Macrophage Extracellular Trap Formation in Response to M. Haemolytica or its Leukptoxin is altered by Co-Incubation with Bovine Herpes Virus-1 Infected Bronchiolar Epithelial Cells

Bovine respiratory disease (BRD) is the primary cause of morbidity in the U.S. beef and dairy industry. BRD is a multifactorial disease that is caused by viral and bacterial agents leading to a severe pleuropneumonia in cattle. BRD is characterized by inflammation, intense neutrophil infiltration, consolidation and recently, extensive amounts of extracellular DNA in the lungs. One possible source of the DNA is from leukocytes that release fibrillar networks of antimicrobial protein-studded DNA matrices referred to as extracellular traps (ETs). Recently, we have demonstrated that neutrophils and macrophages produce ETs in response to Mannheimia haemolytica, an important member of the BRD complex. Previous data has demonstrated that conditioned media removed from bovine herpes virus (BHV)-1 infected bovine bronchiolar epithelial (BBE) cells contain several cytokines. Here, we examined if conditioned media from BHV-1 infected BBE cells could alter ET formation from bovine neutrophils and macrophages. We observed that bovine macrophages pre-incubated with conditioned media from BHV-1 infected BBE cells had a reduced ability to produce ETs when incubated with the leukotoxin (LKT) in comparison to the control macrophages pre-incubated with conditioned media from uninfected BBE cells. In contrast, we observed that bovine macrophages treated with conditioned media demonstrated an increase in ET formation in response to intact M. haemolytica cells. However, conditioned media-treated bovine neutrophils were unaltered in their ability to produce ETs in response to M. haemolytica or LKT. Our findings suggest that BHV infection may alter macrophage production of ETs in response to M. haemolytica or LKT, which could alter host defense

Female Neutrophils Produce Less Neutrophil Extracellular Traps in Response to the Escherichia Coli Hemolysin than Male Neutrophils

Urinary tract infections (UTIs) have been estimated to cost the U.S. approximately 1.6 billion dollars in health care costs. The majority of UTIs affect women more than men where fifty to sixty percent of women will experience a UTI in their lifetime. Uropathogenic Escherichia coli is the leading cause of UTIs. When uropathogenic E. coli is incubated in human urine, E. coli produce significantly more of the hemolysin, an RTX toxin that can lyse a wide variety of cell types. Up regulation of the hemolysin is believed to be important in E. coli colonization of bladder and kidney epithelial cells. During, a UTI there is an influx of neutrophils into the bladder, which have been demonstrated to produce neutrophil extracellular traps (NETs). NETs are composed of extracellular DNA studded with antimicrobial proteins that trap and kill various pathogens. The hypothesis of this research is that female neutrophils produce significantly less NETs in response to the E. coli hemolysin in comparison to male neutrophils. Human neutrophils and macrophages were isolated from whole blood taken from healthy male and female volunteers and were incubated for various times or with various concentrations of the E. coli hemolysin. Extracellular DNA was then quantified using PicoGreen. Red blood cells were also incubated with various concentrations of the hemolysin and red blood cell lysis was determined by the quantification of the release of heme. We observed a significant decrease in the amount of NETs produced by female neutrophils in response to the E. coli hemolysin in comparison to male neutrophils. Similarly, we observed significantly more lysis of female red blood cells as compared to male red blood cells. Preliminary results also suggest that male macrophages produce more macrophage extracellular traps (METs) than female macrophages. Our findings suggest that female neutrophils, which have a reduced ability to produce NETs and METs in response to the E. coli hemolysin, may contribute to the significant difference in the amount of UTIs women experience in comparison to men

Colonization of Methicillin-Resistant Staphylococcus Aureus in Dormitory Versus Non-Dormitory Populations

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that has developed resistance to numerous antibiotics such as methicillin, a commonly used antibiotic for the treatment of Staphylococcus infections. In the community, MRSA is widespread, and it is believed that 1% of the population is a carrier. People are potentially at a greater risk in areas where factors such as close skin-to-skin contact occur, cuts or abrasions in the skin occur, contaminated items and surfaces are present, or where overcrowded living conditions and poor hygiene are common. MRSA’s occurrence in people without risk factors who live in communities is also increasing, and some of these infections are not typically associated with staphylococci bacteria. MRSA carriage rates are important to monitor in populations where individuals are in close contact and have crowded living conditions, such as a dormitory. The goal of this research is to identify MRSA carriers in the dormitory and non-dormitory populations in order to quantify nasal carrier rates. Frequent athletic facility attendance was also monitored to determine if this is an additional risk factor. Nasal swabs samples were collected from volunteers over the age of eighteen. Background information was gathered from volunteers at the time of swabbing by means of an anonymous survey. Background information that was taken into account when analyzing data included: place of living (dormitory versus non-dormitory); if they ever lived in a dormitory and timeframe (how long and how long ago); if they had a MRSA infection previously (confirmed by a physician); how often they visited a gym facility; and their age. Our results demonstrate that approximately 10% of Winona State University student were caring MRSA intranasally. We found a strong correlation between carriage rates and dormitory status and gym usage. Similarly, dormitory populations who used a gym facility frequently were at an even higher risk of MRSA carriage. We believe these findings indicate an increase in carriage rate among WSU’s dormitory student population. These data indicate a need to increase WSU’s surveillance of MRSA carriage rates and infections

Characterization of replication of a satellite RNA associated with the barley yellow dwarf virus-RPV

The focus of this study is a satellite RNA associated with the RPV barley yellow dwarf luteovirus. The (+) strand hammerhead of this satellite RNA has several unique features, one of which is the ability to form an alternative pseudoknot structure which inhibits self-cleavage. The role of this pseudoknot in replication was examined first by generating mutants that either knocked-out or restored pseudoknot formation. As expected, destroying pseudoknot formation (thus favoring the hammerhead) greatly increased self-cleavage but, unexpectedly, decreased replication efficiency. Restoring pseudoknot formation resulted in a decreased cleavage rate and enhanced replication efficiency. Thus, either pseudoknot formation itself or a slow cleavage rate (or both) are required for replication competency. Another aspect of this study focused on answering this question. To do this, additional mutants were generated and tested for cleavage rates and replication abilities. One of these mutants had the unique characteristic of possessing both pseudoknot formation and an increased cleavage rate. The results from this experiment, although not entirely conclusive as to the function of the pseudo knot structure, clearly rule out the possibility that cleavage rate is a determinant of satellite replication

Relevance of macrophage extracellular traps in C. albicans killing

Candida albicans causes systemic life-threatening infections, particularly in immunocompromised individuals, such as patients in intensive care units, patients undergoing chemotherapy, and post-surgical and neutropenic patients. The proliferation of invading Candida cells is mainly limited by the action of the human innate immune system, in which phagocytic cells play a fundamental role. This function is, however, limited in neutropenic patients, who rely mainly on the protective immunity mediated by macrophages. Macrophages have been shown to release extracellular DNA fibers, known as macrophage extracellular traps (METs), which can entrap and kill various microbes by a process called ETosis. In this study, we observed that, upon contact with C. albicans, macrophages became active in phagocyting and engulfing yeast cells. ETosis was induced in 6% of macrophages within the first 30 min of contact, and this percentage increased with the multiplicity of infection until a plateau was reached. After 2.5 h incubation, the presence of extracellular macrophage DNA was observed in approximately half of the cells. This study suggests that the formation of METs occurs before pyroptosis (first 6-8 h) and macrophage cell death (up to 24 h), and thus, METs could be included in models describing C. albicans-macrophage interactions. We also observed that macrophage ETosis and phagocytosis can occur simultaneously and that, in the first hours of infection, both processes are similarly important in controlling the proliferation of yeast cells, this being critical in neutropenic patients. Finally, it can also be concluded that, since C. albicans can degrade DNA, the structural component of METs, yeast extracellular DNase activity can be considered as an important virulence factor.This work was supported by the strategic program UID/BIA/04050/2019 funded by national funds through the FCT I.