Project description:Neutrophils patrol healthy tissues and confer local defense upon perturbations. Recent evidence suggests that they participate in tissue-specific structural programs in addition to their indispensable role in immunity.The goal of this study is to evaluate the effect of neutrophils in healthy mouse skin by comparing the transcriptional landscape of naive skin to neutrophil-depleted skin.
Project description:Neutrophils patrol healthy skin at steady state. Here we evaluate whether the antibody-mediated depletion of neutrophils affect global transcriptome of the skin.
Project description:To investigate the effect of the absence of neutrophils in the transcriptome of fibroblast and macrophage from lung and skin we injected DT to Mrp8 CRE+ iDTR and Mrp8 CRE- iDTR mice every two days for a week and isolated lung and skin fibroblasts and macrophages by cell sorting . We then performed gene expression profiling analysis using data obtained from RNA-seq of macrophages and fibroblast 7 days after DT treatment and compared the transcriptomes of macrophages and fibroblasts in control or neutrophil depletion animals. We focused on the role of neutrophils on the regulation of extracelular matrix genes in fibroblasts and macrophages and found that neutrophil depletion does not significantly affect the RNA level of matrix-related transcripts in those cells.
Project description:To elucidate the role of neutrophils in the molecular alterations observed after I/R, we performed an experiment of I/R in neutrophil-depleted mice. Neutrophil depletion was performed by anti-Ly6G injection and IgG was used as vehicle. We have performed an unbiased, open-search analysis of the oxidative PTMs and quantitative multiplexed proteomics that take place in the mouse heart tissue at 24h after I/R. Neutrophil depletion was performed in 3 mice by Ly6G injection 24h and 48 h prior to I/R and sacrificed 24 hours after reperfusion. A second group consisting on 3 mice that were injected with IgG antibody as vehicle, and subjected to I/R and sacrificed 24h after reperfusion. A third group of 3 mice with no injection were also sacrificed 24h after reperfusion. 12 additional mice from the three previous groups were sacrificed without I/R intervention: control without injection (n=6), IgG injection (n=3) and Ly6G injection (n=3). Whole hearts tissue was collected for proteomics evaluation.
Project description:Interrogating the tumor immune compartment generates new prognostic and treatment modalities. We used the poorly immunogenic oncogenic Kras driven mouse model of lung cancer, to challenge the predictive value of an immune signature, using a new algorithm called MegaClust. It highlighted a predominant negative impact of neutrophil infiltration on multiple immune populations. Human samples and depletion experiments in mice revealed that neutrophils promote tumor growth by altering angiogenesis, associated to increased PI3K/Akt/mTOR signaling in tumor cells and profound immunosubversion. Together with blood vessel spreading and normalization, neutrophil depletion was associated with T cell redistribution into tumor mass and sensitized tumors to anti-PD1 immunotherapy in this refractory model. Our observations illustrate a mechanism of immune exclusion protecting tumor cells from destruction that relies on neutrophil infiltration
Project description:We previously reported that neutrophils infiltrate most tissues in a circadian fashion, as they clear out from the circulation (Casanova‑Acebes et al., 2018). In our current study we hypothesized that neutrophils are relevant contributors to the matrix of barrier tissues and that then their elimination should leave a temporal footprint in the circadian pattern of expression of matrix-associated genes. To answer this question, we generated a circadian transcriptional dataset from the bone marrow, lung, liver, intestine and skin in wild type and neutrophil-depleted animals. The anlysis of the circadian patern of matrix related genes obtained from this datasets indicates that their temporal expression patterns in the analysed tissues is disrupted following neutrophil depletion. This results directly implicate neutrophils in the circadian biology of the matrix organization in various tissues.
Project description:Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.