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Helicobacter pylori (H. pylori) is a bacterial pathogen that exclusively colonizes the human gastric mucosa and can cause persistent infection. In this process, H. pylori employs various strategies to avoid recognition by the human immune system. These range from passive defense strategies (e.g., altered LPS or flagellin structures) that prevent recognition by pattern recognition receptors (PRRs) to more active approaches, such as inhibition of IL-2 secretion and proliferation of T cells via VacA. Despite the growing evidence that H. pylori actively manipulates the human immune system for its own benefit, the direct interaction of H. pylori with immune cells in situ is poorly studied. Here, we present a novel intravital imaging model of the murine stomach gastric mucosa and show for the first time the in situ recruitment of neutrophils during infection and a direct H. pylori-macrophage interaction. For this purpose, we applied multiphoton intravital microscopy adapted with live drift correction software (VivoFollow) on LysM-eGFP and CX3CR1-eGFP reporter mice strains in which specific subsets of leukocytes are fluorescently labeled. Multiphoton microscopy is proving to be an excellent tool for characterizing interactions between immune cells and pathogens in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ishikawa-Ankerhold et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neutrophils, the most abundant white blood cells in the human circulation, play crucial roles in various diseases, including kidney disease. Traditionally viewed as short-lived pro-inflammatory phagocytes that release reactive oxygen species, cytokines and neutrophil extracellular traps, recent studies have revealed their complexity and heterogeneity, thereby challenging this perception. Neutrophils are now recognized as transcriptionally active cells capable of proliferation and reverse migration, displaying phenotypic and functional heterogeneity. They respond to a wide range of signals and deploy various cargo to influence the activity of other cells in the circulation and in tissues. They can regulate the behavior of multiple immune cell types, exhibit innate immune memory, and contribute to both acute and chronic inflammatory responses while also promoting inflammation resolution in a context-dependent manner. Here, we explore the origin and heterogeneity of neutrophils, their functional diversity, and the cues that regulate their effector functions. We also examine their emerging role in infectious and non-infectious diseases with a particular emphasis on kidney disease. Understanding the complex behavior of neutrophils during tissue injury and inflammation may provide novel insights, thereby paving the way for potential therapeutic strategies to manage acute and chronic conditions. By deciphering their multifaceted role, targeted interventions can be developed to address the intricacies of neutrophil-mediated immune responses and improve disease outcomes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression., Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling., Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells., Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression., (© 2023. The Author(s).)

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Signaling via β 2 integrins (CD11/CD18) as well as TCRs and BCRs involves similar pathways. However, the activation of the same signaling molecule can result in opposing effects. One such example is the hematopoietic progenitor kinase 1 (HPK1), which negatively regulates T and B cell activation but enforces neutrophil adhesion via β 2 integrins. This difference may be defined by specific HPK1 interacting networks in different leukocyte subsets which have already been described in the adaptive immune system. Here, we set out to identify interacting proteins of HPK1 in neutrophil-like differentiated HL-60 cells exposed to immobilized fibrinogen and left nonactivated or Mn 2+ -activated to allow β 2 integrin-dependent adhesion. Co-IP experiments followed by mass spectrometry led to the identification of 115 HPK1-interacting proteins. A total of 58 proteins were found only in nonactivated cells and 39 proteins only in Mn 2+ -activated adherent cells. From these results, we decoded a pre-existing signaling cluster of HPK1 in nonactivated cells encompassing proteins essential for β 2 integrin-mediated signaling during neutrophil trafficking, namely DNAX-activation protein 12 (DAP12), spleen tyrosine kinase (Syk), and Rac1. Thus, our study provides novel insights into the complex architecture of the signaling processes during neutrophil activation and the complex signaling profile of HPK1 in leukocytes., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The lumen of blood vessels is covered by endothelial cells, which regulate their permeability to ions and solutes. Endothelial permeability depends on the vascular bed and cell phenotype, and is influenced by different disease states. Most characterization of endothelial permeability has been carried out using isolated cells in culture. While analysis of cultured cells is a valuable approach, it does not account for factors of the native cell environment. Building on Ussing chamber studies of intact tissue specimens, here we describe a method to measure the electrophysiological properties of intact arteriole and venule endothelia, including transendothelial electrical resistance (TEER) and ion permselectivity. As an example, vessels isolated from the mesentery were treated ex vivo , then mounted in a custom-made tissue cassette that enable their analysis by classical approaches with an Ussing chamber. This method enables a detailed analysis of electrophysiological vessel responses to stresses such as proinflammatory cytokines, in the context of an intact vessel. Graphic abstract., Competing Interests: Competing interestsThe authors declare that we have no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α 4 β 1 and α 4 β 7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β 2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β 2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae ( Danio rerio ) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo , the functional impact of β 2  integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β 2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2 , the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bader, Gao, Rivière, Schmid, Walzog and Maier-Begandt.)

