Roles for the Aryl Hydrocarbon Receptor in the Immune Response to Toxoplasma Gondii

One of the major challenges faced by the immune system involves mounting an inflammatory response to control pathogen growth while limiting immune-mediated damage to the host. In order to achieve this balance, responding immune cells need to detect signals from the environment and react appropriately by promoting or attenuating inflammation. Cells of the immune system employ an array of sensors to respond to environmental cues, such as nuclear hormone receptors, cytokine receptors, and Toll-like receptors. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, provides immune cells with an additional means of detecting and responding to environmental signals to promote immunity. The work presented in this thesis examines the effects of AHR signaling during infection with the protozoan parasite Toxoplasma gondii, a medically significant pathogen that naturally infects mice. A variety of AHR ligands are produced by the host and the parasite during toxoplasmosis, which raised the question of whether AHR activity influences the immune response in this setting. Chapter 2 of this thesis describes a role for the AHR in promoting natural killer cell production of IL-10 in vitro and in vivo following infection. NK cells basally expressed the AHR and IL-12 stimulation increased AHR levels in these cells. Inhibition of the AHR led to impaired NK cell IL-10 production in vitro, and NK cells isolated from T. gondii-infected Ahr-/- mice had defective expression of IL-10. Chapter 3 demonstrates context-dependent roles for the AHR during oral and chronic toxoplasmosis. Orally infected Ahr-/- animals exhibited more severe weight loss and increased intestinal tissue pathology compared to wild-type mice, which was associated with CD4+ T cell hyperactivation. Chronically infected Ahr-/- mice developed elevated parasite burdens, but the CD4+ T cell responses in these animals were comparable to those in wild-type animals. Collectively these studies indicate that the AHR has multiple context dependent roles in the immune response to T. gondii

Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses

Reduced Pathology following Infection with Transgenic Leishmania major Expressing Murine CD40 Ligand▿

Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40 ligand (CD40L) with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice (G. Chen, P. A. Darrah, and D. M. Mosser, Infect. Immun. 69:3255-3263, 2001). In the present study, we developed transgenic L. major organisms which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high-dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity

<i>Ahr</i>^<i>-/-</i> mice exhibit increased T cell activation following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with <i>T</i>. <i>gondii</i> for 9 days. <b>(A)</b> Group 3 ILC frequency in the lamina propria of wild type or <i>Ahr</i>^<i>-/-</i> mice following infection. The plots on the left are gated on live CD90.2⁺CD11c^-B220^- cells. (<b>B)</b> Weight loss was monitored at various days post-infection. Data are pooled from 2 experiments with 6–8 mice per group. <b>(C)</b> H&E staining of small intestinal tissue sections. <b>(D)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse. The Peyer’s patch exhibits severe lymphocytolysis (*) and the lamina propria of adjacent villi is expanded by primarily lymphocytes and plasma cells (➜). <b>(E)</b> A higher magnification image of the section in Fig 2C shows that the Peyer’s patch exhibits severe lymphocytolysis characterized by pyknosis and other cellular debris. <b>(F)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse shows crypt loss (*) and multifocal necrotic enterocytes (➜). The lamina propria of the villi is expanded by lymphocytes and plasma cells (❋). <b>(G)</b> Expression of T-bet and Ki67 by FoxP3^- CD4⁺ T cells in the mesenteric lymph nodes of infected mice. Results are pooled from 3 separate experiments. <b>(H)</b> Cytokine production by CD4⁺ T cells following stimulation with PMA/ionomycin. Data are pooled from 2 experiments.</p

Increased T cell responses to <i>Toxoplasma</i> antigen and crude commensal antigen in <i>Ahr</i>^<i>-/-</i> mice following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with 100 <i>T</i>. <i>gondii</i> cysts for 7 days. <b>(A, B)</b> Splenocytes were stimulated with the indicated antigen preparations for 5 hours and then incubated overnight with brefeldin A. The cells were stained to assay IFN-γ expression by CD4⁺ T cells. The graph shows pooled data from 3 independent experiments. <b>(C)</b> Parasite burdens in the terminal ileum were assayed by RT-PCR. Results are pooled from 2 separate experiments.</p

Treg phenotype and IL-10 production in orally infected <i>Ahr</i>^<i>-/-</i> mice.

<p><i>Ahr</i>^<i>-/-</i> mice or wild type controls were infected orally with 20 Me49 cysts for 9 days. Results are pooled from 2–3 separate experiments. <b>(A)</b> Frequency of Tregs in the indicated tissues of wild type or <i>Ahr</i>^<i>-/-</i> mice. In the lamina propria, the plots shown are gated on live CD45⁺CD3⁺CD4⁺ cells, and Tregs were gated as live CD45⁺CD3⁺CD4⁺FoxP3⁺ cells. For the mesenteric lymph node and spleen, the plots shown are gated on CD3⁺CD4⁺ cells, and Tregs were gated as CD3⁺CD4⁺FoxP3⁺ cells. <b>(B)</b> Expression of T-bet, CXCR3, and Ki67 on Tregs in the spleens of wild type or <i>Ahr</i>^<i>-/-</i> mice. The plots are gated on CD3⁺CD4⁺FoxP3⁺ cells. <b>(C)</b> IL-10 secretion by cells isolated from the lamina propria or the spleen following restimulation with soluble <i>Toxoplasma</i> antigen. Results are pooled from 2 separate experiments with a total of 5–7 mice per group.</p

IL-27 Limits Type 2 Immunopathology Following Parainfluenza Virus Infection

<div><p>Respiratory paramyxoviruses are important causes of morbidity and mortality, particularly of infants and the elderly. In humans, a T helper (Th)2-biased immune response to these infections is associated with increased disease severity; however, little is known about the endogenous regulators of these responses that may be manipulated to ameliorate pathology. IL-27, a cytokine that regulates Th2 responses, is produced in the lungs during parainfluenza infection, but its role in disease pathogenesis is unknown. To determine whether IL-27 limits the development of pathogenic Th2 responses during paramyxovirus infection, IL-27-deficient or control mice were infected with the murine parainfluenza virus Sendai virus (SeV). Infected IL-27-deficient mice experienced increased weight loss, more severe lung lesions, and decreased survival compared to controls. IL-27 deficiency led to increased pulmonary eosinophils, alternatively activated macrophages (AAMs), and the emergence of Th2 responses. In control mice, IL-27 induced a population of IFN-γ⁺/IL-10⁺ CD4⁺ T cells that was replaced by IFN-γ⁺/IL-17⁺ and IFN-γ⁺/IL-13⁺ CD4⁺ T cells in IL-27-deficient mice. CD4⁺ T cell depletion in IL-27-deficient mice attenuated weight loss and decreased AAMs. Elimination of STAT6 signaling in IL-27-deficient mice reduced Th2 responses and decreased disease severity. These data indicate that endogenous IL-27 limits pathology during parainfluenza virus infection by regulating the quality of CD4⁺ T cell responses and therefore may have therapeutic potential in paramyxovirus infections.</p></div