Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first post-transplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. Natural killer (NK) cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. Together, these data support an aberrant NK cell-neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.
Daniel R. Calabrese, Tasha Tsao, Mélia Magnen, Colin Valet, Ying Gao, Beñat Mallavia, Jennifer J. Tian, Emily A. Aminian, Kristin M. Wang, Avishai Shemesh, Elman B. Punzalan, Aartik Sarma, Carolyn S. Calfee, Stephanie A. Christenson, Charles R. Langelier, Steven R. Hays, Jeff A. Golden, Lorriana E. Leard, Mary E. Kleinhenz, Nicholas A. Kolaitis, Rupal J. Shah, Aida Venado, Lewis L. Lanier, John R. Greenland, David M. Sayah, Abbas Ardehali, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Jonathan P Singer, Mark R. Looney
Mutations in the BRCA1 tumor suppressor gene, such as 5382insC (BRCA15382insC), give carriers an increased risk for breast, ovarian, prostate and pancreatic cancers. We have previously reported that, in mice, Brca1 deficiency in the hematopoietic system leads to pancytopenia and, as a result, early lethality. Here we explore the cellular consequences of Brca1 null and BRCA1 5382insC alleles in combination with Tp53 deficiency in the murine hematopoietic system. We find that Brca1 and Tp53 co-deficiency leads to a highly penetrant erythroproliferative disorder that is characterized by hepatosplenomegaly and expanded megakaryocyte erythroid progenitor (MEP) and immature erythroid blast populations. The expanded erythroid progenitor populations in both bone marrow and spleen have the capacity to transmit the disease into secondary mouse recipients, suggesting Brca1 and Tp53 co-deficiency provides a new murine model of hematopoietic neoplasia. This Brca1/Tp53 model replicates Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib sensitivity seen in existing Brca1/Tp53 breast cancer models and has the benefits of monitoring disease progression and drug responses via peripheral blood analyses without sacrificing experimental animals. In addition, this erythroid neoplasia develops much faster than murine breast cancer, allowing for increased efficiency of future preclinical studies.
Gerardo Lopez-Perez, Ranjula Wijayatunge, Kelly B. McCrum, Sam R. Holmstrom, Victoria E. Mgbemena, Theodora S. Ross
Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but the possibility of heterogeneous responses between muscles and across time remains unclear. Using mice inoculated with Colon-26 (C26) cancer, we demonstrate that specific force production was reduced in quadriceps and diaphragm at 2 weeks in the absence of atrophy. At this time, pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. By 4 weeks, atrophy occurred in both muscles, but specific force production increased to control levels in quadriceps such that reductions in absolute force were due entirely to atrophy. Specific force production remained reduced in diaphragm. Mitochondrial respiration increased and H2O2 emission was unchanged in both muscles vs control while mitochondrial creatine sensitivity was reduced in quadriceps. These findings indicate muscle weakness precedes atrophy and is linked to heterogeneous mitochondrial alterations that could involve adaptive responses to metabolic stress. Eventual muscle-specific restorations in force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscle to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.
Luca J. Delfinis, Catherine A. Bellissimo, Shivam Gandhi, Sara N. DiBenedetto, Madison C. Garibotti, Arshdeep K. Thuhan, Stavroula Tsitkanou, Megan E. Rosa-Caldwell, Fasih A. Rahman, Arthur J. Cheng, Michael P. Wiggs, Uwe Schlattner, Joe Quadrilatero, Nicholas P. Greene, Christopher G.R. Perry
Leukocyte Adhesion Deficiency Type-1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common β-chain of the ß2 integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. At the same time, LAD-1 patients are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ regulatory T cells (Treg) are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell – dendritic cell (DC) interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extra-lymphatic organs but resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs, and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.
Tanja Klaus, Alicia S. Wilson, Elisabeth Vicari, Eva Hadaschik, Matthias Klein, Sara Salome Helbich, Nadine Kamenjarin, Katrin Hodapp, Jenny Schunke, Maximilian Haist, Florian Butsch, Hans C. Probst, Alexander H. Enk, Karsten Mahnke, Ari Waisman, Monika Bednarczyk, Matthias Bros, Tobias Bopp, Stephan Grabbe
Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The syndrome of inappropriate secretion of anti-diuretic hormone (vasopressin) raised the question if vasopressin could be produced outside of the brain and whether the kidney could be a source of vasopressin. We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects the pre-pro-vasopressin, we found that immunoreactive pre-pro-vasopressin protein is found in mouse and human kidneys. Moreover, we found that murine collecting duct cells make biologically active vasopressin which increases in response to NaCl mediated hypertonicity, and that water restriction increases the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.
Juan P. Arroyo, Andrew S. Terker, Yvonne Zuchowski, Jason A. Watts, Fabian Bock, Cameron Meyer, Wentian Luo, Meghan E. Kapp, Edward R. Gould, Adam X Miranda, Joshua Carty, Ming Jiang, Roberto M. Vanacore, Elizabeth Hammock, Matthew H. Wilson, Roy Zent, Mingzhi Zhang, Gautam Bhave, Raymond C. Harris
Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us towards a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxp/loxp SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid installation. Studies in genetic models linked Pfkfb3 expression and function to hypoxia-inducible factor Hif1a. Intra-tracheal pyruvate instillation not only reconstituted Pfkfb3loxp/loxp or Hif1aloxp/loxp SPC ER Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies confirmed increased PFKFB3 staining in injured lungs and co-localized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counter-balancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.
Christine U. Vohwinkel, Nana Burns, Ethan Coit, Xiaoyi Yuan, Eszter K. Vladar, Christina Sul, Eric P. Schmidt, Peter Carmeliet, Kurt Stenmark, Eva S. Nozik, Rubin M. Tuder, Holger K. Eltzschig
There is limited data on the link between cardiac autonomic neuropathy (CAN) and severe hypoglycemia, in type 2 diabetes. We evaluated the associations of CAN with severe hypoglycemia among 7,421 adults with type 2 diabetes from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study. CAN was defined using electrocardiogram-derived measures. Cox and Andersen-Gill regression models were used to generate hazard ratios (HRs) for first and recurrent severe hypoglycemic episodes, respectively. Over 4.7 years, there were 558 first and 811 recurrent hypoglycemic events. Participants with CAN had increased risks of first (HR 1.23, 95%CI 1.01-1.50) or recurrent (HR: 1.46, 95%CI 1.16-1.84) episodes of severe hypoglycemia. The intensity of glycemic management modified the CAN association with hypoglycemia (P for interaction <0.05). In the standard glycemic management group, compared to participants without CAN, HRs for first severe hypoglycemia and recurrent hypoglycemia were 1.58 (95%CI 1.13-2.23) and 1.96 (1.33-2.90). In the intensive glycemic management group, HRs for first severe hypoglycemia and recurrent hypoglycemia were 1.10 (0.86-1.40) and 1.24 (0.93-1.65). In summary, CAN was independently associated with higher risks of first and recurrent hypoglycemia among adults with type 2 diabetes, with the highest risk observed among those on standard glycemic management.
Arnaud D. Kaze, Matthew F. Yuyun, Rexford S. Ahima, Michael R. Rickels, Justin B. Echouffo-Tcheugui
Acute and chronic intestinal inflammation is associated with epithelial damage, resulting in mucosal wounds in the forms of erosions and ulcers in the intestinal tract. Intestinal epithelial cells (IECs) and immune cells in the wound milieu secrete cytokines and lipid mediators to influence repair. Leukotriene B4 (LTB4), a lipid chemokine, binds to its receptor BLT1 and promotes migration of immune cells to sites of active inflammation, however a role for intestinal epithelial BLT1 during mucosal wound repair is not known. Here we report that BLT1 is expressed in IECs both in vitro and in vivo, where it functions as a receptor not only for LTB4 but also for another ligand Resolvin E1. Intestinal epithelial BLT1 expression is increased when epithelial cells are exposed to an inflammatory microenvironment. Using human and murine primary colonic epithelial cells, we reveal that LTB4-BLT1 axis promotes epithelial migration and proliferation leading to accelerated epithelial wound repair. Furthermore, in vivo intestinal wound repair experiments in BLT1-deficient mice and bone marrow chimeras demonstrate an important contribution of epithelial BLT1 during colonic mucosal wound repair. Taken together, our findings show a novel pro-repair in IEC mechanism mediated by BLT1 signaling.
Shusaku Hayashi, Chithra K. Muraleedharan, Makito Oku, Sunil Tomar, Simon P. Hogan, Miguel Quiros, Charles A. Parkos, Asma Nusrat
The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, these drug development efforts have been unsuccessful in mouse models. Here, we demonstrate unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We establish that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the ER folding environment. Moreover, we demonstrate direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum folding environment by affecting Ca++ concentration. Finally, we develop a panel of isogenic human NPC1 iNeurons expressing wild type, I1061T-, and R934L-NPC1 and demonstrate their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1’s response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.
Mark L. Schultz, Kylie J. Schache, Ruth D. Azaria, Esmée Q. Kuiper, Steven Erwood, Evgueni A. Ivakine, Nicole Y. Farhat, Forbes D. Porter, Koralege C. Pathmasiri, Stephanie M. Cologna, Michael D. Uhler, Andrew P. Lieberman
Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-sequencing, cancer and T cell profiling, and pharmacologic perturbations. RNA-sequencing analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared to localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, which was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.
Jacques Greenberg, Jessica Limberg, Akanksha Verma, David Kim, Xiang Chen, Yeon J. Lee, Maureen D. Moore, Timothy M. Ullmann, Jessica W. Thiesmeyer, Zachary Loewenstein, Kevin J. Chen, Caitlin E. Egan, Dessislava Stefanova, Rohan Bareja, Rasa Zarnegar, Brendan M. Finnerty, Theresa Scognamiglio, Yi-Chieh Nancy Du, Olivier Elemento, Thomas J. Fahey III, Irene M. Min
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