Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. ...Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177
TI Treg population that may serve as a target for TI Treg-specific immunotherapy.
Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of ...disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors.
We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19.
Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19.
These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.
Neutrophil migration across tissue barriers to the site of injury involves integration of complex danger signals and is critical for host survival. Numerous studies demonstrate that these ...environmental signals fundamentally alter the responses of extravasated or "primed" neutrophils. Triggering receptor expressed on myeloid cells 1 (TREM-1) plays a central role in modulating inflammatory signaling and neutrophil migration into the alveolar airspace. Using a genetic approach, we examined the role of TREM-1 in extravasated neutrophil function. Neutrophil migration in response to chemoattractants is dependent upon multiple factors, including reactive oxygen species (ROS) generated either extracellularly by epithelial cells or intracellularly by NADPH oxidase (NOX). We, therefore, questioned whether ROS were responsible for TREM-1-mediated regulation of migration. Thioglycollate-elicited peritoneal neutrophils isolated from wild-type (WT) and TREM-1-deficient mice were stimulated with soluble and particulate agonists. Using electron paramagnetic resonance spectroscopy, we demonstrated that NOX2-dependent superoxide production is impaired in TREM-1-deficient neutrophils. Consistent with these findings, we confirmed with Clark electrode that TREM-1-deficient neutrophils consume less oxygen. Next, we demonstrated that TREM-1 deficient neutrophils have impaired directional migration to fMLP and zymosan-activated serum as compared to WT neutrophils and that deletion or inhibition of NOX2 in WT but not TREM-1-deficient neutrophils significantly impaired direction sensing. Finally, TREM-1 deficiency resulted in decreased protein kinase B (AKT) activation. Thus, TREM-1 regulates neutrophil migratory properties, in part, by promoting AKT activation and NOX2-dependent superoxide production. These findings provide the first mechanistic evidence as to how TREM-1 regulates neutrophil migration.
Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB.
Two major SC compartments in airways are ...submucosal glands (SMGs) and surface airway p63 (also known as TP63 tumor protein 63)-positive/K5 (also known as KRT5 keratin 5)-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB.
Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay.
Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63
/K5
and increase in K5
/K14
and K14
BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model.
SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.
This case study over time describes five years of experience with interventions to improve laboratory test utilization at an academic medical center. The high-frequency laboratory tests showing the ...biggest declines in order volume post intervention were serum albumin (36%) and erythrocyte sedimentation rate (17%). Introduction of restrictions for 170 high-cost send-out tests resulted in a 23% decline in order volume. Targeted interventions reduced mis-orders involving several "look-alike" tests: 1,25-dihydroxyvitamin D, 25-hydroxyvitamin D; manganese, magnesium; beta-2-glycoprotein, beta-2-microglobulin. Lastly, targeted alerts reduced duplicate orders of germline genetic testing and orders of hepatitis B surface antigen within 2 weeks of hepatitis B vaccination.
TREM proteins are a family of cell surface receptors that participate in diverse cell processes, including inflammation, bone homeostasis, neurological development and coagulation. TREM-1, the first ...to be identified, acts to amplify inflammation. Other TREM proteins regulate the differentiation and function of macrophages, microglia, dendritic cells, osteoclasts and platelets. Here we discuss the state of the field, putative ligands of TREM proteins and the challenges that remain in understanding TREM biology.
Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced ...chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1
mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1
neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1
neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming.
Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune ...mechanisms in vitro.
Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs.
Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1.
While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.
The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in ...the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.
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