Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. Myotonic dystrophy is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of myotonic dystrophy in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2 which is characterized by nucleotide repeat expansions often greater than 5000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, MBNL foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls, DM1 and DM2 subjects and differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. IPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High resolution imaging revealed tight association between MBNL clusters and RNA FISH foci in DM1. Ca2+ transients differed between DM1 and DM2 IPSC-derived cardiomyocytes and each differed from healthy control cells. RNA-sequencing from DM1 and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression as well as differential aberrant splicing patterns. Together these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.
Ellis Y. Kim, David Y. Barefield, Andy H. Vo, Anthony M. Gacita, Emma J. Schuster, Eugene J. Wyatt, Janel L. Davis, Biqin Dong, Cheng Sun, Patrick Page, Lisa Dellefave-Castillo, Alexis Demonbreun, Hao F. Zhang, Elizabeth M. McNally
Although Type-2 (T2) induced epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4β isoform were found to be increased in airway specimens from asthmatic patients, in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4β, thereby altering its function and that the β-galactoside α-2,6-sialyltranferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs), we demonstrated that IL-13 increased sialylation of MUC4β under control of ST6GAL1, and that both were increased in asthma, particularly in those with elevated T2 biomarkers. ST6GAL1 induced sialylation of MUC4β altered its lectin binding and secretion. Both ST6GAL1 and MUC4β inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1 induced sialylation of MUC4β in epithelial dysfunction associated with T2-High asthma, thereby identifying specific sialylation pathways as potential targets in asthma.
Xiuxia Zhou, Carol L. Kinlough, Rebecca P. Hughey, Mingzhu Jin, Hideki Inoue, Emily Etling, Brian D. Modena, Naftali Kaminski, Eugene R. Bleecker, Deborah A. Meyers, Nizar N. Jarjour, John B. Trudeau, Fernando Holguin, Anuradha Ray, Sally E. Wenzel
The circadian clock network is an evolutionally conserved system involved in the regulation of metabolic homeostasis; however, its impacts on skeletal metabolism remain largely unknown. We herein demonstrated that circadian clock network in the intestines plays pivotal roles in skeletal metabolism such that the lack of Bmal1 gene in the intestines (Bmal1Int-/- mice) caused bone loss with bone resorption being activated and bone formation suppressed. Mechanistically, Clock interaction with Vitamin D receptor (Vdr) accelerated its binding to VDR response element by enhancing histone acetylation in a circadian-dependent manner, and this was lost in Bmal1Int-/- mice because nuclear translocation of Clock required the presence of Bmal1. Accordingly, the rhythmic expression of Vdr-target genes involved in transcellular calcium (Ca) absorption was created, and this was not observed in Bmal1Int-/- mice. As a result, transcellular Ca absorption was impaired and bone resorption was activated in Bmal1Int-/- mice. Additionally, sympathetic tone, the activation of which suppresses bone formation, was elevated through afferent vagal nerves in Bmal1Int-/- mice, the blockade of which partially recovered bone loss by increasing bone formation and suppressing bone resorption in Bmal1Int-/- mice. These results demonstrate that the intestinal circadian system regulates skeletal bone homeostasis.
Masanobu Kawai, Saori Kinoshita, Miwa Yamazaki, Keiko Yamamoto, Clifford J. Rosen, Shigeki Shimba, Keiichi Ozono, Toshimi Michigami
Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs anti-tumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment. Circulating PMN from patients were not suppressive, but acquired a suppressor phenotype (defined as ≥ 1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20/31) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites supernatant removal and re-stimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naïve, central memory, and effector memory T cells, and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the tumor microenvironment and the potential for complement inhibition to abrogate this barrier to anti-tumor immunity.
Kelly L. Singel, Tiffany R. Emmons, ANM Nazmul H. Khan, Paul C. Mayor, Shichen Shen, Jerry T. Wong, Kayla Morrell, Kevin H. Eng, Jaron Mark, Richard B. Bankert, Junko Matsuzaki, Richard C. Koya, Anna M. Blom, Kenneth R. McLeish, Jun Qu, Sanjay Ram, Kirsten B. Moysich, Scott I. Abrams, Kunle Odunsi, Emese Zsiros, Brahm H. Segal
BACKGROUND. Multiple therapeutic strategies to restore immune regulation and slow type 1 diabetes (T1D) progression are in development and testing. A major challenge has been defining biomarkers to prospectively identify subjects likely to benefit from immunotherapy and/or measure intervention effects. We previously found that compared to healthy controls, Tregs from children with new-onset T1D have an altered Treg gene signature (TGS), suggesting this could be an immunoregulatory biomarker. METHODS. nanoString was used to assess the TGS in sorted Tregs (CD4+CD25hiCD127lo) or Peripheral Blood Mononuclear Cells (PBMC) from individuals with T1D or type 2 diabetes, healthy controls, or T1D recipients of immunotherapy. Biomarker discovery pipelines were developed and applied to various sample group comparisons. RESULTS. Compared to controls, the TGS in isolated Tregs or PBMCs is altered in adult new-onset and cross-sectional T1D cohorts, with sensitivity and specificity of biomarkers increased by including T1D-associated single nucleotide polymorphisms in algorithms. The TGS was distinct in T1D versus type 2 diabetes, indicating disease-specific alterations. TGS measurement at the time of T1D onset revealed an algorithm that accurately predicted future rapid versus slow C-peptide decline, as determined by longitudinal analysis of placebo arms of START and T1DAL trials. The same algorithm stratified participants in a phase I/II clinical trial of ustekinumab (αIL-12/23p40) for future rapid versus slow C-peptide decline. CONCLUSION. These data suggest that biomarkers based on measuring Treg gene signatures could be a new approach to stratify patients and monitor autoimmune activity in T1D.
Anne M. Pesenacker, Virginia Chen, Jana Gillies, Cate Speake, Ashish K. Marwaha, Annika C. Sun, Samuel Chow, Rusung Tan, Thomas Elliott, Jan P. Dutz, Scott J. Tebbutt, Megan K. Levings
The routes by which antibody-based therapeutics reach malignant cells are poorly defined. Tofacitinib, an FDA-approved JAK inhibitor, reduced tumor-associated inflammatory cells and allowed increased delivery of antibody-based agents to malignant cells. Alone, tofacitinib exhibited no antitumor activity, but combinations with immunotoxins or an antibody drug conjugate resulted in increased anti-tumor responses. Quantification using flow cytometry revealed that antibody-based agents accumulated in malignant cells at higher percentages following tofacitinib treatment. Profiling of tofacitinib-treated tumor-bearing mice indicated that cytokine transcripts and various proteins involved in chemotaxis were reduced compared to vehicle-treated mice. Histological analysis revealed significant changes to the composition of the tumor microenvironment, with reductions in monocytes, macrophages and neutrophils. Tumor-associated inflammatory cells contributed to non-target uptake of antibody-based therapeutics; with mice treated with tofacitinib showing decreased accumulation of therapeutics in intratumoral inflammatory cells and increased delivery to malignant cells. Present findings serve as a rationale for conducting trials where short-term treatments with tofacitinib could be administered in combination with antibody-based therapies.
Nathan Simon, Antonella Antignani, Stephen M. Hewitt, Massimo Gadina, Christine Alewine, David FitzGerald
Paracrine interactions between pancreatic islet cells have been proposed as a mechanism to regulate hormone secretion and glucose homeostasis. Here, we demonstrate the importance of proglucagon-derived peptides (PGDPs) for α- to β-cell communication and control of insulin secretion. Signaling through this system occurs through both the glucagon-like peptide receptor (Glp1r) and glucagon receptor (Gcgr). Loss of PGDPs, or blockade of their receptors, decreases insulin secretion in response to both metabolic and non-metabolic stimulation of mouse and human islets. This effect is due to reduced β-cell cAMP and affects the quantity but not dynamics of insulin release, indicating that PGDPs dictate the magnitude of insulin output in an isolated islet. In healthy mice, additional factors that stimulate cAMP can compensate for loss of PGDP signaling; however, input from α-cells is essential to maintain glucose tolerance during the metabolic stress induced by high-fat feeding. These findings demonstrate an essential role for α-cell regulation of β-cells, raising the possibility that abnormal paracrine signaling contributes to impaired insulin secretion in diabetes. Moreover, these findings support reconsideration of the role for α-cells in postprandial glucose control.
