Migration Of Innate Immune Cells Research Articles

The anti-platelet drug cilostazol enhances heart rate and interrenal steroidogenesis and exerts a scant effect on innate immune responses in zebrafish.

Cilostazol, an anti-platelet phosphodiesterase-3 inhibitor used for the treatment of intermittent claudication, is known for its pleiotropic effects on platelets, endothelial cells and smooth muscle cells. However, how cilostazol impacts the endocrine system and the injury-induced inflammatory processes remains unclear. We used the zebrafish, a simple transparent model that demonstrates rapid development and a strong regenerative ability, to test whether cilostazol influences heart rate, steroidogenesis, and the temporal and dosage effects of cilostazol on innate immune cells during tissue damage and repair. While dosages of cilostazol from 10 to 100 μM did not induce any noticeable morphological abnormality in the embryonic and larval zebrafish, the heart rate was increased as measured by ImageJ TSA method. Moreover, adrenal/interrenal steroidogenesis in larval zebrafish, analyzed by whole-mount 3β-Hsd enzymatic activity and cortisol ELISA assays, was significantly enhanced. During embryonic fin amputation and regeneration, cilostazol treatments led to a subtle yet significant effect on reducing the aggregation of Mpx-expressing neutrophil at the lesion site, but did not affect the immediate injury-induced recruitment and retention of Mpeg1-expressing macrophages. Our results indicate that cilostazol has a significant effect on the heart rate and the growth as well as endocrine function of steroidogenic tissue; with a limited effect on the migration of innate immune cells during tissue damage and repair.

Functional and Therapeutic Relevance of Rho GTPases in Innate Immune Cell Migration and Function during Inflammation: An In Silico Perspective.

Systematic regulation of leukocyte migration to the site of infection is a vital step during immunological responses. Improper migration and localization of immune cells could be associated with disease pathology as seen in systemic inflammation. Rho GTPases act as molecular switches during inflammatory cell migration by cycling between Rho-GDP (inactive) to Rho-GTP (active) forms and play an essential role in the precise regulation of actin cytoskeletal dynamics as well as other immunological functions of leukocytes. Available reports suggest that the dysregulation of Rho GTPase signaling is associated with various inflammatory diseases ranging from mild to life-threatening conditions. Therefore, it is crucial to understand the step-by-step activation and inactivation of GTPases and the functioning of different Guanine Nucleotide Exchange Factors (GEFs) and GTPase-Activating Proteins (GAPs) that regulate the conversion of GDP to GTP and GTP to GDP exchange reactions, respectively. Here, we describe the molecular organization and activation of various domains of crucial elements associated with the activation of Rho GTPases using solved PDB structures. We will also present the latest evidence available on the relevance of Rho GTPases in the migration and function of innate immune cells during inflammation. This knowledge will help scientists design promising drug candidates against the Rho-GTPase-centric regulatory molecules regulating inflammatory cell migration.

Cell Migration Guided by Cell-Cell Contacts in Innate Immunity.

The directed migration of leukocytes to sites of damage or infection is necessary for a productive immune response. There is substantial evidence supporting a key role for chemoattractants in directed migration, however, less is known about how cell-cell contacts affect the migratory behavior of leukocytes in innate immunity. Here, we explore how cell-cell contacts can affect the directed migration of innate immune cells, including their role in attracting, repelling, or stopping cell motility. Further investigation of cell contact dynamics as guidance cues may yield new insights into the regulation of innate immunity.

N-acetylcysteine inhibits bacterial lipopeptide-mediated neutrophil transmigration through the choroid plexus in the developing brain

The etiology of neurological impairments associated with prematurity and other perinatal complications often involves an infectious or pro-inflammatory component. The use of antioxidant molecules have proved useful to protect the neonatal brain from injury. The choroid plexuses-CSF system shapes the central nervous system response to inflammation at the adult stage, but little is known on the neuroimmune interactions that take place at the choroidal blood-CSF barrier during development. We previously described that peripheral administration to neonatal mice of the TLR2 ligand PAM3CSK4 (P3C), a prototypic Gram-positive bacterial lipopeptide, induces the migration of innate immune cells to the CSF. Here we showed in neonatal rats exposed to P3C that the migration of neutrophils into the CSF, which occurred through the choroid plexuses, is abolished following administration of the antioxidant drug N-acetylcysteine. Combining light sheet microscopy imaging of choroid plexus, a differentiated model of the blood-CSF barrier, and multiplex cytokine assays, we showed that the choroidal epithelium responds to the bacterial insult by a specific pattern of cytokine secretion, leading to a selective accumulation of neutrophils in the choroid plexus and to their trafficking into CSF. N-acetylcysteine acted by blocking neutrophil migration across both the endothelium of choroidal stromal vessels and the epithelium forming the blood-CSF barrier, without interfering with neutrophil blood count, neutrophil tropism for choroid plexus, and choroidal chemokine-driven chemotaxis. N-acetylcysteine reduced the injury induced by hypoxia-ischemia in P3C-sensitized neonatal rats. Overall, the data show that a double endothelial and epithelial check point controls the transchoroidal migration of neutrophils into the developing brain. They also point to the efficacy of N-acetylcysteine in reducing the deleterious effects of inflammation-associated perinatal injuries by a previously undescribed mechanism, i.e. the inhibition of innate immune cell migration across the choroid plexuses, without interfering with the systemic inflammatory response to infection.

Learning from Mother Nature: Innovative Tools to Boost Endogenous Repair of Critical or Difficult-to-Heal Large Tissue Defects.

For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to a broad application of cell therapy and tissue engineering approaches, i.e., transplantation of “ex vivo” expanded autologous stem/progenitor cells, alone or associated with carrier biomaterials. To enable a large number of patients to benefit, new strategies should be considered. One of the main goals of contemporary regenerative medicine is to develop new regenerative therapies, inspired from Mother Nature. In all injured tissues, when platelets are activated by tissue contact, their released factors promote innate immune cell migration to the wound site. Platelet-derived factors and factors secreted by migrating immune cells create an inflammatory microenvironment, in turn, causing the activation of angiogenesis and vasculogenesis processes. Eventually, repair or regeneration of the injured tissue occurs via paracrine signals activating, mobilizing or recruiting to the wound site cells with healing potential, such as stem cells, progenitors, or undifferentiated cells derived from the reprogramming of tissue differentiated cells. This review, largely based on our studies, discusses the identification of new tools, inspired by cellular and molecular mechanisms overseeing physiological tissue healing, that could reactivate dormant endogenous regeneration mechanisms lost during evolution and ontogenesis.

Modulation of infection-mediated migration of neutrophils and CXCR2 trafficking by osteopontin.

Osteopontin (OPN) is a pro-inflammatory protein that paradoxically protects against inflammation and bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. Using the air pouch as a model of endodontic infection in mice, we showed that neutrophil accumulation at the site of infection with a mixture of endodontic pathogens is significantly reduced in OPN-deficient mice. Reduced neutrophil accumulation in the absence of OPN was accompanied by an increase in bacterial load. OPN-deficiency did not affect neutrophil survival, CXCR2 ligand expression, or the production of inflammatory cytokines in the air pouch. In vitro, OPN enhanced neutrophil migration to CXCL1, whereas in vivo, inhibition of CXCR2 suppressed cellular infiltration in air pouches of infected wild-type mice by > 50%, but had no effect in OPN-deficient mice. OPN increased cell surface expression of CXCR2 on bone marrow neutrophils in an integrin-αv -dependent manner, and suppressed the internalization of CXCR2 in the absence of ligand. Together, these results support a model where the protective effect of OPN results from enhanced initial neutrophil accumulation at sites of infection resulting in optimal bacterial killing. We describe a novel mechanism for this effect of OPN: integrin-αv -dependent suppression of CXCR2 internalization in neutrophils, which increases the ability of these cells to migrate to sites of infection in response to CXCR2 ligands.

Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation.

BackgroundExosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)].MethodsNasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC–MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes.ResultsFirstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression.ConclusionsNasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0927-4) contains supplementary material, which is available to authorized users.

Osteopontin regulates Cxcr2 expression and neutrophil recruitment in endodontic infection (INC6P.302)

Abstract The integrin binding protein osteopontin (OPN) protects against bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. We used the subcutaneous chamber and air pouch as models of endodontic infection in mice. In vitro analysis of CXCR2 expression was determined by FACS and migration was assessed using transwell assay. Using both the chamber and a modified air-pouch infection model, we showed that neutrophil accumulation at the site of infection is significantly reduced in OPN KO mice, but there are very few macrophages and monocytes, and there is little effect of OPN on their accumulation. OPN does not affect neutrophil survival. Reduced neutrophil accumulation is accompanied by lower levels of myeloperoxidase activity and an increase in bacterial load in the absence of osteopontin. In vitro, OPN increases the ability of these cells to migrate in transwell assays when added in either to the upper or the lower chambers. Furthermore, OPN increases expression of CXCR2 on bone marrow neutrophils, in an alphav-integrin dependent manner. Biotinylation assays show that OPN suppresses the internalization of CXCR2. Together, these results support a model whereby OPN binding to the alphav integrin regulates CXCR2 internalization on neutrophils, increasing the ability of these cells to migrate in response to CXCR2 ligands.

Increased neutralization capacity of TNF alpha in sera of MS patients may play a role in — MS pathogenesis and is reversed by therapy with interferon-γ

in vivo analysis. Lymphocyte migration in vitro was evaluated in transwell assays using CD4 T cells purified from anti-muCD52-treated mice. In vivo migration was assessed using an acute CNS inflammation model in which mice were injected intracerebroventricularly with lipopolysaccharide (LPS) following anti-muCD52 treatment. Similarly, migration of innate immune cell subsets was evaluated in a peritonitis model of inflammation, in which animals were injected intraperitoneally with thioglycollate 3 days following anti-muCD52 treatment. The numbers of CNS-infiltrating lymphocytes or peritoneum-infiltrating cells were measured by polychromatic flow cytometry. Results: Immune cells from anti-muCD52-treated mice retained their ability to migrate. In vitro, CD4 T cells were able to migrate in response to SDF-1alpha as efficiently as cells from vehicle-treated animals. In vivo, lymphocytes in anti-muCD52-treated animals were able to migrate into the CNS following local induction of inflammation by LPS. Similarly, innate immune cell migration was not affected by anti-muCD52 treatment, as similar numbers and types of cells were observed in the inflamed peritoneum of anti-muCD52and vehicle-treated animals. Conclusions: These results indicate that following anti-CD52 treatment, immune cells retain their ability to migrate. These findings suggest that anti-CD52 treatment may not compromise immune surveillance, but further studies are required. Study Supported by: Genzyme, a Sanofi company.

TLR4 Signaling Augments Monocyte Chemotaxis by Regulating G Protein–Coupled Receptor Kinase 2 Translocation

Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein-coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphorylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS-TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1-induced monocyte migration. We also show that LPS, through p38 MAPK signaling, induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.

In vitro effect of anti-human immunodeficiency virus CCR5 antagonist maraviroc on chemotactic activity of monocytes, macrophages and dendritic cells

Compounds targeting the chemokine receptor CCR5 have recently been approved for treatment of human immunodeficiency virus (HIV) infection. Given the central role of CCR5 in inflammation and recruitment of antigen-presenting cells (APC), it is important to investigate the immunological consequences of pharmacological inhibition of CCR5. We evaluated the in vitro effect of different concentrations of CCR5 antagonist maraviroc (MVC) on cell migration of monocytes, macrophages (MO) and monocyte-derived dendritic cells (MDC) towards peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and chemokines regulated upon activation normal T cell expressed and secreted (RANTES) and CCL4/macrophage inflammatory protein-1 (MIP-1β) and CCL2/monocyte chemotactic protein-1 (MCP-1). Results of flow cytometric analysis showed that monocytes treated in vitro with MVC exhibited a significant dose-dependent reduction of chemotaxis towards MIP-1β and MCP-1. fMLP-induced chemotactic activity decreased only at higher concentration (1 µM and 10 µM of MVC). In addition, all concentrations of MVC (0·1, 1 and 10 µM) induced in vitro a significant inhibition of chemotaxis of MO and MDC in response to all tested chemoattractants. No change in phenotype (CD1a and CD14) and CCR1, CCR4, CCR5 and formyl peptide receptor (FPR) expression was seen after in vitro treatment with MVC. These findings suggest that CCR5 antagonist MVC may have the in vitro ability of inhibiting the migration of innate immune cells by mechanism which could be independent from the pure anti-HIV effect. The drug might have a potential role in the down-regulation of HIV-associated chronic inflammation by blocking the recirculation and trafficking of MO and MDC.

