Novel Evidence That Alternative Pathway of Complement Cascade Activation is Required for Optimal Homing and Engraftment of Hematopoietic Stem/progenitor Cells

We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, trans-endothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs. Graphical Abstract

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Pathway Association Studies Reveal Gene Loci and Pathway Networks that Associated With Plasma Cystatin C Levels

As a marker for glomerular filtration, plasma cystatin C level is used to evaluate kidney function. To decipher genetic factors that control the plasma cystatin C level, we performed genome-wide association and pathway association studies using United Kingdom Biobank data. One hundred fifteen loci yielded p values less than 1 × 10−100, three genes (clusters) showed the most significant associations, including the CST8-CST9 cluster on chromosome 20, the SH2B3-ATXN2 gene region on chromosome 12, and the SHROOM3-CCDC158 gene region on chromosome 4. In pathway association studies, forty significant pathways had FDR (false discovery rate) and or FWER (family-wise error rate) ≤ 0.001: spermatogenesis, leukocyte trans-endothelial migration, cell adhesion, glycoprotein, membrane lipid, steroid metabolic process, and insulin signaling pathways were among the most significant pathways that associated with the plasma cystatin C levels. We also performed Genome-wide association studies for eGFR, top associated genes were largely overlapped with those for cystatin C.

Zyxin Mediates Vascular Repair via Endothelial Migration Promoted by Forskolin in Mice

Background and Purpose: Endothelial repair upon vascular injury is critical for the protection of vessel integrity and prevention of the development of vascular disorders, but the underlying mechanisms remain poorly understood. In this study, we investigated the role of zyxin and its associated cyclic adenosine monophosphate (cAMP) signaling in the regulation of re-endothelialization after vascular injury.Experimental Approach: In zyxin-/- and wild-type mice, wire injury of the carotid artery was carried out, followed by Evans blue staining, to evaluate the re-endothelialization. Mice with endothelium-specific zyxin knockout were used to further determine its role. An in vitro wound-healing assay was performed in primary human endothelial cells (ECs) expressing zyxin-specific short-hairpin RNAs (shRNAs) or scrambled controls by measuring cell migration and proliferation. The effects of the cAMP signaling agonist forskolin were assessed.Key Results: The re-endothelialization of the injured carotid artery was impaired in zyxin-deficient mice, whereas the rate of cell proliferation was comparable with that in wild-type controls. Furthermore, endothelium-specific deletion of zyxin led to similar phenotypes. Knockdown of zyxin by shRNAs in primary human ECs significantly reduced cell migration in the wound-healing assay. Notably, forskolin enhanced endothelial migration in a dose-dependent manner, and this was dependent on zyxin through its interaction with vasodilator-stimulated phosphoprotein. In addition, forskolin promoted the re-endothelialization of the injured carotid artery, and this was compromised by zyxin deficiency.Conclusion and Implications: This study reveals zyxin as a new player in endothelial repair, which is promoted by forskolin, after vascular injury. Thus, zyxin-mediated signaling might be a potential treatment target for diseases involving vascular injury.

Autophagy Induced by Palmitic Acid Regulates Neutrophil Adhesion Through the Granule-Dependent Degradation of αMβ2 Integrin in Dairy Cows With Fatty Liver

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized–ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Structure of the integrin receptor M2 headpiece in complex with a function-modulating nanobody

The integrin receptor M2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human M2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 Å. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H M variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 Å. The nanobody binds to the I domain of the M subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the M subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound M2 with iC3b suggesting that it represents a novel high-affinity proteinaceous M2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to M2. Furthermore, our data suggest that the iC3b-M2 complex may be more dynamic than predicted from the crystal structure of the core complex.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Maternal Nematode Infection Upregulates Expression of Th2/Treg and Diapedesis Related Genes In The Neonatal Brain

Intestinal nematode infections common during pregnancy have recently been shown to have impacts that extend to their uninfected offspring including altered brain gene expression. If maternal immune signals reach the neonatal brain, they might alter neuroimmune development. We explored expression of genes associated with four distinct types of T cells (Th1, Th2, Th17, Treg) and with leukocyte trans-endothelial migration and endocytosis transport across the blood-brain barrier (BBB) in the postnatal brain of offspring of nematode-infected mice, through secondary analysis of a whole brain gene expression database. Th1/Th17 expression was lowered by maternal infection as evidenced by down-regulated expression of IL-1β, Th1 receptors and related proteins, and of IL22 and several Th17 genes associated immunopathology. In contrast, Th2/Treg related pathways were upregulated as shown by higher expression of IL-4 and TGF-β family genes. Maternal infection also upregulated expression of pathways and integrin genes involved in transport of leukocytes in between endothelial cells but downregulated endosome vesicle formation related genes that are necessary for endocytosis of immunoglobulins across the BBB. Taken together, pup brain gene expression indicates that maternal nematode infection enhanced movement of leukocytes across the neonatal BBB and promoted a Th2/Treg environment that presumably minimizes the proinflammatory Th1 response in the pup brain.