Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF-α caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay.

Alteration of lung tissues proteins in birch pollen induced asthma mice before and after SCIT

Subcutaneous immunotherapy (SCIT) is a classic form of allergen-specific immunotherapy that is used to treat birch pollen induced allergic asthma. To investigate the underlying molecular mechanisms of SCIT, we aimed to profile lung samples to explore changes in the differential proteome before and after SCIT in mice with allergic asthma. Fresh lungs were collected from three groups of female BALB/c mice: 1) control mice, 2) birch pollen-induced allergic mice, and 3) birch pollen-induced allergic mice with SCIT. Tandem mass tag (TMT) labelling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the lung proteome in the mice. Ingenuity pathway analysis (IPA) and Gene Ontology (GO) classification analysis were applied to identify differentially expressed proteins (DEPs) and crucial pathways. The screened DEPs were validated by immunohistochemistry analysis. A total of 317 proteins were upregulated and 184 proteins were downregulated in the asthma group compared to those of the control group. In contrast, 639 DEPs (163 upregulated and 456 downregulated proteins) were identified after SCIT in comparison with those of the asthma group. Among the 639 DEPs, 277 proteins returned to similar levels as those of the relative non-asthma condition. Bioinformatic analysis revealed that the 277 proteins played a significant role in the leukocyte extravasation signaling pathway. The leukocyte extravasation signaling pathway and related DEPs were of crucial importance in birch pollen SCIT.

Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF-α caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of leukocyte migration across the vessel wall, into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay.

2.5D Tractions in monocytes reveal mesoscale mechanics of podosomes during substrate indenting cell protrusion

Degradation and protrusion are key to cellular barrier breaching in cancer metastasis and leukocyte extravasation. Cancerous invadopodia and myelomonocytic podosomes are widely considered as structural tools facilitating these processes and are thus summarized under the term invadosomes. Despite similar behaviour on the individual scale, substantial differences have been reported to arise on the collective scale. They are considered to be a result of podosome mesoscale-connectivity. In this study, we investigated global in-plane and out-of-plane mechanical forces of podosome clusters in ER-Hoxb8 cell derived monocytes. We are able to correlate these forces with the interpodosomal connectivity. The observed traction and protrusion patterns fail to be explained by summation of single podosome mechanics. Instead, they appear to originate from superimposed mesoscale effects. Based on mechanistic and morphological similarities with epithelial monolayer mechanics, we propose a spatiotemporal model of podosome cluster mechanics capable of relating single to collective podosome mechanical behaviour. Our results suggest that network contraction-driven (in-plane) tractions lead to a buckling instability that contributes to the out-of-plane indentation into the substrate. First assigning an active mechanical role to the dorsal podosome actomyosin network, we aim at translating actomyosin hierarchy into scale dependency of podosome mechanics.

POS0402 IDENTIFYING MECHANISMS UNDERLYING THE ASSOCIATION BETWEEN RHEUMATOID ARTHRITIS AND PERIODONTITIS USING IN SILICO ANALYSIS OF CANONICAL PATHWAYS

Background:Periodontal pathogens such as Porphyromonas gingivalis (P.g.) has been proposed too involve in rheumatoid arthritis (RA) progression via citrullinating and producing exogenously citrullinated human and bacterial epitopes.Objectives:We identified pathways downstream to periodontitis onset that involved in RA progression with RNA-sequencing data.Methods:Canonical pathway analysis was conducted by comparing mRNA expression data from whole-transcriptome expression profiling using z-score and p-value visualization to identify underlying mechanisms among patients with RA (n=7) and periodontitis (n=9). Collected biopsies included human blood samples for RA and human gingival tissues for periodontitis. RNA-seq data with -log10(P) values larger than 1.3 were considered significant, and positive z-scores indicated up-regulation. The statistical software was Ingenuity Pathway Analysis (QIAGEN).Results:Among all significantly enriched (-log10(P) > 1.3) periodontitis-associated pathways underlying RA progression identified from the recruited cases, the production of nitric oxide and reactive oxygen species in macrophages, B cell receptor signaling, osteoarthritis pathway, HOTAIR regulatory pathway, IL-8 signaling, LPS/IL-1 mediated inhibition of RXR function, leukocyte extravasation signaling, and neuroinflammation signaling pathway, were up-regulated in RA patients and patients with periodontitis, when compared with patients without either disease. The z-scores of production of nitric oxide and reactive oxygen species in macrophages were 2.45 for RA (-log10(P) = 1.41), 3.89 for periodontitis (-log10(P) = 4.25); the z-scores of B cell receptor signaling were 2.44 for RA (-log10(P) = 1.93), 2.65 for periodontitis (-log10(P) = 6.97); the z-scores of osteoarthritis pathway were 1.89 for RA (-log10(P) = 3.28), 2.33 for periodontitis (-log10(P) = 4.31); the z-scores of HOTAIR regulatory pathway were 1.63 for RA (-log10(P) = 1.71), 3.40 for periodontitis (-log10(P) = 3.68); the z-scores of IL-8 signaling were 1.34 for RA (-log10(P) = 1.30), 4.43 for periodontitis (-log10(P) = 6.05); the z-scores of LPS/IL-1 mediated inhibition of RXR function were 1.33 for RA (-log10(P) = 2.00), 1.27 for periodontitis (-log10(P) = 3.44); and the z-scores of leukocyte extravasation signaling were 1.13 for RA (-log10(P) = 1.80), 3.68 for periodontitis (-log10(P) = 18.14).Conclusion:Our findings suggest that infection-driven activation of macrophages and B cells is involved in RA pathogenesis. This occurs primarily through upregulating cellular activities involving iNOS-presenting macrophages and B cell receptor signaling, which may be associated with the pathogenesis of P.g.-triggered RA.References:[1]Jenning M, Marklein B, Ytterberg J, et al. Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production. Ann Rheum Dis 2020;79:1194–202.doi:10.1136/annrheumdis-2019-216919Figure 1.Canonical pathway analysis on transcriptome of blood samples for patients with RA and gingival tissues for periodontitis.Disclosure of Interests:None declared

Prevention of Melanoma Extravasation as a New Treatment Option Exemplified by p38/MK2 Inhibition

Melanoma releases numerous tumor cells into the circulation; however, only a very small fraction of these cells is able to establish distant metastasis. Intravascular survival of circulating tumor cells is limited through hemodynamic forces and by the lack of matrix interactions. The extravasation step is, thus, of unique importance to establish metastasis. Similar to leukocyte extravasation, this process is under the control of adhesion molecule pairs expressed on melanoma and endothelial cells, and as for leukocytes, ligands need to be adequately presented on cell surfaces. Based on melanoma plasticity, there is considerable heterogeneity even within one tumor and one patient resulting in a mixture of invasive or proliferative cells. The molecular control for this switch is still ill-defined. Recently, the balance between two kinase pathways, p38 and JNK, has been shown to determine growth characteristics of melanoma. While an active JNK pathway induces a proliferative phenotype with reduced invasive features, an active p38/MK2 pathway results in an invasive phenotype and supports the extravasation step via the expression of molecules capable of binding to endothelial integrins. Therapeutic targeting of MK2 to prevent extravasation might reduce metastatic spread.