Christophe Cataisson

HIF-1α regulates epithelial inflammation by cell autonomous NFκB activation and paracrine stromal remodeling

Hypoxia inducible factor-1 (HIF-1) is a master regulatory transcription factor controlling multiple cell-autonomous and non-cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal keratinocytes and demonstrated a signaling pathway from HIF-1 to nuclear factor kappa B (NFkappaB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 12-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1-induced NFkappaB activation was composed of 2 elements, IkappaB hyperphosphorylation and phosphorylation of Ser276 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFkappaB transcriptional targets macrophage inflammatory protein-2 (MIP-2/CXCL2/3), keratinocyte chemokine (KC/CXCL1), and tumor necrosis factor [alfa] (TNFalpha) were constitutively up-regulated and further increased after TPA challenge both in cultured keratinocytes and in transgenic mice. Whole animal KC, MIP-2, or TNFalpha immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta growth factor (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFkappaB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression.

Inflammasome Sensor Nlrp1b-Dependent Resistance to Anthrax Is Mediated by Caspase-1, IL-1 Signaling and Neutrophil Recruitment

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1bS/S or Nlrp1bR/R, respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1bS/S alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1bR/R alleles (which cannot activate caspase-1 in response to toxin). Nlrp1bS-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1bS/S mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1bS/S mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1bS/S,Nlrp1bR/ R and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection.

Activation of Cutaneous Protein Kinase Cα Induces Keratinocyte Apoptosis and Intraepidermal Inflammation by Independent Signaling Pathways

Skin keratinocytes are major mediators of host immune responses. The skin is also a target for immunologically based inflammation in many pathological states. Activation of protein kinase C (PKC) can induce cutaneous inflammation, but the precise role of each of six cutaneous PKC isoforms (α, δ, ε, η, ζ, μ) that regulate normal skin homeostasis or contribute to skin pathology has not been clarified. We generated transgenic mice that overexpress PKCα in the basal layer of the epidermis and the outer root sheath of hair follicles under the regulation of the bovine keratin 5 promoter. K5-PKCα transgenic mice exhibit severe intraepidermal neutrophilic inflammation and disruption of the epidermis and upper hair follicles when treated topically with 12-O-tetradecanoylphorbol-13-acetate (TPA). Both TPA and UVB cause apoptosis in transgenic skin, but only TPA evokes intraepidermal inflammation. TPA also induces apoptosis in cultured transgenic keratinocytes, and this is prevented by an AP-1 dominant-negative construct. However, inhibiting AP-1 in vivo does not abrogate intraepidermal inflammation. Transcripts for specific cytokines and chemokines are elevated in TPA-treated cultured transgenic keratinocytes, and conditioned culture medium from these cells promotes neutrophil migration in vitro. Chemokine expression and neutrophil migration are not diminished by inhibiting AP-1. Thus, PKCα activation induces keratinocyte apoptosis via an AP-1-dependent pathway and mediates chemokine induction and intraepidermal inflammation independently. This model system will be useful to define specific chemokines regulated by PKCα that promote intraepidermal neutrophilic inflammation, a condition that characterizes several human cutaneous diseases such as pustular psoriasis and acute generalized exanthematous pustulosis.

CXCR2 ligands and G-CSF mediate PKCα-induced intraepidermal inflammation

Transgenic mice overexpressing PKCα in the epidermis (K5-PKCα mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCα is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCα increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCα keratinocytes also released KC and MIP-2 into culture supernatants through an NF-κB–dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCα mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCα-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCα-selective inhibitor. Inhibiting PKCα also reduced the basal and TNF-α– or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCα activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCα could be therapeutic targets for inhibition of cutaneous inflammation.

Protein kinase C alpha-mediated chemotaxis of neutrophils requires NF-kappa B activity but is independent of TNF alpha signaling in mouse skin in vivo.

