Myeloid expression of the anti-apoptotic protein Mcl1 is required in anti-myeloperoxidase vasculitis but myeloperoxidase inhibition is not protective.

Antibodies to neutrophil and monocyte myeloperoxidase and proteinase 3 are a feature of anti-neutrophil cytoplasmic antibody vasculitis, a disease with significant morbidity for which new treatments are needed. Mice with a myeloid-specific deletion of the anti-apoptotic protein Mcl1 have reduced numbers of circulating neutrophils. Here, we assessed if myeloid-specific Mcl1 was required in murine anti-myeloperoxidase vasculitis and whether inhibition of myeloperoxidase was protective. In a murine model of anti-neutrophil cytoplasmic antibody vasculitis, induced by anti-myeloperoxidase antibody, mice with a myeloid-specific deletion of Mcl1 were protected from disease. They had fewer crescents, neutrophils, and macrophages in the glomeruli, lower serum creatinine levels and reduced albuminuria compared with controls. At baseline and day six after disease induction they had fewer circulating neutrophils than controls. At day six there were also fewer circulating monocytes. Myeloperoxidase inhibition with AZD5904 had no effect on histological or biochemical parameters of disease, and there was also no reduction in albuminuria at day one, two, five or seven after disease induction. These findings persisted when disease was induced without granulocyte-colony stimulating factor, which increases disease severity. A second myeloperoxidase inhibitor, AZM198, also showed no evidence of an effect, although both AZD5904 and AZM198 inhibited human neutrophil extracellular trap formation in vitro. Thus, our results show that while myeloid-specific Mcl1 is required in this model of anti-myeloperoxidase vasculitis, myeloperoxidase inhibition is not protective.

Antimyeloperoxidase antibodies modulate inflammatory responses and activate profibrotic pathways in human monocytes.

Antimyeloperoxidase (anti-MPO) and antiproteinase 3 (anti-PR3) antibodies are found in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). We investigated the effect of both anti-MPO and anti-PR3 IgG on human monocytes. Peripheral blood monocytes were cultured under a range of conditions that included TLR agonists, anti-MPO IgG and anti-PR3 IgG with appropriate controls. Experiments included whole transcriptome profiling and an assessment of the role of Fc receptors. When monocytes were stimulated with LPS or R848, anti-MPO but not anti-PR3 IgG, caused a reduction in IL-10 secretion and had a profound effect on cell-surface marker expression. Anti-MPO but not anti-PR3 IgG enhanced monocyte survival in the absence of TLR stimulation. These effects depended on the Fc receptor CD32a. With TLR stimulation, the effect of anti-MPO but not anti-PR3 IgG on the transcriptional response at 6 h was variable, but we identified a core set of transcripts likely to be important. Without TLR stimulation, there was a robust effect of anti-MPO but not anti-PR3 IgG on the transcriptional response at 24 h, and there was a highly significant enrichment of genes encoding extracellular matrix and extracellular matrix-associated proteins. Analysis with nCounter confirmed many of the differentially expressed transcripts and supported a role for CD32a. These data show that anti-MPO, but not anti-PR3 IgG, from patients with AAV has wide-ranging effects on monocytes which depend on CD32a. The activation of a profibrotic transcriptional response by anti-MPO but not anti-PR3 IgG may give insights into the differences in disease phenotype.

Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays.

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis-cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.

Phosphatidylinositol 3-Kinase δ Deficiency Protects From Antimyeloperoxidase Vasculitis.

