Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Shuttle Peptide Delivers Base Editor RNPs to Rhesus Monkey Airway Epithelial Cells In Vivo.

Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, to improve base editor RNP delivery, we optimized S10 to derive the S315 peptide. Following intratracheal aerosol of Cy5-labeled peptide cargo in rhesus macaques, we confirmed delivery throughout the respiratory tract. Subsequently, we targeted CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieved editing efficiencies of up to 5.3% in rhesus airway epithelia. Moreover, we documented persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restored anion channel function in cultured human airway epithelial cells. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

Vegetation Formation in Staphylococcus Aureus Endocarditis Inversely Correlates With RNAIII and sarA Expression in Invasive Clonal Complex 5 Isolates.

Infective endocarditis (IE) is one of the most feared and lethal diseases caused by Staphylococcus aureus. Once established, the infection is fast-progressing and tissue destructive. S. aureus of the clonal complex 5 (CC5) commonly cause IE yet are severely understudied. IE results from bacterial colonization and formation of tissue biofilms (known as vegetations) on injured or inflamed cardiac endothelium. S. aureus IE is promoted by adhesins, coagulases, and superantigens, with the exotoxins and exoenzymes likely contributing to tissue destruction and dissemination. Expression of the large repertoire of virulence factors required for IE and sequelae is controlled by complex regulatory networks. We investigated the temporal expression of the global regulators agr (RNAIII), rot, sarS, sarA, sigB, and mgrA in 8 invasive CC5 isolates and established intrinsic expression patterns associated with IE outcomes. We show that vegetation formation, as tested in the rabbit model of IE, inversely correlates with RNAIII and sarA expression during growth in Todd-Hewitt broth (TH). Large vegetations with severe sequelae arise from strains with high-level expression of colonization factors but slower transition towards expression of the exotoxins. Overall, strains proficient in vegetation formation, a hallmark of IE, exhibit lower expression of RNAIII and sarA. Simultaneous high expression of RNAIII, sarA, sigB, and mgrA is the one phenotype assessed in this study that fails to promote IE. Thus, RNAIII and sarA expression that provides for rheostat control of colonization and virulence genes, rather than an on and off switch, promote both vegetation formation and lethal sepsis.

SEC is an antiangiogenic virulence factor that promotes Staphylococcus aureus endocarditis independent of superantigen activity.

The superantigen staphylococcal enterotoxin C (SEC) is critical for Staphylococcus aureus infective endocarditis (SAIE) in rabbits. Superantigenicity, its hallmark function, was proposed to be a major underlying mechanism driving SAIE but was not directly tested. With the use of S. aureus MW2 expressing SEC toxoids, we show that superantigenicity does not sufficiently account for vegetation growth, myocardial inflammation, and acute kidney injury in the rabbit model of native valve SAIE. These results highlight the critical contribution of an alternative function of superantigens to SAIE. In support of this, we provide evidence that SEC exerts antiangiogenic effects by inhibiting branching microvessel formation in an ex vivo rabbit aortic ring model and by inhibiting endothelial cell expression of one of the most potent mediators of angiogenesis, VEGF-A. SEC's ability to interfere with tissue revascularization and remodeling after injury serves as a mechanism to promote SAIE and its life-threatening systemic pathologies.

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors.

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Association of Novel Streptococcus sanguinis Virulence Factors With Pathogenesis in a Native Valve Infective Endocarditis Model.

Streptococcus sanguinis (S. sanguinis) is an abundant oral commensal which can cause disseminated human infection if it gains access to the bloodstream. The most important among these diseases is infective endocarditis (IE). While virulence phenotypes of S. sanguinis have been correlated to disease severity, genetic factors mediating these phenotypes, and contributing to pathogenesis are largely uncharacterized. In this report, we investigate the roles of 128 genes in virulence-related phenotypes of S. sanguinis and characterize the pathogenic potential of two selected mutants in a left-sided, native valve IE rabbit model. Assays determining the ability of our mutant strains to produce a biofilm, bind to and aggregate platelets, and adhere to or invade endothelial cells identified sixteen genes with novel association to these phenotypes. These results suggest the presence of many uncharacterized genes involved in IE pathogenesis which may be relevant for disease progression. Two mutants identified by the above screening process - SSA_1099, encoding an RTX-like protein, and mur2, encoding a peptidoglycan hydrolase - were subsequently evaluated in vivo. Wild type (WT) S. sanguinis reliably induced cardiac vegetations, while the SSA_1099 and mur2 mutants produced either no vegetation or vegetations of small size. Splenomegaly was reduced in both mutant strains compared to WT, while pathology of other distal organs was indistinguishable. Histopathology analyses suggest the cardiac lesions and vegetations in this model resemble those observed in humans. These data indicate that SSA_1099 and mur2 encode virulence factors in S. sanguinis which are integral to pathogenesis of IE.

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

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-1ß. 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.

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

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. ß-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 ß-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 ß-toxin, SMase-deficient ß-toxin (H289N), and biofilm ligase-deficient ß-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. ß-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. ß-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with ß-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. ß-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 ß-toxin, are highly associated with IE. Considering the strong association and established contribution of ß-toxin in animal models of IE, determining how ß-toxin directly affects human cell types, including endothelial cells and platelets, is important. In this study, we demonstrate that ß-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.

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

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.

Chronic superantigen exposure induces systemic inflammation, elevated bloodstream endotoxin, and abnormal glucose tolerance in rabbits: possible role in diabetes.

UNLABELLED: 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.

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.

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.

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.

Natural Tregs, CD4+CD25+ inhibitory hybridomas, and their cell contact dependent suppression.

The natural CD4+CD25+ T regulatory (Treg) lymphocyte has emerged as a critical cell for controlling immune responses to self, foreign proteins, and pathogens. Identified initially by the constitutive expression of CD4 and CD25, natural Tregs suppress a variety of immune cells and responses, including CD4+CD25- proliferation and IL-2 production, and CD8 cell proliferation, IFNgamma production and CTL activity. Although natural Tregs require activation with specific antigen to attain their suppressive phenotype, once activated they execute inhibition in an antigen specific as well as non-specific (bystander) fashion. Treg suppression depends on IL-2, CD25, and cell:cell contact. The use of live cell imaging in vivo and in vitro to visualize the dynamic cell:cell interactions involving natural Tregs as well as the CD4+CD25+ Treg inhibitory hybridoma RD6 has refined the mechanistic models of contact dependent Treg suppression.

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.

Early exposure to a nonhygienic environment alters pulmonary immunity and allergic responses.

The hygiene hypothesis suggests that early life exposure to a nonhygienic environment that contains endotoxin reduces the risk of developing allergic diseases. The mechanisms underlying the hygiene hypothesis are unclear and may involve subtle immune system interactions that occur during maturation. Experimental objectives of this study were to use a novel animal model to test the hygiene hypothesis and to characterize early life immune system responses to a nonhygienic environment. Mice were reared in corn dust, a grain-processing byproduct with a high-endotoxin content and microbial products or in a low-endotoxin environment. The influence of early or later life exposure to corn dust on a subsequent allergen stimulus (ovalbumin) was assessed by bronchoalveolar lavage (BAL) cell analysis, lung histology, serum IgE, and BAL cytokine measurements. The influence of the corn dust environment on the developing pulmonary immune system was assessed by BAL cell analysis and immunostaining of lung tissue. The corn dust environment contained significantly more endotoxin (P < 0.001), and the dust exposures attenuated the cellular inflammatory response to ovalbumin in the adult mouse (P < 0.01) but did not reduce serum IgE levels or alter baseline BAL fluid proinflammatory cytokine levels. The corn dust environment did not induce significant neutrophilia in lavage fluid but significantly increased the number of antigen-presenting cells in alveolar walls early in life by approximately 37%. In conclusion, exposure to a nonhygienic environment did not induce significant airway neutrophilia, yet altered the population of immunologically active cells in the lung and reduced subsequent allergic inflammation.