Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Background: Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. Methods: A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Results: Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. Conclusions: These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Extracellular Vesicle-associated GARP/TGFß:LAP Mediates "Infectious" Allo-tolerance.

Here we test the hypothesis that, like CD81-associated "latent" IL35, the transforming growth factor (TGF)ß:latency-associated peptide (LAP)/glycoprotein A repetitions predominant (GARP) complex was also tethered to small extracellular vesicles (sEVs), aka exosomes, produced by lymphocytes from allo-tolerized mice. Once these sEVs are taken up by conventional T cells, we also test whether TGFß could be activated suppressing the local immune response. Methods: C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFß:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFß:LAP and to activation from its latent form, as well as various TGFß receptors; and (c) TGFß-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay. Results: After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFß:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFß:LAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFß:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface. Conclusions: Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFß:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFß:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFß:LAP along with Treg-derived GARP as part of the infectious tolerance network.

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.

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.

Amelioration of Clostridium difficile Infection in Mice by Dietary Supplementation With Indole-3-carbinol.

OBJECTIVE: To determine the therapeutic effects of dietary supplementation on Clostridium difficile infection (CDI). BACKGROUND: With limited treatment options, the rise of C. difficile-associated disease has spurred on the search for novel therapies. Recent data define a role for the aryl hydrocarbon receptor (AHR) and diet-derived AHR ligands in mucosal immunity. We investigated the efficacy of indole-3-carbinol (I3C), a dietary supplement, and AHR precursor ligand in a murine model of CDI. METHODS: C57BL/6 (B6), AHR, and AHR mice were placed on either grain-based or semipurified diets with or without I3C before and during CDI. Mice were followed clinically for a minimum of 6 days or euthanized between days 0 and 4 of inoculation for analysis of the inflammatory response and microbiota. RESULTS: B6 mice fed an AHR ligand-deficient, semipurified diet have significantly increased disease severity (P<0.001) and mortality (P < 0.001) compared with mice fed on diet containing I3C. The addition of I3C to the diet of AHR null mice had less of an impact than in AHR heterozygous littermates, although some protection was seen. Mice on semipurified I3C-diet had increased cecal Tregs, ILC3s, and γδ T cells and an increased neutrophilic response without increased inflammation or bacterial translocation compared with controls. CONCLUSIONS: I3C is a powerful treatment to reduce impact of CDI in mice. The findings indicate I3C may be acting through both AHR-dependent and -independent mechanisms in this model. Dietary supplementation with I3C is a potential new therapy for prevention and amelioration of C. difficile disease.

Gut Lymphocyte Phenotype Changes After Parenteral Nutrition and Neuropeptide Administration.

OBJECTIVE: To define gut-associated lymphoid tissue (GALT) phenotype changes with parenteral nutrition (PN) and PN with bombesin (BBS). BACKGROUND: PN reduces respiratory tract (RT) and GALT Peyer patch and lamina propria lymphocytes, lowers gut and RT immunoglobulin A (IgA) levels, and destroys established RT antiviral and antibacterial immunity. BBS, an enteric nervous system neuropeptide, reverses PN-induced IgA and RT immune defects. METHODS: Experiment 1: Intravenously cannulated ICR mice received chow, PN, or PN + BBS injections for 5 days. LSR-II flow cytometer analyzed Peyer patches and lamina propria isolated lymphocytes for homing phenotypes (L-selectin and LPAM-1) and state of activation (CD25, CD44) in T (CD3)-cell subsets (CD4 and CD8) along with homing phenotype (L-selectin and LPAM-1) in naive B (IgD) and antigen-activated (IgD or IgM) B (CD45R/B220) cells. Experiment 2: Following the initial experiment 1 protocol, lamina propria T regulatory cell phenotype was evaluated by Foxp3 expression. RESULTS: Experiment 1: PN significantly reduced lamina propria (1) CD4CD25 (activated) and (2) CD4CD25LPAM-1 (activated cells homed to the lamina propria) T cells, whereas PN-BBS assimilated chow levels. PN significantly reduced lamina propria (1) IgD (naive), (2) IgDLPAM (antigen-activated homed to the lamina propria) and CD44 memory B cells, whereas PN-BBS assimilated chow levels. Experiment 2: PN significantly reduced lamina propria CD4CD25Foxp3 T regulatory cells compared with chow-fed mice, whereas PN + BBS assimilated chow levels. CONCLUSIONS: PN reduces lamina propria activated and T regulatory cells and also naive and memory B cells. BBS addition to PN maintains these cell phenotypes, demonstrating the intimate involvement of the enteric nervous system in mucosal immunity.

The aryl hydrocarbon receptor meets immunology: friend or foe? A little of both.

The aryl hydrocarbon receptor (AHR) has long been studied by toxicologists as a ligand-activated transcription factor that is activated by dioxin and other environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs). The hallmark of AHR activation is the upregulation of the cytochrome P450 enzymes that metabolize many of these toxic compounds. However, recent findings demonstrate that both exogenous and endogenous AHR ligands can alter innate and adaptive immune responses including effects on T-cell differentiation. Kynurenine, a tryptophan breakdown product, is one such endogenous ligand of the AHR. Expression of indoleamine 2,3-dioxygenase by dendritic cells causes accumulation of kynurenine and results in subsequent tolerogenic effects including increased regulatory T-cell activity. At the same time, PAHs found in pollution enhance Th17 differentiation in the lungs of exposed mice via the AHR. In this perspective, we will discuss the importance of the AHR in the immune system and the role this might play in normal physiology and response to disease.