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 β2 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.

Dasatinib is a tyrosine kinase inhibitor used to treat imatinib-resistant chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. At present, little is known about how dasatinib influences nonmalignant cells. In the present study, we tested the effect of dasatinib on functional responses of normal mature human neutrophils. Dasatinib completely blocked integrin- and Fc-receptor-mediated neutrophil functions, with the lowest IC(50) values below 10nM under serum-free conditions. Dasatinib caused a partial inhibition of neutrophil responses triggered by G-protein-coupled receptors and had a moderate effect on neutrophil responses triggered by microbial compounds. Whereas dasatinib inhibited neutrophil chemotaxis under static conditions in 2 dimensions, it did not affect migration under flow conditions or in 3-dimensional environments. Dasatinib did not have any major effect on phagocytosis or killing of bacteria by neutrophils. Adhesion of human neutrophils in the presence of whole serum was significantly inhibited by 50-100nM dasatinib, which corresponds to the reported serum concentrations in dasatinib-treated patients. Finally, ex vivo adhesion of mouse peripheral blood neutrophils was strongly reduced after oral administration of 5 mg/kg of dasatinib. Those results suggest that dasatinib treatment may affect the proinflammatory functions of mature neutrophils and raise the possibility that dasatinib-related compounds may provide clinical benefit in neutrophil-mediated inflammatory diseases.

The p53-inducible and death domain-containing PIDD/LRDD protein has been described as an adaptor protein, which forms large protein complexes with RAIDD, another death domain-containing protein, leading to recruitment, and activation of the initiator caspase-2, and p53-mediated apoptosis. Here, we describe in further detail the proteolytic processing of PIDD/LRDD that occurs in healthy cells before induction of apoptosis. We could demonstrate that the C-terminal fragment containing the PIDD death domain shuttles into the nucleoli. This translocation is mediated by or leads to the interaction of the PIDD death domain with nucleolin, a protein important for rRNA processing within nucleoli and possibly involved in the DNA damage response. Ectopically expressed LRDD and endogenous nucleolin co-localized within the nucleoli, and overexpression of both full-length LRDD and the LRDD death domain sensitized cells for UV-induced apoptosis. When expressed alone, the PIDD/LRDD death domain tended to form large filamentous structures resembling so-called death filaments. The functional consequences of the identified PIDD/nucleolin interaction remain to be elucidated, but may be related to a recently discovered new role for PIDD in the activation of NF-kappaB upon genotoxic stress.

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms - a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition - apart from its radiosensitizing potential - obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.

The colonic mucosal barrier protects against infection, inflammation, and tissue ulceration. Composed primarily of Mucin-2, proteolytic erosion of this barrier is an invariant feature of colitis; however, the molecular mechanisms are not well understood. We have applied a recurrent food poisoning model of acquired inflammatory bowel disease using Salmonella enterica Typhimurium to investigate mucosal barrier erosion. Our findings reveal an innate Toll-like receptor 4-dependent mechanism activated by previous infection that induces Neu3 neuraminidase among colonic epithelial cells concurrent with increased Cathepsin-G protease secretion by Paneth cells. These anatomically separated host responses merge with the desialylation of nascent colonic Mucin-2 by Neu3 rendering the mucosal barrier susceptible to increased proteolytic breakdown by Cathepsin-G. Depletion of Cathepsin-G or Neu3 function using pharmacological inhibitors or genetic-null alleles protected against Mucin-2 proteolysis and barrier erosion and reduced the frequency and severity of colitis, revealing approaches to preserve and potentially restore the mucosal barrier.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ 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 in vitro and in vivo. 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 in vivo. 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.

The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.

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.

Although an inflammatory microenvironment is required for successful implantation, an inflammatory overreaction is one of the causes of unexplained recurrent pregnancy losses (uRPL). Prostaglandin E2 (PGE2) plays a pivotal role in regulating immune balance during early pregnancy, and it can stimulate inflammatory reactions via prostaglandin E2 receptor 3 (EP3). However, the role of PGE2 receptor signaling in the uRPL remains unknown. We aimed to investigate whether EP3 signaling is involved in the mechanism of uRPL. Via immunohistochemistry we could show that the expression of cyclooxygenase-2, EP3 and G protein alpha inhibitor 1 (Gi1) was enhanced in the decidua of the uRPL group in comparison to the control group in first-trimester placentas. In vitro, we demonstrated that sulprostone (an EP1/EP3 agonist) inhibited the secretion of beta-hCG and progesterone in JEG-3 cells and the secretion of beta-hCG in HTR-8/SVneo cells while it induced the expression of plasminogen activator inhibitor type 1 in JEG-3 cells. In addition, PGE2/sulprostone was able to stimulate the expression of Gi1, phosphorylated-extracellular signal-regulated kinases 1/2 (p-ERK1/2) and p53. L-798,106 (an EP3-specific antagonist) suppressed the expression of EP3 and p-ERK1/2 without affecting the secretion of beta-hCG. Elevated activation of EP3 signaling in first-trimester placentas plays an important role in regulating the inflammatory microenvironment, the hormone secretion of extravillous trophoblasts and the remodeling of extracellular matrix in the fetal-maternal interface. L-798,106 might be a 'potential therapeutic candidate' for the treatment of uRPL.

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate β2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P3) and Gαq, PLCβ and Ca(2+). Intra-arterial S1P administration increases leukocyte rolling, while S1P3 deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P3. Histamine and epinephrine require S1P3 for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P1 inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P1(-/-) mice. In agreement with a dominant pro-rolling effect of S1P3, FTY720 inhibits rolling in control and S1P1(-/-) but not in S1P3(-/-) mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.

Rapid β2-integrin activation is indispensable for leukocyte adhesion and recruitment to sites of infection and is mediated by chemokine- or P-selectin glycoprotein ligand-1-induced inside-out signaling. Here we uncovered a novel pathway for rapid activation of integrin-dependent leukocyte adhesion, triggered by toll-like receptor (TLR)-mediated signaling. TLR2 or TLR5 ligation rapidly activated integrin-dependent leukocyte adhesion to immobilized ICAM-1 and fibronectin. Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model. TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes. TLR2- and TLR5-triggered integrin activation in leukocytes required enhanced Rap1 GTPase activity, which was mediated by Rac1 activation and NADPH oxidase-2-dependent reactive oxygen species production. This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.