Formyl Peptide Receptor Ligands Promote Wound Closure in Lung Epithelial Cells

Mitochondrial antigens released from damaged cells act as "danger signals" capable of promoting innate immune cell migration and activation via formyl peptide receptors (FPRs). Lung epithelial cells are equipped to migrate and mount innate immune responses in the context of acute lung injury. The goal of this study was to determine whether lung epithelial cells express FPRs, which are capable of responding to mitochondrial antigens to promote wound closure and inflammation. Using human Beas2B lung epithelial cells grown to confluency and subjected to linear scratch injury, it was found that mitochondrial antigens enhanced epithelial wound closure, and this phenomenon was inhibited by cyclosporin H, a selective inhibitor of FPR. Although mitochondrial antigens also promoted IL-8 release, this release was not FPR dependent and was unrelated to FPR-induced lung epithelial cell wound closure. The expression of functional FPR was confirmed in Beas2B and primary human tracheobronchial epithelial cells, particularly in lamellipodia at the leading edge of the closing wound. The expression of FPR was increased in response to TNF-α, LPS, scratch injury, and mitochondrial antigen treatment. Considered together, these data confirm that human lung epithelial cells express functional FPRs, which are capable of responding to endogenous mitochondrial danger signals, to promote wound closure.

Superantigen-Activated Regulatory T Cells Inhibit the Migration of Innate Immune Cells and the Differentiation of Naive T Cells

Regulatory T cells can be used as tools to suppress pathogenic T cells in autoimmunity, graft-vs-host-disease, and transplantation. But even when high numbers of Ag-specific regulatory T cells are available, it is still possible under certain in vivo and in vitro conditions for effector T cells to escape effective control. Current reports suggest that the degree of suppression is modulated by the inflammatory milieu, which can induce resistance to suppression in effector T cells or subvert the inhibitory function of the regulatory T cells. Cells of the innate immune system integrate early signals of injury and infection and have a major impact on the ensuing inflammation. Hence, the modification of these initial events can be key to allowing suppression to dominate. The approach we took here was to test whether the in vivo preactivation of endogenous regulatory T cells with a superantigen could enhance their suppressive potency. We provide evidence that this not only proved effective in expanding the pool of preactivated regulatory T cells but also in preventing the migration of NK cells and granulocytes upon sensitization with matured dendritic cells. The attenuation of innate immune activation was accompanied by linked suppression of adoptively transferred OVA-specific T cells when APC coexpressing OVA and the superantigen were injected. These data suggest that the preactivation of regulatory T cells is a promising approach to increase their potency.

In Vivo Interstitial Migration of Primitive Macrophages Mediated by JNK-Matrix Metalloproteinase 13 Signaling in Response to Acute Injury

Interstitial cell migration through extracellular matrix is a hallmark of the inflammation response, tumor invasion, and metastasis. We have established a stable zebrafish transgenic line expressing enhanced GFP under the lysozyme C promoter for visualizing and measuring primitive macrophage migration in vivo. We show that tissue-resident primitive macrophages migrate rapidly through extracellular matrix to the site of acute injury induced by tail transection. Mechanistically, the specific inhibition of JNK, but not p38 and ERK, dramatically abolished the chemotactic migration in a dose-dependent manner, suppressing the trauma-induced recruitment of phosphorylated C-Jun transcription factor to proximal AP-1 sites in the promoter of matrix metalloproteinase 13 (mmp13), a gene specifically expressed in primitive macrophages during embryogenesis and required for the interstitial migration. Furthermore, dexamethasone suppressed the trauma-induced JNK phosphorylation and macrophage migration accompanied by simultaneous up-regulation of mkp-1, a well-known phosphatase capable of inactivating phosphorylated JNK. The results indicate that the JNK-Mmp13 signaling pathway plays an essential role in regulating the innate immune cell migration in response to severe injury in vivo.

A Small Molecule α4β1 Antagonist Prevents Development of Murine Lyme Arthritis without Affecting Protective Immunity

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of alpha4beta1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of alpha4beta(1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the alpha4beta1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the alpha4beta1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.