Abstract
Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase–mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc– and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2fl/fl) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2fl/fl). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting cofactors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell–extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.
Authors
Steven Tessier, Alexandra C. Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
Abstract
Lesch-Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). LND is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected with LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic–based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency–associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S-adenosylmethionine, a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to patients with LND via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.
Authors
Valentin Ruillier, Johana Tournois, Claire Boissart, Marie Lasbareilles, Gurvan Mahé, Laure Chatrousse, Michel Cailleret, Marc Peschanski, Alexandra Benchoua
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.
Authors
Sonu Kashyap, Kyaw Zaw Hein, Claudia C.S. Chini, Jorgo Lika, Gina M. Warner, Laurie K. Bale, Vicente E. Torres, Peter C. Harris, Claus Oxvig, Cheryl A. Conover, Eduardo N. Chini
Abstract
Pulmonary Langerhans cell histiocytosis (PLCH) is a rare smoking-related lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of patients with PLCH harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models have impeded the study of PLCH pathogenesis. Here, we establish a cigarette smoke–exposed (CS-exposed) BRAF-V600E–mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant cell accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E–dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of myelomonocytic cells and the BRAF-V600E mutation and CS exposure in PLCH pathogenesis and provides a platform to develop biomarkers and therapeutic targets.
Authors
Huan Liu, Andrew R. Osterburg, Jennifer Flury, Zulma Swank, Dennis W. McGraw, Nishant Gupta, Kathryn A. Wikenheiser-Brokamp, Ashish Kumar, Abdellatif Tazi, Yoshikazu Inoue, Masaki Hirose, Francis X. McCormack, Michael T. Borchers
Abstract
Adequate iron supply during pregnancy is essential for fetal development. However, how fetal or amniotic fluid iron levels are regulated during healthy pregnancy, or pregnancies complicated by intraamniotic infection or inflammation (IAI), is unknown. We evaluated amniotic fluid and fetal iron homeostasis in normal and complicated murine, macaque, and human pregnancy. In mice, fetal iron endowment was affected by maternal iron status, but amniotic fluid iron concentrations changed little during maternal iron deficiency or excess. In murine and macaque models of inflamed pregnancy, the fetus responded to maternal systemic inflammation or IAI by rapidly upregulating hepcidin and lowering iron in fetal blood, without altering amniotic fluid iron. In humans, elevated cord blood hepcidin with accompanying hypoferremia was observed in pregnancies with antenatal exposure to IAI compared with those that were nonexposed. Hepcidin was also elevated in human amniotic fluid from pregnancies with IAI compared with those without IAI, but amniotic fluid iron levels did not differ between the groups. Our studies in mice, macaques, and humans demonstrate that amniotic fluid iron is largely unregulated but that the rapid induction of fetal hepcidin by inflammation and consequent fetal hypoferremia are conserved mechanisms that may be important in fetal host defense.
Authors
Allison L. Fisher, Veena Sangkhae, Pietro Presicce, Claire A. Chougnet, Alan H. Jobe, Suhas G. Kallapur, Sammy Tabbah, Catalin S. Buhimschi, Irina A. Buhimschi, Tomas Ganz, Elizabeta Nemeth
Abstract
The HIV latent reservoir in resting memory CD4+ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo, the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR+CD4+ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.
Authors
Adit Dhummakupt, Jessica H. Rubens, Thuy Anderson, Laura Powell, Bareng A.S. Nonyane, Lilly V. Siems, Aleisha Collinson-Streng, Tricia Nilles, R. Brad Jones, Vicki Tepper, Allison Agwu, Deborah Persaud
Abstract
Community-acquired pneumonia is a widespread disease with significant morbidity and mortality. Alveolar macrophages are tissue-resident lung cells that play a crucial role in innate immunity against bacteria that cause pneumonia. We hypothesized that alveolar macrophages display adaptive characteristics after resolution of bacterial pneumonia. We studied mice 1 to 6 months after self-limiting lung infections with Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Alveolar macrophages, but not other myeloid cells, recovered from the lung showed long-term modifications of their surface marker phenotype. The remodeling of alveolar macrophages was (a) long-lasting (still observed 6 months after infection), (b) regionally localized (observed only in the affected lobe after lobar pneumonia), and (c) associated with macrophage-dependent enhanced protection against another pneumococcal serotype. Metabolomic and transcriptomic profiling revealed that alveolar macrophages of mice that recovered from pneumonia had new baseline activities and altered responses to infection that better resembled those of adult humans. The enhanced lung protection after mild and self-limiting bacterial respiratory infections includes a profound remodeling of the alveolar macrophage pool that is long-lasting; compartmentalized; and manifest across surface receptors, metabolites, and both resting and stimulated transcriptomes.
Authors
Antoine Guillon, Emad I. Arafa, Kimberly A. Barker, Anna C. Belkina, Ian Martin, Anukul T. Shenoy, Alicia K. Wooten, Carolina Lyon De Ana, Anqi Dai, Adam Labadorf, Jaileene Hernandez Escalante, Hans Dooms, Hélène Blasco, Katrina E. Traber, Matthew R. Jones, Lee J. Quinton, Joseph P. Mizgerd
Abstract
In recent years, chimeric antigen receptor–modified T cell (CAR T cell) therapy has proven to be a promising approach against cancer. Nonetheless, this approach still faces multiple challenges in eliminating solid tumors, one of which being the immunosuppressive tumor microenvironment (TME). Here, we demonstrated that knocking out the endogenous TGF-β receptor II (TGFBR2) in CAR T cells with CRISPR/Cas9 technology could reduce the induced Treg conversion and prevent the exhaustion of CAR T ce lls. Meanwhile, TGFBR2-edited CAR T cells had better in vivo tumor elimination efficacy, both in cell line–derived xenograft and patient-derived xenograft solid tumor models, whether administered locally or systemically. In addition, the TGFBR2-edited CAR T cells could eliminate contralaterally reinoculated xenografts in mice effectively, with an increased proportion of memory subsets within circulating CAR T cells of central memory and effector memory subsets. In conclusion, we greatly improved the in vitro and in vivo function of CAR T cells in TGF-β–rich tumor environments by knocking out endogenous TGFBR2 and propose a potentially new method to improve the efficacy of CAR T cell therapy for treating solid tumors.
Authors
Na Tang, Chen Cheng, Xingying Zhang, Miaomiao Qiao, Na Li, Wei Mu, Xiao-Fei Wei, Weidong Han, Haoyi Wang
Abstract
Some effector CD4+ T cell subsets display cytotoxic activity, thus breaking the functional dichotomy of CD4+ helper and CD8+ cytotoxic T lymphocytes. However, molecular mechanisms regulating CD4+ cytotoxic T lymphocyte (CD4+ CTL) differentiation are poorly understood. Here we show that levels of histone deacetylases 1 and 2 (HDAC1-HDAC2) are key determinants of CD4+ CTL differentiation. Deletions of both Hdac1 and 1 Hdac2 alleles (HDAC1cKO-HDAC2HET) in CD4+ T cells induced a T helper cytotoxic program that was controlled by IFN-γ–JAK1/2–STAT1 signaling. In vitro, activated HDAC1cKO-HDAC2HET CD4+ T cells acquired cytolytic activity and displayed enrichment of gene signatures characteristic of effector CD8+ T cells and human CD4+ CTLs. In vivo, murine cytomegalovirus–infected HDAC1cKO-HDAC2HET mice displayed a stronger induction of CD4+ CTL features compared with infected WT mice. Finally, murine and human CD4+ T cells treated with short-chain fatty acids, which are commensal-produced metabolites acting as HDAC inhibitors, upregulated CTL genes. Our data demonstrate that HDAC1-HDAC2 restrain CD4+ CTL differentiation. Thus, HDAC1-HDAC2 might be targets for the therapeutic induction of CD4+ CTLs.
Authors
Teresa Preglej, Patricia Hamminger, Maik Luu, Tanja Bulat, Liisa Andersen, Lisa Göschl, Valentina Stolz, Ramona Rica, Lisa Sandner, Darina Waltenberger, Roland Tschismarov, Thomas Faux, Thorina Boenke, Asta Laiho, Laura L. Elo, Shinya Sakaguchi, Günter Steiner, Thomas Decker, Barbara Bohle, Alexander Visekruna, Christoph Bock, Birgit Strobl, Christian Seiser, Nicole Boucheron, Wilfried Ellmeier
Abstract
Accumulation of amyloid β protein (Aβ) due to increased generation and/or impaired degradation plays an important role in Alzheimer’s disease (AD) pathogenesis. In this report, we describe the identification of rare coding mutations in the endothelin-converting enzyme 2 (ECE2) gene in 1 late-onset AD family, and additional case-control cohort analysis indicates ECE2 variants associated with the risk of developing AD. The 2 mutations (R186C and F751S) located in the peptidase domain in the ECE2 protein were found to severely impair the enzymatic activity of ECE2 in Aβ degradation. We further evaluated the effect of the R186C mutation in mutant APP–knockin mice. Overexpression of wild-type ECE2 in the hippocampus reduced amyloid load and plaque formation, and improved learning and memory deficits in the AD model mice. However, the effect was abolished by the R186C mutation in ECE2. Taken together, the results demonstrated that ECE2 peptidase mutations contribute to AD pathogenesis by impairing Aβ degradation, and overexpression of ECE2 alleviates AD phenotypes. This study indicates that ECE2 is a risk gene for AD development and pharmacological activation of ECE2 could be a promising strategy for AD treatment.
Authors
Xinxin Liao, Fang Cai, Zhanfang Sun, Yun Zhang, Juelu Wang, Bin Jiao, Jifeng Guo, Jinchen Li, Xixi Liu, Lina Guo, Yafang Zhou, Junling Wang, Xinxiang Yan, Hong Jiang, Kun Xia, Jiada Li, Beisha Tang, Lu Shen, Weihong Song
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