Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology, characterized by elevated intracranial pressure frequently manifesting with chronic headaches and visual loss. Similar to polycystic ovary syndrome (PCOS), IIH predominantly affects obese women of reproductive age. In this study, we comprehensively examined the systemic and cerebrospinal fluid (CSF) androgen metabolome in women with IIH in comparison to sex-, body mass index- and age-matched control groups with either simple obesity and PCOS, i.e. obesity and androgen excess. IIH women showed a pattern of androgen excess distinct to that observed in PCOS and simple obesity, with increased serum testosterone, and increased CSF testosterone and androstenedione. Human choroid plexus expressed the androgen receptor, alongside the androgen-activating enzyme aldoketoreductase type 1C3. We show that in a rat choroid plexus cell line testosterone significantly enhanced the activity of Na+/K+ ATPase, a surrogate of CSF secretion. We demonstrate that IIH patients have a unique signature of androgen excess and provide evidence that androgens can modulate CSF secretion via the choroid plexus. These findings implicate androgen excess as a potential causal driver and therapeutic target in IIH.
Michael W. O'Reilly, Connar S.J. Westgate, Catherine Hornby, Hannah Botfield, Angela E. Taylor, Keira Markey, James L. Mitchell, William J. Scotton, Susan P. Mollan, Andreas Yiangou, Carl Jenkinson, Lorna C. Gilligan, Mark Sherlock, James Gibney, Jeremy W. Tomlinson, Gareth G. Lavery, David J. Hodson, Wiebke Arlt, Alexandra J. Sinclair
Chimeric antigen receptor (CAR) technology can be used to engineer the antigen-specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from non-human antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain Fv derived from a mouse antibody, we developed a panel of 20 humanized (h)A2-CARs. Systematic testing demonstrated variations in expression, ability to bind HLA-A*02:01, and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen-specificity of CARs, revealing that humanization reduced HLA-A cross reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs which can be used to engineer specificity and homing of therapeutic Tregs.
Nicholas A.J. Dawson, Caroline Lamarche, Romy E. Hoeppli, Peter Bergqvist, Vivian Fung, Emma McIver, Qing Huang, Jana Gillies, Madeleine Speck, Paul C. Orban, Jonathan W. Bush, Majid Mojibian, Megan K. Levings
Recovery from acute lung injury (ALI) is an active process. Foxp3+ regulatory T cells (Tregs) contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared to those from the spleen, indicating that organ-specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into two identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each play a role in Treg-promoted ALI resolution. This study provides novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment-specific transcriptional differences through which Tregs direct their effects.
Jason R. Mock, Catherine F. Dial, Miriya K. Tune, Dustin L. Norton, Jessica R. Martin, John C. Gomez, Robert S. Hagan, Hong Dang, Claire M. Doerschuk
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
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
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