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.

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.

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.

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.

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.

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.

Functional Beta2-Integrins Restrict Skin Inflammation In Vivo.

Beta2-integrins and the important integrin regulator kindlin-3 are essential for leukocyte trafficking, but the role of beta2-integrins in regulating inflammation is still incompletely understood. Here, we have investigated skin inflammation in a mouse model where the kindlin-3 binding site in the beta2-integrin has been mutated (TTT/AAA-beta2-integrin knock-in), leading to expressed but dysfunctional integrins. We show that, surprisingly, neutrophil trafficking into the inflamed skin in a contact hypersensitivity model is normal in these mice, although trafficking of T cells and eosinophils into the skin is reduced. Instead, expression of dysfunctional integrins leads to increased mast cell and dendritic cell numbers in the skin, increased inflammatory cytokine production in the inflamed skin in vivo, and in mast cells in vitro. Furthermore, expression of dysfunctional integrins leads to increased dendritic cell activation and migration to lymph nodes and increased Th1 responses in vivo. Therefore, the kindlin-3/integrin interaction is important for trafficking of T cells and eosinophils but not absolutely required for neutrophil trafficking into the inflamed skin. Functional beta2-integrins also have a major role in restricting the immune response in the inflamed skin and lymph nodes in vivo, likely through effects on mast cell and dendritic cell numbers and activation.

A crucial role for ß2 integrins in podosome formation, dynamics and Toll-like-receptor-signaled disassembly in dendritic cells.

The dynamic properties of podosomes, their ability to degrade the underlying matrix and their modulation by Toll-like receptor (TLR) signaling in dendritic cells (DCs) suggests they have an important role in migration. Integrins are thought to participate in formation and dynamics of podosomes but the multiplicity of integrins in podosomes has made this difficult to assess. We report that murine DCs that lack ß2 integrins fail to form podosomes. Re-expression of ß2 integrins restored podosomes but not when the membrane proximal or distal NPxF motifs, or when an intervening triplet of threonine residues were mutated. We show that ß2 integrins are remarkably long-lived in podosome clusters and form a persistent framework that hosts multiple actin-core-formation events at the same or adjacent sites. When ß2 integrin amino acid residues 745 or 756 were mutated from Ser to Ala, podosomes became resistant to dissolution mediated through TLR signaling. TLR signaling did not detectably modulate phosphorylation at these sites but mutation of either residue to phospho-mimetic Asp increased ß2 integrin turnover in podosomes, indicating that phosphorylation at one or both sites establishes permissive conditions for TLR-signaled podosome disassembly.

Kindlin-3 in the immune system.

Kindlin-3 is a member of the kindlin family of focal adhesion proteins which bind to integrin beta-chain cytoplasmic domains to regulate integrin function. In contrast to kindlin-1 and kindlin-2 proteins, kindlin-3 is expressed mainly in the hematopoietic system. Mutations in kindlin-3 result in the rare genetic disorder, leukocyte adhesion deficiency type III (LAD-III), which is characterized by bleeding and recurrent infections due to deficient beta1, beta2 and beta3 integrin activation in platelets and leukocytes. Here, we review the role of kindlin-3 in integrin activation and in different immune cell functions.

The spontaneously adhesive leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells mediates rapid actin- and calmodulin-dependent adhesion strengthening to ligand under shear flow.

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.

Protein kinase D isoforms are dispensable for integrin-mediated lymphocyte adhesion and homing to lymphoid tissues.

Leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins are essential for lymphocyte adhesion, trafficking and effector functions. Protein kinase D (PKD) has previously been implicated in lymphocyte integrin regulation through regulation of Rap1 activity. However, the true role of PKD in integrin regulation in primary lymphocytes has not previously been investigated. The major PKD isoform in lymphocytes is PKD2. Here we employed PKD2-deficient mice, a specific PKD kinase inhibitor, as well as PKD-null DT40 B cells to investigate the role of PKD in integrin regulation in lymphocytes. We report that PKD2-deficient lymphocytes bound normally to integrin ligands in static and shear flow adhesion assays. They also homed normally to lymphoid organs after adoptive transfer into wild-type mice. DT40 B cells devoid of any PKD isoforms and primary lymphocytes pretreated with a specific PKD inhibitor bound normally to integrin ligands, indicating that multiple PKD isoforms do not redundantly regulate lymphocyte integrins. In addition, PKD2-deficient lymphocytes, as well as DT40 cells devoid of any PKD isoforms, could activate Rap1 in response to B-cell receptor ligation or phorbol ester treatment. Together, these results show that the PKD family does not play a critical role in lymphocyte integrin-mediated cell adhesion or lymphocyte trafficking in vivo.

A systemic lupus erythematosus-associated R77H substitution in the CD11b chain of the Mac-1 integrin compromises leukocyte adhesion and phagocytosis.

The Mac-1 integrin is expressed mainly on myeloid cells and binds several ligands, including members of the ICAM family and the complement factor iC3b. It is involved in essential immunological processes, such as leukocyte extravasation and phagocytosis. In addition, Mac-1 has been described to negatively regulate immune cell signaling. Recently, a single nucleotide polymorphism conferring an amino acid change in the Mac-1 integrin extracellular domain, R77H, was shown to be associated with systemic lupus erythematosus. Here, we demonstrate that the R77H-substituted Mac-1 can be expressed on the cell surface in transfected cells and can undergo conformational changes in response to integrin activation. The affinity of the integrin for ICAMs is only partially reduced, but cell adhesion to ICAM-1 and ICAM-2 is severely compromised, and Jß2.7 cells expressing R77H substituted integrins are deficient in adhesion to ICAM-1 under shear flow conditions. Importantly, cell adhesion to the complement factor iC3b is also diminished, and COS cells expressing R77H-substituted integrins display reduced iC3b-dependent phagocytosis. In addition, U937 cells expressing R77H-CD11b display increased IL-6 production as compared with WT-CD11b-expressing cells. These results suggest that the R77H substitution results in the deficiency of the mutated integrin to mediate cell adhesion to ligands such as ICAMs and iC3b. These deficiencies may ultimately lead to detrimental effects on the immune system and contribute to the development of systemic lupus erythematosus.