The endothelial cell barrier regulates the passage of fluid between the bloodstream and underlying tissues, and barrier function impairment exacerbates the severity of inflammatory insults. To understand how inflammation alters vessel permeability, we studied the effects of the proinflammatory cytokine TNFα on transendothelial permeability and electrophysiology in ex vivo murine veins and arteries. We found that TNFα specifically decreased the barrier function of venous endothelium without affecting that of arterial endothelium. On the basis of RNA expression profiling and protein analysis, we found that claudin-11 (CLDN11) was the predominant claudin in venous endothelial cells and that there was little, if any, CLDN11 in arterial endothelial cells. Consistent with a difference in claudin composition, TNFα increased the permselectivity of Cl - over Na + in venous but not arterial endothelium. The vein-specific effects of TNFα also required the activation of Pannexin 1 (Panx1) channels and the CD39-mediated hydrolysis of ATP to adenosine, which subsequently stimulated A 2A adenosine receptors. Moreover, the increase in vein permeability required the activation of the Ca 2+ channel TRPV4 downstream of Panx1 activation. Panx1-deficient mice resisted the pathologic effects of sepsis induced by cecal ligation and puncture on life span and lung vascular permeability. These data provide a targetable pathway with the potential to promote vein barrier function and prevent the deleterious effects of vascular leak in response to inflammation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Actin-dependent leukocyte trafficking and activation are critical for immune surveillance under steady state conditions and during disease states. Proper immune surveillance is of utmost importance in mammalian homeostasis and it ensures the defense against pathogen intruders, but it also guarantees tissue integrity through the continuous removal of dying cells or the elimination of tumor cells. On the cellular level, these processes depend on the precise reorganization of the actin cytoskeleton orchestrating, e.g., cell polarization, migration, and vesicular dynamics in leukocytes. The fine-tuning of the actin cytoskeleton is achieved by a multiplicity of actin-binding proteins inducing, e.g., the organization of the actin cytoskeleton or linking the cytoskeleton to membranes and their receptors. More than a decade ago, the family of leucine-rich repeat (LRR) and calponin homology (CH) domain-containing (LRCH) proteins has been identified as cytoskeletal regulators. The LRR domains are important for protein-protein interactions and the CH domains mediate actin binding. LRR and CH domains are frequently found in many proteins, but strikingly the simultaneous expression of both domains in one protein only occurs in the LRCH protein family. To date, one LRCH protein has been described in drosophila and four LRCH proteins have been identified in the murine and the human system. The function of LRCH proteins is still under investigation. Recently, LRCH proteins have emerged as novel players in leukocyte function. In this review, we summarize our current understanding of LRCH proteins with a special emphasis on their function in leukocyte biology., (Copyright © 2020 Rivière, Bader, Pogoda, Walzog and Maier-Begandt.)

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., (Copyright © 2020 Werner, Weckbach, Salvermoser, Pitter, Cao, Maier-Begandt, Forné, Schnittler, Walzog and Montanez.)