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

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-α 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-α-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

MECANISMES ET ROLES DE L'HYPEREXPRESSION DE LA PROTEINE APPARENTEE A L'HORMONE PARATHYROIDIENNE (PTHRP) AU COURS DE LA TUMORIGENESE MAMMAIRE

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment

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

Murine models of HRAS-mediated cutaneous skeletal hypophosphatemia syndrome suggest bone as the FGF23 excess source

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and FGF23-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels. This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation–bearing bone or skin lesions. Toward this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia, and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23

Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant.

CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling