The role of integrins in brain health and neurodegenerative diseases.

Integrins are heterodimeric membrane proteins expressed on the surface of most cells. They mediate adhesion and signaling processes relevant for a wealth of physiological processes, including nervous system development and function. Interestingly, integrins are also recognized therapeutic targets for inflammatory diseases, such as multiple sclerosis. Here, we discuss the role of integrins in brain development and function, as well as in neurodegenerative diseases affecting the brain (Alzheimer's disease, multiple sclerosis, stroke). Furthermore, we discuss therapeutic targeting of these adhesion receptors in inflammatory diseases of the brain.

Loss of ß2-integrin function results in metabolic reprogramming of dendritic cells, leading to increased dendritic cell functionality and anti-tumor responses.

Dendritic cells (DCs) are the main antigen presenting cells of the immune system and are essential for anti-tumor responses. DC-based immunotherapies are used in cancer treatment, but their functionality is not optimized and their clinical efficacy is currently limited. Approaches to improve DC functionality in anti-tumor immunity are therefore required. We have previously shown that the loss of ß2-integrin-mediated adhesion leads to epigenetic reprogramming of bone marrow-derived DCs (BM-DCs), resulting in an increased expression of costimulatory markers (CD86, CD80, and CD40), cytokines (IL-12) and the chemokine receptor CCR7. We now show that the loss of ß2-integrin-mediated adhesion of BM-DCs also leads to a generally suppressed metabolic profile, with reduced metabolic rate, decreased ROS production, and lowered glucose uptake in cells. The mRNA levels of glycolytic enzymes and glucose transporters were reduced, indicating transcriptional regulation of the metabolic phenotype. Surprisingly, although signaling through a central regulator of immune cell metabolisms, the mechanistic target of rapamycin (mTOR), was increased in BM-DCs with dysfunctional integrins, rapamycin treatment revealed that mTOR signaling was not involved in suppressing DC metabolism. Instead, bioinformatics and functional analyses showed that the Ikaros transcription factor may be involved in regulating the metabolic profile of non-adhesive DCs. Inversely, we found that induction of metabolic stress through treatment of cells with low levels of an inhibitor of glycolysis, 2-deoxyglucose (2DG), led to increased BM-DC activation. Specifically, 2DG treatment led to increased levels of Il-12 and Ccr7 mRNA, increased production of IL-12, increased levels of cell surface CCR7 and increased in vitro migration and T cell activation potential. Furthermore, 2DG treatment led to increased histone methylation in cells (H3K4me3, H3K27me3), indicating metabolic reprogramming. Finally, metabolic stress induced by 2DG treatment led to improved BM-DC-mediated anti-tumor responses in vivo in a melanoma cancer model, B16-OVA. In conclusion, our results indicate a role for ß2-integrin-mediated adhesion in regulating a novel type of metabolic reprogramming of DCs and DC-mediated anti-tumor responses, which may be targeted to enhance DC-mediated anti-tumor responses in cancer immunotherapy.

ß2-Integrins Regulate Microglial Responses and the Functional Outcome of Hemorrhagic Stroke In Vivo.

Stroke is one of the leading causes of death and long-term disabilities worldwide. In addition to interruption of blood flow, inflammation is widely recognized as an important factor mediating tissue destruction in stroke. Depending on their phenotype, microglia, the main leukocytes in the CNS, are capable of either causing further tissue damage or promoting brain restoration after stroke. ß2-integrins are cell adhesion molecules that are constitutively expressed on microglia. The function of ß2-integrins has been investigated extensively in animal models of ischemic stroke, but their role in hemorrhagic stroke is currently poorly understood. We show in this study that dysfunction of ß2-integrins is associated with improved functional outcome and decreased inflammatory cytokine expression in the brain in a mouse model of hemorrhagic stroke. Furthermore, ß2-integrins affect microglial phenotype and cytokine responses in vivo. Therefore, our findings suggest that targeting ß2-integrins in hemorrhagic stroke may be beneficial.

ß2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional ß2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional ß2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, ß2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting ß2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

Molecular Mechanisms of Leukocyte Migration and Its Potential Targeting-Lessons Learned From MKL1/SRF-Related Primary Immunodeficiency Diseases.

Megakaryoblastic leukemia 1 (MKL1) deficiency is one of the most recently discovered primary immunodeficiencies (PIDs) caused by cytoskeletal abnormalities. These immunological "actinopathies" primarily affect hematopoietic cells, resulting in defects in both the innate immune system (phagocyte defects) and adaptive immune system (T-cell and B-cell defects). MKL1 is a transcriptional coactivator that operates together with serum response factor (SRF) to regulate gene transcription. The MKL/SRF pathway has been originally described to have important functions in actin regulation in cells. Recent results indicate that MKL1 also has very important roles in immune cells, and that MKL1 deficiency results in an immunodeficiency affecting the migration and function of primarily myeloid cells such as neutrophils. Interestingly, several actinopathies are caused by mutations in genes which are recognized MKL(1/2)-dependent SRF-target genes, namely ACTB, WIPF1, WDR1, and MSN. Here we summarize these and related (ARPC1B) actinopathies and their effects on immune cell function, especially focusing on their effects on leukocyte adhesion and migration. Furthermore, we summarize recent therapeutic efforts targeting the MKL/SRF pathway in disease.

Examining the Effect of Kindlin-3 Binding Site Mutation on LFA-1-ICAM-1 Bonds by Force Measuring Optical Tweezers.

Integrins in effector T cells are crucial for cell adhesion and play a central role in cell-mediated immunity. Leukocyte adhesion deficiency (LAD) type III, a genetic condition that can cause death in early childhood, highlights the importance of integrin/kindlin interactions for immune system function. A TTT/AAA mutation in the cytoplasmic domain of the ß2 integrin significantly reduces kindlin-3 binding to the ß2 tail, abolishes leukocyte adhesion to intercellular adhesion molecule 1 (ICAM-1), and decreases T cell trafficking in vivo. However, how kindlin-3 affects integrin function in T cells remains incompletely understood. We present an examination of LFA-1/ICAM-1 bonds in both wild-type effector T cells and those with a kindlin-3 binding site mutation. Adhesion assays show that effector T cells carrying the kindlin-3 binding site mutation display significantly reduced adhesion to the integrin ligand ICAM-1. Using optical trapping, combined with back focal plane interferometry, we measured a bond rupture force of 17.85 ±0.63 pN at a force loading rate of 30.21 ± 4.35 pN/s, for single integrins expressed on wild-type cells. Interestingly, a significant drop in rupture force of bonds was found for TTT/AAA-mutant cells, with a measured rupture force of 10.08 ± 0.88pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the ß2-tail directly affects catch bond formation and bond strength of integrin-ligand bonds. As a consequence of this reduced binding, CD8+ T cell activation in vitro is also significantly reduced.

ß2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of ß2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of ß2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two ß2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting ß2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

Cell Adhesion Molecules and Their Roles and Regulation in the Immune and Tumor Microenvironment.

The immune system and cancer have a complex relationship with the immune system playing a dual role in tumor development. The effector cells of the immune system can recognize and kill malignant cells while immune system-mediated inflammation can also promote tumor growth and regulatory cells suppress the anti-tumor responses. In the center of all anti-tumor responses is the ability of the immune cells to migrate to the tumor site and to interact with each other and with the malignant cells. Cell adhesion molecules including receptors of the immunoglobulin superfamily and integrins are of crucial importance in mediating these processes. Particularly integrins play a vital role in regulating all aspects of immune cell function including immune cell trafficking into tissues, effector cell activation and proliferation and the formation of the immunological synapse between immune cells or between immune cell and the target cell both during homeostasis and during inflammation and cancer. In this review we discuss the molecular mechanisms regulating integrin function and the role of integrins and other cell adhesion molecules in immune responses and in the tumor microenvironment. We also describe how malignant cells can utilize cell adhesion molecules to promote tumor growth and metastases and how these molecules could be targeted in cancer immunotherapy.

A ß2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation.

ß2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of ß2-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the ß2-integrin (TTT/AAA-ß2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-ß2-integrin KI dendritic cells, which leads to a failure of MRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of ß2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of ß2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.

Beta2-Integrins and Interacting Proteins in Leukocyte Trafficking, Immune Suppression, and Immunodeficiency Disease.

Beta2-integrins are complex leukocyte-specific adhesion molecules that are essential for leukocyte (e.g., neutrophil, lymphocyte) trafficking, as well as for other immunological processes such as neutrophil phagocytosis and ROS production, and T cell activation. Intriguingly, however, they have also been found to negatively regulate cytokine responses, maturation, and migratory responses in myeloid cells such as macrophages and dendritic cells, revealing new, and unexpected roles of these molecules in immunity. Because of their essential role in leukocyte function, a lack of expression or function of beta2-integrins causes rare immunodeficiency syndromes, Leukocyte adhesion deficiency type I, and type III (LAD-I and LAD-III). LAD-I is caused by reduced or lost expression of beta2-integrins, whilst in LAD-III, beta2-integrins are expressed but dysfunctional because a major integrin cytoplasmic regulator, kindlin-3, is mutated. Interestingly, some LAD-related phenotypes such as periodontitis have recently been shown to be due to an uncontrolled inflammatory response rather than to an uncontrolled infection, as was previously thought. This review will focus on the recent advances concerning the regulation and functions of beta2-integrins in leukocyte trafficking, immune suppression, and immune deficiency disease.

Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking.

T cells traffic from the bloodstream into tissues to perform their functions in the immune system and are therefore subjected to a range of different mechanical forces. Integrins are essential for T cell trafficking into the tissues, as they mediate firm adhesion between the T cell and the endothelium under shear flow conditions. In addition, integrins are important for the formation of the contact between the T cell and the APC required for T cell activation. The actin-binding protein filamin A (FlnA) provides an important link between the integrin and the actin cytoskeleton. FlnA has been reported to function as an integrin inhibitor by competing with talin. However, its role in regulating integrin-dependent immune functions in vivo is currently poorly understood. In this study, we have investigated the role of FlnA in T cells, using T cell-specific FlnA knockout mice. We report that FlnA is required for the formation of strong integrin-ligand bonds under shear flow and for the generation of integrin-mediated T cell traction forces on ligand-coated hydrogels. Consequently, absence of FlnA leads to a reduction in T cell adhesion to integrin ligands under conditions of shear flow, as well as reduced T cell trafficking into lymph nodes and sites of skin inflammation. In addition, FlnA is not needed for T cell activation in vivo, which occurs in shear-free conditions in lymphoid organs. Our results therefore reveal a role of FlnA in integrin force transmission and T cell trafficking in vivo.

