Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF

This work was supported by the British Heart Foundation (FS/09/022/27354 funded the PhD training of M. Finsterbusch), Arthritis Research-UK (19913 to M.-B. Voisin), and The Wellcome Trust (098291/Z/12/Z to S. Nourshargh)

Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage

20 páginas, 4 figuras, 3 tablas y 7 tablas en material suplementario.Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.This work was performed in partial fulfillment of the requirements for the PhD degree for Cristina Herrera at Universidad de Costa Rica.Peer reviewe

Visceral adipose tissue immune homeostasis is regulated by the crosstalk between adipocytes and dendritic cell subsets

Visceral adipose tissue (VAT) has multiple roles in orchestrating whole-body energy homeostasis. In addition, VAT is now considered an immune site harboring an array of innate and adaptive immune cells with a direct role in immune surveillance and host defense. We report that conventional dendritic cells (cDCs) in VAT acquire a tolerogenic phenotype through upregulation of pathways involved in adipocyte differentiation. While activation of the Wnt/β-catenin pathway in cDC1 DCs induces IL-10 production, upregulation of the PPARγ pathway in cDC2 DCs directly suppresses their activation. Combined, they promote an anti-inflammatory milieu in vivo delaying the onset of obesity-induced chronic inflammation and insulin resistance. Under long-term over-nutrition, changes in adipocyte biology curtail β-catenin and PPARγ activation, contributing to VAT inflammation

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia

This work was supported by funds from the Wellcome Trust (098291/Z/12/Z and 101604/Z/13/Z) (S.N.) and the British Heart Foundation (FS/11/19/28761) (A.W.)

Effects of PI and PIII Snake Venom Haemorrhagic Metalloproteinases on the Microvasculature: A Confocal Microscopy Study on the Mouse Cremaster Muscle

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.This work was supported by funds from the British Heart Foundation (FS/14/3/30518 to T.G. and S.N.), the People Programme (Marie Curie Actions) of the EU’s 7th Framework Programme (FP7/2007-2013) under REA grant agreement 608765 (to T.G. and S.N.), and by the Wellcome Trust (098291/Z/12/Z to S.N.). D.S. is supported by the CNIC, SAF2016-79040-R from the Spanish Ministerio de Ciencia, and ERC-2016-CoG 725091 from the European Research Council. M.T. and A.R. are supported by the Sinergia grant CRSII3_160719 of the Swiss National Science Foundation. G.G. is supported by the Wellcome Trust and the MRC. U.H.v.A. and A.T. are supported by the Ragon Institute of MGH, MIT and Harvard and the HMS Center for Immune Imaging

Dose and time dependency of BaP1 effects on type IV collagen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with different amounts of BaP1 (10, 30 and 100 μg) for either 5 or 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type: (A) arterioles, (B) capillaries, and (C) PCV per cremaster (n = 4). Below each graph, representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for BaP1 (30 μg) and control at 15 min. The images show a decrease in fluorescence intensity for type IV collagen in BM of capillaries of treated tissues as compared to control, whereas no significant reduction was observed in arterioles and PCV. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; Col IV: type IV collagen; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on type IV collagen from vascular BM on mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with either (A) 30 μg of BaP1 or (C) 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either (B) 60 μg of BaP1 or (D) 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per animal (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for CsH1 30 μg and control applied in anesthetized mice (i.e. model with blood flow). The images show a decrease in fluorescence intensity for type IV collagen in BM of arterioles, capillaries, and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Col IV: type IV collagen; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on size and density of gaps between adjacent smooth muscle and pericytes on mouse cremaster muscle vasculature.

<p>Isolated cremaster muscles were incubated with either 30 μg of BaP1 or 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either 60 μg of BaP1 or 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of the gaps between adjacent smooth muscle and pericytes. Results are expressed as the mean ± SEM of the (A, B) gap size and (C, D) gap density (number of gaps per vessel area) of at least five images of arterioles and PCV per cremaster (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for actin α smooth muscle are shown for BaP1 30 μg and control in the model without blood flow. Notice the increase in the gap size (arrows) in arterioles and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on vascular permeability after intradermical application.

<p>Mice received an intravenous injection of 200 μl of Evans Blue dye (6 mg/ml). After 5 min, mice were injected by intradermal route, in the ventral abdominal region, with either 2 μg of BaP1 or 1 μg of CsH1. PBS was injected as control in another group of animals. After 15 min mice were sacrificed by cervical dislocation, their skin was removed and the area of extravasation was measured. (A) Results are expressed as the mean ± SEM (n = 5). **p < 0.001 as compared to CsH1. (B) Figure shows representative images from five animals analyzed.</p