Katarina Kulhankova

Health Effects of Airborne Exposures from Concentrated Animal Feeding Operations

Toxic gases, vapors, and particles are emitted from concentrated animal feeding operations (CAFOs) into the general environment. These include ammonia, hydrogen sulfide, carbon dioxide, malodorous vapors, and particles contaminated with a wide range of microorganisms. Little is known about the health risks of exposure to these agents for people living in the surrounding areas. Malodor is one of the predominant concerns, and there is evidence that psychophysiologic changes may occur as a result of exposure to malodorous compounds. There is a paucity of data regarding community adverse health effects related to low-level gas and particulate emissions. Most information comes from studies among workers in CAFO installations. Research over the last decades has shown that microbial exposures, especially endotoxin exposure, are related to deleterious respiratory health effects, of which cross-shift lung function decline and accelerated decline over time are the most pronounced effects. Studies in naïve subjects and workers have shown respiratory inflammatory responses related to the microbial load. This working group, which was part of the Conference on Environmental Health Impacts of Concentrated Animal Feeding Operations: Anticipating Hazards—Searching for Solutions, concluded that there is a great need to evaluate health effects from exposures to the toxic gases, vapors, and particles emitted into the general environment by CAFOs. Research should focus not only on nuisance and odors but also on potential health effects from microbial exposures, concentrating on susceptible subgroups, especially asthmatic children and the elderly, since these exposures have been shown to be related to respiratory health effects among workers in CAFOs.

MD-2–Dependent Pulmonary Immune Responses to Inhaled Lipooligosaccharides: Effect of Acylation State

Endotoxins represent one of the most potent classes of microbial immunoactive components that can cause pulmonary inflammation. The aim of this study was to compare the inflammatory potency of two types of Neisseria meningitidis endotoxins (lipooligosaccharides) in lungs: wild type (hexaacylated, LOS(wt)) and mutant type (pentaacylated, LOS(msbB)), and to determine the importance of MD-2 in endotoxin responses in lungs in vivo. Endotoxin-normoresponsive mice (BALB/c) were exposed to selected doses of penta- and hexaacylated lipooligosaccharides (LOS) by nasal aspiration. Cellular and cytokine/chemokine inflammatory responses in bronchoalveolar lavage were measured at 1-, 4-, 8-, 16-, 24-, and 48-hour time points. MD-2-null mice were exposed to one dose of hexaacylated LOS and inflammatory responses were measured after 4 and 24 hours. Inhalation of hexaacylated LOS resulted in strong inflammatory responses, while pentaacylated LOS was much less potent in inducing increases of neutrophils, TNF-alpha, macrophage inflammatory protein-1 alpha, IL-6, granulocyte colony-stimulating factor, and IL-1 beta concentration in bronchoalveolar lavage. Similar kinetics of inflammatory responses in lungs were found in both types of endotoxin exposures. Inhalation of hexaacylated LOS in MD-2-null mice resulted in significantly lower numbers of neutrophils in bronchoalveolar lavage than in normoresponsive mice. Markedly lower inflammatory potency of pentaacylated LOS was observed compared with hexaacylated LOS. Hyporesponsiveness in MD-2-null mice after nasal aspiration of wild-type LOS indicate its essential role in airway responsiveness to endotoxin.

Staphylococcal toxic shock syndrome: superantigen-mediated enhancement of endotoxin shock and adaptive immune suppression

Infectious diseases caused by Staphylococcus aureus present a significant clinical and public health problem. S. aureus causes some of the most severe hospital-associated and community-acquired illnesses. Specifically, it is the leading cause of infective endocarditis and osteomyelitis, and the second leading cause of sepsis in the USA. While pathogenesis of S. aureus infections is at the center of current research, many questions remain about the mechanisms underlying staphylococcal toxic shock syndrome (TSS) and associated adaptive immune suppression. Both conditions are mediated by staphylococcal superantigens (SAgs)—secreted staphylococcal toxins that are major S. aureus virulence factors. Toxic shock syndrome toxin-1 (TSST-1) is the SAg responsible for almost all menstrual TSS cases in the USA. TSST-1, staphylococcal enterotoxin B and C are also responsible for most cases of non-menstrual TSS. While SAgs mediate all of the hallmark features of TSS, such as fever, rash, hypotension, and multi-organ dysfunction, they are also capable of enhancing the toxic effects of endogenous endotoxin. This interaction appears to be critical in mediating the severity of TSS and related mortality. In addition, interaction between SAgs and the host immune system has been recognized to result in a unique form of adaptive immune suppression, contributing to poor outcomes of S. aureus infections. Utilizing rabbit models of S. aureus infective endocarditis, pneumonia and sepsis, and molecular genetics techniques, we aim to elucidate the mechanisms of SAg and endotoxin synergism in the pathogenesis of TSS, and examine the cellular and molecular mechanisms underlying SAg-mediated immune dysfunction.

