Inflammation
Abstract
Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define “purinergic Th2-type cytokine bias” as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.
Authors
Jennifer C. Yu, Gene Lin, Joshua J. Field, Joel Linden
Abstract
Poorly controlled diabetes leads to comorbidities and enhanced susceptibility to infections. While the immune components involved in wound healing in diabetes have been studied, the components involved in susceptibility to skin infections remain unclear. Here, we examined the effects of the inflammatory lipid mediator leukotriene B4 (LTB4) signaling through its receptor B leukotriene receptor 1 (BLT1) in the progression of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in 2 models of diabetes. Diabetic mice produced higher levels of LTB4 in the skin, which correlated with larger nonhealing lesion areas and increased bacterial loads compared with nondiabetic mice. High LTB4 levels were also associated with dysregulated cytokine and chemokine production, excessive neutrophil migration but impaired abscess formation, and uncontrolled collagen deposition. Both genetic deletion and topical pharmacological BLT1 antagonism restored inflammatory response and abscess formation, followed by a reduction in the bacterial load and lesion area in the diabetic mice. Macrophage depletion in diabetic mice limited LTB4 production and improved abscess architecture and skin host defense. These data demonstrate that exaggerated LTB4/BLT1 responses mediate a derailed inflammatory milieu that underlies poor host defense in diabetes. Prevention of LTB4 production/actions could provide a new therapeutic strategy to restore host defense in diabetes.
Authors
Stephanie L. Brandt, Sue Wang, Naiara N. Dejani, Nathan Klopfenstein, Seth Winfree, Luciano Filgueiras, Brian P. McCarthy, Paul R. Territo, C. Henrique Serezani
Abstract
Hemorrhagic shock depletes nicotinamide adenine dinucleotide (NAD) and causes metabolic derangements that, in severe cases, cannot be overcome, even after restoration of blood volume and pressure. However, current strategies to treat acute blood loss do not target cellular metabolism. We hypothesized that supplemental nicotinamide mononucleotide (NMN), the immediate biosynthetic precursor to NAD, would support cellular energetics and enhance physiologic resilience to hemorrhagic shock. In a rodent model of decompensated hemorrhagic shock, rats receiving NMN displayed significantly reduced lactic acidosis and serum IL-6 levels, two strong predictors of mortality in human patients. In both livers and kidneys, NMN increased NAD levels and prevented mitochondrial dysfunction. Moreover, NMN preserved mitochondrial function in isolated hepatocytes cocultured with proinflammatory cytokines, indicating a cell-autonomous protective effect that is independent from the reduction in circulating IL-6. In kidneys, but not in livers, NMN was sufficient to prevent ATP loss following shock and resuscitation. Overall, NMN increased the time animals could sustain severe shock before requiring resuscitation by nearly 25% and significantly improved survival after resuscitation (P = 0.018), whether NMN was given as a pretreatment or only as an adjunct during resuscitation. Thus, we demonstrate that NMN substantially mitigates inflammation, improves cellular metabolism, and promotes survival following hemorrhagic shock.
Authors
Carrie A. Sims, Yuxia Guan, Sarmistha Mukherjee, Khushboo Singh, Paul Botolin, Antonio Davila Jr., Joseph A. Baur
Abstract
Brain death that occurs in the setting of deceased organ donation for transplantation is associated with systemic inflammation of unknown origin. It has recently been recognized that mitochondria-derived damage-associated molecular patterns (mtDAMPs) released into the circulation in the setting of trauma and tissue injury are associated with a systemic inflammatory response. We examined the blood of deceased organ donors and found elevated levels of inflammatory cytokines and chemokines that correlated with levels of mtDAMPs. We also found that donor neutrophils are activated and that donor plasma contains a neutrophil-activating factor that is blocked by cyclosporin H, a formyl peptide receptor-1 antagonist. Examination of donor plasma by electron microscopy and flow cytometry revealed that free- and membrane-bound mitochondria are elevated in donor plasma. Interestingly, we demonstrated a correlation between donor plasma mitochondrial DNA levels and early allograft dysfunction in liver transplant recipients, suggesting a role for circulating mtDAMPs in allograft outcomes. Current approaches to prolong allograft survival focus on immune suppression in the transplant recipient; our data indicate that targeting inflammatory factors in deceased donors prior to organ procurement is another potential strategy for improving transplant outcomes.
