The role of the proinflammatory cytokines IL-­17, IL-­6 and IL-­1 in (IMQ-­induced) psoriasis

Psoriasis vulgaris is the most common form of the inflammatory skin disease psoriasis and leads to the formation of red, scaly plaques, mainly on the knees, elbows and scalp. Psoriasis affects 2-4% of the population worldwide and is associated with involvement of different immune cells (e.g. T cells and myeloid cells) and proinflammatory cytokines, for example IL-17, IL-6 or IL-1. Here, we used different transgene mouse models to examine the function of these cytokines and the respective receptors in more detail for their role in psoriatic disease. Our lab recently established a novel mouse model where IL-17A is specifically overexpressed in keratinocytes (K14-IL-17Aind/ mice), which leads to skin inflammation showing many hallmarks of human psoriasis. As it was already shown that neutralization of IL-6 in those mice led to an improvement of the psoriatic phenotype, we also investigated the impact of anti-IL-17A treatment in this thesis. We could demonstrate that also the neutralization of IL-17A resulted in a partially improved disease severity. Moreover, the sera of treated K14-IL-17Aind/ mice displayed significantly reduced IL-17A levels compared to controls. Further, the treatment did result in an improved PASI score and less spleen-infiltrating myeloid cells. Hence, the results indicate a positive effect of the treatment. This we also confirmed by an anti-IL-17A treatment of wild type (wt) mice in the model of IMQ-induced psoriasis where the treatment also reduced the PASI score. IMQ is the active compound of AldaraTM cream and serves as a TLR7/8 agonist. Its topical application to the skin leads to a psoriasis-like skin disease, also in terms of the involved cytokines and infiltrating cells. The role of IL-6 signaling in the psoriasis-like disease was investigated by deleting the membrane-bound IL-6Rα specifically in myeloid cells (IL-6RαΔmyel). Treating these mice with IMQ resulted in comparable disease severity as in wt mice. These findings could indicate that the effect of the deletion might be compensated by the soluble form of the receptor or by other cytokines of the gp130 family with redundant activities. Interestingly, IL-1 signaling also seems to have a strong involvement in psoriasis-like disease, as Il1r2-/- mice treated with IMQ exhibited significantly more myeloid cells than wt mice. To identify which cells are responsible for the observed phenotype, mice with a tissue specific deletion of IL-1R2 in T cells (Il1r2ΔT) and neutrophils (Il1r2ΔN) were analyzed in the same model. As none of these mouse strains showed differences compared to wt mice during IMQ treatment, neither neutrophils, nor T cells seemed to be responsible for the initial observed findings. Also here compensation by the soluble form of IL-1R2 should be considered. The results of this thesis underline the neutralization of IL-17A being a promising treatment option for psoriasis. In addition, our findings regarding the role of the cytokines IL-6 and IL-1, could lead to the discovery of new drug targets for the treatment of this inflammatory skin disease.Psoriasis vulgaris ist die häufigste Form der entzündlichen Hautkrankheit Psoriasis und führt zur Bildung von roten schuppigen Plaques hauptsächlich an den Knien, Ellenbogen und der Kopfhaut. Etwa 2-4% der weltweiten Bevölkerung sind von dieser Krankheit betroffen, welche mit der Beteiligung von verschiedenen Immunzellen (z.B. T Zellen und myeloide Zellen) und entzündlichen Zytokinen, wie IL-17, IL-6 und IL-1 einhergeht. In dieser Doktorarbeit wurden verschiedene transgene Mausmodelle verwendet, um die Funktion dieser Zytokine und deren Rezeptoren im Zusammenhang mit der Entwicklung von Psoriasis im Detail zu untersuchen. Vor einiger Zeit wurde von unserem Labor ein Mausmodell veröffentlicht, in welchem IL-17A spezifisch in Keratinozyten überexprimiert ist (K14-IL-17Aind/ ). Diese Überexpression führt zu einer entzündlichen Hautreaktion mit vielen Merkmalen der menschlichen Psoriasis. Nachdem eine anti-IL-6 Behandlung dieser Mäuse bereits sehr erfolgreich war, konnten wir in dieser Doktorarbeit zeigen, dass sich auch durch Behandlung mit einem anti-IL-17A Antikörper eine Verbesserung des Hautphänotyps der K14-IL-17Aind/ Mäuse eingestellt hat. Dies ging mit einem signifikant reduzierten Serumlevel von IL-17A einher. Außerdem resultierte die Behandlung in einem verbesserten PASI score und reduzierter Infiltration myeloider Zellen in die Milz. Somit deuten die Ergebnisse auf einen positiven Effekt der Behandlung hin. Dies konnte durch eine anti-IL-17A Behandlung von wildtyp (wt) Mäusen im IMQ Modell bestätigt werden da auch hier die Behandlung zu einem reduzierten PASI score geführt hat. IMQ ist ein TLR7/8 Agonist und als solcher der Wirkstoff in Aldara Creme. Seine Anwendung auf der Haut von Mäusen führt zu Entzündungsreaktionen ähnlich der menschlichen Psoriasis, auch bezüglich der involvierten Zytokine und infiltrierenden Effektorzellen. Die Funktion von IL-6 im psoriasis-ähnlichen Modell wurde durch die Deletion des membranständigen IL-6Rα in myeloiden Zellen (IL-6RαΔmyel) untersucht. Im Vergleich zu wt Mäusen resultierte die Behandlung der IL-6RαΔmyel Mäuse mit IMQ in der selben Schwere der Krankheit. Eine mögliche Erklärung hierfür könnte sein, dass die Deletion durch den löslichen IL-6R oder andere Zytokine der gp130 Familie kompensiert wird. Interessanterweise hat auch IL-1 einen großen Einfluss auf den Verlauf der psoriasis-ähnlichen Hautkrankheit, da IMQ behandelte Il1r2-/- Mäuse signifikant mehr myeloide Zellen in der Haut und Milz aufwiesen als wt Mäuse. Um zu ermitteln welche Zellen für den beobachteten Phänotyp verantwortlich sind, wurden auch Mäuse mit einer spezifischen Deletion für den IL-1R2 in T Zellen (Il1r2ΔT) und Neutrophilen (Il1r2ΔN) im IMQ Modell untersucht. Allerdings zeigte keiner dieser Stämme Unterschiede gegenüber wt Mäusen auf. Dies deutet darauf hin, dass weder T Zellen noch Neutrophile für den beobachteten Phänotyp verantwortlich sind. Auch hier sollte aber eine Kompensation durch den löslichen IL-1R2 in Betracht gezogen werden. Die hier dargestellten Ergebnisse untermauern die Relevanz der Neutralisierung von IL-17A als Behandlungsmöglichkeit für Psoriasis. Des Weiteren könnten die Ergebnisse dieser Arbeit zur Rolle der Zytokine IL-6 und IL-1 im Psoriasismodell den Grundstein für die Entwicklung neuer vielversprechender Behandlungsmöglichkeiten dieser entzündlichen Hautkrankheit legen.VI, 133 Blätte

The role of the proinflammatory cytokines IL-­17, IL-­6 and IL-­1 in (IMQ-­induced) psoriasis

Psoriasis vulgaris is the most common form of the inflammatory skin disease psoriasis and leads to the formation of red, scaly plaques, mainly on the knees, elbows and scalp. Psoriasis affects 2-4% of the population worldwide and is associated with involvement of different immune cells (e.g. T cells and myeloid cells) and proinflammatory cytokines, for example IL-17, IL-6 or IL-1. Here, we used different transgene mouse models to examine the function of these cytokines and the respective receptors in more detail for their role in psoriatic disease. Our lab recently established a novel mouse model where IL-17A is specifically overexpressed in keratinocytes (K14-IL-17Aind/ mice), which leads to skin inflammation showing many hallmarks of human psoriasis. As it was already shown that neutralization of IL-6 in those mice led to an improvement of the psoriatic phenotype, we also investigated the impact of anti-IL-17A treatment in this thesis. We could demonstrate that also the neutralization of IL-17A resulted in a partially improved disease severity. Moreover, the sera of treated K14-IL-17Aind/ mice displayed significantly reduced IL-17A levels compared to controls. Further, the treatment did result in an improved PASI score and less spleen-infiltrating myeloid cells. Hence, the results indicate a positive effect of the treatment. This we also confirmed by an anti-IL-17A treatment of wild type (wt) mice in the model of IMQ-induced psoriasis where the treatment also reduced the PASI score. IMQ is the active compound of AldaraTM cream and serves as a TLR7/8 agonist. Its topical application to the skin leads to a psoriasis-like skin disease, also in terms of the involved cytokines and infiltrating cells. The role of IL-6 signaling in the psoriasis-like disease was investigated by deleting the membrane-bound IL-6Rα specifically in myeloid cells (IL-6RαΔmyel). Treating these mice with IMQ resulted in comparable disease severity as in wt mice. These findings could indicate that the effect of the deletion might be compensated by the soluble form of the receptor or by other cytokines of the gp130 family with redundant activities. Interestingly, IL-1 signaling also seems to have a strong involvement in psoriasis-like disease, as Il1r2-/- mice treated with IMQ exhibited significantly more myeloid cells than wt mice. To identify which cells are responsible for the observed phenotype, mice with a tissue specific deletion of IL-1R2 in T cells (Il1r2ΔT) and neutrophils (Il1r2ΔN) were analyzed in the same model. As none of these mouse strains showed differences compared to wt mice during IMQ treatment, neither neutrophils, nor T cells seemed to be responsible for the initial observed findings. Also here compensation by the soluble form of IL-1R2 should be considered. The results of this thesis underline the neutralization of IL-17A being a promising treatment option for psoriasis. In addition, our findings regarding the role of the cytokines IL-6 and IL-1, could lead to the discovery of new drug targets for the treatment of this inflammatory skin disease.Psoriasis vulgaris ist die häufigste Form der entzündlichen Hautkrankheit Psoriasis und führt zur Bildung von roten schuppigen Plaques hauptsächlich an den Knien, Ellenbogen und der Kopfhaut. Etwa 2-4% der weltweiten Bevölkerung sind von dieser Krankheit betroffen, welche mit der Beteiligung von verschiedenen Immunzellen (z.B. T Zellen und myeloide Zellen) und entzündlichen Zytokinen, wie IL-17, IL-6 und IL-1 einhergeht. In dieser Doktorarbeit wurden verschiedene transgene Mausmodelle verwendet, um die Funktion dieser Zytokine und deren Rezeptoren im Zusammenhang mit der Entwicklung von Psoriasis im Detail zu untersuchen. Vor einiger Zeit wurde von unserem Labor ein Mausmodell veröffentlicht, in welchem IL-17A spezifisch in Keratinozyten überexprimiert ist (K14-IL-17Aind/ ). Diese Überexpression führt zu einer entzündlichen Hautreaktion mit vielen Merkmalen der menschlichen Psoriasis. Nachdem eine anti-IL-6 Behandlung dieser Mäuse bereits sehr erfolgreich war, konnten wir in dieser Doktorarbeit zeigen, dass sich auch durch Behandlung mit einem anti-IL-17A Antikörper eine Verbesserung des Hautphänotyps der K14-IL-17Aind/ Mäuse eingestellt hat. Dies ging mit einem signifikant reduzierten Serumlevel von IL-17A einher. Außerdem resultierte die Behandlung in einem verbesserten PASI score und reduzierter Infiltration myeloider Zellen in die Milz. Somit deuten die Ergebnisse auf einen positiven Effekt der Behandlung hin. Dies konnte durch eine anti-IL-17A Behandlung von wildtyp (wt) Mäusen im IMQ Modell bestätigt werden da auch hier die Behandlung zu einem reduzierten PASI score geführt hat. IMQ ist ein TLR7/8 Agonist und als solcher der Wirkstoff in Aldara Creme. Seine Anwendung auf der Haut von Mäusen führt zu Entzündungsreaktionen ähnlich der menschlichen Psoriasis, auch bezüglich der involvierten Zytokine und infiltrierenden Effektorzellen. Die Funktion von IL-6 im psoriasis-ähnlichen Modell wurde durch die Deletion des membranständigen IL-6Rα in myeloiden Zellen (IL-6RαΔmyel) untersucht. Im Vergleich zu wt Mäusen resultierte die Behandlung der IL-6RαΔmyel Mäuse mit IMQ in der selben Schwere der Krankheit. Eine mögliche Erklärung hierfür könnte sein, dass die Deletion durch den löslichen IL-6R oder andere Zytokine der gp130 Familie kompensiert wird. Interessanterweise hat auch IL-1 einen großen Einfluss auf den Verlauf der psoriasis-ähnlichen Hautkrankheit, da IMQ behandelte Il1r2-/- Mäuse signifikant mehr myeloide Zellen in der Haut und Milz aufwiesen als wt Mäuse. Um zu ermitteln welche Zellen für den beobachteten Phänotyp verantwortlich sind, wurden auch Mäuse mit einer spezifischen Deletion für den IL-1R2 in T Zellen (Il1r2ΔT) und Neutrophilen (Il1r2ΔN) im IMQ Modell untersucht. Allerdings zeigte keiner dieser Stämme Unterschiede gegenüber wt Mäusen auf. Dies deutet darauf hin, dass weder T Zellen noch Neutrophile für den beobachteten Phänotyp verantwortlich sind. Auch hier sollte aber eine Kompensation durch den löslichen IL-1R2 in Betracht gezogen werden. Die hier dargestellten Ergebnisse untermauern die Relevanz der Neutralisierung von IL-17A als Behandlungsmöglichkeit für Psoriasis. Des Weiteren könnten die Ergebnisse dieser Arbeit zur Rolle der Zytokine IL-6 und IL-1 im Psoriasismodell den Grundstein für die Entwicklung neuer vielversprechender Behandlungsmöglichkeiten dieser entzündlichen Hautkrankheit legen