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.

Early-life co-administration of cockroach allergen and endotoxin augments pulmonary and systemic responses

Background Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early‐life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease.

Chronic Superantigen Exposure Induces Systemic Inflammation, Elevated Bloodstream Endotoxin, and Abnormal Glucose Tolerance in Rabbits: Possible Role in Diabetes

ABSTRACT Excessive weight and obesity are associated with the development of diabetes mellitus type 2 (DMII) in humans. They also pose high risks of Staphylococcus aureus colonization and overt infections. S. aureus causes a wide range of severe illnesses in both healthy and immunocompromised individuals. Among S. aureus virulence factors, superantigens are essential for pathogenicity. In this study, we show that rabbits that are chronically exposed to S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1) experience impaired glucose tolerance, systemic inflammation, and elevated endotoxin levels in the bloodstream, all of which are common findings in DMII. Additionally, such DMII-associated findings are also seen through effects of TSST-1 on isolated adipocytes. Collectively, our findings suggest that chronic exposure to S. aureus superantigens facilitates the development of DMII, which may lead to therapeutic targeting of S. aureus and its superantigens. IMPORTANCE Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo. Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes. Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo. Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes.

Phenotypes and Virulence among Staphylococcus aureus USA100, USA200, USA300, USA400, and USA600 Clonal Lineages.

S. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia (D. R. Murdoch et al., Arch Intern Med 169:463–473, 2009, http://dx.doi.org/10.1001/archinternmed.2008.603 , and H. Wisplinghoff et al., Clin Infect Dis 39:309–317, 2004, http://dx.doi.org/10.1086/421946 ). Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% (S. Y. Tong et al., Clin Microbiol Rev 28:603–661, 2015, http://dx.doi.org/10.1128/CMR.00134-14 ). S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains. ABSTRACT Staphylococcus aureus diseases affect ~500,000 individuals per year in the United States. Worldwide, the USA100, USA200, USA400, and USA600 lineages cause many of the life-threatening S. aureus infections, such as bacteremia, infective endocarditis, pneumonia, toxic shock syndrome, and surgical site infections. However, the virulence mechanisms associated with these clonal lineages, in particular the USA100 and USA600 isolates, have been severely understudied. We investigated the virulence of these strains, in addition to strains in the USA200, USA300, and USA400 types, in well-established in vitro assays and in vivo in the rabbit model of infective endocarditis and sepsis. We show in the infective endocarditis and sepsis model that strains in the USA100 and USA600 lineages cause high lethality and are proficient in causing native valve infective endocarditis. Strains with high cytolytic activity or producing toxic shock syndrome toxin 1 (TSST-1) or staphylococcal enterotoxin C (SEC) caused lethal sepsis, even with low cytolytic activity. Strains in the USA100, USA200, USA400, and USA600 lineages consistently contained genes that encode for the enterotoxin gene cluster proteins, SEC, or TSST-1 and were proficient at causing infective endocarditis, while the USA300 strains lacked these toxins and were deficient in promoting vegetation growth. The USA100, USA200, and USA400 strains in our collection formed strong biofilms in vitro, whereas the USA200 and USA600 strains exhibited increased blood survival. Hence, infective endocarditis and lethal sepsis are multifactorial and not intrinsic to any one individual clonal group, further highlighting the importance of expanding our knowledge of S. aureus pathogenesis to clonal lineages causative of invasive disease. IMPORTANCE S. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia (D. R. Murdoch et al., Arch Intern Med 169:463–473, 2009, http://dx.doi.org/10.1001/archinternmed.2008.603 , and H. Wisplinghoff et al., Clin Infect Dis 39:309–317, 2004, http://dx.doi.org/10.1086/421946 ). Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% (S. Y. Tong et al., Clin Microbiol Rev 28:603–661, 2015, http://dx.doi.org/10.1128/CMR.00134-14 ). S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains.

Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities

ABSTRACT Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like “vegetations” composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an S. aureus virulence factor that contributes to the microorganism’s ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N), and biofilm ligase-deficient β-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity. IMPORTANCE Each year there are up to 100,000 cases of infective endocarditis (IE) in the United States. S. aureus is the most common pathogen in patients with health care-associated IE and the leading cause of community-associated IE in the developed world. Multiple clonal group strains as defined by the Centers for Disease Control and Prevention, particularly USA200 and other clones encoding β-toxin, are highly associated with IE. Considering the strong association and established contribution of β-toxin in animal models of IE, determining how β-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that β-toxin functions to modulate endothelial cells and platelets by both toxin sphingomyelinase and biofilm ligase activities. Our data suggest that these activities modulate inflammation and increase infection severity. Each year there are up to 100,000 cases of infective endocarditis (IE) in the United States. S. aureus is the most common pathogen in patients with health care-associated IE and the leading cause of community-associated IE in the developed world. Multiple clonal group strains as defined by the Centers for Disease Control and Prevention, particularly USA200 and other clones encoding β-toxin, are highly associated with IE. Considering the strong association and established contribution of β-toxin in animal models of IE, determining how β-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that β-toxin functions to modulate endothelial cells and platelets by both toxin sphingomyelinase and biofilm ligase activities. Our data suggest that these activities modulate inflammation and increase infection severity.

Endotoxin inhalation alters lung development in neonatal mice.

BACKGROUND Childhood asthma is a significant public health problem. Epidemiologic evidence suggests an association between childhood asthma exacerbations and early life exposure to environmental endotoxin. Although the pathogenesis of endotoxin-induced adult asthma is well studied, questions remain about the impact of environmental endotoxin on pulmonary responsiveness in early life. METHODS We developed a murine model of neonatal/juvenile endotoxin exposures approximating those in young children and evaluated the lungs inflammatory and remodeling responses. RESULTS Persistent lung inflammation induced by the inhalation of endotoxin in early life was demonstrated by the influx of inflammatory cells and pro-inflammatory mediators to the airways and resulted in abnormal alveolarization. CONCLUSIONS Results of this study advance the understanding of the impact early life endotoxin inhalation has on the lower airways, and demonstrates the importance of an experimental design that approximates environmental exposures as they occur in young children.

The Superantigen Toxic Shock Syndrome Toxin 1 Alters Human Aortic Endothelial Cell Function

ABSTRACT Staphylococcus aureus infective endocarditis (IE) is a fast-progressing and tissue-destructive infection of the cardiac endothelium. The superantigens (SAgs) toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin C (SEC), and the toxins encoded by the enterotoxin gene cluster (egc) play a novel and essential role in the etiology of S. aureus IE. Recent studies indicate that SAgs act at the infection site to cause tissue pathology and promote vegetation growth. The underlying mechanism of SAg involvement has not been clearly defined. In SAg-mediated responses, immune cell priming is considered a primary triggering event leading to endothelial cell activation and altered function. Utilizing immortalized human aortic endothelial cells (iHAECs), we demonstrated that TSST-1 directly activates iHAECs, as documented by upregulation of vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1). TSST-1-mediated activation results in increased monolayer permeability and defects in vascular reendothelialization. Yet stimulation of iHAECs with TSST-1 fails to induce interleukin-8 (IL-8) and IL-6 production. Furthermore, simultaneous stimulation of iHAECs with TSST-1 and lipopolysaccharide (LPS) inhibits LPS-mediated IL-8 and IL-6 secretion, even after pretreatment with either of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β. IL-8 suppression is not mediated by TSST-1 binding to its canonical receptor major histocompatibility complex class II (MHC-II), supporting current evidence for a nonhematopoietic interacting site on SAgs. Together, the data suggest that TSST-1 differentially regulates cell-bound and secreted markers of endothelial cell activation that may result in dysregulated innate immune responses during S. aureus IE. Endothelial changes resulting from the action of SAgs can therefore directly contribute to the aggressive nature of S. aureus IE and development of life-threatening complications.