The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Genetic background but not prostatic epithelial beta-catenin influences susceptibility of male mice to testosterone and estradiol-induced urinary dysfunction.

Urinary voiding dysfunction in aging men can cause bothersome symptoms and irreparable tissue damage. Underlying mechanisms are not fully known. We previously demonstrated that subcutaneous, slow-release testosterone and estradiol implants (T+E2) drive a pattern of urinary voiding dysfunction in male mice that resembles that of aging men. The initial goal of this study was to test the hypothesis that prostatic epithelial beta-catenin (Ctnnb1) is required for T+E2-mediated voiding dysfunction. Targeted Ctnnb1 deletion did not significantly change voiding function in control or T+E2 treated mice but led to the surprising discovery that the C57BL/6J × FVB/NJ × 129S1 mixed genetic background onto which Ctnnb1 loss of function alleles were maintained is profoundly susceptible to voiding dysfunction. The mixed background mice develop a more rapid T+E2-mediated increase in spontaneous urine spotting, are more impaired in ability to initiate bladder contraction, and develop larger and heavier bladders than T+E2 treated C57BL/6J pure bred mice. To better understand mechanisms, we separately evaluated contributions of T and E2 and found that E2 mediates voiding dysfunction. Our findings that genetic factors serve as modifiers of responsiveness to T and E2 demonstrate the need to control for genetic background in studies of male voiding dysfunction. We also show that genetic factors could control severity of voiding dysfunction. We demonstrate the importance of E2 as a key mediator of voiding impairment, and show that the concentration of E2 in subcutaneous implants determines the severity of voiding dysfunction in mice, demonstrating that the mouse model is tunable, a factor which is important for future pharmacological intervention studies.

A uropathogenic E. coli UTI89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate.

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation using transurethral instillations of Escherichia coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributed exclusively to the bladder, 100- and 200-µL volumes distributed to the bladder and prostate, and a 500-µL volume distributed to the bladder, prostate, and ureter. A threshold optical density of 0.4 E. coli UTI89 in the instillation fluid was necessary for significant (P < 0.05) prostate colonization. E. coli UTI89 infection resulted in a low frequency, high volume spontaneous voiding pattern. This phenotype was due to exposure to E. coli UTI89, not catheterization alone, and was minimally altered by a 50-µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation was isolated to the dorsal prostate and was accompanied by increased collagen density. This was partnered with increased density of protein tyrosine phosphatase receptor type C+, procollagen type I-α1+ copositive cells and decreased density of α2-smooth muscle actin+, procollagen type I-α1+ copositive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

Real-world PM extracts differentially enhance Th17 differentiation and activate the aryl hydrocarbon receptor (AHR).

Atmospheric particulate matter (PM) is a complex component of air pollution and the leading environmental risk factor for death worldwide. PM is formed from a combination of primary sources that emit PM directly into the atmosphere and secondary sources that emit gaseous precursors which oxidize in the atmosphere to form PM and composed of both inorganic and organic components. Currently, all PM is regulated by total mass. This regulatory strategy does not consider individual chemical constituents and assumes all PM is equally toxic. The chemically-extracted organic fraction (OF) of PM retains most organic constituents such as polycyclic aromatic hydrocarbons (PAHs) and excludes inorganics. PAHs are ubiquitous environmental toxicants and known aryl hydrocarbon receptor (AHR) ligands. This study addressed the role of individual components, specifically PAHs, of PM and how differences in chemical composition of real-world samples drive differential responses. This study tested the hypothesis that real-world extracts containing different combinations and concentrations of PAHs activate the AHR and enhance T helper (Th)17 differentiation to different degrees based on specific PAHs present in each sample, and not simply the mass of PM. The ability of the real-world OF, with different PAH compositions, to enhance Th17 differentiation and activate the AHR was tested in vitro. Cumulatively, the results identified PAHs as possible candidate components of PM contributing to increased inflammation and demonstrated that the sum concentration of PAHs does not determine the extent to which each PM activates the AHR and enhances Th17 differentiation suggesting individual components of each PM, and interactions of those components with others in the mixture, contribute to the inflammatory response. This demonstrates that not all PM are the same and suggests that when regulating PM based on its ability to cause human pathology, a strategy based on PM mass may not reduce pathologic potential of exposures.

Ambient urban dust particulate matter reduces pathologic T cells in the CNS and severity of EAE.

