Abstract
Susceptibility to chronic beryllium (Be) disease (CBD) is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the β-chain (βGlu69), with the most prevalent βGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2–transgenic (HLA-DP2–Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2–restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2–Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2–Tg mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest what we believe is a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.
Authors
Shaikh M. Atif, Douglas G. Mack, Amy S. McKee, Javier Rangel-Moreno, Allison K. Martin, Andrew Getahun, Lisa A. Maier, John C. Cambier, Rubin Tuder, Andrew P. Fontenot
Abstract
Monosomy 7 and deletion of 7q, known as del(7q), are common clonal cytogenetic abnormalities associated with high-grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current nontransplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGF-β pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild-type cells, consistent with hyperactivation of the TGF-β pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGF-β pathway rescued hematopoiesis in SDS iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS predisposition syndrome and highlighted the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.
Authors
Melisa Ruiz-Gutierrez, Özge Vargel Bölükbaşı, Gabriela Alexe, Adriana G. Kotini, Kaitlyn Ballotti, Cailin E. Joyce, David W. Russell, Kimberly Stegmaier, Kasiani Myers, Carl D. Novina, Eirini P. Papapetrou, Akiko Shimamura
Abstract
Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex, respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator–activated receptor γ coactivator 1 α gene (PPARGC1A, encoding PGC1α). Both TGF-β and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of the CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGF-β– and matrix stiffness–promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.
Authors
Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y.S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin
Abstract
Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain insight into BPH. By RNA-Seq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules bone morphogenetic protein 5 (BMP5) and CXC chemokine ligand 13 (CXCL13) were enriched in BPH while estrogen-regulated pathways were depleted. Notably, BMP5’s addition to cultured prostatic myofibroblasts altered their expression profile toward a BPH profile that included the BPH stromal signature. RNA-Seq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNA-Seq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets but also depletion of neuroendocrine cells and an estrogen receptor–positive fibroblast cell type residing near the epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling) and reveals BPH to be not merely a hyperplasia, but rather a fundamental relandscaping of cell types.
Authors
Lance W. Middleton, Zhewei Shen, Sushama Varma, Anna S. Pollack, Xue Gong, Shirley Zhu, Chunfang Zhu, Joseph W. Foley, Sujay Vennam, Robert T. Sweeney, Karen Tu, Jewison Biscocho, Okyaz Eminaga, Rosalie Nolley, Robert Tibshirani, James D. Brooks, Robert B. West, Jonathan R. Pollack
Abstract
Despite current immunosuppressive strategies, long-term lung transplant outcomes remain poor due to rapid allogenic responses. Using a stringent mouse model of allo-airway transplantation, we identified the CCR4-ligand axis as a central node driving secondary lymphoid tissue homing and activation of the allogeneic T cells that prevent long-term allograft survival. CCR4 deficiency on transplant recipient T cells diminished allograft injury and when combined with CTLA4-Ig led to lung allograft accommodation lasting longer than in any previous study to our knowledge. Thus, we identify CCR4-ligand interactions as a central mechanism driving allogeneic transplant rejection and suggest it as a potential target to enhance long-term lung transplant survival.
Authors
Vyacheslav Palchevskiy, Ying Ying Xue, Rita Kern, Stephen S. Weigt, Aric L. Gregson, Sophie X. Song, Michael C. Fishbein, Cory M. Hogaboam, David M. Sayah, Joseph P. Lynch III, Michael P. Keane, David G. Brooks, John A. Belperio
Abstract
Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and impaired glucose-dependent facilitation of insulin exocytosis. We show in β cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion events, visualized by total internal reflection fluorescence microscopy in 24 of these donors, demonstrated that these events are nonrandom across the surface of β cells from donors with no diabetes. The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose dependent and notably impaired in T2D β cells. Mechanistically, multichannel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion “hotspots,” while SUMOylation sites at the channel N- (K145) and C-terminus (K470) determine the relative proportion of fusion events occurring within these regions. Thus, a glucose-dependent compartmentalization of fusion, regulated in part by a structural role for Kv2.1, is disrupted in β cells from donors with T2D.
Authors
Jianyang Fu, John Maringa Githaka, Xiaoqing Dai, Gregory Plummer, Kunimasa Suzuki, Aliya F. Spigelman, Austin Bautista, Ryekjang Kim, Dafna Greitzer-Antes, Jocelyn E. Manning Fox, Herbert Y. Gaisano, Patrick E. MacDonald
Abstract
TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform–specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
Authors
Tatsuya Katsuno, Inna A. Belyantseva, Alexander X. Cartagena-Rivera, Keisuke Ohta, Shawn M. Crump, Ronald S. Petralia, Kazuya Ono, Risa Tona, Ayesha Imtiaz, Atteeq Rehman, Hiroshi Kiyonari, Mari Kaneko, Ya-Xian Wang, Takaya Abe, Makoto Ikeya, Cristina Fenollar-Ferrer, Gavin P. Riordan, Elisabeth A. Wilson, Tracy S. Fitzgerald, Kohei Segawa, Koichi Omori, Juichi Ito, Gregory I. Frolenkov, Thomas B. Friedman, Shin-ichiro Kitajiri
Abstract
Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin–dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell–dependent autoimmune diseases.
Authors
Michihito Kono, Kayaho Maeda, Irina Stocton-Gavanescu, Wenliang Pan, Masataka Umeda, Eri Katsuyama, Catalina Burbano, Seo Yeon K. Orite, Milena Vukelic, Maria G. Tsokos, Nobuya Yoshida, George C. Tsokos
Abstract
African Americans develop end-stage renal disease at a higher rate compared with European Americans due to 2 polymorphisms (G1 and G2 risk variants) in the apolipoprotein L1 (APOL1) gene common in people of African ancestry. Although this compelling genetic evidence provides an exciting opportunity for personalized medicine in chronic kidney disease, drug discovery efforts have been greatly hindered by the fact that APOL1 expression is lacking in rodents. Here, we describe a potentially novel physiologically relevant genomic mouse model of APOL1-associated renal disease that expresses human APOL1 from the endogenous human promoter, resulting in expression in similar tissues and at similar relative levels as humans. While naive APOL1-transgenic mice did not exhibit a renal disease phenotype, administration of IFN-γ was sufficient to robustly induce proteinuria only in APOL1 G1 mice, despite inducing kidney APOL1 expression in both G0 and G1 mice, serving as a clinically relevant “second hit.” Treatment of APOL1 G1 mice with IONIS-APOL1Rx, an antisense oligonucleotide (ASO) targeting APOL1 mRNA, prior to IFN-γ challenge robustly and dose-dependently inhibited kidney and liver APOL1 expression and protected against IFN-γ–induced proteinuria, indicating that the disease-relevant cell types are sensitive to ASO treatment. Therefore, IONIS-APOL1Rx may be an effective therapeutic for APOL1 nephropathies and warrants further development.
Authors
Mariam Aghajan, Sheri L. Booten, Magnus Althage, Christopher E. Hart, Anette Ericsson, Ingela Maxvall, Joseph Ochaba, Angela Menschik-Lundin, Judith Hartleib, Steven Kuntz, Danielle Gattis, Christine Ahlström, Andrew T. Watt, Jeffery A. Engelhardt, Brett P. Monia, Maria Chiara Magnone, Shuling Guo
Abstract
The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1–/– zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced–obesity zebrafish model was used to analyze glo1–/– zebrafish under high nutrient intake. Glo1–/– zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1–/– zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1–/– zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake.
Authors
Elisabeth Lodd, Lucas M. Wiggenhauser, Jakob Morgenstern, Thomas H. Fleming, Gernot Poschet, Michael Büttner, Christoph T. Tabler, David P. Wohlfart, Peter P. Nawroth, Jens Kroll
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