Megan E. Capozzi, Berit Svendsen, Sara E. Encisco, Sophie L. Lewandowski, Mackenzie D. Martin, Haopeng Lin, Justin L. Jaffe, Reilly W. Coch, Jonathan M. Haldeman, Patrick E. MacDonald, Matthew J. Merrins, David A. D'Alessio, Jonathan E. Campbell
Increased airway vagal sensory C-fiber activity contributes to the symptoms of inflammatory airway diseases. The KCNQ/Kv7/M-channel is a well-known determinant of neuronal excitability, yet whether it regulates the activity of vagal bronchopulmonary C-fibers and airway reflex sensitivity remain unknown. Here we addressed this issue using single-cell RT-PCR, patch clamp technique, extracellular recording of single vagal nerve fibers innervating the mouse lungs, and telemetric recording of cough in free-moving mice. Single-cell mRNA analysis and biophysical properties of M-current (IM) indicate that KCNQ3/Kv7.3 is the major M-channel subunit in mouse nodose neurons. The M-channel opener retigabine negatively shifted the voltage-dependent activation of IM, leading to membrane hyperpolarization, increased rheobase and suppression of both evoked and spontaneous action potential (AP) firing in nodose neurons in the M-channel inhibitor XE991-sensitive manner. Retigabine also markedly suppressed the α,β-methylene ATP-induced AP firing in nodose C-fiber terminals innervating the mouse lungs, and irritant gases-evoked coughing in awake mice. In conclusion, KCNQ/M-channels play a role in regulating the excitability of vagal airway C-fibers at both the cell soma and nerve terminals. Drugs that open M-channels in airway sensory afferents may relieve the sufferings associated with pulmonary inflammatory diseases such as chronic coughing.
Hui Sun, An-Hsuan Lin, Fei Ru, Mayur J. Patil, Sonya Meeker, Lu-Yuan Lee, Bradley J. Undem
Macrophages are well-recognized for their dual roles in orchestrating inflammatory responses and regulating tissue repair. In almost all acutely inflamed tissues, two main subclasses of macrophages co-exist. These include embryonically-derived resident tissue macrophages and bone marrow-derived recruited macrophages. While it is clear that macrophage subsets categorized in this fashion display distinct transcriptional and functional profiles, whether all cells within these categories and in the same inflammatory microenvironment share similar functions or whether further specialization exists has not been determined. To investigate inflammatory macrophage heterogeneity on a more granular level, we induced acute lung inflammation in mice and performed single cell RNA sequencing of macrophages isolated from the airspaces during health, peak inflammation, and resolution of inflammation. In doing so, we confirm that cell origin is the major determinant of AM programing and describe two previously uncharacterized, transcriptionally distinct subdivisions of AMs based on proliferative capacity and inflammatory programing.
Kara J. Mould, Nathan D. Jackson, Peter M. Henson, Max A. Seibold, William J. Janssen
While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti-VEGF therapy include the upregulation of other pro-angiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signalling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor-induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularisation in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signalling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularisation, whilst enhancing reparative angiogenesis in the ischemic retina.
Sadaf Ashraf, Samuel Bell, Caitriona O'Leary, Paul Canning, Ileana Micu, Jose A. Fernandez, Michael O'Hare, Peter Barabas, Hannah McCauley, Derek P. Brazil, Alan W. Stitt, J. Graham McGeown, Tim M. Curtis
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