Neutrophil extracellular traps (NET): not only antimicrobial but also modulators of innate and adaptive immunities in inflammatory autoimmune diseases.

Polymorphonuclear neutrophils (PMN) represent one of the first lines of defence against invading pathogens and are the most abundant leucocytes in the circulation. Generally described as pro-inflammatory cells, recent data suggest that PMN also have immunomodulatory capacities. In response to certain stimuli, activated PMN expel neutrophil extracellular traps (NET), structures made of DNA and associated proteins. Although originally described as an innate immune mechanism fighting bacterial infection, NET formation (or probably rather an excess of NET together with impaired clearance of NET) may be deleterious. Indeed, NET have been implicated in the development of several inflammatory and autoimmune diseases as rheumatoid arthritis or systemic lupus erythematosus, as well as fibrosis or cancer. They have been suggested as a source of (neo)autoantigens or regulatory proteins like proteases or to act as a physical barrier. Different mechanisms of NET formation have been described, leading to PMN death or not, depending on the stimulus. Interestingly, NET may be both pro-inflammatory and anti-inflammatory and this probably partly depends on the mechanism, and thus the stimuli, triggering NET formation. Within this review, we will describe the pro-inflammatory and anti-inflammatory activities of NET and especially how NET may modulate immune responses.

The exposome in rheumatoid arthritis.

The exposome integrates the variety and accumulation of exposures (external and internal) to which an individual is submitted to from conception to death. Exposome may therefore be a useful tool for understanding the diversity of these factors and their role in the pathophysiology of rheumatoid arthritis (RA). Life is perceived as a continuum of cumulative changes, with key periods of disruption (e.g. birth, adolescence, pregnancy, prolonged treatment). The combination of these changes and the external signals that cause them constitute an individual's exposome, which is constantly changing and expanding throughout life. Thus, measuring the exposome requires specific tools and approaches as well as a global perspective. RA, a complex, heterogeneous, pro-inflammatory autoimmune disease with a genetic component and for which a large number of environmental factors have already been incriminated is an appropriate field of application for the exposome. The aim of this review is to define the exposome concept, outline the different analytic tools available for its study and finally apply them to the field of RA.

Increased Concentrations of Circulating Soluble MHC Class I-Related Chain A (sMICA) and sMICB and Modulation of Plasma Membrane MICA Expression: Potential Mechanisms and Correlation With Natural Killer Cell Activity in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease of unknown etiology. The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are stress-inducible cell surface molecules. MICA and MICB label malfunctioning cells for their recognition by cytotoxic lymphocytes such as natural killer (NK) cells. Alterations in this recognition have been found in SLE. MICA/MICB can be shed from the cell surface, subsequently acting either as a soluble decoy receptor (sMICA/sMICB) or in CD4+ T-cell expansion. Conversely, NK cells are frequently defective in SLE and lower NK cell numbers have been reported in patients with active SLE. However, these cells are also thought to exert regulatory functions and to prevent autoimmunity. We therefore investigated whether, and how, plasma membrane and soluble MICA/B are modulated in SLE and whether they influence NK cell activity, in order to better understand how MICA/B may participate in disease development. We report significantly elevated concentrations of circulating sMICA/B in SLE patients compared with healthy individuals or a control patient group. In SLE patients, sMICA concentrations were significantly higher in patients positive for anti-SSB and anti-RNP autoantibodies. In order to study the mechanism and the potential source of sMICA, we analyzed circulating sMICA concentration in Behcet patients before and after interferon (IFN)-α therapy: no modulation was observed, suggesting that IFN-α is not intrinsically crucial for sMICA release in vivo. We also show that monocytes and neutrophils stimulated in vitro with cytokines or extracellular chromatin up-regulate plasma membrane MICA expression, without releasing sMICA. Importantly, in peripheral blood mononuclear cells from healthy individuals stimulated in vitro by cell-free chromatin, NK cells up-regulate CD69 and CD107 in a monocyte-dependent manner and at least partly via MICA-NKG2D interaction, whereas NK cells were exhausted in SLE patients. In conclusion, sMICA concentrations are elevated in SLE patients, whereas plasma membrane MICA is up-regulated in response to some lupus stimuli and triggers NK cell activation. Those results suggest the requirement for a tight control in vivo and highlight the complex role of the MICA/sMICA system in SLE.

Deoxyribonuclease 1-Mediated Clearance of Circulating Chromatin Prevents From Immune Cell Activation and Pro-inflammatory Cytokine Production, a Phenomenon Amplified by Low Trap1 Activity: Consequences for Systemic Lupus Erythematosus.

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo, after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro, delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression.

Origins of rheumatoid arthritis.

While the exact cause of rheumatoid arthritis is unknown, several mechanisms have been described extensively. The genetic predisposition for this autoimmune disease is largely attributed to MHC class II genes, especially the main polymorphism in the HLA shared epitope. Non-genetic factors account for the rest. The best known are autoantigens to citrullinated or carbmylated proteins, although there are many others. They are recognized by an immune system with defective control mechanisms, in which regulator T-cells are unable to prevent inflammation and the destruction of tissue, joint and vascular structures (among others). Polymorphonuclear neutrophils, which are very abundant at sites of inflammation, interfere with attempts at regulation. Cell metabolism, which typically participates in fighting against the autoantigen attack, does not respond correctly to the demands, making the inflammatory phenomenon worse. This is also the case for environmental factors such as atmospheric pollution, dust, diet (especially salt intake) and infections. Inflammatory cytokines such as TNF-α, IL-1 and IL-17, are certain implicated, but not initially. They appear as a common execution pathway for a lengthy sentence following an unfortunate encounter between genetic predisposition and a harmful environment.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

OBJECTIVE: To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). METHODS: The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. RESULTS: Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. CONCLUSION: TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Extracellular Chromatin Triggers Release of Soluble CEACAM8 Upon Activation of Neutrophils.

Increased concentrations of extracellular chromatin are observed in cancer, sepsis, and inflammatory autoimmune diseases like systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). In SLE and RA, extracellular chromatin may behave as a danger-associated molecular pattern (DAMP). Polymorphonuclear neutrophils (PMN) are described as typical pro-inflammatory cells but possess also immunoregulatory properties. They are activated in SLE and RA but surprisingly remain moderately studied in these diseases, and especially the disease-associated stimuli triggering PMN activation are still not completely characterized. PMN express plasma membrane carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 8 (CD66b) and secrete a soluble form of CEACAM8 after activation. Soluble CEACAM8 has in turn immunoregulatory functions. However, few natural stimuli inducing soluble CEACAM8 secretion by PMN have been identified. Here we demonstrate for the first time that extracellular chromatin triggers secretion of soluble CEACAM8 by primary human PMN. Priming of PMN was not required. Secretion was associated with activation of PMN. Similar induction of soluble CEACAM8 release was observed with purified mono-nucleosomes as well as long chromatin fragments and occurred in a time-dependent and concentration-dependent manner. Results indicate that chromatin induces both neo-synthesis of soluble CEACAM8 and release of soluble CEACAM8 through degranulation. In addition, we report the presence of soluble CEACAM8 at high concentration in the synovial fluid of RA patients. Thus, we describe here a novel mechanism by which a natural DAMP, with inflammatory properties in SLE and RA, induces soluble CEACAM8 secretion by activated PMN with potential immunoregulatory consequences on other immune cells, including PMN.

Neutrophil extracellular traps exert both pro- and anti-inflammatory actions in rheumatoid arthritis that are modulated by C1q and LL-37.

OBJECTIVE: Neutrophil extracellular traps (NET), produced by activated polymorphonuclear neutrophils (PMN), are supposed to play a role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease characterized by anti-citrullinated protein antibodies (ACPA). Indeed, NET contain citrullinated autoantigens and some RA autoantibodies recognize NET. However, the mechanisms by which NET trigger or perpetuate the inflammatory process in RA are hitherto not elucidated. We hypothesized that, in addition to citrullination, NET might also contain stimulatory proteins and directly activate inflammatory target cells, as PMN and macrophages. METHODS: NET antigenic and inflammatory properties were analyzed in 157 healthy donors (HD) and RA patients, the largest analysis reported so far. Primary PMN and monocyte-derived macrophages were isolated and immunoglobulin G (IgG) purified. NET were induced (NETosis), isolated and quantified. NET antigenicity was analyzed by fluorescence microscopy. PMN and macrophages were stimulated with NET with/without ACPA, C1q, LL-37 or lipopolysaccharide (LPS) and cell activation was estimated by flow cytometry and ELISA. RESULTS: PMN from RA patients produced more NET than HD PMN. We next dissected how NET mechanistically affect inflammatory cells. Particularly, we show for the first time that RA and HD NET activated both resting macrophages and PMN, but importantly RA NET were more stimulatory, leading to secretion of inflammatory cytokines and up-regulation of HLA/CD86/CD11b. IgG from ACPA-positive RA patients specifically recognized RA and even HD NET. Nevertheless, NET-induced cell activation occurs independently of immune complex formation with ACPA. Likewise, endosomal acidification was not required. Notably, we also report that complement C1q increased the NET stimulatory activity on macrophages only, due to higher expression of C1q receptors, which was further supported by the LL-37 antimicrobial peptide. In contrast, NET specifically inhibited interleukin (IL)-6 secretion by LPS-activated macrophages and not PMN, especially with C1q/LL-37. This inhibition was not mediated by NET-derived proteases or LPS neutralization and was associated with the simultaneous induction of IL-10 secretion. CONCLUSION: We show that NET possess both pro- and anti-inflammatory properties depending on target cells, their activation levels and C1q/LL-37. Thus, independently of ACPA, NET modulate RA chronic inflammation via this new dual activity we identified. In addition, NET may trigger autoimmunity in RA as ACPA recognize NET antigens but not non-activated PMN. Therefore, we conclude that excess of NETosis together with enhanced NET activity participate to RA pathogenesis at different levels.

Tumor Necrosis Factor-Alpha Targeting Can Protect against Arthritis with Low Sensitization to Infection.

Tumor necrosis factor-alpha (TNF-α) blockade is an effective treatment for rheumatoid arthritis (RA) and other inflammatory diseases, but in patients, it is associated with reduced resistance to the infectious agents Mycobacterium tuberculosis and Listeria monocytogenes, among others. Our goal was to model infection and arthritis in mice and to compare etanercept, a currently used anti-TNF-α inhibitor, to an anti-TNF-α vaccine. We developed a murine surrogate of the TNF-α kinoid and produced an anti-murine TNF-α vaccine (TNFKi) composed of keyhole limpet hemocyanin conjugated to TNF-α, which resulted in anti-TNF-α antibody production in mice. We also used etanercept (a soluble receptor of TNF commonly used to treat RA) as a control of TNF neutralization. In a mouse model of collagen-induced arthritis, TNFKi protected against inflammation similar to etanercept. In a mouse model of acute L. monocytogenes infection, all TNFKi-treated mice showed cleared bacterial infection and survived, whereas etanercept-treated mice showed large liver granulomas and quickly died. Moreover, TNFKi mice infected with the virulent H37Rv M. tuberculosis showed resistance to infection, in contrast with etanercept-treated mice or controls. Depending on the TNF-α blockade strategy, treating arthritis with a TNF-α inhibitor could result in a different profile of infection suceptibility. Our TNFKi vaccine allowed for a better remaining host defense than did etanercept.

Arthritis models: usefulness and interpretation.

Animal models of arthritis are used to better understand pathophysiology of a disease or to seek potential therapeutic targets or strategies. Focusing on models currently used for studying rheumatoid arthritis, we show here in which extent models were invaluable to enlighten different mechanisms such as the role of innate immunity, T and B cells, vessels, or microbiota. Moreover, models were the starting point of in vivo application of cytokine-blocking strategies such as anti-TNF or anti-IL-6 treatments. The most popular models are the different types of collagen-induced arthritis and arthritis in KBN mice. As spontaneous arthritides, human TNF-α transgenic mice are a reliable model. It is mandatory to use animal models in the respect of ethical procedure, particularly regarding the number of animals and the control of pain. Moreover, design of experiments should be of the highest level, animal models of arthritis being dedicated to exploration of well-based novelties, and never used for confirmation or replication of already proven concepts. The best interpretations of data in animal models of arthritis suppose integrated research, including translational studies from animals to humans.

In Vivo Expansion of Activated Foxp3+ Regulatory T Cells and Establishment of a Type 2 Immune Response upon IL-33 Treatment Protect against Experimental Arthritis.

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33-treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33-treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39(high) Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

TLR9 independent interferon α production by neutrophils on NETosis in response to circulating chromatin, a key lupus autoantigen.

OBJECTIVES: Interferon (IFN) α is a key immunoregulatory cytokine secreted by activated plasmacytoid dendritic cells (PDC) that constitute less than 1% of leucocytes. IFNα plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Nevertheless, the natural IFNα inducers in SLE as well as the different IFNα secreting cell types are only partially characterised. METHODS: Chromatin was purified from calf thymus. Human peripheral blood mononuclear cells (PBMC), neutrophils and mouse bone marrow neutrophils were purified and cultured with different stimuli. IFNα production was estimated by flow cytometry, ELISA and a bioassay, and gene expression by quantitative real time PCR. Neutrophil activation and NETosis were analysed by flow cytometry, ELISA and confocal microscopy. RESULTS: Neutrophils produced a bioactive IFNα on stimulation with purified chromatin. IFNα secretion was observed with steady state neutrophils purified from 56 independent healthy individuals and autoimmune patients in response to free chromatin and not chromatin containing immune complexes. Chromatin induced IFNα secretion occurred independently of Toll-like receptor 9 (TLR9). Neutrophil priming by granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor or IFNα was not necessary but PBMC sustained IFNα secretion by neutrophils. PDC were 27 times more efficient than neutrophils but blood neutrophils were 100 times more frequent than PDC. Finally, neutrophil activation by chromatin was associated with NETosis and DNA sensor upregulation. CONCLUSIONS: Neutrophils have the capability of producing IFNα on selective triggering, and we identified a natural lupus stimulus involved, unveiling a new mechanism involved in SLE. Neutrophils represent another important source of IFNα and important targets for future therapies aimed at influencing IFNα levels.

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence.

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation-requiring, pH-stable and heat-stable nuclease that we named Nuclease A (NucA). Substitution of the histidine(148) by alanine reduced nuclease activity of the GBS wild-type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA(H148A) mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.

Primary blood neutrophils express a functional cell surface Toll-like receptor 9.

Polymorphonuclear leukocytes (PMNs) represent one of the first lines of defense against pathogens. TLR9 is normally expressed in endosomes/lysosomes where it is activated by pathogen-derived DNA. Here we show that freshly isolated human and mouse primary PMNs express TLR9 at the cell surface ex vivo. Moreover, surface TLR9 expression is upregulated upon activation of PMNs with different stimuli and not only TLR9 agonists. Importantly, surface TLR9 is processed, active, and functional. TLR9 ligands, oligo-nucleotides containing unmethylated CpG motifs, indeed bind to surface TLR9 and binding was strongly observed at the cell surface of human cells expressing surface TLR9 and at the surface of WT but not TLR9-deficient mouse PMNs. Finally, CpG oligonucleotides cross-linked onto a solid phase and having no access to intracellular TLR9 are able to trigger cell surface TLR9 and induce neutrophil activation, even when endosomal acidification is inhibited. This is the first demonstration of a functional TLR9 expressed at the cell surface of human primary cells. This pathway may be triggered when pathogen-derived TLR9 ligands cannot reach the endosome, offering a rescue mechanism for neutrophil activation.

The mouse dendritic cell marker CD11c is down-regulated upon cell activation through Toll-like receptor triggering.

Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC.

Pain and immunity.

Chronic neuropathic and inflammatory pain is a major public health problem. Nociceptors undergo sensitization, first in peripheral tissues then in the central nervous sytem, via neuroimmune interactions linking neurons, glial cells (microglia and astrocytes), and immune cells. These interactions may either exacerbate or attenuate the pain and inflammation, which normally reach a state of equilibrium. With more powerful or longer lasting stimuli, specific profiles of microglial and, subsequently, astrocytic activation in the dorsal horn play a key role in neuronal plasticity and transition to chronic pain. Recent insights into the interactions between the nervous system and the immune system suggest a large number of potential therapeutic targets that could be influenced either by targeted inhibition or by directing the neuroimmune response toward the antiinflammatory and analgesic end of its spectrum.

Neutrophils and interferon-α-producing cells: who produces interferon in lupus?

Interferon-α plays a crucial role in the pathogenesis of systemic lupus erythematosus. Nevertheless, the different human cell types producing this cytokine as well as the stimuli inducing its production have not been completely characterized. So far, a subpopulation of dendritic cells activated by immune complexes has been identified as major producers of interferon-α in patients with lupus. However, those cells represent a minor population and some studies have reported the secretion of interferon-α by other cells. On the other hand, more than 50% of blood leukocytes are neutrophils and their functions are still not fully understood. Recent data suggest that neutrophils, though usually not considered interferon-α-producing cells, may represent an unexpected source of this cytokine in response to some lupus stimuli.

Nucleosome-induced neutrophil activation occurs independently of TLR9 and endosomal acidification: implications for systemic lupus erythematosus.

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome-induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro-inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL-8/IL-6/TNF secretion and CD11b up-regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up-regulation and act synergistically with TLR9 ligands. Nucleosome-induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9-KO mice were activated by nucleosomes, as detected by MIP-2 secretion and CD11b up-regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome-induced innate immunity and anti-nucleosome B-cell autoimmunity.

Neurological symptoms in patients with biopsy proven celiac disease.

In celiac disease (CD), the gut is the typical manifestation site but atypical neurological presentations are thought to occur in 6 to 10% with cerebellar ataxia being the most frequent symptom. Most studies in this field are focused on patients under primary neurological care. To exclude such an observation bias, patients with biopsy proven celiac disease were screened for neurological disease. A total of 72 patients with biopsy proven celiac disease (CD) (mean age 51 +/- 15 years, mean disease duration 8 +/- 11 years) were recruited through advertisements. All participants adhered to a gluten-free diet. Patients were interviewed following a standard questionnaire and examined clinically for neurological symptoms. Medical history revealed neurological disorders such as migraine (28%), carpal tunnel syndrome (20%), vestibular dysfunction (8%), seizures (6%), and myelitis (3%). Interestingly, 35% of patients with CD reported of a history of psychiatric disease including depression, personality changes, or even psychosis. Physical examination yielded stance and gait problems in about one third of patients that could be attributed to afferent ataxia in 26%, vestibular dysfunction in 6%, and cerebellar ataxia in 6%. Other motor features such as basal ganglia symptoms, pyramidal tract signs, tics, and myoclonus were infrequent. 35% of patients with CD showed deep sensory loss and reduced ankle reflexes in 14%. Gait disturbances in CD do not only result from cerebellar ataxia but also from proprioceptive or vestibular impairment. Neurological problems may even develop despite strict adherence to a gluten-free diet.