Dendritic cell progenitor trafficking and identification and functional analyses of dendritic cells with distinct developmental origin

Conventional dendritic cells (cDCs), are the major antigen-presenting cell type that bridges the innate and adaptive immune system. DCs are constantly replenished from myeloid bone marrow progenitors which latest stage, pre-cDC, leave the BM, seeds the peripheral tissues and further differentiates into two functionally and developmentally distinct subsets, cDC1 and cDC2. This study aimed to investigate DC development by assessing the trafficking of pre-cDCs and by analyzing the effect of a specific depletion of DC progenitors. The signals that regulate the recruitment of pre-cDCs to different peripheral organs are poorly understood. Therefore, this study aimed to identify pre-cDCs in different peripheral organs and to find differences in expression pattern of trafficking receptors. In this study 39 trafficking receptors have been identified to be expressed on pre-cDCs of the analysed tissues and showed differences in the expression patterns between peripheral organs. These receptors are interesting candidates to further study differences in the recruitment of pre-cDCs to different peripheral tissues This can provide possibilities to influence the recruitment of pre-cDCs in certain diseases, where the replenishment of cDCs is accelerated. To generate a DC deficient model, DNGR-1/ CLEC9A expressing cells and its progeny were depleted by crossing Clec9a-Cre mice to Rosa-lox-STOP-lox- diphtheria toxin (DTA) mice. Despite cDC progenitors being diminished in these mice, as expected, cells that phenotypically resemble cDC2 arise independent of conventional DC progenitors. As these cells show somatic rearrangements of the Ig-heavy gene locus, typical for lymphoid cells, they were termed lymphoid DC2. A lymphoid origin of DCs has been shown in vitro as well as in adoptive transfer studies, however, the reason for this dual origin and under which physiological settings lymphoid derived DC2 develop and replenish myeloid-derived cDCs is unknown. To test the hypothesis that lymphoid DCs represent a subset of cells with distinct functions that replace myeloid-derived DCs in certain types of diseases, functional analyses were performed. Indeed, less lymphoid DC2 showed TNFα expression after LPS stimulation compared to DC2 from control mice. Furthermore, less lymphoid DC2 showed migration towards CCR7 ligands suggesting a migration defect. Additionally, increased cell death of lymphoid DC2 compared to DC2 from control mice was found in vitro. Increased cell death, on the one hand, provides evidence that lymphoid DC2 behave different from bona fide cDC2, on the other hand it impedes the interpretation of quantitative functional analyses, such as migration assays. Taken together, depletion of myeloid DC progenitors in Clec9aCreRosaDTA mice provides an artificially induced situation in which DC2 like cells can develop in the absence of myeloid DC progenitor. Furthermore, preliminary findings indicate that lymphoid DC2 show functional differences to bona fide cDC2 which argues for the requirement of a redundant developmental pathway to create a situation adapted repertoire of cells

Nuclear Deformation During Neutrophil Migration at Sites of Inflammation

Cell migration is indispensable for various biological processes including angiogenesis, wound healing, and immunity. In general, there are two different migration modes described, the mesenchymal migration mode and the amoeboid migration mode. Neutrophils rapidly migrate toward the sites of injury, infection, and inflammation using the amoeboid migration mode which is characterized by cell polarization and a high migration velocity. During site-directed trafficking of neutrophils from the blood stream into the inflamed tissue, neutrophils must first withstand shear stress while migrating on the 2-dimensional endothelial surface. Subsequently, they have to cross different physical barriers during the extravasation process including the squeezing through the compact endothelial monolayer that comprises the blood vessel, the underlining basement membrane and then the 3-dimensional meshwork of extracellular matrix (ECM) proteins in the tissue. Therefore, neutrophils have to rapidly switch between distinct migration modes such as intraluminal crawling, transmigration, and interstitial migration to pass these different confinements and mechanical barriers. The nucleus is the largest and stiffest organelle in every cell and is therefore the key cellular element involved in cellular migration through variable confinements. This review highlights the importance of nuclear deformation during neutrophil crossing of such confinements, with a focus on transendothelial migration and interstitial migration. We discuss the key molecular components involved in the nuclear shape changes that underlie neutrophil motility and squeezing through cellular and ECM barriers. Understanding the precise molecular mechanisms that orchestrate these distinct neutrophil migration modes introduces an opportunity to develop new therapeutic concepts for controlling pathological neutrophil-driven inflammation

