B-1b Cells Have Unique Functional Traits Compared to B-1a Cells at Homeostasis and in Aged Hyperlipidemic Mice With Atherosclerosis.

Immunoglobulin M (IgM) to oxidation specific epitopes (OSE) are inversely associated with atherosclerosis in mice and humans. The B-1b subtype of B-1 cells secrete IgM to OSE, and unlike B-1a cells, are capable of long-lasting IgM memory. What attributes make B-1b cells different than B-1a cells is unknown. Our objectives were to determine how B-1b cells produce more IgM compared to B-1a cells at homeostatic condition and to see the differences in the B-1a and B-1b cell distribution and IgM CDR-H3 sequences in mice with advanced atherosclerosis. Here, in-vivo studies demonstrated greater migration to spleen, splenic production of IgM and plasma IgM levels in ApoE-/-Rag1-/- mice intraperitoneally injected with equal numbers of B-1b compared to B-1a cells. Bulk RNA seq analysis and flow cytometry of B-1a and B-1b cells identified CCR6 as a chemokine receptor more highly expressed on B-1b cells compared to B-1a. Knockout of CCR6 resulted in reduced B-1b cell migration to the spleen. Moreover, B-1b cell numbers were significantly higher in spleen of aged atherosclerotic ApoE-/- mice compared to young ApoE-/- mice. Single cell sequencing results of IgHM in B-1a and B-1b cells from peritoneal cavity and spleen of atherosclerotic aged ApoE-/- mice revealed significantly more N additions at the V-D and D-J junctions, greater diversity in V region usage and CDR-H3 sequences in B-1b compared to B-1a cells. In summary, B-1b cells demonstrated enhanced CCR6-mediated splenic migration, IgM production, and IgM repertoire diversification compared to B-1a cells. These findings suggest that potential strategies to selectively augment B-1b cell numbers and splenic trafficking could lead to increased and more diverse IgM targeting OSE to limit atherosclerosis.

Mesenchymal Stem Cells Alter MicroRNA Expression and Attenuate Thoracic Aortic Aneurysm Formation.

BACKGROUND: Thoracic aortic aneurysms (TAA) are a progressive disease characterized by inflammation, smooth muscle cell activation and matrix degradation. We hypothesized that mesenchymal stem cells (MSCs) can immunomodulate vascular inflammation and remodeling via altered microRNA (miRNAs) expression profile to attenuate TAA formation. MATERIALS AND METHODS: C57BL/6 mice underwent topical elastase application to form descending TAAs. Mice were also treated with MSCs on days 1 and 5 and aortas were analyzed on day 14 for aortic diameter. Cytokine array was performed in aortic tissue and total RNA was tagged and hybridized for miRNAs microarray analysis. Immunohistochemistry was performed for elastin degradation and leukocyte infiltration. RESULTS: Treatment with MSCs significantly attenuated aortic diameter and TAA formation compared to untreated mice. MSC administration also attenuated T-cell, neutrophil and macrophage infiltration and prevented elastic degradation to mitigate vascular remodeling. MSC treatment also attenuated aortic inflammation by decreasing proinflammatory cytokines (CXCL13, IL-27, CXCL12 and RANTES) and upregulating anti-inflammatory interleukin-10 expression in aortic tissue of elastase-treated mice. TAA formation demonstrated activation of specific miRNAs that are associated with aortic inflammation and vascular remodeling. Our results also demonstrated that MSCs modulate a different set of miRNAs that are associated with decrease leukocyte infiltration and vascular inflammation to attenuate the aortic diameter and TAA formation. CONCLUSIONS: These results indicate that MSCs immunomodulate specific miRNAs that are associated with modulating hallmarks of aortic inflammation and vascular remodeling of aortic aneurysms. Targeted therapies designed using MSCs and miRNAs have the potential to regulate the growth and development of TAAs.

An Integrated Transcriptomic Approach to Identify Molecular Markers of Calcineurin Inhibitor Nephrotoxicity in Pediatric Kidney Transplant Recipients.

Calcineurin inhibitors are highly efficacious immunosuppressive agents used in pediatric kidney transplantation. However, calcineurin inhibitor nephrotoxicity (CNIT) has been associated with the development of chronic renal allograft dysfunction and decreased graft survival. This study evaluated 37 formalin-fixed paraffin-embedded biopsies from pediatric kidney transplant recipients using gene expression profiling. Normal allograft samples (n = 12) served as negative controls and were compared to biopsies exhibiting CNIT (n = 11). The remaining samples served as positive controls to validate CNIT marker specificity and were characterized by other common causes of graft failure such as acute rejection (n = 7) and interstitial fibrosis/tubular atrophy (n = 7). MiRNA profiles served as the platform for data integration. Oxidative phosphorylation and mitochondrial dysfunction were the top molecular pathways associated with overexpressed genes in CNIT samples. Decreased ATP synthesis was identified as a significant biological function in CNIT, while key toxicology pathways included NRF2-mediated oxidative stress response and increased permeability transition of mitochondria. An integrative analysis demonstrated a panel of 13 significant miRNAs and their 33 CNIT-specific gene targets involved with mitochondrial activity and function. We also identified a candidate panel of miRNAs/genes, which may serve as future molecular markers for CNIT diagnosis as well as potential therapeutic targets.

Chemokine Receptor-6 Promotes B-1 Cell Trafficking to Perivascular Adipose Tissue, Local IgM Production and Atheroprotection.

Chemokine receptor-6 (CCR6) mediates immune cell recruitment to inflammatory sites and has cell type-specific effects on diet-induced atherosclerosis in mice. Previously we showed that loss of CCR6 in B cells resulted in loss of B cell-mediated atheroprotection, although the B cell subtype mediating this effect was unknown. Perivascular adipose tissue (PVAT) harbors high numbers of B cells including atheroprotective IgM secreting B-1 cells. Production of IgM antibodies is a major mechanism whereby B-1 cells limit atherosclerosis development. Yet whether CCR6 regulates B-1 cell number and production of IgM in the PVAT is unknown. In this present study, flow cytometry experiments demonstrated that both B-1 and B-2 cells express CCR6, albeit at a higher frequency in B-2 cells in both humans and mice. Nevertheless, B-2 cell numbers in peritoneal cavity (PerC), spleen, bone marrow and PVAT were no different in ApoE-/-CCR6-/- compared to ApoE-/-CCR6+/+ mice. In contrast, the numbers of atheroprotective IgM secreting B-1 cells were significantly lower in the PVAT of ApoE-/-CCR6-/- compared to ApoE-/-CCR6+/+ mice. Surprisingly, adoptive transfer (AT) of CD43- splenic B cells into B cell-deficient µMT-/-ApoE-/- mice repopulated the PerC with B-1 and B-2 cells and reduced atherosclerosis when transferred into ApoE-/-CCR6+/+sIgM-/- mice only when those cells expressed both CCR6 and sIgM. CCR6 expression on circulating human B cells in subjects with a high level of atherosclerosis in their coronary arteries was lower only in the putative human B-1 cells. These results provide evidence that B-1 cell CCR6 expression enhances B-1 cell number and IgM secretion in PVAT to provide atheroprotection in mice and suggest potential human relevance to our murine findings.

Clinical and Genetic Risk Factors of Recurrent Nonalcoholic Fatty Liver Disease After Liver Transplantation.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) has been increasingly reported among recipients of liver transplantation (LT). We aimed to identify clinical and genetic risk factors responsible for the development of early recurrent NAFLD in nonalcoholic steatohepatitis transplant recipients. METHODS: Forty-six total single nucleotide polymorphisms with known association with NAFLD were tested among both recipient and donor liver samples in 66 LT recipients with nonalcoholic steatohepatitis to characterize influences on NAFLD recurrence at ∼1 year post-LT (median interval from LT to biopsy: 377 days). RESULTS: Recurrent NAFLD was identified in 43 (65.2%) patients, 20 (30.3%) with mild recurrence, and 23 (34.8%) with moderate to severe NAFLD. On adjusted analysis, change in the body mass index (BMI) (ΔBMI) was significantly associated with NAFLD recurrence, whereas post-LT diabetes mellitus was associated with increased severity of NAFLD recurrence. ADIPOR1 rs10920533 in the recipient was associated with increased risk of moderate to severe NAFLD recurrence, whereas the minor allele of SOD2 rs4880 in the recipient was associated with reduced risk. Similar reduced risk was noted in the presence of donor SOD2 rs4880 and HSD17B13 rs6834314 polymorphism. DISCUSSION: Increased BMI post-LT is strongly associated with NAFLD recurrence, whereas post-LT diabetes mellitus was associated with increased severity of NAFLD recurrence. Both donor and recipient SOD2 rs4880 and donor HSD17B13 rs6834314 single nucleotide polymorphisms may be associated with reduced risk of early NAFLD recurrence, whereas presence of the minor allele form of ADIPOR1 rs10920533 in the recipient is associated with increased severity NAFLD recurrence.

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.

Complement C1r serine protease contributes to kidney fibrosis.

We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-ß), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.

Adenosine 2A Receptor Activation Attenuates Ischemia Reperfusion Injury During Extracorporeal Cardiopulmonary Resuscitation.

OBJECTIVE: We tested the hypothesis that systemic administration of an A2AR agonist will reduce multiorgan IRI in a porcine model of ECPR. SUMMARY BACKGROUND DATA: Advances in ECPR have decreased mortality after cardiac arrest; however, subsequent IRI contributes to late multisystem organ failure. Attenuation of IRI has been reported with the use of an A2AR agonist. METHODS: Adult swine underwent 20 minutes of circulatory arrest, induced by ventricular fibrillation, followed by 6 hours of reperfusion with ECPR. Animals were randomized to vehicle control, low-dose A2AR agonist, or high-dose A2AR agonist. A perfusion specialist using a goal-directed resuscitation protocol managed all the animals during the reperfusion period. Hourly blood, urine, and tissue samples were collected. Biochemical and microarray analyses were performed to identify differential inflammatory markers and gene expression between groups. RESULTS: Both the treatment groups demonstrated significantly higher percent reduction from peak lactate after reperfusion compared with vehicle controls. Control animals required significantly more fluid, epinephrine, and higher final pump flow while having lower urine output than both the treatment groups. The treatment groups had lower urine NGAL, an early marker of kidney injury (P = 0.01), lower plasma aspartate aminotransferase, and reduced rate of troponin rise (P = 0.01). Pro-inflammatory cytokines were lower while anti-inflammatory cytokines were significantly higher in the treatment groups. CONCLUSIONS: Using a novel and clinically relevant porcine model of circulatory arrest and ECPR, we demonstrated that a selective A2AR agonist significantly attenuated systemic IRI and warrants clinical investigation.

Mechanistic analyses in kidney transplant recipients prospectively randomized to two steroid free regimen-Low dose Tacrolimus with Everolimus versus standard dose Tacrolimus with Mycophenolate Mofetil.

Calcineurin inhibitors (CNI), the cornerstone of immunosuppression after transplantation are implicated in nephrotoxicity and allograft dysfunction. We hypothesized that combined low doses of CNI and Everolimus (EVR) may result in better graft outcomes and greater tolerogenic milieu. Forty adult renal transplant recipients were prospectively randomized to (steroid free) low dose Tacrolimus (TAC) and EVR or standard dose TAC and Mycophenolate (MMF) after Alemtuzumab induction. Baseline characteristics were statistically similar. EVR levels were maintained at 3-8 ng/ml. TAC levels were 4.5±1.9 and 6.4±1.5 ng/ml in the TAC+EVR and TAC+MMF group respectively. Follow up was 14±4 and 17±5 months respectively and included protocol kidney biopsies at 3 and 12 months post-transplantation. Rejection-rate was lower in the TAC+EVR group. However patient and overall graft survival, eGFR and incidence of adverse events were similar. TAC+EVR induced expansion of CD4+CD25hiFoxp3+ regulatory T cells as early as 3 months and expansion of IFN-γ+CD4+CD25hiFoxp3+ regulatory T cells at 12 months post-transplant. Gene expression profile showed a trend toward decreased inflammation, angiogenesis and connective tissue growth in the TAC+EVR Group. Thus, greater tolerogenic mechanisms were found to be operating in patients with low dose TAC+EVR and this might be responsible for the lower rejection-rate than in patients on standard dose TAC+MMF. However, further studies with longer follow up and evaluating impact on T regulatory cells are warranted.

Publisher Correction: ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

In the version of this article originally published, a sentence was erroneously included in the author contributions, and information regarding second shared authorship was missing from the author contributions. The following should not have been included in the author contributions: "C.W. and A.J.R.H. supervised the work presented in Figs. 1, 2, 5, 6; P.Z. and C.S. supervised the work presented in Figs. 3, 4." Additionally, this sentence should have appeared at the beginning of the author contributions: "These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H." The errors have been corrected in the print, PDF and HTML versions of the article.

ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aß plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aß plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aß-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.

Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b+ macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.

Messengers of tolerance.

The use of immunosuppressant drugs after organ transplantation has brought great success in the field of organ transplantation with respect to short-term outcome. However, major challenges (i.e., limited improvement of long-term survival, immunosuppressant toxicity, infections and carcinoma) demand alternate treatment approaches that minimizes the use of immunosuppressants. Interestingly, few studies have identified groups of transplant patients who developed operational tolerance and thereby keep their allograft without complications in absence of immunosuppressants. These rare groups of patients are of particular interest as study subjects for understanding mechanisms of graft tolerance that could be leveraged in future for inducing tolerance and for understanding mechanisms involved in improving long-term allograft outcomes. Also, biomarkers from these studies could benefit the larger transplant population by their application in immunosuppressant tailoring and identification of tolerant patients among patients with stably functioning allografts. This review compiles several gene expression studies performed in samples from tolerant patients in different solid organ transplantations to identify key genes and associated molecular pathways relevant to tolerance. This review is aimed at putting forth all this important work done thus far and to identify research gaps that need to be filled, in order to achieve the greater purpose of these studies.

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

Artery Tertiary Lymphoid Organs Control Multilayered Territorialized Atherosclerosis B-Cell Responses in Aged ApoE-/- Mice.

OBJECTIVE: Explore aorta B-cell immunity in aged apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Transcript maps, fluorescence-activated cell sorting, immunofluorescence analyses, cell transfers, and Ig-ELISPOT (enzyme-linked immunospot) assays showed multilayered atherosclerosis B-cell responses in artery tertiary lymphoid organs (ATLOs). Aging-associated aorta B-cell-related transcriptomes were identified, and transcript atlases revealed highly territorialized B-cell responses in ATLOs versus atherosclerotic lesions: ATLOs showed upregulation of bona fide B-cell genes, including Cd19, Ms4a1 (Cd20), Cd79a/b, and Ighm although intima plaques preferentially expressed molecules involved in non-B effector responses toward B-cell-derived mediators, that is, Fcgr3 (Cd16), Fcer1g (Cd23), and the C1q family. ATLOs promoted B-cell recruitment. ATLO B-2 B cells included naive, transitional, follicular, germinal center, switched IgG1(+), IgA(+), and IgE(+) memory cells, plasmablasts, and long-lived plasma cells. ATLOs recruited large numbers of B-1 cells whose subtypes were skewed toward interleukin-10(+) B-1b cells versus interleukin-10(-) B-1a cells. ATLO B-1 cells and plasma cells constitutively produced IgM and IgG and a fraction of plasma cells expressed interleukin-10. Moreover, ApoE(-/-) mice showed increased germinal center B cells in renal lymph nodes, IgM-producing plasma cells in the bone marrow, and higher IgM and anti-MDA-LDL (malondialdehyde-modified low-density lipoprotein) IgG serum titers. CONCLUSIONS: ATLOs orchestrate dichotomic, territorialized, and multilayered B-cell responses in the diseased aorta; germinal center reactions indicate generation of autoimmune B cells within the diseased arterial wall during aging.

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro.

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.