Type 2 immune polarization is associated with cardiopulmonary disease in preterm infants.

Postnatal maturation of the immune system is poorly understood, as is its impact on illnesses afflicting term or preterm infants, such as bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension. These are both cardiopulmonary inflammatory diseases that cause substantial mortality and morbidity with high treatment costs. Here, we characterized blood samples collected from 51 preterm infants longitudinally at five time points, 20 healthy term infants at birth and age 3 to 16 weeks, and 5 healthy adults. We observed strong associations between type 2 immune polarization in circulating CD3+CD4+ T cells and cardiopulmonary illness, with odds ratios up to 24. Maternal magnesium sulfate therapy, delayed hepatitis B vaccination, and increasing fetal, but not maternal, chorioamnionitis severity were associated with attenuated type 2 polarization. Blocking type 2 mediators such as interleukin-4 (IL-4), IL-5, IL-13, or signal transducer and activator of transcription 6 (STAT6) in murine neonatal cardiopulmonary disease in vivo prevented changes in cell type composition, increases in IL-1ß and IL-13, and losses of pulmonary capillaries, but not gains in larger vessels. Thereby, type 2 blockade ameliorated lung inflammation, protected alveolar and vascular integrity, and confirmed the pathological impact of type 2 cytokines and STAT6. In-depth flow cytometry and single-cell transcriptomics of mouse lungs further revealed complex associations between immune polarization and cardiopulmonary disease. Thus, this work advances knowledge on developmental immunology and its impact on early life disease and identifies multiple therapeutic approaches that may relieve inflammation-driven suffering in the youngest patients.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of ß2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

Microscopy Methods for Imaging MIF and Its Interaction Partners.

Fluorescence microscopy has become a powerful tool to investigate proteins in their natural environment. Well-established techniques like widefield and confocal fluorescence microscopy have commonly been used for decades to visualize biomolecules in single cells and tissue sections. Live cell microscopy allows for the investigation of biomolecular trafficking, and other specialized techniques, such as proximity ligation assays (PLA) and fluorescence lifetime imaging microscopy (FLIM), can be used to study interactions between biomolecules of interest. Finally, with the most recent rise of optical super-resolution microscopy, we can investigate target biomolecules in situ with unprecedented detail on the nanometer scale. Here, we discuss various optical microscopy techniques that have successfully been used to image MIF. We highlight applications, advantages, and limitations of each technique. The techniques described here can easily be adapted to investigate other target proteins, their localization, interaction partners, and mechanisms of action.

Molecular signature of interleukin-22 in colon carcinoma cells and organoid models.

Interleukin (IL)-22 activates STAT (signal transducer and activator of transcription) 3 and antiapoptotic and proproliferative pathways; but beyond this, the molecular mechanisms by which IL-22 promotes carcinogenesis are poorly understood. Characterizing the molecular signature of IL-22 in human DLD-1 colon carcinoma cells, we observed increased expression of 26 genes, including NNMT (nicotinamide N-methyltransferase, ≤10-fold) and CEA (carcinoembryonic antigen, ≤7-fold), both known to promote intestinal carcinogenesis. ERP27 (endoplasmic reticulum protein-27, function unknown, ≤5-fold) and the proinflammatory ICAM1 (intercellular adhesion molecule-1, ≤4-fold) were also increased. The effect on CEA was partly STAT3-mediated, as STAT3-silencing reduced IL-22-induced CEA by ≤56%. Silencing of CEA or NNMT inhibited IL-22-induced proliferation/migration of DLD-1, Caco-2, and SW480 colon carcinoma cells. To validate these results in primary tissues, we assessed IL-22-induced gene expression in organoids from human healthy colon and colon cancer patients, and from normal mouse small intestine and colon. Gene regulation by IL-22 was similar in DLD-1 cells and human and mouse healthy organoids. CEA was an exception with no induction by IL-22 in organoids, indicating the 3-dimensional organization of the tissue may produce signals absent in 2D cell culture. Importantly, augmentation of NNMT was 5-14-fold greater in human cancerous compared to normal organoids, supporting a role for NNMT in IL-22-mediated colon carcinogenesis. Thus, NNMT and CEA emerge as mediators of the tumor-promoting effects of IL-22 in the intestine. These data advance our understanding of the multifaceted role of IL-22 in the gut and suggest the IL-22 pathway may represent a therapeutic target in colon cancer.

