A number of studies in model animal systems and in the clinic have established that RANKL promotes bone resorption. Paradoxically, we found that pulsing ovariectomized mice with low-dose RANKL suppressed bone resorption, decreased the levels of proinflammatory effector T cells and led to increased bone mass. This effect of RANKL is mediated through the induction of FoxP3+CD25+ regulatory CD8+ T cells (TcREG) by osteoclasts. Here, we show that pulses of low-dose RANKL are needed to induce TcREG, as continuous infusion of identical doses of RANKL by pump did not induce TcREG. We also show that low-dose RANKL can induce TcREG at 2, 3, 6, and 10 weeks after ovariectomy. Our results show that low-dose RANKL treatment in ovariectomized mice is optimal at once-per-month doses to maintain the bone mass. Finally, we found that treatment of ovariectomized mice with the Cathepsin K inhibitor odanacatib also blocked TcREG induction by low-dose RANKL. We interpret this result to indicate that antigens presented to CD8+ T cells by osteoclasts are derived from the bone protein matrix because Cathepsin K degrades collagen in the bone. Taken together, our studies provide a basis for using low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis.
Anna Cline-Smith, Jesse Gibbs, Elena Shashkova, Zachary S. Buchwald, Rajeev Aurora
Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.
Sarah D. Ahadome, David J. Abraham, Suryanarayana Rayapureddi, Valerie P. Saw, Daniel R. Saban, Virginia L. Calder, Jill T. Norman, Markella Ponticos, Julie T. Daniels, John K. Dart
Current methods of drug screening in human blood focus on the immediate products of the affected pathway and mostly rely on approaches that lack sensitivity and the capacity for multiplex analysis. We have developed a sensitive and selective method based on ultra-performance liquid chromatography–tandem mass spectrometry to scan the effect of drugs on the bioactive eicosanoid lipidome in vitro and ex vivo. Using small sample sizes, we can reproducibly measure a broad spectrum of eicosanoids in human blood and capture drug-induced substrate rediversion and unexpected shifts in product formation. Microsomal prostaglandin E synthase-1 (mPGES-1) is an antiinflammatory drug target alternative to COX-1/-2. Contrasting effects of targeting mPGES-1 versus COX-1/-2, due to differential substrate shifts across the lipidome, were observed and can be used to rationalize and evaluate drug combinations. Finally, the in vitro results were extrapolated to ex vivo studies by administration of the COX-2 inhibitor, celecoxib, to volunteers, illustrating how this approach can be used to integrate preclinical and clinical studies during drug development.
Liudmila L. Mazaleuskaya, John A. Lawson, Xuanwen Li, Gregory Grant, Clementina Mesaros, Tilo Grosser, Ian A. Blair, Emanuela Ricciotti, Garret A. FitzGerald
It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant–mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2+ monocyte–derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury.
