Polyfunctional and IFN-γ monofunctional human CD4⁺ T cell populations are molecularly distinct

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Abstract

Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4⁺ T cells induced during Plasmodium falciparum (P. falciparum) blood-stage infection in humans have a unique transcriptomic profile compared with IFN-γ monofunctional CD4⁺ T cells and, thus, are molecularly distinct. The 14-gene signature revealed in P. falciparum–reactive polyfunctional T cells is associated with cytokine signaling and lymphocyte chemotaxis, and systems biology analysis identified IL-27 as an upstream regulator of the polyfunctional gene signature. Importantly, the polyfunctional gene signature is largely conserved in Influenza-reactive polyfunctional CD4⁺ T cells, suggesting that polyfunctional T cells have core characteristics independent of pathogen specificity. This study provides the first evidence to our knowledge that consistent molecular differences exist between polyfunctional and monofunctional CD4⁺ T cells.

Authors

Julie G. Burel, Simon H. Apte, Penny L. Groves, James S. McCarthy, Denise L. Doolan

BET inhibitors block pancreatic stellate cell collagen I production and attenuate fibrosis in vivo

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Abstract

The fibrotic reaction, which can account for over 70%–80% of the tumor mass, is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of collagen I production and fibrosis in vivo. In this report, we show that members of the bromodomain and extraterminal (BET) family of proteins are expressed in primary PSCs isolated from human PDAC tumors, with BRD4 positively regulating, and BRD2 and BRD3 negatively regulating, collagen I expression in primary cancer-associated PSCs. We show that the inhibitory effect of pan-BET inhibitors on collagen I expression in primary cancer-associated PSCs is through blocking of BRD4 function. Importantly, we show that FOSL1 is repressed by BRD4 in primary cancer-associated PSCs and negatively regulates collagen I expression. While BET inhibitors do not affect viability or induce PSC apoptosis or senescence, BET inhibitors induce primary cancer-associated PSCs to become quiescent. Finally, we show that BET inhibitors attenuate stellate cell activation, fibrosis, and collagen I production in the EL-Kras^G12D transgenic mouse model of pancreatic tumorigenesis. Our results demonstrate that BET inhibitors regulate fibrosis by modulating the activation and function of cancer-associated PSCs.

Authors

Krishan Kumar, Brian T. DeCant, Paul J. Grippo, Rosa F. Hwang, David J. Bentrem, Kazumi Ebine, Hidayatullah G. Munshi

Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma

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Abstract

Excessive ROS promote allergic asthma, a condition characterized by airway inflammation, eosinophilic inflammation, and increased airway hyperreactivity (AHR). The mechanisms by which airway ROS are increased and the relationship between increased airway ROS and disease phenotypes are incompletely defined. Mitochondria are an important source of cellular ROS production, and our group discovered that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is present in mitochondria and activated by oxidation. Furthermore, mitochondrial-targeted antioxidant therapy reduced the severity of allergic asthma in a mouse model. Based on these findings, we developed a mouse model of CaMKII inhibition targeted to mitochondria in airway epithelium. We challenged these mice with OVA or Aspergillus fumigatus. Mitochondrial CaMKII inhibition abrogated AHR, inflammation, and eosinophilia following OVA and A. fumigatus challenge. Mitochondrial ROS were decreased after agonist stimulation in the presence of mitochondrial CaMKII inhibition. This correlated with blunted induction of NF-κB, the NLRP3 inflammasome, and eosinophilia in transgenic mice. These findings demonstrate a pivotal role for mitochondrial CaMKII in airway epithelium in mitochondrial ROS generation, eosinophilic inflammation, and AHR, providing insights into how mitochondrial ROS mediate features of allergic asthma.

Authors

Sara C. Sebag, Olha M. Koval, John D. Paschke, Christopher J. Winters, Omar A. Jaffer, Ryszard Dworski, Fayyaz S. Sutterwala, Mark E. Anderson, Isabella M. Grumbach

Corticosteroids inhibit anti-IgE activities of specialized proresolving mediators on B cells from asthma patients

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Abstract

Specialized proresolving mediators (SPMs) promote the resolution of inflammation and exert beneficial effects in animal models of chronic inflammatory diseases, including asthma. Previously, we have shown that certain SPMs reduce IgE production in B cells from healthy individuals, which has a critical role in allergic asthma. Here, we investigated the effects of SPMs on B cell IgE production in asthma patients. Peripheral blood mononuclear cells from asthma patients were treated with 17-HDHA or RvD1, and IgE levels were measured. RvD1 and 17-HDHA dampened IgE production in B cells from most asthma patients, whereas B cells from a subset of patients taking oral steroids were refractory to SPM treatment. Molecular mechanisms underlying the interaction between corticosteroids and SPMs were investigated by treating B cells from nonasthmatic donors with corticosteroids in vitro. Corticosteroids blocked the inhibitory effects of 17-HDHA and RvD1 on B cell IgE production by abolishing the suppressive activity of these mediators on IgE class switching. Corticosteroids decreased the expression of transcriptional repressor Bcl-6 as well as its suppressive activity on epsilon germline transcription. We conclude that 17-HDHA and RvD1 can reduce IgE production in asthma patients not taking high doses of steroids but that corticosteroids interfere with the ability of B cells to respond to proresolving mediators.

