Siponimod enriches regulatory T and B lymphocytes in secondary progressive multiple sclerosis

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Abstract

BACKGROUND. Siponimod (BAF312) is a selective sphingosine 1-phosphate receptor 1 and 5 (S1PR1, S1PR5) modulator recently approved for active secondary progressive multiple sclerosis (SPMS). The immunomodulatory effects of siponimod in SPMS have not been previously described. METHODS. We conducted a multi-centered randomized, double-blind, placebo-controlled AMS04 mechanistic study with 36 SPMS participants enrolled in the EXPAND trial. Gene expression profiles were analyzed using RNA derived from whole blood with Affymetrix Human Gene ST 2.1 microarray technology. We performed flow cytometry based assays to analyze the immune cell composition and microarray gene expression analysis on peripheral blood from siponimod-treated participants with SPMS relative to baseline and placebo during the first year randomization phase. RESULTS. Microarray analysis showed that immune-associated genes involved in T and B cell activation and receptor signaling were largely decreased by siponimod, which is consistent with the reduction of CD4+ T cells, CD8+ T cells, and B cells. Analysis done by flow cytometry showed that within the remaining lymphocyte subsets, there was a reduction in the frequencies of CD4 and CD8 naïve T cells and central memory cells, while T effector memory cells, anti-inflammatory Th2, and T regulatory (Treg) cells were enriched. Transitional Bregs (CD24hiCD38hi) and B1 cell subsets (CD43+CD27+) were enriched, shifting the balance in favor of regulatory B cells over memory B cells. The pro-regulatory shift driven by siponimod treatment included a higher proliferative potential of Tregs compared with non-Tregs, and upregulated expression of PD-1 on Tregs. Additionally, a positive correlation was found between regulatory T cells and regulatory B cells in siponimod treated participants. CONCLUSION. The shift toward an anti-inflammatory and suppressive homeostatic immune system may contribute to the clinical efficacy of siponimod in SPMS. TRIAL REGISTRATION. NCT02330965.

Authors

Qi Wu, Elizabeth A. Mills, Qin Wang, Catherine A. Dowling, Caitlyn Fisher, Britany Kirch, Steven K. Lundy, David A. Fox, Yang Mao-Draayer

Association of persistent wild-type measles virus RNA with long-term humoral immunity in rhesus macaques

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Abstract

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.

Authors

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

Stiff matrix instigates type I collagen biogenesis by mammalian cleavage factor I complex-mediated alternative polyadenylation

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Abstract

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.

Authors

Zijing Zhou, Jing Qu, Li He, Yi Zhu, Shanzhong Yang, Feng Zhang, Ting Guo, Hong Peng, Ping Chen, Yong Zhou

Regeneration after acute kidney injury requires PTIP mediated epigenetic modifications

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Abstract

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.

Authors

Abdul Soofi, Ana P. Kutschat, Mohammad H. Azam, Ann M. Laszczyk, Gregory R. Dressler

Reciprocal immune enhancement of dengue and Zika virus infection in human skin

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Abstract

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.

Authors

Priscila M.S. Castanha, Geza Erdos, Simon C. Watkins, Louis D. Falo, Jr., Ernesto T.A. Marques, Simon M. Barratt-Boyes

Circadian rhythm phase shifts caused by timed exercise vary with chronotype

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Abstract

BACKGROUND. The circadian system entrains behavioral and physiological rhythms to environmental cycles and modern lifestyles disrupt this entrainment. We investigated a timed exercise intervention to phase shift the internal circadian rhythm. METHODS. In fifty-two young, sedentary adults, dim light melatonin onset (DLMO) was measured before and after five days of morning (10h after DLMO; n = 26) or evening (20h after DLMO; n = 26) exercise. Phase shifts were calculated as the difference in DLMO before and after exercise. RESULTS. Morning exercise induced phase advance shifts (0.62 ± 0.18h) that were significantly greater than phase shifts from evening exercise (-0.02 ± 0.18h; P = 0.01). Chronotype also influenced the effect of timed exercise. For later chronotypes, both morning and evening exercise induced phase advances (0.54 ± 0.29h and 0.46 ±0.25h, respectively). In contrast, earlier chronotypes had phase advances from morning exercise (0.49 ± 0.25h), but phase delays from evening exercise (-0.41 ± 0.29h). CONCLUSION. Late chronotypes, who experience the most severe circadian misalignment, may benefit from phase advances induced by exercise in the morning or evening, but evening exercise may exacerbate circadian misalignment in early chronotypes. Thus, personalized exercise timing prescription based on chronotype could alleviate circadian misalignment in young adults. TRIAL REGISTRATION. www.clinicaltrials.gov, NCT # NCT04097886.FUNDING. National Institutes of Health grants UL1TR001998 and TL1TR001997, the Barnstable Brown Diabetes and Obesity Center, the Pediatric Exercise Physiology Laboratory Endowment, the Arvle and Ellen Turner Thacker Research Fund, and the University of Kentucky.

Authors

J. Matthew Thomas, Philip A. Kern, Heather M. Bush, Kristen J. McQuerry, W. Scott Black, Jody L. Clasey, Julie S. Pendergast

Association of urine mitochondrial DNA with clinical measures of COPD in the SPIROMICS cohort

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Abstract

BACKGROUND. Mitochondrial dysfunction, a proposed mechanism of COPD pathogenesis, is associated with the leakage of mitochondrial DNA (mtDNA), which may be detected extracellularly in various bodily fluids. Despite evidence for the increased prevalence of chronic kidney disease in COPD subjects and for mitochondrial dysfunction in the kidneys of murine COPD models, whether urine mtDNA (u-mtDNA) associates with measures of disease severity in COPD is unknown. METHODS. Cell-free u-mtDNA, defined as copy number of mitochondrially-encoded NADH dehydrogenase-1 (MTND1) gene, was measured by real-time quantitative PCR and normalized to urine creatinine in cell-free urine samples from participants in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Urine albumin/creatinine ratios (UACR) were measured in the same samples. Associations between u-mtDNA and UACR and clinical disease parameters, including FEV1 % predicted, clinical measures of exercise tolerance, respiratory symptom burden, and chest CT measures of lung structure were examined. RESULTS. U-mtDNA and UACR levels were measured in never smokers (n = 64), smokers without airflow obstruction (n = 109), participants with mild/moderate COPD (n = 142), and participants with severe COPD (n = 168). U-mtDNA was associated with increased respiratory symptom burden, especially among smokers without COPD. Significant sex differences in u-mtDNA levels were observed with females having higher u-mtDNA levels across all study subgroups. U-mtDNA associated with worse spirometry and CT emphysema in males only, and worse respiratory symptoms in females only. Similar associations were not found with UACR. CONCLUSION. U-mtDNA levels may help to identify distinct clinical phenotypes and underlying pathobiological differences in males versus females with COPD.

Authors

William Z. Zhang, Michelle C. Rice, Katherine L. Hoffman, Clara Oromendia, Igor Barjaktarevic, J. Michael Wells, Annette T. Hastie, Wassim W. Labaki, Christopher B. Cooper, Alejandro P. Comellas, Gerard J. Criner, Jerry A. Krishnan, Robert Paine III, Nadia N. Hansel, Russell P. Bowler, R. Graham Barr, Stephen P. Peters, Prescott G. Woodruff, Jeffrey L. Curtis, Meilan K. Han, Karla V. Ballman, Fernando J. Martinez, Augustine M.K. Choi, Kiichi Nakahira, Suzanne M. Cloonan, Mary E. Choi

Ultrasound-induced microbubble cavitation via a transcanal or transcranial approach facilitates inner ear drug delivery

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Abstract

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.

Authors

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

Genetic and Pathogenic-Diversity of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) in South Korea

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Abstract

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

Authors

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

Effects of MYBPC3 loss of function mutations preceding hypertrophic cardiomyopathy

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Abstract

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.

Authors

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