Protein kinase C (PKC) isoforms are major regulators of cutaneous homeostasis and mediate inflammation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We have previously reported that transgenic mice overexpressing PKCα in the skin exhibit severe intraepidermal neutrophilic inflammation and keratinocyte apoptosis when treated topically with TPA. Activation of PKCα increases the production of TNFα and the transcription of chemotactic factors (MIP-2, KC, S100A8/A9), vascular endothelial growth factor, and GM-CSF in K5-PKCα keratinocytes. In response to PKCα activation, NF-κB translocates to the nucleus and this is associated with IκB phosphorylation and degradation. Preventing IκB degradation reduces both the expression of inflammation-associated genes and chemoattractant release. To determine whether TNFα mediated NF-κB translocation and subsequent expression of proinflammatory factors, K5-PKCα mice were treated systemically with a dimeric soluble form of p75 TNFR (etanercept) or crossed with mice deficient for both TNFR isoforms, and keratinocytes were cultured in the presence of TNFα-neutralizing Abs. The in vivo treatment and TNFR deficiency did not prevent inflammation, and the in vitro treatment did not prevent NF-κB nuclear translocation after TPA. Together these results implicate PKCα as a regulator of a subset of cutaneous cytokines and chemokines responsible for intraepidermal inflammation independent of TNFα. PKCα inhibition may have therapeutic benefit in some human inflammatory skin disorders.

IL-1R–MyD88 signaling in keratinocyte transformation and carcinogenesis

Keratinocyte MyD88 is a component of an IL-1α–IL-1R autocrine loop that drives Ras-mediated transformation in vitro and contributes to skin tumor formation in vivo.

EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo.

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-alpha to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-alpha-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation.

Inducible Cutaneous Inflammation Reveals a Protumorigenic Role for Keratinocyte CXCR2 in Skin Carcinogenesis

Transgenic mice that overexpress PKCalpha in the epidermis (K5-PKCalpha mice) exhibit acute CXCR2-mediated intraepidermal neutrophilic inflammation and a strong epidermal hyperplasia in response to application of 12-O-tetradecanoylphorbol-13-acetate (TPA). We now show that hyperplasia is independent of infiltrating neutrophils. Furthermore, when K5-PKCalpha mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and promoted with a low dose of TPA, 58% of K5-PKCalpha mice developed skin papillomas that progressed to carcinoma, whereas wild-type mice did not develop tumors. We confirmed that CXCR2 is expressed by keratinocytes and showed that transformation by oncogenic ras (a hallmark of DMBA initiation) or TPA exposure induced all CXCR2 ligands. Ras induction of CXCR2 ligands was mediated by autocrine activation of epidermal growth factor receptor and nuclear factor-kappaB, and potentiated by PKCalpha. Oncogenic ras also induced CXCR2 ligands in keratinocytes genetically ablated for CXCR2. However, ras transformed CXCR2 null keratinocytes formed only small skin tumors in orthotopic skin grafts to CXCR2 intact hosts, whereas transformed wild-type keratinocytes produced large tumors. In vitro, CXCR2 was essential for CXCR2 ligand-stimulated migration of ras-transformed keratinocytes and for ligand activation of the extracellular signal-regulated kinase (ERK) and Akt pathways. Both migration and activation of ERK and Akt were restored by CXCR2 reconstitution of CXCR2 null keratinocytes. Thus, activation of CXCR2 on ras-transformed keratinocytes has both promigratory and protumorigenic functions. The up-regulation of CXCR2 ligands after initiation by oncogenic ras and promotion with TPA in the mouse skin model provides a mechanism to stimulate migration by both autocrine and paracrine pathways and contribute to tumor development.

TGF-beta signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3.