Interleukin-1 Receptor Antagonist Protects Newborn Mice Against Pulmonary Hypertension.

Pulmonary hypertension secondary to bronchopulmonary dysplasia (BPD-PH) represents a major complication of BPD in extremely preterm infants for which there are currently no safe and effective interventions. The abundance of interleukin-1 (IL-1) is strongly correlated with the severity and long-term outcome of BPD infants and we have previously shown that IL-1 receptor antagonist (IL-1Ra) protects against murine BPD; therefore, we hypothesized that IL-1Ra may also be effective against BPD-PH. We employed daily injections of IL-1Ra in a murine model in which BPD/BPD-PH was induced by antenatal LPS and postnatal hyperoxia of 65% O2. Pups reared in hyperoxia for 28 days exhibited a BPD-PH-like disease accompanied by significant changes in pulmonary vascular morphology: micro-CT revealed an 84% reduction in small vessels (4-5 µm diameter) compared to room air controls; this change was prevented by IL-1Ra. Pulmonary vascular resistance, assessed at day 28 of life by echocardiography using the inversely-related surrogate marker time-to-peak-velocity/right ventricular ejection time (TPV/RVET), increased in hyperoxic mice (0.27 compared to 0.32 in air controls), and fell significantly with daily IL-1Ra treatment (0.31). Importantly, in vivo cine-angiography revealed that this protection afforded by IL-1Ra treatment for 28 days is maintained at day 60 of life. Despite an increased abundance of mediators of pulmonary angiogenesis in day 5 lung lysates, namely vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), no difference was detected in ex vivo pulmonary vascular reactivity between air and hyperoxia mice as measured in precision cut lung slices, or by immunohistochemistry in alpha-smooth muscle actin (α-SMA) and endothelin receptor type-A (ETA) at day 28. Further, on day 28 of life we observed cardiac fibrosis by Sirius Red staining, which was accompanied by an increase in mRNA expression of galectin-3 and CCL2 (chemokine (C-C motif) ligand 2) in whole hearts of hyperoxic pups, which improved with IL-1Ra. In summary, our findings suggest that daily administration of the anti-inflammatory IL-1Ra prevents the increase in pulmonary vascular resistance and the pulmonary dysangiogenesis of murine BPD-PH, thus pointing to IL-1Ra as a promising candidate for the treatment of both BPD and BPD-PH.

SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm.

dsRNA is a common by-product of viral replication and acts as a potent trigger of antiviral immunity. SIDT1 and SIDT2 are closely related members of the SID-1 transmembrane family. SIDT2 functions as a dsRNA transporter and is required to traffic internalized dsRNA from endocytic compartments into the cytosol for innate immune activation, but the role of SIDT1 in dsRNA transport and in the innate immune response to viral infection is unclear. In this study, we show that Sidt1 expression is upregulated in response to dsRNA and type I IFN exposure and that SIDT1 interacts with SIDT2. Moreover, similar to SIDT2, SIDT1 localizes to the endolysosomal compartment, interacts with the long dsRNA analog poly(I:C), and, when overexpressed, enhances endosomal escape of poly(I:C) in vitro. To elucidate the role of SIDT1 in vivo, we generated SIDT1-deficient mice. Similar to Sidt2-/- mice, SIDT1-deficient mice produced significantly less type I IFN following infection with HSV type 1. In contrast to Sidt2-/- mice, however, SIDT1-deficient animals showed no impairment in survival postinfection with either HSV type 1 or encephalomyocarditis virus. Consistent with this, we observed that, unlike SIDT2, tissue expression of SIDT1 was relatively restricted, suggesting that, whereas SIDT1 can transport extracellular dsRNA into the cytoplasm following endocytosis in vitro, the transport activity of SIDT2 is likely to be functionally dominant in vivo.

Macrophage migration inhibitory factor is required for NLRP3 inflammasome activation.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1ß, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

The ß-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.

Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury.