Authors

Nina Kim, Thomas H. Thatcher, Patricia J. Sime, Richard P. Phipps

Foxp3 drives oxidative phosphorylation and protection from lipotoxicity

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Abstract

Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation–fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is “hardwired” by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy–based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs’ increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid β-oxidation also results in selective protection of Foxp3⁺ cells from fatty acid–induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically.

Authors

Duncan Howie, Stephen Paul Cobbold, Elizabeth Adams, Annemieke Ten Bokum, Andra Stefania Necula, Wei Zhang, Honglei Huang, David J. Roberts, Benjamin Thomas, Svenja S. Hester, David J. Vaux, Alexander G. Betz, Herman Waldmann

Real-time genomic profiling of histiocytoses identifies early-kinase domain BRAF alterations while improving treatment outcomes

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Abstract

Many patients with histiocytic disorders such as Langerhans cell histiocytosis (LCH) or Erdheim-Chester disease (ECD) have treatment-refractory disease or suffer recurrences. Recent findings of gene mutations in histiocytoses have generated options for targeted therapies. We sought to determine the utility of prospective sequencing of select genes to further characterize mutations and identify targeted therapies for patients with histiocytoses. Biopsies of 72 patients with a variety of histiocytoses underwent comprehensive genomic profiling with targeted DNA and RNA sequencing. Fifteen patients (21%) carried the known BRAF V600E mutation, and 11 patients (15%) carried various mutations in MAP2K1, which we confirm induce constitutive activation of extracellular signal–regulated kinase (ERK) and were sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK, the product of MAP2K1). We also identified recurring ALK rearrangements, and 4 LCH patients with an uncommon in-frame deletion in BRAF (N486_P490del or N486_T491>K), resulting in constitutive activation of ERK with resistance to V600E-specific inhibitors. We subsequently describe clinical cases where patients with aggressive multisystem LCH experience dramatic and sustained responses to monotherapy with either dabrafenib or trametinib. These findings support our conclusion that comprehensive genomic profiling should be regularly applied to these disorders at diagnosis, and can positively impact clinical care.

Authors

Lynn H. Lee, Anjelika Gasilina, Jayeeta Roychoudhury, Jason Clark, Francis X. McCormack, Joseph Pressey, Michael S. Grimley, Robert Lorsbach, Siraj Ali, Mark Bailey, Philip Stephens, Jeffrey S. Ross, Vincent A. Miller, Nicolas N. Nassar, Ashish R. Kumar

Mitochondrial quality-control dysregulation in conditional HO-1^–/– mice

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Abstract

The heme oxygenase-1 (Hmox1; HO-1) pathway was tested for defense of mitochondrial quality control in cardiomyocyte-specific Hmox1 KO mice (HO-1[CM]^–/–) exposed to oxidative stress (100% O₂). After 48 hours of exposure, these mice showed persistent cardiac inflammation and oxidative tissue damage that caused sarcomeric disruption, cardiomyocyte death, left ventricular dysfunction, and cardiomyopathy, while control hearts showed minimal damage. After hyperoxia, HO-1(CM)^–/– hearts showed suppression of the Pgc-1α/nuclear respiratory factor-1 (NRF-1) axis, swelling, low electron density mitochondria by electron microscopy (EM), increased cell death, and extensive collagen deposition. The damage mechanism involves structurally deficient autophagy/mitophagy, impaired LC3II processing, and failure to upregulate Pink1- and Park2-mediated mitophagy. The mitophagy pathway was suppressed through loss of NRF-1 binding to proximal promoter sites on both genes. These results indicate that cardiac Hmox1 induction not only prevents heme toxicity, but also regulates the timing and registration of genetic programs for mitochondrial quality control that limit cell death, pathological remodeling, and cardiac fibrosis.

