Unlocking the power of NOX2: A comprehensive review on its role in immune regulation.

Reactive oxygen species (ROS) are a family of highly reactive molecules with numerous, often pleiotropic functions within the cell and the organism. Due to their potential to destroy biological structures such as membranes, enzymes and organelles, ROS have long been recognized as harmful yet unavoidable by-products of cellular metabolism leading to "oxidative stress" unless counterbalanced by cellular anti-oxidative defense mechanisms. Phagocytes utilize this destructive potential of ROS released in high amounts to defend against invading pathogens. In contrast, a regulated and fine-tuned release of "signaling ROS" (sROS) provides essential intracellular second messengers to modulate central aspects of immunity, including antigen presentation, activation of antigen presenting cells (APC) as well as the APC:T cell interaction during T cell activation. This regulated release of sROS is foremost attributed to the specialized enzyme NADPH-oxidase (NOX) 2 expressed mainly in myeloid cells such as neutrophils, macrophages and dendritic cells (DC). NOX-2-derived sROS are primarily involved in immune regulation and mediate protection against autoimmunity as well as maintenance of self-tolerance. Consequently, deficiencies in NOX2 not only result in primary immune-deficiencies such as Chronic Granulomatous Disease (CGD) but also lead to auto-inflammatory diseases and autoimmunity. A comprehensive understanding of NOX2 activation and regulation will be key for successful pharmaceutical interventions of such ROS-related diseases in the future. In this review, we summarize recent progress regarding immune regulation by NOX2-derived ROS and the consequences of its deregulation on the development of immune disorders.

Molecular analysis of Annexin expression in cancer.

BACKGROUND: Uptake of apoptotic cells induces a tolerogenic phenotype in phagocytes and promotes peripheral tolerance. The highly conserved Annexin core domain, present in all members of the Annexin family, becomes exposed on the apoptotic cell-surface and triggers tolerogenic signalling in phagocytes via the Dectin-1 receptor. Consequently, Annexins exposed on tumour cells upon cell death are expected to induce tolerance towards tumour antigens, inhibiting tumour rejection. METHODS: Expression analysis for all Annexin family members was conducted in cancer cell lines of diverse origins. Presentation of Annexins on the cell surface during apoptosis of cancer cell lines was investigated using surface washes and immunoblotting. Expression data from the GEO database was analysed to compare Annexin levels between malignant and healthy tissue. RESULTS: Six Annexins at least were consistently detected on mRNA and protein level for each investigated cell line. AnxA1, AnxA2 and AnxA5 constituted the major part of total Annexin expression. All expressed Annexins translocated to the cell surface upon apoptosis induction in all cell lines. Human expression data indicate a correlation between immune infiltration and overall Annexin expression in malignant compared to healthy tissue. CONCLUSIONS: This study is the first comprehensive analysis of expression, distribution and presentation of Annexins in cancer.

Annexin-coated particles induce antigen-specific immunosuppression.

Apoptotic cells mediate the development of tolerogenic dendritic cells (DC) and thus facilitate induction and maintenance of peripheral tolerance. Following the identification of the evolutionary conserved annexin core domain (Anx) as a specific signal on apoptotic cells which antagonises Toll-like receptor (TLR) signalling, we examined whether the tolerogenic capacity of Anx can be exploited to downregulate antigen-specific immune responses. The treatment of bone marrow-derived dendritic cells (BMDC) with particles harbouring Anx as well as the model antigen ovalbumin (OVA) attenuated the response of OVA-specific OT-II T cells. The co-culture of Anx-particle-treated DC and T cells resulted in an anergy-like phenotype characterized by reduced proliferation and cytokine secretion. Here we demonstrate that the anti-inflammatory effects of Anx which are mediated through DC can be used as a tool to generate a particle-based antigen delivery system that promotes antigen-specific immunosuppression. Such Anx-particles may be a new therapeutic approach for the treatment of autoimmune diseases.

Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells.

Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). In this protocol, a robust method to determine low intracellular ROS production using H2DCFDA was validated by several ROS-specific as well as NOX-2-specific inhibitors. Cells were treated with inhibitors or control substances prior to treatment with the ROS-inducer of interest. H2DCFDA was added only for the last 30 min of the treatment schedule. To terminate its conversion, we used a ROS-specific inhibitor until analysis by flow cytometry within the FITC-channel (Ex: 488 nm/Em: 519 nm). In summary, this protocol allows the detection of signaling-relevant intracellular ROS production in cell lines and primary immune cells (e.g., Mono Mac 6 cells and Bone marrow-derived dendritic cells, respectively). Using this method in combination with specific inhibitors, we were able to validate even exceptionally low amounts of ROS produced by NOX-2 and relevant for immune-regulatory signaling.

Dectin-1 Binding to Annexins on Apoptotic Cells Induces Peripheral Immune Tolerance via NADPH Oxidase-2.

Uptake of apoptotic cells (ACs) by dendritic cells (DCs) and induction of a tolerogenic DC phenotype is an important mechanism for establishing peripheral tolerance to self-antigens. The receptors involved and underlying signaling pathways are not fully understood. Here, we identify Dectin-1 as a crucial tolerogenic receptor binding with nanomolar affinity to the core domain of several annexins (annexin A1, A5, and A13) exposed on ACs. Annexins bind to Dectin-1 on a site distinct from the interaction site of pathogen-derived ß-glucans. Subsequent tolerogenic signaling induces selective phosphorylation of spleen tyrosine kinase (SYK), causing activation of NADPH oxidase-2 and moderate production of reactive oxygen species. Thus, mice deficient for Dectin-1 develop autoimmune pathologies (autoantibodies and splenomegaly) and generate stronger immune responses (cytotoxic T cells) against ACs. Our data describe an important immunological checkpoint system and provide a link between immunosuppressive signals of ACs and maintenance of peripheral immune tolerance.

More than just innate affairs - on the role of annexins in adaptive immunity.

In more than 30 years of research annexins have been demonstrated to regulate immune responses. The prototype member of this family, annexin (Anx) A1, has been widely recognized as an anti-inflammatory mediator affecting migration and cellular responses of various cell types of the innate immune system. Evidently, effects on innate immune cells also impact on the course of adaptive immune responses. Innate immune cells provide a distinct cytokine milieu during initiation of adaptive immunity which regulates the development of T cell responses. Moreover, innate immune cells such as monocytes can differentiate into dendritic cells and take an active part in T cell stimulation. Accumulating evidence shows a direct role for annexins in adaptive immunity. Anx A1, the annexin protein studied in most detail, has been shown to influence antigen presentation as well as T cells directly. Moreover, immune modulatory roles have been described for several other annexins such as Anx A2, Anx A4, Anx A5 and Anx A13. This review will focus on the involvement of Anx A1 and other annexins in central aspects of adaptive immunity, such as recruitment and activation of antigen presenting cells, T cell differentiation and the anti-inflammatory removal of apoptotic cells.

Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate.

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-ß in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-ß, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.

The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain.

Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow-derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Anx-expressing ACs prevented induction of Ag-specific CD8(+) T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders.

Photosensitivity, apoptosis, and cytokines in the pathogenesis of lupus erythematosus: a critical review.

The underlying pathomechanisms of lupus erythematosus (LE), a multifactorial autoimmune disease, remain elusive. Due to the clinical evidence demonstrating a clear relationship between ultraviolet (UV) light exposure and skin lesions of LE, photosensitivity has been proven to be an important factor in the pathogenesis of the disease. Standardised photoprovocation with UVA and UVB irradiation has been shown to be a reliable model for evaluating photosensitivity in patients with cutaneous LE (CLE) and analysing the underlying medical conditions of the disease. In this respect, UV irradiation can cause aberrant induction of apoptosis in keratinocytes and contribute to the appearance of excessive apoptotic cells in the skin of CLE patients. Moreover, apoptotic cells that cannot be cleared by phagocytes may undergo secondary necrosis and release proinflammatory compounds and potential autoantigens, which may contribute to the inflammatory micromilieu that leads to formation of skin lesions in the disease. In addition to UV-mediated induction of apoptosis, the molecular and cellular factors that may cause the abnormal long-lasting photoreactivity in CLE include mediators of inflammation, such as cytokines and chemokines. In particular, interferons (IFNs) are important players in the early activation of the immune system and have a specific role in the immunological interface between the innate and the adaptive immune system. The fact that treatment with recombinant type I IFNs (α and ß) can induce not only systemic organ manifestations but also LE-like skin lesions provides additional evidence for a pathogenetic role of these IFNs in the disease.

Interferon-α abrogates the suppressive effect of apoptotic cells on dendritic cells in an in vitro model of systemic lupus erythematosus pathogenesis.

OBJECTIVE: An increased incidence of apoptotic cells and an increased activation of dendritic cells (DC) may be involved in the pathogenesis of systemic lupus erythematosus (SLE). We investigated the characteristics of apoptotic neutrophils and monocyte-derived DC of patients with SLE, their interaction, and the influence of autoantibodies and inflammatory cytokines on this interaction. METHODS: Kinetics of neutrophil apoptosis and DC activation were studied by flow cytometry. To analyze the interaction of apoptotic cells with phagocytes, crossover coculture experiments were performed with DC from patients with SLE and apoptotic Jurkat T cells as well as with apoptotic neutrophils from patients with SLE and the monocytic cell line U937. SLE serum and cytokines were added to this coculture, and activation and suppression of DC were quantified by levels of inflammatory cytokine secretion. RESULTS: Apoptotic neutrophils and DC from patients with SLE showed no inherent defects compared to healthy controls, and the suppressive nature of their interaction was not affected. Autoantibodies as well as the inflammatory cytokines interleukin 17 (IL-17) and IL-1ß had no influence on the interaction in this setup. Interferon (IFN)-α, however, substantially reduced the suppressive effect of apoptotic cells on DC. CONCLUSION: The data suggest that aberrant immune reactivity in SLE is not generally due to an intrinsic defect in apoptotic cells, their processing, or their interaction with DC, but likely arises from the milieu in which this interaction takes place. Our study highlights the importance of IFN-α during early stages of SLE and its potential as a therapeutic target.

Annexin A1 on the surface of early apoptotic cells suppresses CD8+ T cell immunity.

Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer.

Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIPshort.

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIP(long) was only slightly down-regulated in sensitized T cells, c-FLIP(short) became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIP(short), rather than c-FLIP(long), confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.