OBJECTIVE: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a systemic autoimmune disease in which glomerulonephritis is an important manifestation. Antibodies against myeloperoxidase (MPO) or proteinase 3 are thought to be important in pathogenesis. Phosphoinositide 3-kinase δ (PI3Kδ) mediates a number of effects in lymphocytes, but its role in myeloid cell responses is less clear. Therefore, this study was undertaken to assess this in a preclinical model of glomerulonephritis induced by the transfer of antibodies to MPO. METHODS: D910A mice with inactive PI3Kδ were compared with wild-type controls. Disease protocols allowed for a comparison of experimental groups in the setting of both mild and more severe disease. Adoptive transfer experiments were performed, with flow cytometric analysis of digested kidneys taken at the end of the experiment. RESULTS: With mild disease, D910A mice had fewer glomerular macrophages, fewer glomerular neutrophils, and reduced albuminuria compared with wild-type controls. With more severe disease, they also had fewer glomerular crescents and lower serum creatinine levels, indicating protection from acute kidney injury. Adoptive transfer experiments showed a defect in the recruitment of D910A monocytes to the diseased kidney. CONCLUSION: Mice with inactive PI3Kδ were protected from anti-MPO vasculitis. This is due to cell intrinsic defect in the recruitment of monocytes to the kidney. These findings suggest that PI3Kδ is a potential therapeutic target in AAV.

Experimentally Induced Anti-Myeloperoxidase Vasculitis Is Not Attenuated in Factor B or VISTA Deficient Mice.

Background: Anti-neutrophil cytoplasmic antibody vasculitis is characterized by antibodies to myeloperoxidase or proteinase 3. Previous work in murine anti-myeloperoxidase vasculitis has shown a role for the alternative pathway complement component factor B and the anaphylatoxin C5a. However, mice deficient in properdin, which stabilizes the alternative pathway convertase, were not protected. V-Type immunoglobulin domain-containing suppressor of T-cell activation (VISTA)-deficient mice were protected in the nephrotoxic nephritis model but the role of VISTA in anti-myeloperoxidase vasculitis is unknown. Objectives: This study had 2 aims. First, we attempted to reproduce previous findings on the role of factor B in anti-myeloperoxidase vasculitis. Second, we examined the role of VISTA in this model, in order to see if the protection in the nephrotoxic nephritis model extended to anti-myeloperoxidase vasculitis. Methods: Anti-myeloperoxidase vasculitis was induced in wild type, factor B, or VISTA deficient mice. Disease was assessed by quantifying glomerular crescents and macrophages, in addition to albuminuria and serum creatinine. Results: When wild type and factor B deficient mice were compared, there were no differences in any of the histological or biochemical parameters of disease assessed. Similarly, when wild type or VISTA deficient mice were compared, there were no differences. Conclusions: Factor B deficient mice were not protected which is in contrast to previous studies. Therefore alternative pathway activation is not essential in this model, under the conditions used in this study. VISTA deficient mice were not protected, suggesting that therapies targeting VISTA may not be effective in vasculitis.

Evaluation of genotoxicity in gills and hemolymph of clam Ruditapes decussatus fed with the toxic dinoflagellate Prorocentrum lima.

Diarrheic shellfish poisoning (DSP) is a gastrointestinal (GIT) disease that appears a few hours after ingesting okadaic acid (OA)-contaminated mollusks; okadaic acid is present in dinoflagellates of the genera Dinophysis and Prorocentrum. Toxic manifestations occur all year round at a higher or lesser intensity, and as a consequence, extractive production factories need to be closed during these periods which affects the economy of aquaculture industries. Although the concentration of harmful algae is usually found at high levels in clam digestive gland, bivalve mortality was not increased. In this study, the genotoxic effects produced by OA in clam Ruditapes decussatus were determined using the comet assay. In vitro (exposing hemocytes to different concentrations of OA) and in vivo (feeding clams with toxic dinoflagellate Prorocentrum lima) experiments were conducted in order to determine the genotoxic effects of OA on bivalve cells. Hemocytes and gill cells were analyzed by in vivo and in vitro approaches. While the in vitro study showed a rapid effect of OA on hemocytes, data obtained in the in vivo experiment reflected contradictory results dependent upon the concentration of OA and cell type evaluated. An increase in DNA damage was observed at the lower concentration and only in gill tissue. The results obtained may contribute to a better understanding of the mechanisms underlying genotoxic effects induced by OA on bivalves.

Comparison between two bivalve species as tools for the assessment of pollution levels in an estuarian environment.

Estuaries are semi-enclosed marine areas with water short residence times. Estuary ecosystems show a higher susceptibility to contamination, as historically these sites are linked to urban and industrial development. Polycyclic aromatic hydrocarbons (PAH) are ubiquitous contaminants present in high quantities in these marine environments. Chemical analyses of sediments provides information regarding PAH pollution levels but not a direct measure of the toxicological effects attributed to these contaminants. Samples of sediments and of two bivalve species, Cerastoderma edule and Mytilus galloprovincialis, were collected from two locations (Corcubión and A Concha) in an estuary from northwestern Spain. The PAH levels in sediment and bivalve species and possible sources were determined. A moderate level and a low level of pyrogenic PAH contamination were observed in Corcubión and in A Concha, respectively. Genotoxic damage was evaluated in gills and hemocytes from mussels and cockles by means of the comet assay. DNA damage measured as DNAt values showed a reliable relationship with pollution load levels of the two sampling sites. The higher sensitivity of C. edule compared to M. galloprovincialis enables one to recommend including another species coupled with mussels for biomonitoring estuarine environments.

Unbiased high-throughput characterization of mussel transcriptomic responses to sublethal concentrations of the biotoxin okadaic acid.

Background. Harmful Algal Blooms (HABs) responsible for Diarrhetic Shellfish Poisoning (DSP) represent a major threat for human consumers of shellfish. The biotoxin Okadaic Acid (OA), a well-known phosphatase inhibitor and tumor promoter, is the primary cause of acute DSP intoxications. Although several studies have described the molecular effects of high OA concentrations on sentinel organisms (e.g., bivalve molluscs), the effect of prolonged exposures to low (sublethal) OA concentrations is still unknown. In order to fill this gap, this work combines Next-Generation sequencing and custom-made microarray technologies to develop an unbiased characterization of the transcriptomic response of mussels during early stages of a DSP bloom. Methods. Mussel specimens were exposed to a HAB episode simulating an early stage DSP bloom (200 cells/L of the dinoflagellate Prorocentrum lima for 24 h). The unbiased characterization of the transcriptomic responses triggered by OA was carried out using two complementary methods of cDNA library preparation: normalized and Suppression Subtractive Hybridization (SSH). Libraries were sequenced and read datasets were mapped to Gene Ontology and KEGG databases. A custom-made oligonucleotide microarray was developed based on these data, completing the expression analysis of digestive gland and gill tissues. Results. Our findings show that exposure to sublethal concentrations of OA is enough to induce gene expression modifications in the mussel Mytilus. Transcriptomic analyses revealed an increase in proteasomal activity, molecular transport, cell cycle regulation, energy production and immune activity in mussels. Oppositely, a number of transcripts hypothesized to be responsive to OA (notably the Serine/Threonine phosphatases PP1 and PP2A) failed to show substantial modifications. Both digestive gland and gill tissues responded similarly to OA, although expression modifications were more dramatic in the former, supporting the choice of this tissue for future biomonitoring studies. Discussion. Exposure to OA concentrations within legal limits for safe consumption of shellfish is enough to disrupt important cellular processes in mussels, eliciting sharp transcriptional changes as a result. By combining the study of cDNA libraries and a custom-made OA-specific microarray, our work provides a comprehensive characterization of the OA-specific transcriptome, improving the accuracy of the analysis of expresion profiles compared to single-replicated RNA-seq methods. The combination of our data with related studies helps understanding the molecular mechanisms underlying molecular responses to DSP episodes in marine organisms, providing useful information to develop a new generation of tools for the monitoring of OA pollution.

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis.

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1ß p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.

Effects of okadaic acid on haemocytes from Mytilus galloprovincialis: a comparison between field and laboratory studies.

Individuals of Mytilus galloprovincialis, contaminated with Diarrheic Shellfish Poisoning (DSP) toxins, were studied with the aim to correlate the okadaic acid (OA) body burden and the percentage of damaged haemocytes by quantifying annexin V positive cells by flow cytometry. Results showed less percentage of damaged haemocytes in high OA contaminated samples. These data were compared with results of in vitro assays of mussel haemocytes exposed to increased concentrations of OA. Similarly, haemocytes exposed to the most concentrated OA solution were less damaged.