Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (R(i)) from 19 to 7 h and improved organ dysfunction with enhanced alveolar–capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (R(i); 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation

An Essential Role of Caspase 1 in the Induction of Murine Lupus and Its Associated Vascular Damage

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102036/1/art38225.pd

The Role of Neutrophils during Mild and Severe Influenza Virus Infections of Mice

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6high neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1+ cells, not neutrophils, limit disease severity during mild influenza infections

Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production

The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB(4), and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study

Non-canonical PI3K–Cdc42–Pak–Mek-Erk Signaling Promotes Immune-Complex-Induced Apoptosis in Human Neutrophils

SummaryNeutrophils are peripheral blood leukocytes that represent the first line of immune cell defense against bacterial and fungal infections but are also crucial players in the generation of the inflammatory response. Many neutrophil cell surface receptors regulate important cellular processes via activation of agonist-activated PI3Ks. We show here that activation of human neutrophils with insoluble immune complexes drives a previously uncharacterized, PI3K-dependent, non-canonical, pro-apoptotic signaling pathway, FcγR-PI3Kβ/δ-Cdc42-Pak-Mek-Erk. This is a rare demonstration of Ras/Raf-independent activation of Erk and of PI3K-mediated activation of Cdc42. In addition, comparative analysis of immune-complex- and fMLF-induced signaling uncovers key differences in pathways used by human and murine neutrophils. The non-canonical pathway we identify in this study may be important for the resolution of inflammation in chronic inflammatory diseases that rely on immune-complex-driven neutrophil activation

Signalisation intracellulaire dans la régulation des fonctions bactéricides chez les polynucléaires neutrophiles de souris

Since the creation of the transgenic mice, the study of the innate immunity of this animal has received renewed interest. To exploit this experimental model as well as possible, it was initially necessary to know its normal function in order to compare to our current knowledge of the human immune system. The aim is to apprehend and define the limits of the murine model. We isolated neutrophils from mouse blood and bone marrow and compared their bactericidal functions. We have shown that mouse bone marrow contains a large population of neutrophils which are morphologically and functionally as mature as blood neutrophils. In addition, the discovery of new molecules aiming at stimulating the microbicidal activity of neutrophils as well as the study of intracellular signalling pathways leading to either the activation of these cells, their survival or their accelerated death, allowed us to better understand their regulation.Depuis la création des souris transgéniques, l'étude de l'immunité innée de cet animal a connu un regain d'intérêt. Pour exploiter au mieux ce modèle expérimental, il fallait d'abord connaître son fonctionnement normal afin de comparer les connaissances ainsi acquises chez la souris aux données connues chez l'Homme. Ceci dans le but d'appréhender et de définir les limites du modèle murin. Nous avons isolé des neutrophiles du sang et de la moelle osseuse de souris et comparé leurs fonctions bactéricides. Nous avons montré que la moelle osseuse des souris contient une grande population de neutrophiles morphologiquement et fonctionnellement aussi matures que ceux issus du sang. D'autre part, la découverte de nouvelles molécules visant à stimuler l'activité microbicide des neutrophiles ainsi que l'étude des voies de signalisation intracellulaire aboutissant soit à l'activation de ces cellules, à leur survie ou à leur mort accélérée, nous ont permis de mieux comprendre leur fonctionnement

Signalisation intracellulaire dans la régulation des fonctions bactéricides chez les polynucléaires neutrophiles de souris

Depuis la création des souris transgéniques, l'étude de l'immunité innée de cet animal a connu un regain d'intérêt. Pour exploiter au mieux ce modèle expérimental, il fallait d'abord connaître son fonctionnement normal afin de comparer les connaissances ainsi acquises chez la souris aux données connues chez l'Homme. Ceci dans le but d'appréhender et de définir les limites du modèle murin. Nous avons isolé des neutrophiles du sang et de la moelle osseuse de souris et comparé leurs fonctions bactéricides. Nous avons montré que la moelle osseuse des souris contient une grande population de neutrophiles morphologiquement et fonctionnellement aussi matures que ceux issus du sang. D'autre part, la découverte de nouvelles molécules visant à stimuler l'activité microbicide des neutrophiles ainsi que l'étude des voies de signalisation intracellulaire aboutissant soit à l'activation de ces cellules, à leur survie ou à leur mort accélérée, nous ont permis de mieux comprendre leur fonctionnement.Since the creation of the transgenic mice, the study of the innate immunity of this animal has received renewed interest. To exploit this experimental model as well as possible, it was initially necessary to know its normal function in order to compare to our current knowledge of the human immune system. The aim is to apprehend and define the limits of the murine model. We isolated neutrophils from mouse blood and bone marrow and compared their bactericidal functions. We have shown that mouse bone marrow contains a large population of neutrophils which are morphologically and functionally as mature as blood neutrophils. In addition, the discovery of new molecules aiming at stimulating the microbicidal activity of neutrophils as well as the study of intracellular signalling pathways leading to either the activation of these cells, their survival or their accelerated death, allowed us to better understand their regulation.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF