Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system.

Purpose of review Neutrophil extracellular traps (NETs) can be found at the sites of vascular lesions and in the circulation of patients with active small vessel vasculitis. Neutrophils from vasculitis patients release more NETs in vitro, and NETs have properties that can harm the vasculature both directly and indirectly. There are several ways to interfere with NET formation, which open for new therapeutic options. However, there are several types of NETs and different mechanisms of NET formation, and these might have different effects on inflammation. Here we review recent findings regarding the pathogenesis and therapeutic potentials of NETs in vasculitis. Recent findings Experimental mouse models support a role for NETs in promoting vascular damage, where histones and mitochondrial DNA appear to be driving forces. Impaired formation of NETs, however, in an SLE-like mouse model leads to more severe disease, suggesting that NETs can be important in limiting inflammation. Studies on drug-induced vasculitis reveal that levamisole can induce NETosis via muscarinic receptors, predisposing for the generation of autoantibodies, including antineutrophil cytoplasmic autoantibodies (ANCA). This supports the notion that NETs can bridge the innate and adaptive immune systems. Summary NETs can participate in the pathogenesis of vasculitis, but in some models there also seem to be protective effects of NETs. This complexity needs further evaluation with experimental models that are as specific as possible for human primary vasculitis.

A group of pauci-immune vasculitides, characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of antineutrophil cytoplasmic antibodies (ANCAs), is referred to as ANCA-associated vasculitis (AAV). ANCAs against proteinase 3 (PR3) (PR3-ANCA) or myeloperoxidase (MPO) (MPO-ANCA) are found in over 90% of patients with active disease, and these ANCAs are implicated in the pathogenesis of AAV. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally, it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. AAV patients also possess elevated levels of NETs in the circulation. ANCAs are capable of inducing NETosis in neutrophils, and their potential to do so has been shown to be affinity dependent and to correlate with disease activity. Neutrophils from AAV patients are also more prone to release NETs spontaneously than neutrophils from healthy blood donors. NETs contain proinflammatory proteins and are thought to contribute to vessel inflammation directly by damaging endothelial cells and by activating the complement system and indirectly by acting as a link between the innate and adaptive immune system through the generation of PR3- and MPO-ANCA. Injection of NET-loaded myeloid dendritic cells into mice results in circulating PR3- and MPO-ANCA and the development of AAV-like disease. NETs have also been shown to be essential in a rodent model of drug-induced vasculitis. NETs induced by propylthiouracil could not be degraded by DNaseI, implying that disordered NETs might be important for the generation of ANCAs. NET degradation was also highlighted in another study showing that AAV patients have reduced DNaseI activity resulting in less NET degradation. With this in mind, it might be that prolonged exposure to proteins in the NETs due to the overproduction of NETs and/or reduced clearance of NETs is important in AAV. However, not all ANCAs are pathogenic and some might possibly also aid in the clearance of NETs. A dual role for ANCAs in relation to circulating NET levels has been proposed because a negative correlation was observed between PR3-ANCA and NET remnants in patients in remission.

OBJECTIVES:Neutrophil extracellular traps (NETs) have been visualized at the site of ANCA-associated vasculitis (AAV) lesions. Increased levels of NET remnants in the circulation have been reported in some AAV patients with active disease. The aim of the present study was to analyse NET remnants in a larger cohort of AAV patients with varying degrees of disease activity and to elucidate possible factors responsible for remnant variation.METHODS:Levels of NET remnants in the circulation of healthy controls (HCs; n = 31) and AAV patients (n = 93) were determined with ELISA. NET remnants were then correlated with ANCA levels, spontaneous and induced cell death (NETosis/necrosis) in vitro, neutrophil count and corticosteroid therapy.RESULTS:Patients with active disease showed higher levels of circulating NET remnants compared with patients in remission (P = 0.026) and HCs (P = 0.006). From patients sampled during both remission and active disease, we found increased levels during active disease (P = 0.0010). In remission, ANCA-negative patients had higher levels of NET remnants than ANCA-positive patients and a negative correlation was observed between NET remnants and PR3-ANCA (rs = -0.287, P = 0.048). NET remnants correlated with neutrophil count in HCs (rs = 0.503, P = 0.014) but not in patients during remission. Neutrophils from patients showed enhanced spontaneous cell death (P = 0.043).CONCLUSION:We found increased levels of circulating NET remnants in patients with active AAV. Furthermore, AAV patients exhibited an increased propensity for spontaneous cell death. NET remnant levels seem to be positively related to disease activity and neutrophil count, but inversely related to ANCA at least during remission.

BACKGROUND:Imatinib mesylate (Glivec®, formerly STI-571) is a selective tyrosine kinase inhibitor used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, there are reports suggesting that imatinib could be atheroprotective by lowering plasma low-density lipoprotein (LDL).AIM:To investigate the potential inhibitory effect of imatinib on cholesterol uptake in human macrophages as well as its effect on matrix metalloproteinase (MMP) activity.METHODS AND RESULTS:Uptake of fluorescence-labeled LDL was analyzed using flow cytometry. Macrophages treated with imatinib showed a 23.5%, 27%, and 15% decrease in uptake of native LDL (p<0.05), acetylated LDL (p<0.01), and copper-modified oxidized LDL (p<0.01), respectively. Gel-based zymography showed that secretion and activity of MMP-2 and MMP-9 were inhibited by imatinib. Using GeneChip Whole Transcript Expression array analysis, no obvious gene candidates involved in the mechanisms of cholesterol metabolism or MMP regulation were found to be affected by imatinib. Instead, we found that imatinib up-regulated microRNA 155 (miR155) by 43.8% and down-regulated ADAM metallopeptidase domain 28 (ADAM28) by 41.4%. Both genes could potentially play an atheroprotective role and would be interesting targets in future studies.CONCLUSION:Our results indicate that imatinib causes post-translational inhibition with respect to cholesterol uptake and regulation of MMP-2 and MMP-9. More research is needed to further evaluate the role of imatinib in the regulation of other genes and processes.

A successful pregnancy requires that the maternal immune system adapts to tolerate the semi-allogeneic fetal-placental unit. This adaptation mainly occurs locally, i.e. at the fetal-maternal interface, where fetal-derived tissues come into close contact with maternal cells in the uterine endometrium (called decidua during pregnancy). Macrophages and regulatory T (Treg) cells are maternal immune cells that are enriched in the decidua and they likely play a central role in promoting fetal tolerance. However, the precise function of decidual macrophages and the factors regulating both macrophages and Treg cells in humans are unknown. The aim of this thesis was to characterize the phenotype and function of decidual macrophages from first trimester human pregnancy and to identify factors responsible for inducing tolerogenic properties in both decidual macrophages and Treg cells. CD14+ decidual macrophages showed characteristics of immune suppressive or homeostatic macrophages (expression of CD163, CD206 and CD209), mainly produced immunosuppressive cytokines, like IL-10 and IL-35, while levels of inflammatory cytokines, for instance IL-12 and IL-23, were low. Decidual macrophages also induced the expansion of CD25highFoxp3+ Treg cells, but not of Th1, Th2 and Th17 cells, in vitro. In addition, decidual macrophages preferentially secreted the monocyte- and Treg cell-associated chemokines CCL2 and CCL18, while Th1-, Th2- and Th17-related chemokines were produced at low levels. These results suggest that decidual macrophages contribute to create the unique decidual cell composition and a tolerogenic immune environment that is compatible with fetal development. Further, by comparing decidual macrophages with different in vitro macrophage subsets, we showed that M-CSF and IL-10, but not GM-CSF, Th1 or Th2 stimuli, induced macrophages that resemble decidual macrophages in terms of cell surface marker expression, cytokine andchemokine production and gene expression profile. First trimester placental tissue, in particular placental trophoblast cells, was identified as an important source of M-CSF and IL-10. We also demonstrated that human fetal-derived placental tissue can induce the characteristics of decidual macrophages (CD163+CD206+CD209+IL-10+CCL18+) and the selective expansion of functionally suppressive CD25highFoxp3+ Treg cells, the latter partly mediated through IL-10, TGF-β and TRAIL. The placenta also limited activation of Th cells, for instance by generally reduced cytokine production. Our data show that the placenta has a unique ability to induce tolerogenic immune cells with a reduced inflammatory potential, which is essential for maintaining tissue integrity and preventing inflammation-induced fetal loss.

SummaryAnti-neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA-associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3–ANCA-positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P = 0·019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N-terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.