BACKGROUND: Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs) have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD. METHODS: Lipopolysaccharide was introduced intra-amniotically at day 16 of pregnancy prior to exposure to 65% oxygen (hyperoxia) at birth. hAECs were administered either 12 hours (early) or 4 days (late) after hyperoxia commenced. Collective lung tissues were subjected to histological analysis, multikine ELISA for inflammatory cytokines, FACS for immune cell populations and 3D lung stem cell culture at neonatal stage (postnatal day 7 and 14). Invasive lung function test and echocardiography were applied at 6 and 10 weeks of age. RESULTS: hAECs improved the tissue-to-airspace ratio and septal crest density in a dose-dependent manner, regardless of administration route. Early administration of hAECs, coinciding with the commencement of postnatal hyperoxia, was associated with reduced macrophages, dendritic cells and natural killer cells. This was not the case if hAECs were administered when lung injury was established. Fittingly, early hAEC treatment was more efficacious in reducing interleukin-1ß, tumour necrosis factor alpha and monocyte chemoattractant protein-1 levels. Early hAEC treatment was also associated with reduced airway hyper-responsiveness and normalisation of pressure-volume loops. Pulmonary hypertension and right ventricle hypertrophy were also prevented in the early hAEC treatment group, and this persisted until 10 weeks of age. CONCLUSIONS: Early hAEC treatment appears to be advantageous over late treatment. There was no difference in efficacy between intravenous and intratracheal administration. The benefits of hAEC administration resulted in long-term improvements in cardiorespiratory function.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Near-infrared autofluorescence induced by intraplaque hemorrhage and heme degradation as marker for high-risk atherosclerotic plaques.

Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology that allows identification of atherosclerotic plaques with intraplaque hemorrhage and ultimately holds promise for detection of high-risk plaques in patients.Atherosclerosis diagnosis relies primarily on imaging and early detection of high-risk atherosclerotic plaques is important for risk stratification of patients and stabilization therapies. Here Htun et al. demonstrate that vulnerable atherosclerotic plaques generate near-infrared autofluorescence that can be detected via emission computed tomography.

Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria.

Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i) outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii) the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii) translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.

Ovine multiparity is associated with diminished vaginal muscularis, increased elastic fibres and vaginal wall weakness: implication for pelvic organ prolapse.

Pelvic Organ Prolapse (POP) is a major clinical burden affecting 25% of women, with vaginal delivery a major contributing factor. We hypothesised that increasing parity weakens the vagina by altering the extracellular matrix proteins and smooth muscle thereby leading to POP vulnerability. We used a modified POP-quantification (POP-Q) system and a novel pressure sensor to measure vaginal wall weakness in nulliparous, primiparous and multiparous ewes. These measurements were correlated with histological, biochemical and biomechanical properties of the ovine vagina. Primiparous and multiparous ewes had greater displacement of vaginal tissue compared to nulliparous at points Aa, Ap and Ba and lower pressure sensor measurements at points equivalent to Ap and Ba. Vaginal wall muscularis of multiparous ewes was thinner than nulliparous and had greater elastic fibre content. Collagen content was lower in primiparous than nulliparous ewes, but collagen organisation did not differ. Biomechanically, multiparous vaginal tissue was weaker and less stiff than nulliparous. Parity had a significant impact on the structure and function of the ovine vaginal wall, as the multiparous vaginal wall was weaker and had a thinner muscularis than nulliparous ewes. This correlated with "POP-Q" and pressure sensor measurements showing greater tissue laxity in multiparous compared to nulliparous ewes.

PRC2 is required for extensive reorganization of H3K27me3 during epigenetic reprogramming in mouse fetal germ cells.

BACKGROUND: Defining how epigenetic information is established in the germline during fetal development is key to understanding how epigenetic information is inherited and impacts on evolution and human health and disease. RESULTS: Here, we show that Polycomb Repressive Complex 2 is transiently localized in the nucleus of mouse fetal germ cells, while DNA methylation is removed from the germline. This coincides with significant enrichment of trimethylated lysine 27 on histone 3 near the nuclear lamina that is dependent on activity of the essential PRC2 catalytic proteins, Enhancer of Zeste 1 and/or 2. CONCLUSIONS: Combined, these data reveal a role for Polycomb Repressive Complex 2 and trimethylated lysine 27 on histone 3 during germline epigenetic programming that we speculate is required to repress target sequences while DNA methylation is removed.

Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics.

Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology.

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

Human Endometrial Exosomes Contain Hormone-Specific Cargo Modulating Trophoblast Adhesive Capacity: Insights into Endometrial-Embryo Interactions.

Embryo implantation into receptive endometrium requires synergistic endometrial-blastocyst interactions within the uterine cavity and is essential for establishing pregnancy. We demonstrate that exosomes (40-150 nm nanovesicles) released from endometrial epithelial cells are an important component of these interactions. We defined the proteome of purified endometrial epithelial-derived exosomes (Exos) influenced by menstrual cycle hormones estrogen (E; proliferative phase) and estrogen plus progesterone (EP; receptive phase) and examined their potential to modify trophoblast function. E-/EP-Exos were uniquely enriched with 254 and 126 proteins, respectively, with 35% newly identified proteins not previously reported in exosome databases. Importantly, EP-Exos protein cargo was related to fundamental changes in implantation: adhesion, migration, invasion, and extracellular matrix remodeling. These findings from hormonally treated ECC1 endometrial cancer cells were validated in human primary uterine epithelial cell-derived exosomes. Functionally, exosomes were internalized by human trophoblast cells and enhanced their adhesive capacity, a response mediated partially through active focal adhesion kinase (FAK) signaling. Thus, exosomes contribute to the endometrial-embryo interactions within the human uterine microenvironment essential for successful implantation.

Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.

Myeloperoxidase (MPO) is an important neutrophil lysosomal enzyme, a major autoantigen, and a potential mediator of tissue injury in MPO-ANCA-associated vasculitis (MPO-AAV) and glomerulonephritis. Here we examined MPO deposition in kidney biopsies from 47 patients with MPO-AAV. Leukocyte accumulation and fibrin deposition consistent with cell-mediated immunity was a major feature. Tubulointerstitial macrophage, CD4+ and CD8+ T-cell, and neutrophil numbers correlated with low presenting eGFR. MPO was not detected in kidneys from patients with minimal change or thin basement membrane disease, but was prominent in glomerular, periglomerular, and tubulointerstitial regions in MPO-AAV. Extracellular MPO released from leukocytes was pronounced in all MPO-AAV patients. Similar numbers of neutrophils and macrophages expressed MPO in the kidneys, but colocalization studies identified neutrophils as the major source of extracellular MPO. Extraleukocyte MPO was prominent in neutrophil extracellular traps in the majority of patients; most of which had traps in half or more glomeruli. These traps were associated with more neutrophils and more MPO within glomeruli. Glomerular MPO-containing macrophages generated extracellular trap-like structures. MPO also localized to endothelial cells and podocytes. The presence of the most active glomerular lesions (both segmental necrosis and cellular crescents) correlated with intraglomerular CD4+ cells and MPO+ macrophages. Thus, cellular and extracellular MPO may cause glomerular and interstitial injury.

Analysis of ER-mitochondria contacts using correlative fluorescence microscopy and soft X-ray tomography of mammalian cells.

Mitochondrial fission is important for organelle transport, quality control and apoptosis. Changes to the fission process can result in a wide variety of neurological diseases. In mammals, mitochondrial fission is executed by the GTPase dynamin-related protein 1 (Drp1; encoded by DNM1L), which oligomerizes around mitochondria and constricts the organelle. The mitochondrial outer membrane proteins Mff, MiD49 (encoded by MIEF2) and MiD51 (encoded by MIEF1) are involved in mitochondrial fission by recruiting Drp1 from the cytosol to the organelle surface. In addition, endoplasmic reticulum (ER) tubules have been shown to wrap around and constrict mitochondria before a fission event. Up to now, the presence of MiD49 and MiD51 at ER-mitochondrial division foci has not been established. Here, we combine confocal live-cell imaging with correlative cryogenic fluorescence microscopy and soft x-ray tomography to link MiD49 and MiD51 to the involvement of the ER in mitochondrial fission. We gain further insight into this complex process and characterize the 3D structure of ER-mitochondria contact sites.