Functionality of <i>Il6ra</i>^{<i>Δmyel</i>} mice.

<p>(A) Flow cytometric analysis of IL-6Rα expression of different organs. CD4⁺ cells are pre-gated on living CD90.2⁺ cells. Neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) and macrophages (Gr-1^- F4/80⁺) are pre-gated on living CD90.2^-/B220^- and CD11b⁺ cells. Gray histograms represent IgG2b isotype control for the IL-6R staining. Numbers in upper right corner represent the mean Mean Fluorescent Intensity (MFI) values of Il6ra^Δmyel or <i>wt</i> cells. Shown are representative histograms (n = 7 or 8 (3 independent experiments). (B) MFI of IL-6Rα expression pre-gated on CD4⁺ cells, neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) or macrophages (Gr-1^- F4/80⁺). Data are shown as bar graphs with mean and SEM. *p ≤ 0,05 Significance was calculated using Mann Whitney test. (C) Quantitative RT-PCR from CD11b⁺ MACS purified bone marrow cells for the IL-6Rα gene in <i>wt</i> and <i>Il6ra</i>^{<i>Δmyel</i>} mice. Expression levels are shown relative to the housekeeping gene HPRT (n = 5). Data are shown as bar graphs with mean and SEM. * p ≤ 0,05 Significance was calculated using Mann Whitney test. (D) Serum concentrations of sIL-6Rα examined by ELISA in <i>wt</i> (n = 6) and <i>Il6ra</i>^{<i>Δmyel</i>} (n = 10) mice at the age of 5 weeks to 5 months. Data are shown as bar graphs with mean and SEM. *** p ≤ 0,001 Significance was calculated using Mann Whitney test.</p

No differences of <i>Il6ra^Δmyel</i> mice in myelomonocytic cells and T cells compared to <i>wt</i> in IMQ-induced psoriasis.

<p>(A) Flow cytometric analysis of CD11b⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/B220^- cells. (B) Total cell numbers of CD11b⁺ cells of the indicated organs. Bar graphs are shown with mean and SEM. Significance was calculated using Kruskal-Wallis test. (C) Flow cytometric analysis of Ly6C⁺ and Ly6G⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/B220^- and CD11b⁺ cells. (D) Total cell numbers of Ly6G⁺ cells, pro-inflammatory monocytes (Ly6G^-Ly6C^hi) and resident monocytes (Ly6G^-Ly6C^int). Bar graphs of the indicated organs are shown with mean and SEM. Significances of the spleen were calculated using Kruskal-Wallis test (IMQ groups n = 6, sham groups n = 3). Significances of the ears were calculated with Student’s t-Test (IMQ groups n = 6, sham groups n = 1). (E) Flow cytometric analysis of IL-17A producing γδ T cells of the indicated organs. Cells are pre-gated on living CD45.2⁺/CD3⁺ cells. (F) Total cell numbers of γδ TCR⁺ and IL-17A⁺ γδ TCR⁺. Data are shown as bar graphs with mean and SEM. Significance was calculated using Kruskal-Wallis test.</p

<i>Il6ra</i>^{<i>Δmyel</i>} mice show no effect in clinical scores and histology in IMQ-induced psoriasis compared to <i>wt</i> mice.

<p>(A) Weight, ear/back skin thickness and erythema/scaling was scored daily on a scale from 0 to 4 with a modified PASI from human. IMQ treated groups with n = 6, sham treated group n = 3. (B) Back skin of <i>Il6ra</i>^{<i>Δmyel</i>} and <i>wt</i> mice treated with sham (show one representative) or IMQ (n = 5) was stained by fluorescence-Immunohistochemistry for myeloperoxidase (MPO)⁺. (C) Back skin of <i>Il6ra</i>^{<i>Δmyel</i>} and <i>wt</i> mice treated with sham (show one representative) or IMQ (n = 5) was stained by fluorescence-Immunohistochemistry for F4/80⁺. (magnifications are given from representative stainings), white scale bars = 200μm.</p

<i>Il6ra</i>^{<i>Δmyel</i>} mice show no differences in myelomonocytic cell compartments and T cells compared to <i>wt</i>.

<p>(A) Flow cytometric analysis of CD11b⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/CD19^- cells (n = 5). (B) Percentages and total cell numbers of CD11b⁺ cells of the indicated organs. Bar graphs are shown with mean and SEM. Significance was calculated using Mann Whitney test (n = 5). (C) Flow cytometric analysis of Gr-1⁺ and F4/80⁺ cells of the indicated organs. Cells are pre-gated on living CD90.2^-/CD19^- and CD11b⁺ cells (n = 5). (D) Total cell numbers of neutrophils (Gr-1^hi F4/80^-), monocytes (Gr-1^int F4/80⁺) and macrophages (Gr-1^- F4/80⁺). Data are shown as bar graphs with mean and SEM. Significance was calculated using Mann Whitney test (n = 5). (E) Flow cytometric analysis of IL-17A producing γδ T cells of indicated organs. Cells are pre-gated on living CD45.2⁺/CD3⁺ cells (n = 5). (F) Total cell numbers of γδ TCR⁺ and IL-17A⁺ γδ TCR⁺. Data are shown as bar graphs with mean and SEM. Significance was calculated using Mann Whitney test (n = 5).</p

Data from: Platyzoan paraphyly based on phylogenomic data supports a non-coelomate ancestry of Spiralia

Based on molecular data three major clades have been recognized within Bilateria: Deuterostomia, Ecdysozoa and Spiralia. Within Spiralia, small-sized and simply organized animals such as flatworms, gastrotrichs and gnathostomulids have recently been grouped together as Platyzoa. However, the representation of putative platyzoans was low in the respective molecular phylogenetic studies, in terms of both, taxon number and sequence data. Furthermore, increased substitution rates in platyzoan taxa raised the possibility that monophyletic Platyzoa represents an artefact due to long-branch attraction. In order to overcome such problems, we employed a phylogenomic approach, thereby substantially increasing i) the number of sampled species within Platyzoa and ii) species-specific sequence coverage in datasets of up to 82,162 amino acid positions. Using established and new measures (long-branch score) we disentangled phylogenetic signal from misleading effects such as long-branch attraction. In doing so, our phylogenomic analyses did not recover a monophyletic origin of platyzoan taxa that, instead, appeared paraphyletic with respect to the other spiralians. Platyhelminthes and Gastrotricha formed a monophylum, which we name Rouphozoa. To the exclusion of Gnathifera, Rouphozoa and all other spiralians represent a monophyletic group, which we name Platytrochozoa. Platyzoan paraphyly suggests that the last common ancestor of Spiralia was a simple-bodied organism lacking coelomic cavities, segmentation and complex brain structures, and that more complex animals such as annelids evolved from such a simply organized ancestor. This conclusion contradicts alternative evolutionary scenarios proposing an annelid-like ancestor of Bilateria and Spiralia and several independent events of secondary reduction

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4 and CD8 T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4 T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c and plasmacytoid CD303 dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-kappaB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials