inflammation and thrombosis.
      ). Thus, we predicted that targeted blockade of IL-12/23 or IL-17A would also improve cardiovascular outcomes. KC-Tie2 mice treated with nonblocking isotype IgGs clotted more quickly than control animals (P < 0.05 vs. mouse IgG; P = 0.059 vs. mouse/rat IgG), confirming prior findings (
      • Wang Y.
      • Gao H.
      • Loyd C.M.
      • Fu W.
      • Diaconu D.
      • Liu S.
      • et al.
      Chronic skin-specific inflammation promotes vascular inflammation and thrombosis.
      ,
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ). KC-Tie2 mice treated with antibodies targeting IL-23 or IL-17A/IL-17RA (Figure 1d) showed lengthened times to occlusive thrombus formation and no longer differed from wild-type controls. Furthermore, correlation analyses showed an inverse correlation between acanthosis and clotting times (Figure 1e) (R2 = 0.067), suggesting that skin inflammation, using acanthosis as a surrogate measure, correlates with shortened clotting times. These findings show that improving psoriasis-like skin inflammation in mice by directly targeting IL-23 or IL-17A eliminates the susceptibility to thrombosis. These findings suggest that current biologic approaches targeting IL-23 or IL-17A used to treat cutaneous inflammation in psoriasis patients may lower the risk of future cardiovascular events.
      Although treatment of psoriasis patients with biologics is on the rise, these measures are generally reserved for patients with moderate to severe disease. Therefore, a large subset of patients may not have optimal control of their skin inflammation. The impact of unrestrained skin inflammation versus treatment with biologics and the risk of cardiovascular events, vascular inflammation, and atherogenesis are topics of high interest in the fields of dermatology and cardiology.
      Prospective studies are critical to ascertain major adverse cardiac event outcomes for patients currently taking biologics that target IL-23, IL-12/23, IL-17A, and IL-17RA. Thus far, there are no short- or long-term major adverse cardiac event signals for ustekinumab (
      • Papp K.
      • Gottlieb A.B.
      • Naldi L.
      • Pariser D.
      • Ho V.
      • Goyal K.
      • et al.
      Safety aurveillance for ustekinumab and other psoriasis treatments from the Psoriasis Longitudinal Assessment and Registry (PSOLAR).
      ), and there are no short-term major adverse cardiac event signals for either anti-IL-17A drugs, brodalumab, or anti-p19 drugs. Ongoing studies are examining effects of secukinumab on surrogate biomarkers of CVD (ClinicalTrials.gov NCT02559622) and vascular inflammation using FDG-PET-CT (ClinicalTrials.gov NCT0269070). Furthermore, current randomized clinical trials are prospectively studying the impact of IL-12/23 blockade on vascular inflammation using FDG-PET-CT (ClinicalTrials.gov identifier NCT02187172). Additional studies of extended duration are needed to further assess the impact, either negative or positive, of these drugs on cardiovascular comorbidities.
      Preclinical data are also unclear regarding the pro- versus anti-atherogenic effects of IL-17A (for review, see
      • Taleb S.
      • Tedgui A.
      • Mallat Z.
      IL-17 and Th17 cells in atherosclerosis: subtle and contextual roles.
      ). One report showed that IL-17A inhibition destabilizes established atherosclerotic plaque, potentially leading to thrombotic events (
      • Gistera A.
      • Robertson A.K.
      • Andersson J.
      • Ketelhuth D.F.
      • Ovchinnikova O.
      • Nilsson S.K.
      • et al.
      Transforming growth factor-beta signaling in T cells promotes stabilization of atherosclerotic plaques through an interleukin-17-dependent pathway.
      ). Another showed that low serum IL-17A levels were associated with an increased risk of cardiovascular events in patients with acute myocardial infarction (
      • Simon T.
      • Taleb S.
      • Danchin N.
      • Laurans L.
      • Rousseau B.
      • Cattan S.
      • et al.
      Circulating levels of interleukin-17 and cardiovascular outcomes in patients with acute myocardial infarction.
      ). Others have described distant vessel endothelial dysfunction, increases in systolic blood pressure, and a shorter lifespan in mice overexpressing IL-17A under the keratin 14 promoter (
      • Karbach S.
      • Croxford A.L.
      • Oelze M.
      • Schuler R.
      • Minwegen D.
      • Wegner J.
      • et al.
      Interleukin 17 drives vascular inflammation, endothelial dysfunction, and arterial hypertension in psoriasis-like skin disease.
      ). In our study, mice do not have existent atherosclerosis, suggesting that inhibition of the IL-17 pathway (directly or indirectly) may improve risk, likely as a result of controlling persistent skin-derived inflammation. These studies suggest that aggressive treatment of the primary disease (i.e., psoriasis) will decrease circulating IL-17A levels and confer protection in cases where atherosclerosis is absent. Translating these findings may require psoriasis patient stratification based on atherosclerosis burden before initiating treatment with IL-17A inhibitors.
      Tumor necrosis factor (TNF)-α inhibition is the most common biologic approach and has historically served as the standard-of-care treatment for psoriasis patients. The literature has suggested that TNF-α inhibition decreases the relative risk of CVD in patients (
      • Mazzoccoli G.
      • Notarsanto I.
      • de Pinto G.D.
      • Dagostino M.P.
      • De Cata A.
      • D'Alessandro G.
      • et al.
      Anti-tumor necrosis factor-alpha therapy and changes of flow-mediated vasodilatation in psoriatic and rheumatoid arthritis patients.
      ,
      • Piaserico S.
      • Osto E.
      • Famoso G.
      • Zanetti I.
      • Gregori D.
      • Poretto A.
      • et al.
      Treatment with tumor necrosis factor inhibitors restores coronary microvascular function in young patients with severe psoriasis.
      ,
      • Pina T.
      • Corrales A.
      • Lopez-Mejias R.
      • Armesto S.
      • Gonzalez-Lopez M.A.
      • Gomez-Acebo I.
      • et al.
      Anti-tumor necrosis factor-alpha therapy improves endothelial function and arterial stiffness in patients with moderate to severe psoriasis: A 6-month prospective study.
      ). However, a recent double-blind, placebo-controlled psoriasis trial of adalimumab reported a lack of effect on vascular inflammation in the ascending aorta and carotid arteries as assessed using FDG-PET-CT in psoriasis patients (
      • Bissonnette R.
      • Harel F.
      • Krueger J.G.
      • Guertin M.C.
      • Chabot-Blanchet M.
      • Gonzalez J.
      • et al.
      TNF-alpha antagonist and vascular inflammation in patients with psoriasis vulgaris: a randomized placebo-controlled study.
      ); whether this reflects sensitivity or variability in the methodology for measuring vascular inflammation remains unclear (
      • Lensen K.D.F.
      • van Sijl A.M.
      • Voskuyl A.E.
      • van der Laken C.J.
      • Heymans M.W.
      • Comans E.F.I.
      • et al.
      Variability in quantitative analysis of atherosclerotic plaque inflammation using 18F-FDG PET/CT.
      ). Other studies provide additional support that TNF-α inhibition significantly reduces myocardial infarct risk and incidence (
      • Wu J.J.
      • Poon K.Y.
      Tumor necrosis factor inhibitor therapy and myocardial infarction risk in patients with psoriasis, psoriatic arthritis, or both.
      ,
      • Wu J.J.
      • Poon K.Y.
      • Channual J.C.
      • Shen A.Y.
      Association between tumor necrosis factor inhibitor therapy and myocardial infarction risk in patients with psoriasis.
      ). Because of a lack of murine-specific anti-TNF-α antibodies, we did not examine thrombosis susceptibility after TNF-α inhibition in KC-Tie2 mice; however, cutaneous TNF-α transcript levels decreased in the skin of all treated groups, except mice treated with anti-IL-17A (data not shown).
      We previously showed that elevated circulating proinflammatory monocytes and neutrophils precede shortened times to occlusive thrombus formation in KC-Tie2 mice (
      • Wang Y.
      • Gao H.
      • Loyd C.M.
      • Fu W.
      • Diaconu D.
      • Liu S.
      • et al.
      Chronic skin-specific inflammation promotes vascular inflammation and thrombosis.
      ). In the current study, splenic immune cell populations were examined for expression of CD11b, Ly6C, Ly6G, and CD4 after blockade of IL-12/23, IL-17A, or IL-17RA (Figure 2). CD11b+Ly6G+ neutrophils decreased significantly in each antibody treated KC-Tie2 group versus isotype IgG (Figure 2c) (F(5) = 4.75, P < 0.005). Anti-IL-12p40 was the only treatment to have a significant effect on the monocyte populations, such that CD11b+Ly6Chigh monocytes decreased concomitantly with a reciprocal increase in CD11b+Ly6Clow monocytes (Figure 2a and b), suggesting a role for IL-12p40 in both monocyte and neutrophil regulation. CD4+ T cells increased significantly only in mice treated with anti-IL-23p19 antibodies (Figure 2d) (P < 0.05). These results suggest that decreases in circulating neutrophils correspond better than monocytes or T cells with lengthening of carotid artery thrombosis clotting times and confirm recent reports showing similar decreases in neutrophils in mice protected from thrombosis (
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ). These findings are also consistent with reports showing that psoriasis patients have elevated levels of circulating neutrophils that correlate with vascular inflammation (
      • Naik H.B.
      • Natarajan B.
      • Stansky E.
      • Ahlman M.A.
      • Teague H.
      • Salahuddin T.
      • et al.
      Severity of Psoriasis Associates With Aortic Vascular Inflammation Detected by FDG PET/CT and Neutrophil Activation in a Prospective Observational Study.
      ).
      Figure 2
      Figure 2Neutrophils decrease significantly in KC-Tie2 mice after IL-12/23, IL-17A, and IL-17RA blockade. (a) CD11b+Ly6Chigh monocytes, (b) CD11b+Ly6Clow monocytes, (c) CD11b+Ly6G+ neutrophils, and (d) CD4+ T cells from spleens of KC-Tie2 mice treated with isotype IgG (n = 5, mouse; n = 4, mouse rat), anti-IL-12p40 (n = 5), anti-IL-23p19 (n = 5), anti-IL-17A (n = 5), or anti-IL-17RA (n = 5). Each symbol represents an individual mouse. Solid straight lines indicate the mean for each group. Data were analyzed using a Fishers least significant difference test. P < 0.05 versus isotype IgG.
      The role of neutrophils in thrombosis may reflect neutrophil interactions with platelets and potentially with neutrophil extracellular traps (NETs) (
      • Mocsai A.
      Diverse novel functions of neutrophils in immunity, inflammation, and beyond.
      ). Chronic exposure to inflammation can extend the normal lifespan of a neutrophil in circulation, which can in turn promote inflammatory events at distant sites (
      • Mantovani A.
      • Cassatella M.A.
      • Costantini C.
      • Jaillon S.
      Neutrophils in the activation and regulation of innate and adaptive immunity.
      ). During thrombus formation, neutrophils are one of the first cell types to adhere to the site of vessel wall injury, which precedes platelet activation (
      • Darbousset R.
      • Thomas G.M.
      • Mezouar S.
      • Frere C.
      • Bonier R.
      • Mackman N.
      • et al.
      Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation.
      ). Neutrophil-derived NETs also promote coagulation during thrombosis (
      • Massberg S.
      • Grahl L.
      • von Bruehl M.L.
      • Manukyan D.
      • Pfeiler S.
      • Goosmann C.
      • et al.
      Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.
      ), likely triggered via platelet activation (
      • Fuchs T.A.
      • Brill A.
      • Duerschmied D.
      • Schatzberg D.
      • Monestier M.
      • Myers Jr., D.D.
      • et al.
      Extracellular DNA traps promote thrombosis.
      ). Our prior work showed parallel decreases in neutrophils and platelets in mice protected from thrombosis (
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ), and others have shown that neutrophil depletion reduces thrombosis (
      • Darbousset R.
      • Thomas G.M.
      • Mezouar S.
      • Frere C.
      • Bonier R.
      • Mackman N.
      • et al.
      Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation.
      ). IL-17A has previously been shown to induce NETosis (
      • Khandpur R.
      • Carmona-Rivera C.
      • Vivekanandan-Giri A.
      • Gizinski A.
      • Yalavarthi S.
      • Knight J.S.
      • et al.
      NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.
      ), activate platelets (
      • Maione F.
      • Cicala C.
      • Liverani E.
      • Mascolo N.
      • Perretti M.
      • D'Acquisto F.
      IL-17A increases ADP-induced platelet aggregation.
      ), and contribute to thrombus formation (
      • de Boer O.J.
      • Li X.
      • Teeling P.
      • Mackaay C.
      • Ploegmakers H.J.
      • van der Loos C.M.
      • et al.
      Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction.
      ). Thus, functional inhibition of the neutrophil activation pathway, either directly by blocking IL-17A/17RA or indirectly by suppression of IL-23, may lead to decreases in NETosis-specific initiators and/or neutrophil-platelet interactions. These interactions are critical for thrombus formation and, as a result, lengthen the time required to form a clot in the experimental bioassay used in this study.
      In summary, we show that functional blockade of IL-23 or IL-17A in an established mouse model of psoriasis improves skin inflammation, decreases the number of circulating neutrophils, and lengthens thrombosis clotting times, suggesting that targeting cytokines that mediate psoriatic inflammation may improve cardiovascular comorbidities.

      Methods

      Mice

      K5tTA and TetosTie2 mouse engineering, genotyping, and generation of double transgenic KC-Tie2 mice have been previously described (
      • Wolfram J.A.
      • Diaconu D.
      • Hatala D.A.
      • Rastegar J.
      • Knutsen D.A.
      • Lowther A.
      • et al.
      Keratinocyte but not endothelial cell specific overexpression of Tie2 leads to the development of psoriasis.
      ). Transgenic mice inheriting a single, nonexpressing gene (K5tTA or TetosTie2) or wild-type littermates served as controls.
      Six-week-old male and female KC-Tie2 mice with established skin disease were treated systemically (intraperitoneally) once per week for a 6-week period with antibodies targeting IL-12/23p40 (10 mg/kg), IL-23p19 (2 mg/kg), or mouse IgG isotype (10 mg/kg) for these two antibodies or with antibodies targeting either IL-17A (10 mg/kg) or IL-17RA (20 mg/kg) or mouse/rat IgG isotype control for these two antibodies (at 20 mg/kg). Dosing was determined and antibodies were generously provided by Kristine Kikly (Eli Lilly, Indianapolis, IN). Antibody specificity has been extensively characterized internally at Eli Lilly and previously shown in a murine spondyloarthritis model (
      • Benham H.
      • Rehaume L.M.
      • Hasnain S.Z.
      • Velasco J.
      • Baillet A.C.
      • Ruutu M.
      • et al.
      Interleukin-23 mediates the intestinal response to microbial beta-1,3-glucan and the development of spondyloarthritis pathology in SKG mice.
      ).

      Murine model of carotid arterial thrombosis

      The carotid arterial thrombosis assay was completed as previously described (
      • Wang Y.
      • Gao H.
      • Loyd C.M.
      • Fu W.
      • Diaconu D.
      • Liu S.
      • et al.
      Chronic skin-specific inflammation promotes vascular inflammation and thrombosis.
      ). Time to occlusion was identified as the time point at which blood flow ceased for 10 minutes.

      Tissue collection, histology, and immunostaining analyses

      Skin was collected and processed for hematoxylin and eosin histology and CD4+, CD8+, and F4/80+ immunohistochemistry, with epidermal thickness and immune cell quantification performed as previously described (
      • Wolfram J.A.
      • Diaconu D.
      • Hatala D.A.
      • Rastegar J.
      • Knutsen D.A.
      • Lowther A.
      • et al.
      Keratinocyte but not endothelial cell specific overexpression of Tie2 leads to the development of psoriasis.
      ).

      Flow cytometry

      Spleen cells from a separate cohort of mice that did not undergo the thrombosis assay were isolated and stained for analysis by flow cytometry as described previously (
      • Wang Y.
      • Golden J.B.
      • Fritz Y.
      • Zhang X.
      • Diaconu D.
      • Camhi M.I.
      • et al.
      Interleukin 6 regulates psoriasiform inflammation-associated thrombosis.
      ) (see Supplementary Figure S1 online). Monocytes were classified as expressing CD11b+Ly6GlowLy6Chigh or CD11b+Ly6GlowLy6Clow, whereas neutrophils expressed CD11b+Ly6GhighLy6Clow. T cells were classified as expressing CD4+.

      Statistics

      Comparisons between groups for all outcomes were performed using a one-way analysis of variance. Simple effects were probed with a Fisher least significant difference test, with a focus on planned comparisons of interest (i.e., antibodies vs. control isotype IgG). Probability values less than 0.05 were considered significant.

      Study approval

      Animal protocols were consistent with guidelines issued by the American Association for Accreditation of Laboratory Animal Care and were approved by the Case Western Reserve University Institutional Animal Care and Use Committee.

      ORCIDs

      Conflict of Interest

      Kristy Kikly is an employee of Eli Lilly. The other authors state no conflict of interest.

      Acknowledgments

      This work was supported in part by The Lozick Discovery Grant and the National Psoriasis Foundation, the Murdough Family Center for Psoriasis, the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (P30 AR39750, P50 AR055508, R01 AR063437, R01 AR062546, R21 AR063852, R01 AR069071, T32AR007569), and the National Institute for Heart, Lung, and Blood of the National Institutes of Health (R01 HL057506).

      Author Contributions

      TSM and NLW conceived the idea. JBG, KK, TSM, YW, and NLW designed the experiments. YL, JBG, MIC, XZ, YF, DD, and NLW performed experiments and collected data. YL, JBG, YF, TSM, YW, and NLW analyzed the data. TLI performed the statistics. YL, JBG, TLI, TSM, and NLW generated the figures and wrote and revised the manuscript.

      Supplementary Material

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