Claudio Mauro

ABIN-1 Binds to NEMO/IKKγ and Co-operates with A20 in Inhibiting NF-κB

Nuclear factor κB (NF-κB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-κB is dependent on the phosphorylation of the inhibitory subunit IκBα that is mediated by a multimeric, high molecular weight complex, called IκB kinase (IKK) complex. This is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO/IKKγ. The latter protein is essential for the activation of IKKs and NF-κB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-κB) as a NEMO/IKKγ-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-κB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-κB at the level of the IKK complex and that A20 inhibits activation of NF-κB by de-ubiquitination of NEMO/IKKγ. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKγ and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-κB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKγ and facilitates A20-mediated de-ubiquitination of NEMO/IKKγ, thus resulting in inhibition of NF-κB.

Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions.

Lactate has long been considered a “waste” by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.

Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications.

The blood–brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1−/− mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.

Central Role of the Scaffold Protein Tumor Necrosis Factor Receptor-associated Factor 2 in Regulating Endoplasmic Reticulum Stress-induced Apoptosis

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-κB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-κB in protecting cells from ER stress-induced apoptosis.

Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T cell-mediated immunity

To engage in proliferation, cells need to increase their biomass and replicate their genome. This process presents a substantial bioenergetic challenge: proliferating cells must increase ATP production and acquire or synthesize raw materials, including lipids, proteins and nucleic acids. To do so, proliferating cells actively reprogramme their intracellular metabolism from catabolic mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis and other anabolic pathways. This metabolic reprogramming, which directs nutrient uptake and metabolism during cell activation and proliferation, is under the control of specific signal transduction pathways. The underlying molecular mechanisms of cell metabolism reprogramming and their relevance to physiology and disease are currently under intense study. Several reports have uncovered the mechanisms of metabolic reprogramming that drive high rates of cell proliferation in cancer. Some recent studies have elucidated the physiological role of metabolic reprogramming during T‐cell activation, differentiation and trafficking, which are potentially relevant to inflammatory disorders. This review describes the impact of metabolic reprogramming on the pathogenesis of cancer and the physiology of T‐cell‐mediated immune responses, with an emphasis on the phosphatidyl inositol 3‐kinase–serine/threonine kinase–mammalian target of rapamycin pathway and the recently discovered metabolic processes regulated by nuclear factor‐κB. These discoveries will hopefully translate into a better understanding of the role of metabolic reprogramming as a key regulator of T‐cell‐mediated immune responses and offer novel, immune‐based therapeutic approaches.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction. We propose that these effects are dependent on an as yet unrecognized role for CD31-mediated homophilic interactions between T cells and antigen-presenting cells (APCs) during priming. We show that loss of CD31 interactions leads to enhanced primary clonal expansion, increased killing capacity, and diminished regulatory functions by T cells. Immunomodulation by CD31 signals correlates with a partial inhibition of proximal T-cell receptor (TCR) signaling, specifically Zap-70 phosphorylation. However, CD31-deficient mice do not develop autoimmunity due to increased T-cell death following activation, and we show that CD31 triggering induces Erk-mediated prosurvival activity in T cells either in conjunction with TCR signaling or autonomously. We conclude that CD31 functions as a nonredundant comodulator of T-cell responses, which specializes in sizing the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death.

An immunologist's guide to CD31 function in T-cells.

Summary Although it is expressed by all leukocytes, including T-, B-lymphocytes and dendritic cells, the immunoglobulin-like receptor CD31 is generally regarded by immunologists as a marker of endothelial cell lineage that lacks an established functional role in adaptive immunity. This perception has recently been challenged by studies that reveal a key role for this molecule in the regulation of T-cell homeostasis, effector function and trafficking. The complexity of the biological functions of CD31 results from the integration of its adhesive and signaling functions in both the immune and vascular systems. Signaling by means of CD31 is induced by homophilic engagement during the interactions of immune cells and is mediated by phosphatase recruitment or activation through immunoreceptor tyrosine inhibitory motifs (ITIMs) that are located in its cytoplasmic tail. Loss of CD31 function is associated with excessive immunoreactivity and susceptibility to cytotoxic killing. Here, we discuss recent findings that have brought to light a non-redundant, complex role for this molecule in the regulation of T-cell-mediated immune responses, with large impact on our understanding of immunity in health and disease.