CLIC4 (chloride intracellular channel 4), a multifunctional protein that traffics between the cytoplasm and nucleus, interacts with Schnurri-2, a transcription factor in the bone morphogenetic protein (BMP) signalling pathway. Here we show that transforming growth factor β (TGF-β) promotes the expression of CLIC4 and Schnurri-2 as well as their association in the cytoplasm and their translocation to the nucleus. In the absence of CLIC4 or Schnurri-2, TGF-β signalling is abrogated. Direct nuclear targeting of CLIC4 enhances TGF-β signalling and removes the requirement for Schnurri-2. Nuclear CLIC4 associates with phospho (p)-Smad2 and p-Smad3, protecting them from dephosphorylation by nuclear phosphatases. An intact TGF-β signalling pathway is essential for CLIC4-mediated growth-arrest. These results newly identify Schnurri-2 and CLIC4 as modifiers of TGF-β signalling through their stabilization of p-Smad2 and 3 in the nucleus.

Gene from a psoriasis susceptibility locus primes the skin for inflammation.

Psoriasis candidate genes promote susceptibility to skin inflammation and provide a treatment approach for psoriasis-related inflammation. Tipping the Scales Fashion models and the masses alike crave flawless skin achieved without the help of Photoshop. Yet, for sufferers of psoriasis, an autoimmune disease of the skin, this dream is far out of reach. Psoriasis, which frequently manifests as red or white scaly plaques on the skin, currently has no cure, although the symptoms may be treated by inhibiting either T cells or inflammatory cytokines. Wolf et al. bring us one step closer to a cure by fashioning a mouse model that mimics psoriasis in patients and can be used to develop therapies for the disease. Both immune cells and epidermal cells of the skin contribute to pathogenesis in psoriatic lesions; however, the relative contribution of these systems to disease development remains unknown. To investigate this contribution, Wolf et al. generated a mouse model of psoriasis by conditionally overexpressing the inflammation-associated protein mS100a7a15 in keratinocytes. The related human S100A7 and S100A15 proteins are encoded in a psoriasis susceptibility locus and are involved in cellular signaling during epithelial host defense. This new mouse model is especially relevant to the human disease because the human homologs of mS100a7a15 (hS100A7/S100A15) are overexpressed in the epidermis of inflammatory lesions from psoriasis patients. The model by Wolf et al. mimicked the disease in humans, inducing an inflammatory response after abrasion that consisted of elevated amounts of proinflammatory T cells and cytokines. These effects were mediated by the mS100a7a15 receptor RAGE (receptor of advanced glycation end products), suggesting the S100a7a15-RAGE axis as a therapeutic target for psoriasis. Thus, this model not only links the epidermal and immune contributions of psoriasis but also may tip the scales toward fewer scales (and flawless skin) for psoriasis patients. Psoriasis is a common complex genetic disease characterized by hyperplasia and inflammation in the skin; however, the relative contributions of epidermal cells and the immune system to disease pathogenesis remain unclear. Linkage studies have defined a psoriasis susceptibility locus (PSORS4) on 1q21, the epidermal differentiation complex, which includes genes for small S100 calcium-binding proteins. These proteins are involved in extracellular and intracellular signaling during epithelial host defense, linking innate and adaptive immunity. Inflammation-prone psoriatic skin constitutively expresses elevated concentrations of S100A7 (psoriasin) and S100A15 (koebnerisin) in the epidermis. Here, we report that genetically modified mice expressing elevated amounts of doxycycline-regulated mS100a7a15 in skin keratinocytes demonstrated an exaggerated inflammatory response when challenged by exogenous stimuli such as abrasion (Koebner phenomenon). This immune response was characterized by immune cell infiltration and elevated concentrations of T helper 1 (TH1) and TH17 proinflammatory cytokines, which have been linked to the pathogenesis of psoriasis and were further amplified upon challenge. Both inflammation priming and amplification required mS100a7a15 binding to the receptor of advanced glycation end products (RAGE). mS100a7a15 potentiated inflammation by acting directly as a chemoattractant for leukocytes, further increasing the number of inflammatory cells infiltrating the skin. This study provides a pathogenetic psoriasis model using a psoriasis candidate gene to link the epidermis and innate immune system in inflammation priming, highlighting the S100A7A15-RAGE axis as a potential therapeutic target.