Wenjun Li, Hsi-Min Hsiao, Ryuji Higashikubo, Brian T. Saunders, Ankit Bharat, Daniel R. Goldstein, Alexander S. Krupnick, Andrew E. Gelman, Kory J. Lavine, Daniel Kreisel
Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to
Kong Chen, Brian T. Campfield, Sally E. Wenzel, Jeremy P. McAleer, James L. Kreindler, Geoffrey Kurland, Radha Gopal, Ting Wang, Wei Chen, Taylor Eddens, Kathleen M. Quinn, Mike M. Myerburg, William T. Horne, Jose M. Lora, Brian K. Albrecht, Joseph M. Pilewski, Jay K. Kolls
Dopamine D2 receptor (DRD2) deficiency increases renal inflammation and blood pressure in mice. We show here that long-term renal-selective silencing of
Prasad R. Konkalmatt, Laureano D. Asico, Yanrong Zhang, Yu Yang, Cinthia Drachenberg, Xiaoxu Zheng, Fei Han, Pedro A. Jose, Ines Armando
Conventional memory CD8+ T cells and mucosal-associated invariant T cells (MAIT cells) are found in blood, liver, and mucosal tissues and have similar effector potential following activation, specifically expression of IFN-γ and granzyme B. To better understand each subset’s unique contributions to immunity and pathology, we interrogated inflammation- and TCR-driven activation requirements using human memory CD8+ T and MAIT cells isolated from blood and mucosal tissue biopsies in ex vivo functional assays and single cell gene expression experiments. We found that MAIT cells had a robust IFN-γ and granzyme B response to inflammatory signals but limited responsiveness when stimulated directly via their TCR. Importantly, this is not due to an overall hyporesponsiveness to TCR signals. When delivered together, TCR and inflammatory signals synergize to elicit potent effector function in MAIT cells. This unique control of effector function allows MAIT cells to respond to the same TCR signal in a dichotomous and situation-specific manner. We propose that this could serve to prevent responses to antigen in noninflamed healthy mucosal tissue, while maintaining responsiveness and great sensitivity to inflammation-eliciting infections. We discuss the implications of these findings in context of inflammation-inducing damage to tissues such as BM transplant conditioning or HIV infection.
Chloe K. Slichter, Andrew McDavid, Hannah W. Miller, Greg Finak, Brenda J. Seymour, John P. McNevin, Gabriela Diaz, Julie L. Czartoski, M. Juliana McElrath, Raphael Gottardo, Martin Prlic
Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIghi macrophages differed between patients and in the same patient over time, and a high proportion of CRIghi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIglo macrophages, CRIghi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIghi cells, human macrophages, and mouse F4/80hi resident peritoneal macrophages and among CRIglo macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIghi and CRIglo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.
Katharine M. Irvine, Xuan Banh, Victoria L. Gadd, Kyle K. Wojcik, Juliana K. Ariffin, Sara Jose, Samuel Lukowski, Gregory J. Baillie, Matthew J. Sweet, Elizabeth E. Powell
Reperfusion injury can exacerbate tissue damage in ischemic stroke, but little is known about the mechanisms linking ROS to stroke severity. Here, we tested the hypothesis that protein methionine oxidation potentiates NF-κB activation and contributes to cerebral ischemia/reperfusion injury. We found that overexpression of methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that reverses protein methionine oxidation, attenuated ROS-augmented NF-κB activation in endothelial cells, in part, by protecting against the oxidation of methionine residues in the regulatory domain of calcium/calmodulin-dependent protein kinase II (CaMKII). In a murine model, MsrA deficiency resulted in increased NF-κB activation and neutrophil infiltration, larger infarct volumes, and more severe neurological impairment after transient cerebral ischemia/reperfusion injury. This phenotype was prevented by inhibition of NF-κB or CaMKII. MsrA-deficient mice also exhibited enhanced leukocyte rolling and upregulation of E-selectin, an endothelial NF-κB–dependent adhesion molecule known to contribute to neurovascular inflammation in ischemic stroke. Finally, bone marrow transplantation experiments demonstrated that the neuroprotective effect was mediated by MsrA expressed in nonhematopoietic cells. These findings suggest that protein methionine oxidation in nonmyeloid cells is a key mechanism of postischemic oxidative injury mediated by NF-κB activation, leading to neutrophil recruitment and neurovascular inflammation in acute ischemic stroke.
Sean X. Gu, Ilya O. Blokhin, Katina M. Wilson, Nirav Dhanesha, Prakash Doddapattar, Isabella M. Grumbach, Anil K. Chauhan, Steven R. Lentz
Keisuke Maeshima, Stephanie M. Stanford, Deepa Hammaker, Cristiano Sacchetti, Li-fan Zeng, Rizi Ai, Vida Zhang, David L. Boyle, German R. Aleman Muench, Gen-Sheng Feng, John W. Whitaker, Zhong-Yin Zhang, Wei Wang, Nunzio Bottini, Gary S. Firestein
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