Authors
Justin Pollara, R. Whitney Edwards, Liwen Lin, Victoria A. Bendersky, Todd V. Brennan
Abstract
Polyarticular juvenile idiopathic arthritis (JIA) is among the most challenging of the JIA subtypes to treat. Even with current biologic therapies, the disease remains difficult to control in a substantial subset of patients, highlighting the need for new therapies. The aim of this study was to use the high dimensionality afforded by mass cytometry with phospho-specific antibodies to delineate signaling abnormalities in immune cells from treatment-naive polyarticular JIA patients. Peripheral blood mononuclear cells were isolated from 17 treatment-naive polyarticular JIA patients, 10 of the patients after achieving clinical remission, and 19 healthy controls. Samples were stimulated for 15 minutes with IL-6 or IFN-γ and analyzed by mass cytometry. Following IFN-γ stimulation, increased STAT1 and/or STAT3 phosphorylation was observed in subsets of CD4 T cells and classical monocytes from treatment-naive patients. The enhanced IFN-γ signaling was associated with increased expression of JAK1 and SOCS1 in CD4 T cells. Furthermore, substantial heterogeneity in surface marker expression was observed among the subsets of CD4 T cells and classical monocytes with increased IFN-γ responsiveness. The identification of enhanced IFN-γ signaling in CD4 T cells and classical monocytes from treatment-naive polyarticular JIA patients provides mechanistic support for investigations into therapies that attenuate IFN-γ signaling in this disease.
Authors
Allison A. Throm, Halima Moncrieffe, Amir B. Orandi, Jeanette T. Pingel, Theresa L. Geurs, Hannah L. Miller, Allyssa L. Daugherty, Olga N. Malkova, Daniel J. Lovell, Susan D. Thompson, Alexei A. Grom, Megan A. Cooper, Stephen T. Oh, Anthony R. French
Abstract
Chronic liver disease (CLD) is associated with tissue-destructive fibrosis. Considering that common mechanisms drive fibrosis across etiologies, and that steatosis is an important cofactor for pathology, we performed RNA sequencing on liver biopsies of patients with different fibrosis stages, resulting from infection with hepatitis C virus (HCV) (with or without steatosis) or fatty liver disease. In combination with enhanced liver fibrosis score correlation analysis, we reveal a common set of genes associated with advanced fibrosis, as exemplified by those encoding the transcription factor ETS-homologous factor (EHF) and the extracellular matrix protein versican (VCAN). We identified 17 fibrosis-associated genes as candidate EHF targets and demonstrated that EHF regulates multiple fibrosis-associated genes, including VCAN, in hepatic stellate cells. Serum VCAN levels were also elevated in advanced fibrosis patients. Comparing biopsies from patients with HCV with or without steatosis, we identified a steatosis-enriched gene set associated with advanced fibrosis, validating follistatin-like protein 1 (FSTL1) as an exemplar of this profile. In patients with advanced fibrosis, serum FSTL1 levels were elevated in those with steatosis (versus those without). Liver Fstl1 mRNA levels were also elevated in murine CLD models. We thus reveal a common gene signature for CLD-associated liver fibrosis and potential biomarkers and/or targets for steatosis-associated liver fibrosis.
Authors
Divya Ramnath, Katharine M. Irvine, Samuel W. Lukowski, Leigh U. Horsfall, Zhixuan Loh, Andrew D. Clouston, Preya J. Patel, Kevin J. Fagan, Abishek Iyer, Guy Lampe, Jennifer L. Stow, Kate Schroder, David P. Fairlie, Joseph E. Powell, Elizabeth E. Powell, Matthew J. Sweet
Abstract
BACKGROUND. Necroptosis is a form of programmed necrotic cell death that is rapidly emerging as an important pathophysiological pathway in numerous disease states. Necroptosis is dependent on receptor-interacting protein kinase 3 (RIPK3), a protein shown to play an important role in experimental models of critical illness. However, there is limited clinical evidence regarding the role of extracellular RIPK3 in human critical illness. METHODS. Plasma RIPK3 levels were measured in 953 patients prospectively enrolled in 5 ongoing intensive care unit (ICU) cohorts in both the USA and Korea. RIPK3 concentrations among groups were compared using prospectively collected phenotypic and outcomes data. RESULTS. In all 5 cohorts, extracellular RIPK3 levels in the plasma were higher in patients who died in the hospital compared with those who survived to discharge. In a combined analysis, increasing RIPK3 levels were associated with elevated odds of in-hospital mortality (odds ratio [OR] 1.7 for each log10-unit increase in RIPK3 level, P < 0.0001). When adjusted for baseline severity of illness, the OR for in-hospital mortality remained statistically significant (OR 1.33, P = 0.007). Higher RIPK3 levels were also associated with more severe organ failure. CONCLUSIONS. Our findings suggest that elevated levels of RIPK3 in the plasma of patients admitted to the ICU are associated with in-hospital mortality and organ failure. FUNDING. Supported by NIH grants P01 HL108801, R01 HL079904, R01 HL055330, R01 HL060234, K99 HL125899, and KL2TR000458-10. Supported by Samsung Medical Center grant SMX1161431.
Authors
Kevin C. Ma, Edward J. Schenck, Ilias I. Siempos, Suzanne M. Cloonan, Eli J. Finkelzstein, Maria A. Pabon, Clara Oromendia, Karla V. Ballman, Rebecca M. Baron, Laura E. Fredenburgh, Angelica Higuera, Jin Young Lee, Chi Ryang Chung, Kyeongman Jeon, Jeong Hoon Yang, Judie A. Howrylak, Jin-Won Huh, Gee Young Suh, Augustine M.K. Choi
Abstract
Nonneuronal cell types in the CNS are increasingly implicated as critical players in brain health and disease. While gene expression profiling of bulk brain tissue is routinely used to examine alterations in the brain under various conditions, it does not capture changes that occur within single cell types or allow interrogation of crosstalk among cell types. To this end, we have developed a concurrent brain cell type acquisition (CoBrA) methodology, enabling the isolation and profiling of microglia, astrocytes, endothelia, and oligodendrocytes from a single adult mouse forebrain. By identifying and validating anti-ACSA-2 and anti-CD49a antibodies as cell surface markers for astrocytes and vascular endothelial cells, respectively, and using established antibodies to isolate microglia and oligodendrocytes, we document that these 4 major cell types are isolated with high purity and RNA quality. We validated our procedure by performing acute peripheral LPS challenge, while highlighting the underappreciated changes occurring in astrocytes and vascular endothelia in addition to microglia. Furthermore, we assessed cell type–specific gene expression changes in response to amyloid pathology in a mouse model of Alzheimer’s disease. Our CoBrA methodology can be readily implemented to interrogate multiple CNS cell types in any mouse model at any age.
Authors
Dan B. Swartzlander, Nicholas E. Propson, Ethan R. Roy, Takashi Saito, Takaomi Saido, Baiping Wang, Hui Zheng
Abstract
Cytokines play an important role in dysregulated immune responses to infection, pancreatitis, ischemia/reperfusion injury, burns, hemorrhage, cardiopulmonary bypass, trauma, and many other diseases. Moreover, the imbalance between inflammatory and antiinflammatory cytokines can have deleterious effects. Here, we demonstrated highly selective blood-filtering devices — antibody-modified conduits (AMCs) — that selectively eliminate multiple specific deleterious cytokines in vitro. AMCs functionalized with antibodies against human vascular endothelial growth factor A or tumor necrosis factor α (TNF-α) selectively eliminated the target cytokines from human blood in vitro and maintained them in reduced states even in the face of ongoing infusion at supraphysiologic rates. We characterized the variables that determine AMC performance, using anti–human TNF-α AMCs to eliminate recombinant human TNF-α. Finally, we demonstrated selective cytokine elimination in vivo by filtering interleukin 1 β from rats with lipopolysaccharide-induced hypercytokinemia.
Authors
J. Brian McAlvin, Ryan G. Wylie, Krithika Ramchander, Minh T. Nguyen, Charles K. Lok, Morgan Moroi, Andre Shomorony, Nikolay V. Vasilyev, Patrick Armstrong, Jason Yang, Alexander M. Lieber, Obiajulu S. Okonkwo, Rohit Karnik, Daniel S. Kohane
Abstract
Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II–induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.
Authors
Andrew Willeford, Takeshi Suetomi, Audrey Nickle, Hal M. Hoffman, Shigeki Miyamoto, Joan Heller Brown
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