BACKGROUND: Autoimmune diseases have increased in incidence and prevalence worldwide. While genetic predispositions play a role, environmental factors are a major contributor. Atmospheric particulate matter (PM) is a complex mixture composed of metals, nitrates, sulfates and diverse adsorbed organic compounds like polycyclic aromatic hydrocarbons (PAHs) and dioxins. Exposure to atmospheric PM aggravates autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, among others. PAHs and dioxins are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and directs the balance between effector and regulatory T cells in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify pathways that contribute to autoimmune disease and their potential use as therapeutic targets to alleviate symptoms and the need for global immunosuppression. This study tests the hypothesis that atmospheric PM enhances effector T cell differentiation and aggravates autoimmune disease. RESULTS: An atmospheric ambient urban dust PM sample, standard reference material (SRM)1649b, was tested for its effects on autoimmunity. SRM1649b PM enhanced Th17 differentiation in an AHR-dependent manner in vitro, however intranasal treatment of SRM1649b PM delayed onset of EAE and reduced cumulative and peak clinical scores. Chronic and acute intranasal exposure of SRM1649b PM delayed onset of EAE. Chronic intranasal exposure did not reduce severity of EAE while acute intranasal exposure significantly reduced severity of disease. Acute intranasal treatment of low dose SRM1649b PM had no effect on clinical score or day of onset in EAE. Delayed onset of EAE by intranasal SRM1649b PM was AHR-dependent in vivo. Oral gavage of SRM1649b PM, in the absence of AHR ligands in the diet, had no effect on day of disease onset or severity of EAE. Day 10 analysis of T cells in the CNS after intranasal treatment of SRM1649b PM showed a reduction of pathologic T cell subsets in vivo. Moreover, MOG-specific splenocytes require AHR to generate or maintain IL-10 producing cells and reduce IFNγ producing cells in vitro. CONCLUSIONS: These results identify the AHR pathway as a potential target for driving targeted immunosuppression in the CNS in the context of atmospheric PM-mediated autoimmune disease. The effects of SRM1649b PM on EAE are dependent on route of exposure, with intranasal treatment reducing severity of EAE and delaying disease onset while oral gavage has no effect. Intranasal SRM1649b PM reduces pathologic T cells in the CNS, specifically Th1 cells and Th1Th17 double positive cells, leading to reduced severity of EAE and AHR-dependent delayed disease onset. Additionally, SRM1649b PM treatment of antigen-specific T cells leads to AHR-dependent increase in percent IL-10 positive cells in vitro. These findings may shed light on the known increase of infection after exposure to atmospheric PM and serve as the first step in identifying components of the AHR pathway responsible for Th1-mediated immunosuppression in response to atmospheric PM exposure.

The Aryl Hydrocarbon Receptor as an Immune-Modulator of Atmospheric Particulate Matter-Mediated Autoimmunity.

This review examines the current literature on the effects of atmospheric particulate matter (PM) on autoimmune disease and proposes a new role for the aryl hydrocarbon receptor (AHR) as a modulator of T cells in PM-mediated autoimmune disease. There is a significant body of literature regarding the strong epidemiologic correlations between PM exposures and worsened autoimmune diseases. Genetic predispositions account for 30% of all autoimmune disease leaving environmental factors as major contributors. Increases in incidence and prevalence of autoimmune disease have occurred concurrently with an increase in air pollution. Currently, atmospheric PM is considered to be the greatest environmental health risk worldwide. Atmospheric PM is a complex heterogeneous mixture composed of diverse adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and dioxins, among others. Exposure to atmospheric PM has been shown to aggravate several autoimmune diseases. Despite strong correlations between exposure to atmospheric PM and worsened autoimmune disease, the mechanisms underlying aggravated disease are largely unknown. The AHR is a ligand activated transcription factor that responds to endogenous and exogenous ligands including toxicants present in PM, such as PAHs and dioxins. A few studies have investigated the effects of atmospheric PM on AHR activation and immune function and demonstrated that atmospheric PM can activate the AHR, change cytokine expression, and alter T cell differentiation. Several studies have found that the AHR modulates the balance between regulatory and effector T cell functions and drives T cell differentiation in vitro and in vivo using murine models of autoimmune disease. However, there are very few studies on the role of AHR in PM-mediated autoimmune disease. The AHR plays a critical role in the balance of effector and regulatory T cells and in autoimmune disease. With increased incidence and prevalence of autoimmune disease occurring concurrently with increases in air pollution, potential mechanisms that drive inflammatory and exacerbated disease need to be elucidated. This review focuses on the AHR as a potential mechanistic target for modulating T cell responses associated with PM-mediated autoimmune disease providing the most up-to-date literature on the role of AHR in autoreactive T cell function and autoimmune disease.

Polycyclic aromatic hydrocarbons (PAHs) present in ambient urban dust drive proinflammatory T cell and dendritic cell responses via the aryl hydrocarbon receptor (AHR) in vitro.