Filamin A Regulates Neutrophil Adhesion, Production of Reactive Oxygen Species, and Neutrophil Extracellular Trap Release.

Neutrophils are of fundamental importance in the early immune response and use various mechanisms to neutralize invading pathogens. They kill endocytosed pathogens by releasing reactive oxygen species in the phagosome and release neutrophil extracellular traps (NETs) into their surroundings to immobilize and kill invading micro-organisms. Filamin A (FlnA) is an important actin cross-linking protein that is required for cellular processes involving actin rearrangements, such cell migration. It has also been shown to negatively regulate integrin activation and adhesion. However, its role in the regulation of ß2 integrin-dependent adhesion, as well as in other cellular functions in neutrophils, is poorly understood. Using a transgenic mouse model in which FlnA is selectively depleted in myeloid cells, such as neutrophils, we show that FlnA negatively regulates ß2 integrin adhesion to complement component iC3b and ICAM-1 in shear-free, but not shear-flow, conditions. FlnA deletion does not affect phagocytosis of Escherichia coli or Staphylococcus aureus or their intracellular killing. However, FlnA negatively regulates production of reactive oxygen species upon cell activation. Conversely, neutrophil activation through TLR4, as well as through activation by the Gram-negative bacteria E. coli, results in reduced NET production in FlnA-depleted neutrophils. Thus, FlnA is a negative regulator of ß2 integrin-dependent cell adhesion and reactive oxygen species production but is required for NET production in primary murine neutrophils.

Caveolin-1 Influences LFA-1 Redistribution upon TCR Stimulation in CD8 T Cells.

TCR stimulation by peptide-MHC complexes on APCs requires precise reorganization of molecules into the area of cellular contact to form an immunological synapse from where T cell signaling is initiated. Caveolin (Cav)1, a widely expressed transmembrane protein, is involved in the regulation of membrane composition, cellular polarity and trafficking, and the organization of signal transduction pathways. The presence of Cav1 protein in T cells was identified only recently, and its function in this context is not well understood. We show that Cav1-knockout CD8 T cells have a reduction in membrane cholesterol and sphingomyelin, and upon TCR triggering they exhibit altered morphology and polarity, with reduced effector function compared with Cav1 wild-type CD8 T cells. In particular, redistribution of the ß2 integrin LFA-1 to the immunological synapse is compromised in Cav1-knockout T cells, as is the ability of LFA-1 to form high-avidity interactions with ICAM-1. Our results identify a role for Cav1 in membrane organization and ß2 integrin function in primary CD8 T cells.

Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status.

RATIONALE: The diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood. OBJECTIVE: Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties. METHODS AND RESULTS: In primary hepatocytes from healthy animals, metformin and the IKKß (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α-dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1ß, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/ß activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02-0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22-2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11). CONCLUSION: We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups. CLINICAL TRIAL REGISTRATION: Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.

Blood cell integrins and diseases.

Integrins are adhesion molecules on the surface of cells. In blood cells they are responsible for rapid changes during adhesion of the cell to the endothelium. Deficiency or defective function of integrins will result in severe illnesses. Surprisingly, certain variants of integrins are associated with an increased risk of developing SLE. In autoimmune diseases and as a result of organ transplantations integrins participate in reactions in which leukocytes attack the body's own tissues. This has resulted in the development of drugs in antibody form for inhibition of the action of integrins. These drugs may, however, exhibit severe adverse effects.

Mice Lacking beta2-Integrin Function Remain Glucose Tolerant in Spite of Insulin Resistance, Neutrophil Infiltration and Inflammation.

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function-Associated Antigen-1 and Kindlin-3.

Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA ß2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA ß2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.

A dominant role for the methyl-CpG-binding protein Mbd2 in controlling Th2 induction by dendritic cells.

Dendritic cells (DCs) direct CD4(+) T-cell differentiation into diverse helper (Th) subsets that are required for protection against varied infections. However, the mechanisms used by DCs to promote Th2 responses, which are important both for immunity to helminth infection and in allergic disease, are currently poorly understood. We demonstrate a key role for the protein methyl-CpG-binding domain-2 (Mbd2), which links DNA methylation to repressive chromatin structure, in regulating expression of a range of genes that are associated with optimal DC activation and function. In the absence of Mbd2, DCs display reduced phenotypic activation and a markedly impaired capacity to initiate Th2 immunity against helminths or allergens. These data identify an epigenetic mechanism that is central to the activation of CD4(+) T-cell responses by DCs, particularly in Th2 settings, and reveal methyl-CpG-binding proteins and the genes under their control as possible therapeutic targets for type-2 inflammation.