Authors

Hagir B. Suliman, Jeffrey E. Keenan, Claude A. Piantadosi

A role for muscarinic receptors in neutrophil extracellular trap formation and levamisole-induced autoimmunity

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Abstract

Levamisole, an anthelmintic drug with cholinergic properties, has been implicated in cases of drug-induced vasculitis when added to cocaine for profit purposes. Neutrophil extracellular trap (NET) formation is a cell death mechanism characterized by extrusion of chromatin decorated with granule proteins. Aberrant NET formation and degradation have been implicated in idiopathic autoimmune diseases that share features with levamisole-induced autoimmunity as well as in drug-induced autoimmunity. This study’s objective was to determine how levamisole modulates neutrophil biology and its putative effects on the vasculature. Murine and human neutrophils exposed to levamisole demonstrated enhanced NET formation through engagement of muscarinic subtype 3 receptor. Levamisole-induced NETosis required activation of Akt and the RAF/MEK/ERK pathway, ROS induction through the nicotinamide adenine dinucleotide phosphate oxidase, and peptidylarginine deiminase activation. Sera from two cohorts of patients actively using levamisole-adulterated cocaine displayed autoantibodies against NET components. Cutaneous biopsy material obtained from individuals exposed to levamisole suggests that neutrophils produce NETs in areas of vasculitic inflammation and thrombosis. NETs generated by levamisole were toxic to endothelial cells and impaired endothelium-dependent vasorelaxation. Stimulation of muscarinic receptors on neutrophils by cholinergic agonists may contribute to the pathophysiology observed in drug-induced autoimmunity through the induction of inflammatory responses and neutrophil-induced vascular damage.

Authors

Carmelo Carmona-Rivera, Monica M. Purmalek, Erica Moore, Meryl Waldman, Peter J. Walter, H. Martin Garraffo, Karran A. Phillips, Kenzie L. Preston, Jonathan Graf, Mariana J. Kaplan, Peter C. Grayson

An early-biomarker algorithm predicts lethal graft-versus-host disease and survival

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Abstract

BACKGROUND. No laboratory test can predict the risk of nonrelapse mortality (NRM) or severe graft-versus-host disease (GVHD) after hematopoietic cellular transplantation (HCT) prior to the onset of GVHD symptoms. METHODS. Patient blood samples on day 7 after HCT were obtained from a multicenter set of 1,287 patients, and 620 samples were assigned to a training set. We measured the concentrations of 4 GVHD biomarkers (ST2, REG3α, TNFR1, and IL-2Rα) and used them to model 6-month NRM using rigorous cross-validation strategies to identify the best algorithm that defined 2 distinct risk groups. We then applied the final algorithm in an independent test set (n = 309) and validation set (n = 358). RESULTS. A 2-biomarker model using ST2 and REG3α concentrations identified patients with a cumulative incidence of 6-month NRM of 28% in the high-risk group and 7% in the low-risk group (P < 0.001). The algorithm performed equally well in the test set (33% vs. 7%, P < 0.001) and the multicenter validation set (26% vs. 10%, P < 0.001). Sixteen percent, 17%, and 20% of patients were at high risk in the training, test, and validation sets, respectively. GVHD-related mortality was greater in high-risk patients (18% vs. 4%, P < 0.001), as was severe gastrointestinal GVHD (17% vs. 8%, P < 0.001). The same algorithm can be successfully adapted to define 3 distinct risk groups at GVHD onset. CONCLUSION. A biomarker algorithm based on a blood sample taken 7 days after HCT can consistently identify a group of patients at high risk for lethal GVHD and NRM. FUNDING. The National Cancer Institute, American Cancer Society, and the Doris Duke Charitable Foundation.

Authors

Matthew J. Hartwell, Umut Özbek, Ernst Holler, Anne S. Renteria, Hannah Major-Monfried, Pavan Reddy, Mina Aziz, William J. Hogan, Francis Ayuk, Yvonne A. Efebera, Elizabeth O. Hexner, Udomsak Bunworasate, Muna Qayed, Rainer Ordemann, Matthias Wölfl, Stephan Mielke, Attaphol Pawarode, Yi-Bin Chen, Steven Devine, Andrew C. Harris, Madan Jagasia, Carrie L. Kitko, Mark R. Litzow, Nicolaus Kröger, Franco Locatelli, George Morales, Ryotaro Nakamura, Ran Reshef, Wolf Rösler, Daniela Weber, Kitsada Wudhikarn, Gregory A. Yanik, John E. Levine, James L.M. Ferrara

Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells

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Abstract

Rat and human CD4⁺ and CD8⁺ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4⁺ and CD8⁺ Foxp3⁺ Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RC^high T cells through intrinsic cell signaling but preserved and potentiated CD4⁺ and CD8⁺ CD45RC^low/– Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4⁺ and CD8⁺ CD45RC^low/– Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human.

Authors

Elodie Picarda, Séverine Bézie, Laetitia Boucault, Elodie Autrusseau, Stéphanie Kilens, Dimitri Meistermann, Bernard Martinet, Véronique Daguin, Audrey Donnart, Eric Charpentier, Laurent David, Ignacio Anegon, Carole Guillonneau

Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction

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Abstract

Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle–specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction.

Authors

Evalynn Vasquez, Vivian Cristofaro, Stefan Lukianov, Fiona C. Burkhard, Ali Hashemi Gheinani, Katia Monastyrskaya, Diane R. Bielenberg, Maryrose P. Sullivan, Rosalyn M. Adam

Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease

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Abstract

Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.

Authors

Dominic J. Gessler, Danning Li, Hongxia Xu, Qin Su, Julio Sanmiguel, Serafettin Tuncer, Constance Moore, Jean King, Reuben Matalon, Guangping Gao

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown

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Abstract

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Authors

Shuntaro Ogura, Kaori Kurata, Yuki Hattori, Hiroshi Takase, Toshina Ishiguro-Oonuma, Yoonha Hwang, Soyeon Ahn, Inwon Park, Wataru Ikeda, Sentaro Kusuhara, Yoko Fukushima, Hiromi Nara, Hideto Sakai, Takashi Fujiwara, Jun Matsushita, Masatsugu Ema, Masanori Hirashima, Takashi Minami, Masabumi Shibuya, Nobuyuki Takakura, Pilhan Kim, Takaki Miyata, Yuichiro Ogura, Akiyoshi Uemura

Adipocyte JAK2 mediates growth hormone–induced hepatic insulin resistance

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Abstract

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue–dependent mechanism.

Authors

Kevin C. Corbit, João Paulo G. Camporez, Jennifer L. Tran, Camella G. Wilson, Dylan A. Lowe, Sarah M. Nordstrom, Kirthana Ganeshan, Rachel J. Perry, Gerald I. Shulman, Michael J. Jurczak, Ethan J. Weiss

Diverse repetitive element RNA expression defines epigenetic and immunologic features of colon cancer

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Abstract

There is tremendous excitement for the potential of epigenetic therapies in cancer, but the ability to predict and monitor response to these drugs remains elusive. This is in part due to the inability to differentiate the direct cytotoxic and the immunomodulatory effects of these drugs. The DNA-hypomethylating agent 5-azacitidine (AZA) has shown these distinct effects in colon cancer and appears to be linked to the derepression of repeat RNAs. LINE and HERV are two of the largest classes of repeats in the genome, and despite many commonalities, we found that there is heterogeneity in behavior among repeat subtypes. Specifically, the LINE-1 and HERV-H subtypes detected by RNA sequencing and RNA in situ hybridization in colon cancers had distinct expression patterns, which suggested that these repeats are correlated to transcriptional programs marking different biological states. We found that low LINE-1 expression correlates with global DNA hypermethylation, wild-type TP53 status, and responsiveness to AZA. HERV-H repeats were not concordant with LINE-1 expression but were found to be linked with differences in FOXP3⁺ Treg tumor infiltrates. Together, distinct repeat RNA expression patterns define new molecular classifications of colon cancer and provide biomarkers that better distinguish cytotoxic from immunomodulatory effects by epigenetic drugs.

Authors

Niyati Desai, Dipti Sajed, Kshitij S. Arora, Alexander Solovyov, Mihir Rajurkar, Jacob R. Bledsoe, Srinjoy Sil, Ramzi Amri, Eric Tai, Olivia C. MacKenzie, Mari Mino-Kenudson, Martin J. Aryee, Cristina R. Ferrone, David L. Berger, Miguel N. Rivera, Benjamin D. Greenbaum, Vikram Deshpande, David T. Ting

Knockin mouse with mutant Gα₁₁ mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

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Abstract

Heterotrimeric G proteins play critical roles in transducing extracellular signals generated by 7-transmembrane domain receptors. Somatic gain-of-function mutations in G protein α subunits are associated with a variety of diseases. Recently, we identified gain-of-function mutations in Gα₁₁ in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11^R6OC is partly dependent on coupling to the CASR. Treatment with the Gα_11/q-specific inhibitor YM-254890 increased blood calcium in heterozygous but not in homozygous GNA11^R60C mice, consistent with published crystal structure data showing that Arg60 forms a critical contact with YM-254890. This animal model of ADH2 provides insights into molecular mechanism of this G protein–related disease and potential paths toward new lines of therapy.

Authors

Kelly L. Roszko, Ruiye Bi, Caroline M. Gorvin, Hans Bräuner-Osborne, Xiao-Feng Xiong, Asuka Inoue, Rajesh V. Thakker, Kristian Strømgaard, Thomas Gardella, Michael Mannstadt

Gα₁₁ mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

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Abstract

Heterozygous germline gain-of-function mutations of G-protein subunit α₁₁ (Gα₁₁), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα₁₁ mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα₁₁ to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα₁₁ mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

Authors

Caroline M. Gorvin, Fadil M. Hannan, Sarah A. Howles, Valerie N. Babinsky, Sian E. Piret, Angela Rogers, Andrew J. Freidin, Michelle Stewart, Anju Paudyal, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Tonia L. Vincent, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker