Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (> 95%) of myocardial B cells remain intravascular, whereas few (< 5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.
Luigi Adamo, Cibele Rocha-Resende, Chieh-Yu Lin, Sarah Evans, Jesse W. Williams, Hao Dun, Wenjun Li, Cedric Mpoy, Prabhakar Andhey, Buck E. Rogers, Kory Lavine, Daniel Kreisel, Maxim N. Artyomov, Gwendalyn J. Randolph, Douglas Mann
OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are characterized by muscle inflammation and weakness, myositis specific autoantibodies (MSAs) and extramuscular organ damage. The role of neutrophil dysregulation and neutrophil extracellular traps (NETs) in IIM is unclear. We assessed if pathogenic neutrophil subsets (low-density granulocytes, LDGs) and NETs were elevated in IIM, associated with clinical presentation and MSAs, and their effect on skeletal myoblasts and myotubes.METHODS: Circulating NETs and LDGs were quantified and correlated with clinical measures. Specific MSAs were tested for their ability to induce NETs. NETs and neutrophil gene expression were measured in IIM biopsies. Whether NETs damage skeletal myoblasts and myotubes was tested.RESULTS: Circulating LDGs and NETs were increased in IIM. IIM LDGs had enhanced ability to form NETs. LDGs and NETs correlated with IIM disease activity and muscle damage. The serum MSA anti-MDA5 correlated with circulating and tissue NETs and directly enhanced NET formation. An enhanced neutrophil gene signature was present in IIM muscle and associated with muscle injury and tissue interferon gene signatures. IIM NETs decreased the viability of myotubes in a citrullinated histone-dependent manner. CONCLUSION: Dysregulated neutrophil pathways may play pathogenic roles in IIM through their ability to directly injure muscle cells and other affected tissues.
Nickie L. Seto, Jose Jiram Torres-Ruiz, Carmelo Carmona-Rivera, Iago Pinal-Fernandez, Katherine Pak, Monica M. Purmalek, Yuji Hosono, Catia Fernandes-Cerqueira, Prateek C. Gowda, Nathan Arnett, Alexander Gorbach, Olivier Benveniste, Diana Gómez-Martín, Albert Selva-O'Callaghan, Jose C. Milisenda, Josep M. Grau-Junyent, Lisa Christopher-Stine, Frederick W. Miller, Ingrid E. Lundberg, J. Michelle Kahlenberg, Adam I. Schiffenbauer, Andrew L. Mammen, Lisa G. Rider, Mariana J. Kaplan
Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for six months after infection with WT measles virus (MeV). Infection caused viremia and rash with clearance of infectious virus by 14 days. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H) and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral Tfh cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody to both H and N, with more H-specific ASCs in BM. From 14-21 days 20-100-fold more total ASCs than MeV-specific ASCs appeared in circulation suggesting mobilization of pre-existing ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.
Ashley N. Nelson, Wen-Hsuan W. Lin, Rupak Shivakoti, Nicole E. Putnam, Lisa M. Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3’ UTR cleavage and polyadenylation represents a common characteristic among many disease states including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I (CFIm)-mediated APA in regulating the extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulates expression of CFIm68, CFIm59 and CFIm25 subunits, and promotes APA in favor of the proximal poly(A) site usage in the 3’ UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix-induced APA and overproduction of COL1A1, whereas CFIm does not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits is associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3’ UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.
Zijing Zhou, Jing Qu, Li He, Yi Zhu, Shanzhong Yang, Feng Zhang, Ting Guo, Hong Peng, Ping Chen, Yong Zhou
Adult renal proximal tubules are composed of terminally differentiated epithelial cells that exhibit few signs of proliferation over time. However, upon acute kidney injury (AKI), surviving epithelial cells can re-enter the mitotic cycle and express genes and proteins coincident with a dedifferentiated, more embryonic phenotype. While a stable, terminally differentiated cellular phenotype is thought to be maintained, at least in part, by epigenetic imprints that impart both active and repressive histone marks, it is unclear whether regenerating cells after injury need to replicate such marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes the adaptor protein PTIP, which is part of an Mll3/4 histone H3K4 methyltransferase complex and is essential for embryonic development. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to re-enter mitosis and reactivate regulatory genes such as Sox9, which is necessary for epithelial cell regeneration. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. A transient decrease in trimethylation was also observed in controls after AKI but returned to normal after repair. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4 mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.
Abdul Soofi, Ana P. Kutschat, Mohammad H. Azam, Ann M. Laszczyk, Gregory R. Dressler
Dengue (DENV) and Zika viruses (ZIKV) are closely related mosquito-borne flaviviruses that co-circulate in tropical regions and constitute major threats to global human health. Whether preexisting immunity to one virus affects disease caused by the other during primary or secondary infections is unknown but is critical in preparing for future outbreaks and predicting vaccine safety. Using a human skin explant model, we show that DENV-3 immune sera increased recruitment and infection of Langerhans cells, macrophages and dermal dendritic cells following inoculation with DENV-2 or ZIKV. Similarly, ZIKV immune sera enhanced infection with DENV-2. Immune sera increased migration of infected Langerhans cells to dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in dermis almost tenfold and reduced the amount of virus required to infect a majority of myeloid cells by 100 to 1,000 fold. Enhancement was associated with cross-reactive IgG and induction of IL-10 expression and was mediated by both CD32 and CD64 Fcγ receptors. These findings reveal that preexisting heterotypic immunity greatly enhances DENV and ZIKV infection, replication and spread in human skin. This relevant tissue model will be valuable in assessing the efficacy and risk of dengue and Zika vaccines in humans.
Priscila M.S. Castanha, Geza Erdos, Simon C. Watkins, Louis D. Falo, Jr., Ernesto T.A. Marques, Simon M. Barratt-Boyes
Ultrasound-induced microbubble (USMB) cavitation is widely used to promote drug delivery. Our previous study investigated USMB targeting round window membrane by applying the ultrasound transducer to tympanic bulla. In the present study we further extended the use of this technology to enhance drug delivery to inner ear by introducing the ultrasound transducer into external auditory canal (EAC) or applying it to skull. Using a three-dimensional-printed diffusion apparatus mimicking the pathway for ultrasound passing through and reaching middle ear cavity in vitro, both models simulating the transcanal and transcranial approach demonstrated 4.8-fold and 3.7-fold higher delivery efficiencies, respectively. In vivo model of guinea pigs, by filling tympanic bulla with microbubbles and biotin-fluorescein (biotin-FITC), USMB applied transcanally and transcranially induced 2.8-fold and 1.5-fold increases in biotin-FITC delivery efficiencies, respectively. In addition, the gentamicin uptake by cochlear and vestibular hair cells and gentamicin-induced hair cell loss were significantly enhanced following transcanal application of USMB. On the 28th day after transcanal USMB, safety assessment showed no significant changes in the hearing thresholds and the integrity of cochlea. These are the first results to demonstrate the feasibility and support the potential clinical application of applying USMB via EAC to facilitate drug delivery into inner ear.
Ai-Ho Liao, Chih-Hung Wang, Ping-Yu Weng, Yi-Chun Lin, Hao Wang, Hang-Kang Chen, Hao-Li Liu, Ho-Chiao Chuang, Cheng-Ping Shih
To investigate the nationwide severe fever with thrombocytopenia syndrome virus (SFTSV) infection status, we isolated SFTSVs from severe fever with thrombocytopenia syndrome (SFTS)-suspected patients in 207 hospitals throughout South Korea between 2013 and April of 2017. A total of 116 SFTSVs were isolated from 3,137 SFTS-suspected patients with an overall 21.6% case fatality rate. Genetic characterization revealed that at least six genotypes of SFTSVs are co-circulating in South Korea with multiple reassortments among them. Of these, the genotype B-2 strains were the most prevalent (n = 48, 36.1%) followed by the A and F genotypes. Clinical and epidemiologic investigations revealed that genotype B strains were associated with the highest case-fatality rate (34.8%, 32/92), while genotype A caused only one fatality out of ten patients. Further, ferret infection studies demonstrated varied clinical manifestations and case mortality rates of different strains of SFTSV, which suggests this virus could exhibit genotype-dependent pathogenicity.Keywords: severe fever with thrombocytopenia syndrome virus (SFTSV), clinical manifestations, genotypes, pathogenesis
Seok-Min Yun, Su-Jin Park, Young-Il Kim, Sun-Whan Park, Min-Ah Yu, Hyeok-Il Kwon, Eun-Ha Kim, Kwang-Min Yu, Hye Won Jeong, Jungsang Ryou, Won-Ja Lee, Youngmee Jee, Joo-Yeon Lee, Young Ki Choi
Mutations in cardiac myosin binding protein (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in premature termination codons (PTCs) that cause RNA degradation and a reduction of MyBP-C in HCM patient hearts. However, a reduction in MyBP-C has not been consistently observed in MYBPC3 mutant induced pluripotent stell cell cardiomyocytes (iPSCMs). To determine early MYBPC3 mutation effects, we utilized both patient and genome-engineered iPSCMs. iPSCMs with frameshift mutations were compared to iPSCMs with MYBPC3 promoter and translational start site deletions, revealing that allelic loss of function is the primary inciting consequence of mutations that cause PTCs. Despite a reduction in wild type mRNA in all heterozygous iPSCMs, no reduction in MyBP-C protein was observed, indicating protein-level compensation through a previously uncharacterized mechanism. Although homozygous mutant iPSCMs exhibited contractile dysregulation, heterozygous mutant iPSCMs had normal contractile function in the context of compensated MyBP-C levels. Agnostic RNA-seq analysis revealed differential expression in protein chaperone genes as the only dysregulated gene set. To determine how MYBPC3 mutant iPSCMs achieve compensated MyBP-C levels, sarcomeric protein synthesis and degradation were measured with stable isotope-labeling. Heterozygous mutant iPSCMs showed reduced MyBP-C synthesis rates but with a corresponding reduction in MyBP-C degradation. These findings indicate that cardiomyocytes have an innate capacity to attain normal MyBP-C stoichiometry despite MYBPC3 allelic loss of function due to truncating mutations. Modulating MyBP-C degradation to maintain MyBP-C protein levels may be a novel treatment approach upstream of contractile dysfunction for HCM patients.
Adam S Helms, Vi T. Tang, Thomas S. O'Leary, Sabrina Friedline, Mick Wauchope, Akul Arora, Aaron H Wasserman, Eric D Smith, Lap Man Lee, Xiaoquan Wen, Jordan A. Shavit, Allen P Liu, Michael J Previs, Sharlene M. Day
Genetic variants within/near the interferon regulatory factor 5 (IRF5) locus associate with systemic lupus erythematosus (SLE) across ancestral groups. The major IRF5-SLE risk haplotype is common across populations, yet immune functions for the risk haplotype are undefined. We characterized the global immune-phenotype of healthy donors homozygous for the major risk and non-risk haplotypes and identified cell lineage-specific alterations that mimic pre-symptomatic SLE. Contrary to previous studies in B lymphoblastoid cell lines and SLE immune cells, IRF5 genetic variants had little effect on IRF5 protein levels in healthy donors. Instead, we detected basal IRF5 hyper-activation in the myeloid compartment of risk donors that drives the SLE immune-phenotype. Risk donors were ANA positive with anti-Ro and -MPO specificity, had increased circulating plasma cells and plasmacytoid dendritic cells, and enhanced spontaneous NETosis. The IRF5-SLE immune-phenotype was conserved over time and probed mechanistically by ex vivo co-culture, indicating that risk neutrophils are drivers of the global immune-phenotype. RNA-seq of risk neutrophils revealed increased IRF5 transcript expression, IFN pathway enrichment and decreased expression of ROS pathway genes. Altogether, data support that individuals carrying the IRF5-SLE risk haplotype are more susceptible to environmental/stochastic influences that trigger chronic immune activation, predisposing to the development of clinical SLE.
Dan Li, Bharati Matta, Su Song, Victoria Nelson, Kirsten Diggins, Kim R. Simpfendorfer, Peter K. Gregersen, Peter Linsley, Betsy J. Barnes
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