Atmospheric particulate matter (PM) is a complex component of air pollution that is a composed of inorganic and organic constituents. The chemically-extracted organic fraction (OF) of PM excludes inorganics but retains most organic constituents like polycyclic aromatic hydrocarbons (PAHs). PAHs are ubiquitous environmental toxicants and known aryl hydrocarbon receptor (AHR) ligands. The AHR is a ligand activated transcription factor that responds to endogenous ligands and exogenous ligands including PAHs. Activation of the AHR leads to upregulation of cytochrome P450 (CYP) metabolizing enzymes which are important for the biotransformation of toxicants to less toxic, or in the case of PAHs, more toxic intermediates. Additionally, the AHR plays an important role in balancing regulatory and effector T cell responses. This study aimed to determine whether PAHs present in PM aggravate inflammation by driving inflammatory T cell and dendritic cell (DC) responses and their mechanism of action. This study tests the hypothesis that PAHs present in PM activate the AHR and alter the immune balance shifting from regulation to inflammation. To test this, the effects of SRM1649b OF on T cell differentiation and DC function were measured in vitro. SRM1649b OF enhanced Th17 differentiation in an AHR and CYP-dependent manner and increased the percent of IFNγ positive DCs in an AHR-dependent manner. SRM1649b PAH mixtures enhanced Th17 differentiation in an AHR-dependent but CYP-independent manner and increased the percent of IFNγ positive DCs. Cumulatively, these results suggest that PAHs present in PM are active components that contribute to immune responses in both T cells and BMDCs through the AHR and CYP metabolism. Understanding the role of AHR and CYP metabolism of PAHs in immune cells after PM exposure will shed light on new targets that will shift the immune balance from inflammation to regulation.

Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease.

BACKGROUND: Exposure to particulate matter (PM) has been associated with increased incidence and severity of autoimmune disease. Diesel PM is primarily composed of an elemental carbon core and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and contributes up to 40% of atmospheric PM. The organic fraction (OF) of PM excludes all metals and inorganics and retains most organic compounds, such as PAHs. Both PM and OF increase inflammation in vitro and aggravate autoimmune disease in humans. PAHs are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and effector function in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify whether the total mass or active components of PM are responsible for activating pathways associated with exposure to PM and autoimmune disease. This study tests the hypothesis that active components present in diesel PM and their OF enhance effector T cell differentiation and aggravate autoimmune disease. RESULTS: Two different diesel samples, each characterized for their components, were tested for their effects on autoimmunity. Both diesel PM enhanced effector T cell differentiation in an AHR-dose-dependent manner and suppressed regulatory T cell differentiation in vitro. Both diesel PM aggravated EAE in vivo. Fractionated diesel OFs exhibited the same effects as PM in vitro, but unlike PM, only one diesel OF aggravated EAE. Additionally, both synthetic PAH mixtures that represent specific PAHs found in the two diesel PM samples enhanced Th17 differentiation, however one lost this effect after metabolism and only one required the AHR. CONCLUSIONS: These findings suggest that active components of PM and not total mass are driving T cell responses in vitro, but in vivo the PM matrix and complex mixtures adsorbed to the particles, not just the OF, are contributing to the observed EAE effects. This implies that examining OF alone may not be sufficient in vivo. These data further suggest that bioavailability and metabolism of organics, especially PAHs, may have an important role in vivo.

Modeling the Effect of the Aryl Hydrocarbon Receptor on Transplant Immunity.

BACKGROUND: Exposure to pollutants through inhalation is a risk factor for lung diseases including cancer, asthma, and lung transplant rejection, but knowledge of the effects of inhaled pollutants on pathologies outside of the lung is limited. METHODS: Using the minor-mismatched model of male C57BL/6J (B6) to female B6 skin grafts, recipient mice were treated with an inhaled urban dust particle sample every 3 days before and after grafting. Graft survival time was determined, and analysis of the resulting immune response was performed at time before rejection. RESULTS: Significant prolongation of male skin grafts occurred in recipient female mice treated with urban dust particles compared with controls and was found to be dependent on aryl hydrocarbon receptor (AHR) expression in the recipient mouse. T cell responses to the male histocompatibility antigen (H-Y) Dby were not altered by exposure to pollutants. A reduction in the frequency of IFNγ-producing CD4 T cells infiltrating the graft on day 7 posttransplant was observed. Flow cytometry analysis revealed that AHR expression is upregulated in IFNγ-producing CD4 T cells during immune responses in vitro and in vivo. CONCLUSIONS: Surprisingly, inhalation of a pollutant standard was found to prolong graft survival in a minor-mismatched skin graft model in an AHR-dependent manner. One possible mechanism may be an effect on IFNγ-producing CD4 T cells responding to donor antigen. The increased expression of AHR in this CD4 T cell subset suggests that AHR ligands within the particulate matter may be directly affecting the type 1 T helper cell response in this model.

The exosome complex establishes a barricade to erythroid maturation.

Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.