The NF-κB Transcription Factor Pathway as a Therapeutic Target in Cancer: Methods for Detection of NF-κB Activity

NF-kappaB transcription factors marshal innate and adaptive immunity and inflammation. NF-kappaB also counters programmed cell death (PCD) induced by the proinflammatory cytokine tumor necrosis factor (TNF)alpha, and this activity of NF-kappaB is crucial for organismal physiology, chronic inflammation, and tumorigenesis. Indeed, whereas NF-kappaB contributes to many aspects of oncogenesis, it is now clear that its suppressive action on PCD is central to this process. Notably, recent studies indicate that NF-kappaB represents a crucial link in the well-established association between inflammation and carcinogenesis. In this link, NF-kappaB promotes synthesis of inflammatory mediators (e.g. TNFalpha) that stimulate growth of cancer cells, and upregulates genes that protect these cells against PCD induced by inflammatory signals. Elevated NF-kappaB activity also hampers tumor-cell killing inflicted by radiation and chemotherapeutic drugs, and in so doing, promotes resistance to anticancer therapy. Accordingly, NF-kappaB-targeting drugs are increasingly being used for treatment of human malignancies. Owing to the ubiquitous nature of the NF-kappaB pathway, however, these drugs have serious side effects, which limit their clinical use. Thus, a preferable approach would be to block, rather than NF-kappaB itself, its critical downstream targets that mediate discrete functions in cancer, such as prosurvival functions. Recent discoveries unraveling tissue specificity in the NF-kappaB-inducible mechanism(s) for control of PCD and identifying putative effectors of this control clearly validate this therapeutic approach. Given the emerging role of TNFkappa-induced signals of NF-kappaB activation in cancer and the potential of these signals for yielding new anticancer therapies, we focus herein on the methods most commonly used for analysis of the molecular steps leading from the triggering of TNF-Receptor (TNF-R)1 - the primary receptor of TNFalpha - to the induction of NF-kappaB. Specifically, we review the methods used for analysis of TNF-R1 trafficking, assembly of so-called TNF-R1 complex I, formation and activation of the IkappaB kinase (IKK) complex, phosphorylation and proteolysis of inhibitory IkappaB proteins, post-translational modifications and nuclear translocation of NF-kappaB dimers, induction of NF-kappaB transcriptional activity and binding to specific promoters, and upregulation of NF-kappaB target genes. The analysis of these events in cancerous cells may not only provide a better understanding of the basis for the role of NF-kappaB in carcinogenesis, but also potential new targets for selective anticancer therapy.

Neopterin induces pro‐atherothrombotic phenotype in human coronary endothelial cells

Summary.  Background: Inflammation plays a pivotal role in atherothrombosis. Recent data indicate that serum levels of neopterin, a marker of inflammation and immune modulator secreted by monocytes/macrophages, are elevated in patients with acute coronary syndromes and seem to be a prognostic marker for major cardiovascular events. The aim of the present study was to determine whether neopterin might affect the thrombotic and atherosclerotic characteristics of human coronary artery endothelial cells (HCAECs). Methods and results: In HCAECs, neopterin induced TF‐mRNA transcription as demonstrated by real time polymerase chain reaction and expression of functionally active tissue factor (TF) as demonstrated by procoagulant activity assay, and of cellular adhesion molecules (CAMs) as demonstrated by FACS analysis, in a dose‐dependent fashion. These neopterin effects were prevented by lovastatin, a HMG‐CoA reductase inhibitor. Neopterin‐induced TF and CAMs expression was mediated by oxygen free radicals through the activation of the transcription factor, nuclear factor‐kappa B (NF‐κB), as demonstrated by electrophoretic mobility shift assay and by suppression of CAMs and TF expression by superoxide dismutase and by NF‐κB inhibitor, pyrrolidine‐dithio‐carbamate ammonium. Conclusions: These data indicate that neopterin exerts direct effects on HCAECs by promoting CAMs and TF expression and support the hypothesis that neopterin, besides representing a marker of inflammation, might be an effector molecule able to induce a pro‐atherothrombotic phenotype in cells of the coronary circulation.

Estrogen protects the blood-brain barrier from inflammation-induced disruption and increased lymphocyte trafficking.

Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood-brain barrier (BBB) and it is recognized as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesize that estrogen can limit BBB breakdown following the onset of peripheral inflammation. Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modeled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammation-induced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models. Together, our data provide novel mechanisms for the protective effects of estrogen, and enhance our understanding of the beneficial role it plays in neurovascular/neuroimmune disease.