Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration

C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1β cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation

Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid beta 2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo

Extracellular MRP8/14 is a regulator of β2 integrin-dependent neutrophil slow rolling and adhesion

Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid beta 2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo

Camillo Panciera di Zoppola. Le ricerche della Postumia (1893-1896)

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer and . Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes . Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer

EpCAM^low CD11b^high DCs are the main skin DC subset trafficking to the DLN after BCG infection.

<p>Frequency (<b>A</b>) and total number (<b>B</b>) of CFSE⁺ cells within defined subsets of migratory skin DCs in pLN after BCG infection. (<b>A</b>) Zebra plots showing gating strategy employed (top panel) and the frequency of CFSE⁺ cells within each, defined subset at different time points after BCG infection (bottom panels). The frequency of CFSE⁺ cells within the EpCAMl^ow CD11b^high population in BCG-draining pLN is statistically significant relative to PBS-draining pLN for all three time points analyzed. (<b>B</b>) Total number of CFSE⁺ cells within each subset as defined in (<b>A</b>). For each experiment, at least 5 mice were used for BCG-infected groups and 3 for PBS controls. Bars indicate standard error of the mean. *Denotes statistically significant differences relative to PBS-injected controls. One of three independent experiments shown.</p

CFSE⁺ cells enter the paracortex, co-localize with BCG and are closely apposed to P25 TCRTg cells.

<p>P25 TCRTg cells were isolated from LNs of naïve P25 TCRTg RAG–1^-/- ECFP mice using a CD4 negative selection procedure (Miltenyi Biotec), and transferred i.v. into WT recipients. Recipients were then inoculated with BCG-RFP and the CFSE-based migration assay performed. (<b>A</b>) Distribution of CFSE⁺ cells in the paracortex (T-cell area) of the pLN as determined by CD3 staining on pLN sections from BCG-injected mice. (<b>B</b>) Quantification of CFSE⁺ cells within the T-cell area of the pLN from BCG- and PBS-injected mice expressed as the number of CFSE⁺ cells per area of CD3 staining. (<b>C</b>) Micrograph showing distribution of BCG (red), CFSE⁺ cells (green), p25 TCRTg cells (blue) and HEVs (white) in the pLN. Boxed inserts in the micrograph were magnified to show x/y/z projections of two CFSE⁺ cells with intracellular BCG-RFP. BCG were overexposed in these magnified views to facilitate visualization. Infected cell in lower panel is also in contact with a p25TCRTg ECFP cell (blue). (<b>D</b>) Quantification of the number of CFSE⁺ cells containing BCG-RFP (left graph), found in close apposition with P25 TCRTg ECFP cells (center graph), and containing BCG-RFP and in close apposition with P25 TCRTg ECFP cells (right graph). Data samples represent average obtained from individual pLN from which multiple fields of maximum intensity projections were analyzed. Bars indicate standard error of the mean. White bar on micrographs depict 100 microns. *Denotes statistically significant difference between BCG-infected and PBS controls in (<b>B</b>) and between day 1 and 6 in (<b>D</b>).</p