A Student-Initiated Elective in Medical Ethics: Innovations in Design and Institutionalization

Introduction: This study addresses how to best approach the instruction and evaluation of clinical ethics with preclinical medical students. [See PDF for complete abstract

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

Loss of IL-27 signaling results in increased disease severity and lung immunopathology after SeV.

<p><b>(A)</b> Female mice were infected intranasally with SeV and weighed every two days until 17 dpi. Weights are graphed as percent of starting weight. Plotted weights are of two mice per group (mock) and five mice per group (infected) and are representative of weight trends in five separate experiments <i>(p<0</i>.<i>0001)</i>. <b>(B)</b> Survival of infected males after SeV. Findings are pooled from five experiments and include WT (n = 25) and IL-27Rα^-/- (n = 18) mice <i>(p</i> = <i>0</i>.<i>0009</i>, Mantel-Cox test) <b>(C)</b> Whole lung tissue from infected females was homogenized, and viral load was quantified by titration of infected cells and RT-PCR at 3 and 10 dpi. Data are representative of two separate experiments, three to five mice per group. <b>(D)</b> Sections of lung tissue from WT and IL-27Rα^-/- mice were preserved in formalin and stained by hematoxylin and eosin (H&E) at 10 dpi. Mock indicates intranasal inoculation with PBS (vehicle) <b>(E)</b> Lung pathology was scored for severity of inflammation. Histological scores are pooled from three separate experiments with three to five mice per group (*<i>p<0</i>.<i>0155</i>, Mann-Whitney test).</p

IL-27 limits type 2 innate immune responses in the lung after SeV infection.

<p>Mice were infected intranasally with SeV and control PBS (mock), and lungs were harvested and evaluated for cellular infiltrate at 10 dpi. Frequency of <b>(A)</b> pulmonary eosinophils (CD3^-/CD19^-/NK1.1^-/FcεR^-/CD11b⁺/Siglec F⁺), and <b>(B)</b> alternatively activated macrophages by flow cytometry (CD3^-/CD19^-/NK1.1^-/CD45⁺/CD11c⁺/CD11b⁺/CD206⁺). For AAMs the same results were obtained using the marker CD64 in place of CD11c. Flow plots are of infected mice using groups of male or female mice. Plots are of three to five mice per group and are representative of six experiments (*<i>p<0</i>.<i>05</i>, <i>****p<0</i>.<i>0001</i>).</p

IL-27 is produced in the lung in response to SeV.

<p>Female WT mice were infected intranasally with SeV. <b>(A)</b> After 10 days, total lung tissue was homogenized, and RT PCR was performed to evaluate expression of EBi3 and IL-27p28. Data are representative of three independent experiments with three to five mice per group <b>(B)</b> BALF was evaluated by ELISA to quantify IL-27p28 protein at the time points indicated. Data are representative of three mice per group <b>(C)</b> Cells from the spleen, dLN, and lung were analyzed for IL-27p28 expression by FACS. Populations shown were gated on live cells negative for CD3, CD19, and NK1.1. DC/AM are CD11c^hi, MHC II⁺. MΦ are CD11c^int, MHC II^-. As indicated, data points are representative of 6 and 10 dpi and are representative of two independent experiments with three mice per group.</p

Pathogenic Th2 responses emerge in the absence of IL-27 (A-D).

<p>WT and IL-27Rα^-/- mice were infected with SeV, and monoclonal anti-CD4 antibody versus isotype control was administered at 0, 4, and 7 dpi (data representative of two experiments, three to five mice per group) <b>(A)</b> Weight change in IL-27Rα^-/- plus isotype versus IL-27Rα^-/- plus anti-CD4. <b>(B-C)</b> Lung lymphocytes were isolated at 10 dpi and evaluated by flow cytometry for frequency of <b>(B)</b> eosinophils, and <b>(C)</b> AAMs. <b>(D-E)</b> WT, IL-27Rα^-/-, and IL-27Rα^-/-/STAT6^-/- mice were infected with SeV (data representative of three experiments, three to five mice per group). <b>(D)</b> Weight loss plotted over time in each group (infected mice) <b>(E)</b> Percent survival of infected male mice over time <b>(F-G)</b> Lung tissue was taken for analysis at 10 dpi with frequencies of <b>(F)</b> eosinophils, and <b>(G)</b> AAMs plotted. <b>(H-I)</b> Lung lymphocytes were isolated and restimulated ex vivo with PMA/ionomycin/BFA/monensin for 4 hours, then stained intracellularly for cytokine production. <b>(H)</b> Flow cytometric and <b>(I)</b> Graphical representation of cytokine production by lung CD4⁺ T cells after restimulation for IFN-γ, IL-10, and IL-13. Representative plots are from male mice. (<i>*p<0</i>.<i>05</i>, <i>**p<0</i>.<i>01</i>, <i>***p<0</i>.<i>001</i>, <i>****p<0</i>.<i>0001</i>).</p

Loss of IFN-γ⁺/IL-10⁺ CD4⁺ T cells in IL-27 deficiency is associated with the emergence of Th2 and Th17 responses.

<p>WT and IL-27Rα-/- mice were infected intranasally with SeV or control PBS (mock). Lung lymphocytes were isolated and restimulated ex vivo with PMA/ionomycin/BFA/monensin for 4 hours at 10 dpi, then stained intracellularly for cytokine production. Data shown are representative of five experiments, three to five mice per group. <b>(A)</b> Flow plots and <b>(B)</b> graphical representation of lung CD4⁺ T cells after restimulation for IL-10 and IFN-γ (above). Frequency of IL-10 production by IFN-γ^+ve and IFN-γ^-ve CD4⁺ T cells (below). <b>(C)</b> Flow plots showing IL-13 and IL-17 production as they relate to IFN-γ production by CD4⁺ T cells. <b>(D)</b> Frequency of IL-13 and IL-17 production by IFN-γ^+ve and IFN-γ^-ve CD4⁺ T cells. <b>(E)</b> SPICE analysis representation of CD4⁺ T cell production of IL-13, IL-10, IFN-γ, IL-17 in all cells producing two cytokines. Flow plots are of infected female mice. (<i>*p<0</i>.<i>05</i>, <i>**p<0</i>.<i>01</i>, <i>***p = 0</i>.<i>001</i>).</p