Clec9a-mediated ablation of conventional dendritic cells suggests a lymphoid path to generating dendritic cells In Vivo

Conventional dendritic cells (cDCs) are versatile activators of immune responses that develop as part of the myeloid lineage downstream of hematopoietic stem cells. We have recently shown that in mice precursors of cDCs, but not of other leukocytes, are marked by expression of DNGR-1/CLEC9A. To genetically deplete DNGR-1-expressing cDC precursors and their progeny, we crossed Clec9a-Cre mice to Rosa-lox-STOP-lox-diphtheria toxin (DTA) mice. These mice develop signs of age-dependent myeloproliferative disease, as has been observed in other DC-deficient mouse models. However, despite efficient depletion of cDC progenitors in these mice, cells with phenotypic characteristics of cDCs populate the spleen. These cells are functionally and transcriptionally similar to cDCs in wild type control mice but show somatic rearrangements of Ig-heavy chain genes, characteristic of lymphoid origin cells. Our studies reveal a previously unappreciated developmental heterogeneity of cDCs and suggest that the lymphoid lineage can generate cells with features of cDCs when myeloid cDC progenitors are impaired

Coronin 1B Controls Endothelial Actin Dynamics at Cell-Cell Junctions and Is Required for Endothelial Network Assembly

Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. VE-cadherin (VEcad)-based cell-cell junctions are connected to the actin cytoskeleton and regulated by actin-binding proteins. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia. The role of Coro1B in endothelial cells (ECs) is not fully understood and investigated in this study. Here, we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveals that Coro1B is recruited to, and operated at actin-driven membrane protrusions at cell-cell junctions. Coro1B is recruited to cell-cell junctions via a mechanism that requires the relaxation of the actomyosin cytoskeleton. By analyzing the Coro1B interactome, we identify integrin-linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Functional experiments reveal that depletion of Coro1B causes defects in the actin cytoskeleton and cell-cell junctions. Finally, in matrigel tube network assays, depletion of Coro1B results in reduced network complexity, tube number and tube length. Together, our findings point toward a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junctions and the assembly of endothelial networks

Elevated circulating Hsp70 levels are correlative for malignancies in different mammalian species

Circulating Hsp70 levels were determined in feline and porcine cohorts using two different ELISA systems. These comparative animal models of larger organisms often reflect diseases, and especially malignant tumors, better than conventional rodent models. It is therefore essential to investigate the biology and utility of tumor biomarkers in animals such as cats and pigs. In this study, levels of free Hsp70 in the blood of cats with spontaneously occurring tumors were detected using a commercial Hsp70 ELISA (R&D Systems). Sub-analysis of different tumor groups revealed that animals with tumors of epithelial origin presented with significantly elevated circulating Hsp70 concentrations. In addition to free Hsp70 levels measured with the R&D Systems Hsp70 ELISA, levels of exosomal Hsp70 were determined using the compHsp70 ELISA in pigs. Both ELISA systems detected significantly elevated Hsp70 levels (R&D Systems: median 24.9 ng/mL; compHsp70: median 44.2 ng/mL) in the blood of a cohort of APC$^{1311/+}$ pigs diagnosed with high-grade adenoma polyps, and the R&D Systems Hsp70 ELISA detected also elevated Hsp70 levels in animals with low-grade polyps. In contrast, in flTP53$^{R167H}$ pigs, suffering from malignant osteosarcoma, the compHsp70 ELISA (median 674.32 ng/mL), but not the R&D Systems Hsp70 ELISA (median 4.78 ng/mL), determined significantly elevated Hsp70 concentrations, indicating that in tumor-bearing animals, the dominant form of Hsp70 is of exosomal origin. Our data suggest that both ELISA systems are suitable for detecting free circulating Hsp70 levels in pigs with high-grade adenoma, but only the compHsp70 ELISA can measure elevated, tumor-derived exosomal Hsp70 levels in tumor-bearing animals

Early monocyte response following local ablation in hepatocellular carcinoma

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients’ response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation