reprogramming-induced-pluripotent-stem-cell-lines-from-frozen-buffy

Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

Human pluripotent stem cells (hPSCs) are a valuable tool for disease modeling. Further stem cells can be reprogrammed from adult somatic cells, called ...

University of Georgia

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes.  is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill , it remains largely unknown why PMNs fail to eliminate  in CF. The goal of this study was to observe how the CF airway environment affects  killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of  were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of  tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum,  killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing . Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of  was not diminished. The -induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of  by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill .

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of  upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected -deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice.

Human intelectin-1 (hIntL-1) is a secreted glycoprotein capable of binding exocyclic 1,2-diols within surface glycans of human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, and Helicobacter pylori. For the latter, lectin binding was shown to cause bacterial agglutination and increased phagocytosis, suggesting a role for hIntL-1 in pathogen surveillance. In this study, we investigated the interactions between hIntL-1 and S. pneumoniae, the leading cause of bacterial pneumonia. We show that hIntL-1 also agglutinates S. pneumoniae serotype 43, which displays an exocyclic 1,2-diol moiety in its capsular polysaccharide but is unable to kill in a complement-dependent manner or to promote bacterial killing by peripheral blood mononuclear cells. In contrast, hIntL-1 not only significantly increases serotype-specific S. pneumoniae killing by neutrophils but also enhances the attachment of these bacteria to A549 lung epithelial cells. Taken together, our results suggest that hIntL-1 participates in host surveillance through microbe sequestration and enhanced targeting to neutrophils.

Human Intelectin-1 Promotes Cellular Attachment and Neutrophil Killing of Streptococcus pneumoniae in a Serotype-Dependent Manner.

 (Mtb) is the primary cause of human tuberculosis (TB) and is currently the second most common cause of death due to a singleinfectious agent. The first line of defense against airborne pathogens, including Mtb, is the respiratory epithelium. One of the innate defenses used by respiratory epithelial cells to prevent microbial infection is an oxidative antimicrobial system consisting of the proteins, lactoperoxidase (LPO) and Dual oxidase 1 (Duox1), the thiocyanate anion (SCN-) and hydrogen peroxide (H2O2), which together lead to the generation of antimicrobial hypothiocyanite (OSCN-) in the airway lumen. OSCN- kills bacteria and viruses in vitro, but the role of this Duox1-based system in bacterial infections in vivo remains largely unknown. The goal of this study was to assess whether Duox1 contributes to the immune response against the unique respiratory pathogen, Mtb.

Dual oxidase 1 is dispensable during  infection in mice.

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus.

Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs).  has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant  (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils' oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host-pathogen interactions present in the CF lung.

Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant  by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species.

Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces  PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear.

Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies.

Loss of the flagellum marks the pathoadaptation of  to the cystic fibrosis (CF) airway environment during lung disease. Losing the flagellum is advantageous to the bacterium as the flagellum can be recognized by immune cells. The primary purpose of the flagellum is, however, to provide motility to the bacterium. Our goal was to determine whether the loss of flagellar motility or the loss of flagellum expression contributes to  lung infection in CF. To address this, wild-type and gut-corrected FABP-human cystic fibrosis transmembrane conductance regulator (hCFTR) mice deficient in the murine  gene were infected intratracheally with lethal doses of wild-type or flagellum-deficient . While there was no significant difference in the survival of wild-type mice after infection with either of the bacterial strains, a significantly higher mortality was observed in FABP-hCFTR mice infected with flagellum-deficient , compared to mice infected with their flagellated counterparts. When FABP-hCFTR mice were infected with isogenic, motility-deficient flagellated mutants, animal survival and lung bacterial titers were similar to those observed in mice infected with the wild-type bacterium. Airway levels of neutrophils and the amount neutrophil elastase were similar in mice infected with either the wild-type bacteria or the flagellum-deficient . Our results show that FABP-hCFTR mice have a different response to flagellum loss in  compared to wild-type animals. The loss of flagellum expression, rather than the loss of motility, is the main driver behind the increased virulence of flagellum-deficient  in CF. These observations provide new insight into  virulence in CF.IMPORTANCE, a major respiratory pathogen in cystic fibrosis, is known to lose its flagellum during the course of infection in the airways. Here, we show that the loss of flagellum leads to a more enhanced virulence in Cftr-deficient cystic fibrosis mice than in control animals. Loss of flagellum expression, rather than the loss of flagellar swimming motility, represents the main driver behind this increased virulence suggesting that this appendage plays a specific role in  virulence in cystic fibrosis airways.

Flagellum-deficient  is more virulent than non-motile but flagellated mutants in a cystic fibrosis mouse model.

Cystic Fibrosis (CF) airway disease is characterized by impaired mucociliary clearance, chronic, polymicrobial infections and robust, neutrophil-dominated inflammation. Pulmonary disease is the leading cause of morbidity and mortality in people with CF and is due to progressive airflow obstruction and ultimately respiratory failure. One of the earliest abnormalities in CF airway disease is the recruitment of neutrophils to the lungs. Neutrophil activation leads to the release of their intracellular content, including neutrophil elastase (NE), that damages lung tissues in CF. Our goal is to characterize a known bacterial NE inhibitor, ecotin, in the CF airway environment. Our results indicate that ecotins cloned from four Gram-negative bacterial species (, ,  and  inhibit NE activity in CF sputum samples in a dose-dependent manner. Although we observed differences in the NE-inhibitory activity of the tested ecotins with the  homologs being the most effective in NE inhibition in CF sputa, none of the ecotins impaired the ability of human neutrophils to kill major CF respiratory pathogens,  or , . Overall, we demonstrate that bacterial ecotins inhibit NE activity in CF sputa without compromising bacterial killing by neutrophils.

The bacterial serine protease inhibitor ecotin inhibits neutrophil elastase enzymatic activity in cystic fibrosis sputa.

Cystic fibrosis (CF) lung disease manifests through abnormally thick mucus, persistent bacterial infections and a dysregulated innate immune system that involves significant neutrophilic inflammation. Neutrophils, immune cells essential to fight infections, accumulate in large numbers in CF airways and release neutrophil extracellular traps (NETs) into the airway lumen that deliver extracellular DNA, granule content and cytokines including IL-1β. Interleukin-1β, a powerful, proinflammatory cytokine, represents another, significant component of the innate immune system that is dysregulated in CF. Both defense mechanisms become problematic as NETs and IL-1β are present at elevated levels in CF airways, potentially creating a destructive cycle that exacerbates lung damage rather than protects against infections. Therefore, understanding the interplay between IL-1β and NETs is crucial for addressing CF lung disease progression. This review examines the general mechanisms of IL-1β release and NET formation, with particular focus on their role in CF lung disease, and proposes that a self-perpetuating, positive feedback loop between these two innate immune processes represents a major driving force in disease progression. This understanding suggests potential therapeutic targets for interrupting the cycle of inflammation and tissue damage in CF airways.

Article	Year
Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill . Pathogens (Basel, Switzerland)\| PubMed ID: 34200034	2021
RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice. Frontiers in immunology\| PubMed ID: 34912341	2021
Human Intelectin-1 Promotes Cellular Attachment and Neutrophil Killing of Streptococcus pneumoniae in a Serotype-Dependent Manner. Infection and immunity\| PubMed ID: 35499339	2022
Dual oxidase 1 is dispensable during infection in mice. Frontiers in immunology\| PubMed ID: 36936954	2023
Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 37211502	2023
Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species. Pathogens (Basel, Switzerland)\| PubMed ID: 37764956	2023
Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies. Frontiers in immunology\| PubMed ID: 37908356	2023
Flagellum-deficient is more virulent than non-motile but flagellated mutants in a cystic fibrosis mouse model. Microbiology spectrum\| PubMed ID: 39248473	2024
The bacterial serine protease inhibitor ecotin inhibits neutrophil elastase enzymatic activity in cystic fibrosis sputa. Heliyon\| PubMed ID: 39444402	2024
Neutrophil extracellular traps and interleukin-1β in cystic fibrosis lung disease. Frontiers in immunology\| PubMed ID: 40791588	2025

Kayla Fantone

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