Amelioration of Murine Autoimmune Pancreatitis by Targeted LTßR Inhibition and Anti-CD20 Treatment.

Autoimmune pancreatitis (AIP) is a rare form of chronic pancreatitis, for which treatment options, especially the long-term management, are limited. The only therapy that has been established and accepted so far is corticosteroids, but the relapse rate is significant. In the current study, we discern the effector mechanisms of targeted LTßR pathway inhibition using LTßR-Ig. Furthermore, the efficacy of LTßR-Ig therapy is compared with the depletion of immune cell subsets (CD4+ and CD20+), which are suggested to play a pathological role in AIP development. Three well-established mouse models of AIP were used to examine treatment efficacies and mechanisms. Tg(Ela1-Lta,b) mice represent a genetic model, in which AIP develops spontaneously. In MRL/Mp and IL-10-/- mice, AIP is induced by repeated polyinosinic:polycytidylic acid injection. Mice with AIP were treated with anti-CD20, anti-CD4 mAbs, or targeted LTßR-Ig. LTßR-Ig and anti-CD20 treatment led to significant improvement of AIP, including a decrease in autoantibody production and pancreatic inflammation in Tg(Ela1-Lta,b) and IL-10-/- mice. The molecular mechanism of this beneficial effect possibly involves the downregulation of Stat3 and noncanonical NF-κb activation. Anti-CD4 treatment reduced Th1 and Th2 signature but did not alleviate AIP. Additionally, in contrast to anti-CD20 or anti-CD4 treatments, blocking LTßR signaling disrupted tertiary lymphoid organs in all three models. We demonstrate that treatment with LTßR-Ig or anti-CD20 Ab alleviated murine AIP. LTßR-Ig treatment for AIP was effective in both lymphotoxin-dependent and lymphotoxin-independent AIP models, possibly because of its dual anti-inflammatory and antiautoimmune mechanisms.

Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A.

Excessive or aberrant generation of neutrophil extracellular traps (NETs) has recently become implicated in the underlying aetiology of a number of human pathologies including preeclampsia, systemic lupus erythromatosus, rheumatoid arthritis, auto-antibody induced small vessel vasculitis, coagulopathies such as deep vein thrombosis or pulmonary complications. These results imply that effective pharmacological therapeutic strategies will need to be developed to counter overt NETosis in these and other inflammatory disorders. As calcium flux is implicated in the generation of reactive oxygen species and histone citrullination, two key events in NETosis, we analysed the roles of both extra- and intracellular calcium pools and their modulation by pharmacological agents in the NETotic process in detail. Interleukin-8 (IL-8) was used as a physiological stimulus of NETosis. Our data demonstrate that efficient induction of NETosis requires mobilisation of both extracellular and intracellular calcium pools. Since modulation of the calcineurin pathway by cyclosporine A has been described in neutrophils, we investigated its influence on NETosis. Our data indicate that IL-8 induced NETosis is reduced by ascomycin and cyclosporine A, antagonists of the calcineurin pathway, but not following treatment with rapamycin, which utilizes the mTOR pathway. The action of the G protein coupled receptor phospholipase C pathway appears to be essential for the induction of NETs by IL-8, as NETosis was diminished by treatment with either pertussis toxin, a G-protein inhibitor, the phospholipase C inhibitor, U73122, or staurosporine, an inhibitor of protein kinase C. The data regarding the calcineurin antagonists, ascomycin and cyclosporine A, open the possibility to therapeutically suppress or modulate NETosis. They also provide new insight into the mechanism whereby such immune suppressive drugs render transplant patients susceptible to opportunistic fungal infections.

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death.

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co-culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL-8 by activated EC. Extended neutophil/EC co-culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia.

Decrease in lipid levels of syncytiotrophoblast micro-particles reduced their potential to inhibit endothelial cell proliferation.

BACKGROUND: Preeclampsia is characterized by damage to the maternal endothelium that has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. Previously, we have shown that STBM, prepared by three different methods: mechanical dissection, in vitro placental explants culture and perfusion of placenta, can inhibit endothelial cell proliferation. Only mechanically prepared STBM induced apoptosis in the endothelial cells. Now, we have examined lipid levels in the three STBM preparations and their differential responses on endothelial cells. METHODS: We examined the lipid levels in the three STBM preparations using thin layer chromatography. Furthermore, the effects of reduced lipid levels in the three STBM preparations using the pharmacological agent methyl-beta-cyclodextrin were examined on endothelial cell proliferation and apoptosis. RESULTS: Among the three STBM preparations, mechanical STBM contained highest levels of lipids. The reduction in lipid levels in mechanical STBM reduced their potential to inhibit human umbilical vein endothelial cells (HUVEC) proliferation and blocked their potential to induce apoptosis. No similar effect was observed following lipid reduction in the two other STBM preparations. CONCLUSIONS: As it has been suggested that mechanically derived STBM may more closely resemble placental micro-particles generated in preeclampsia, our data suggest that lipid content may play a role in the anti-endothelial defects present in this disease.

Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis.

BACKGROUND: Increased concentrations of cell-free DNA have been found in several disorders and have been interpreted as evidence of increased rates of cell death or turnover. Evidence from in vitro and animal experiments suggests that DNA may play a role in the pathogenesis of rheumatoid arthritis (RA). METHODS: We measured cell-free DNA in plasma and serum from patients with RA and healthy controls by use of quantitative PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA. We used protein G Sepharosetrade mark bead adsorption of plasma and elution to isolate antibody-bound DNA. RESULTS: In paired plasma and serum samples of 16 healthy controls the median GAPDH copies were 4500 genome equivalents (GE)/mL plasma (range 319-21 000) and in 26 RA patients 17 000 GE/mL plasma (2100-2 375 000, P = 0.0001). In the serum from normal controls the median GAPDH copies were 35 000 GE/mL (1700-239 000) and from RA patients 222 000 GE/mL (21 000-2 375 000, P = 0.004). A median of 81% of the cell-free DNA in RA was associated with antibody compared with 9% in healthy controls (P = 0.001). The concentrations of DNA did not vary with the type of therapy patients received. CONCLUSIONS: These results provide new evidence for a role of cell-free DNA-antibody complexes in the etiology of RA, suggest new avenues for basic research, and may prove to be relevant to diagnosis and assessment of therapy.

Neutrophil NETs: a novel contributor to preeclampsia-associated placental hypoxia?

Recent studies have suggested that the innate immune system is involved in the pathogenesis of preeclampsia. Its pathogenesis involves neutrophil activation and increased levels of cell-free DNA in the maternal plasma. Activation of neutrophils has recently been shown to induce DNA containing neutrophil extracellular traps (NETs) which trap and kill bacteria. Massive NETs induction by the placentally derived factors (IL-8 and placental micro-debris) and their increased presence in preeclamptic placenta suggest that NETs might be involved in the pathogenesis of preeclampsia. Therefore, increased presence of NETs in preeclampsia may play a role in the deficient placental perfusion associated with this disorder.

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFalpha by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFalpha antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Minimal alteration in the ratio of circulatory fetal DNA to fetal corticotropin-releasing hormone mRNA level in preeclampsia.

OBJECTIVES: We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. METHODS: Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. RESULTS: Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (<34 weeks of gestation) and late (>34 weeks of gestation) onset preeclampsia. CONCLUSIONS: Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered.

Analysis of plasma elastase levels in early and late onset preeclampsia.

BACKGROUND: Circulatory neutrophils have been reported to be activated in preeclampsia. It has been suggested that maternal plasma levels of elastase may serve as a possible cell-free marker to quantify such activation. Although plasma elastase levels have been found to be elevated in cases with manifest preeclampsia and eclampsia, this has not yet been examined in cases with early and late onset preeclampsia. We have now examined this aspect. METHODS: In this retrospective study, maternal plasma samples were examined from eight cases with early onset preeclampsia (<34 weeks of gestation), eight cases with late onset preeclampsia (>34 weeks of gestation) and an equal number of gestational age matched normotensive term controls. Plasma concentrations of elastase were measured by ELISA using a commercially available assay. RESULTS: Plasma elastase concentrations were significantly elevated the preeclampsia study group when compared to the normotensive control group (median=139.2 ng/ml versus median=72.1 ng/ml; P=0.0025). These elevations remained significant when the preeclampsia study group was stratified into case with early onset preeclampsia (median=118.8 ng/ml versus median=62.2 ng/ml; P=0.03), but jailed failed to attain significance for those cases with late onset preeclampsia (median=181.3 ng/ml versus median=86.3 ng/ml; P=0.061). CONCLUSIONS: Our data indicate that elastase levels are elevated in both early and late onset forms of preeclampsia, and imply that the activation of neutrophils may be more acute in the former than in the latter (238 words).

Microparticle-free placentally derived soluble factors downmodulate the response of activated T cells.

Soluble placental factors may have immunoregulatory properties and have been demonstrated to inhibit T-lymphocyte proliferation in vitro. On the other hand, placentally derived syncytiotrophoblast microparticles and crude placental homogenates have been demonstrated to inhibit proliferation of mixed lymphocytes in vitro. Because previous studies on placentally derived soluble factors may have been contaminated by the presence of trophoblast-derived microparticles, we prepared microparticle-free placental supernatants. Such supernatants reduced the activation response of T cells, as well as their proliferation and the production of cytokines such as interleukin-2 and interferon gamma, in a dose-dependent manner. This reduction in T-cell proliferation does not appear to be caused by indoleamine 2,3-dioxygenase (IDO) because it was not reversed by the addition of L-tryptophan or an inhibitor of IDO (1-methyl-DL-tryptophan). No evidence was found for the presence of IDO in these supernatants when we used a biochemical assay measuring tryptophan catabolism. We conclude that the placenta produces currently unknown soluble factors that reduce T-cell activation, proliferation, and cytokine production.

Syncytiotrophoblast micro-particles do not induce apoptosis in peripheral T lymphocytes, but differ in their activity depending on the mode of preparation.

Recent studies have suggested that pre-eclampsia may result from endothelial cell damage and overt immune activity triggered by the elevated release of syncytiotrophoblast-derived micro-particles (STBM). In this context, STBM have been reported to inhibit lymphocyte proliferation and induce Jurkat T cell apoptosis. In this study, STBM were prepared by three different in vitro methods (mechanical dissection, villous explant culture, and placental perfusion) and their functional properties were tested on T lymphocytes enriched from peripheral blood samples. Mechanically- and villous explant-derived STBM significantly inhibited activation, proliferation and cytokine release by T lymphocytes, while placental perfusion-derived STBM significantly induced T cell proliferation and a slight increase in IFNgamma release. None of the STBM preparations caused T cell apoptosis. Therefore, STBM prepared by different methods in vitro exhibit different effects on circulating T cells, a feature that will have to be taken into account when considering their potential role in normal pregnancy and pre-eclampsia.

Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia.

Preeclampsia, a severe pregnancy-related disorder, involves an overt activation of the maternal innate immune system, proposed to result from the elevated release of inflammatory syncytiotrophoblast microparticles (STBM) and cytokines from underlying placental anomaly involving abnormal trophoblast differentiation. Activation of circulating neutrophils has recently been shown to lead to the generation of fibrous extracellular lattices containing DNA, termed NETs (neutrophil extracellular traps). Therefore, we examined whether placentally derived factors activated peripheral neutrophils to generate NETs, and whether NETs formation was increased in preeclamptic placenta. Activation of isolated circulatory neutrophils on treatment with placentally derived factors (interleukin-8 and STBM) was assessed by measuring CD11b expression using Flow cytometry. Furthermore, NETs generation by these activated neutrophils in vitro and in vivo in the placental tissue sections were examined using electron microscopy and fluorescence microscopy. We report that placentally-derived interleukin-8 and STBM efficiently activated neutrophils and triggered NETs formation. Large numbers of NETs were present directly in the intervillous space of preeclamptic placentae. NETs, therefore, appear to be an integral part of neutrophil activation, and their increased presence in preeclampsia suggests that NETs may play a role in the underlying pathology.