TLR3 modulates immunopathology during a Schistosoma mansoni egg-driven Th2 response in the lung - PubMed (original) (raw)
TLR3 modulates immunopathology during a Schistosoma mansoni egg-driven Th2 response in the lung
Amrita D Joshi et al. Eur J Immunol. 2008 Dec.
Abstract
We examined the role of TLR3 in Th2-driven pulmonary granulomatous disease, using wildtype (TLR3(+/+)) and TLR3 gene-deficient (TLR3(-/-)) mice in a well-established model of Schistosoma mansoni egg-induced pulmonary granuloma. The intravenous bolus injection of S. mansoni eggs into S. mansoni-sensitized TLR3(+/+) mice was associated with an increase in TLR3 transcript expression in alveolar macrophages and ex vivo spleen and lung cultures at day 8 after egg injection. Lungs from TLR3(-/-) mice showed an increase in granuloma size, greater collagen deposition around the granuloma, and increased Th2 cytokine and chemokine levels compared with similarly sensitized and challenged TLR3(+/+) mice. Macrophages from TLR3(-/-) mice exhibited an M2 phenotype characterized by increased arginase and CCL2 expression. Significantly greater numbers of CD4(+)CD25(+) T cells were present in the lungs of TLR3(-/-) mice compared with TLR3(+/+) mice at day 8 after egg embolization. Cells derived from granulomatous lung and lung draining lymph nodes of TLR3(-/-) mice released significantly higher levels of IL-17 levels relative to TLR3(+/+) cells. Thus, our data suggest that TLR3 has a major regulatory role during a Th2-driven granulomatous response as its absence enhanced immunopathology.
Figures
Figure 1
Transcript and protein expression of TLR3 prior to and after egg embolization in _S. mansoni_-sensitized TLR3+/+ mice. (A) Whole lung cells were analyzed by TAQMAN for TLR3 gene expression. Data are expressed as fold change over TLR3 transcript level in naïve lungs. (B) Membrane fractions derived from whole lungs were subjected to Western blot analysis. Also shown (bottom panel, Fig. 1B) are results of the normalization of TLR3 to GAPDH. Alveolar macrophages obtained from broncho-alveolar lavage fluid were analyzed by TAQMAN for TLR3 gene expression (C). Data are expressed as fold change over transcript levels in naïve alveolar macrophages. Spleen cells (D) and bone marrow derived macrophages (E) harvested from _S. mansoni_-sensitized TLR3+/+ mice were incubated in media alone or SEA for 24 h before RNA extraction for TLR3 gene expression analysis. Data shown are mean +/− SE and are from a representative experiment of two independent experiments. Each time point had at least 3–5 mice per group. One-way ANOVA and Newman-Keuls post-test was used to analyze significance. Significant differences are shown as * P ≤ 0.05, ** P ≤0.01.
Figure 2
IL-4, IL-5, IL-13, and IL-17 levels in cultures of dispersed spleen cells from sensitized TLR3+/+ and TLR3−/− mice. Spleen cells pooled from 3–5 mice per group were cultured in vitro in the presence of SEA for 48 h. Cell-free supernatant was then collected and analyzed by multiplex bead immunoassay. Data shown are mean +/− SE of triplicate cultures from a representative of three separate experiments. Significance was determined by using one-way ANOVA and Newman-Keuls post-test (* P ≤ 0.05, ** P ≤0.01, *** P ≤ 0.001).
Figure 3
Histopathological appearance of egg granulomas in TLR3+/+ and TLR3−/− mice at days 8 and 16 after egg embolization. Masson’s trichrome staining (magnification, 200X) of TLR3+/+ granulomas at day 8 (A) and day 16 (C) and of TLR3−/− granulomas at day 8 (B) and day 16 (D) is depicted. High power micrographs (magnification, 400X) of Masson’s Trichrome staining of TLR3−/− (F) granulomas display chitinase-like crystals, which were absent in TLR3+/+ granulomas (E). Granuloma area (G) and percentage of collagen in each granuloma (H) was quantified using IP lab software. Data shown are mean +/− SE and are from 1 representative experiment of 3 independent experiments. Each time point had at least 3–5 mice per group. Student’s t-test was used to analyze significance. Significant differences are depicted as * P ≤ 0.05.
Figure 4
IL-4, CCL11, CXCL10, and CXCL9 levels in whole lung samples from TLR3+/+ and TLR3−/− mice prior to and at days 8 and 16 after egg embolization. Whole lung lobes were homogenized in triton X-100 containing buffer and centrifuged to yield membrane-free supernatants, which were analyzed by ELISA (see Materials and Methods). Data shown are mean +/− SE from 1 representative of two separate experiments. Each time point had three-five mice per group. Significance was determined by using one-way ANOVA and Newman-Keuls post-test (* P ≤ 0.05,).
Figure 5
CXCL10 and IL-12 generation by TLR3+/+ and TLR3−/− bone marrow macrophages derived from naïve mice following either no activation, or exposure to dsRNA or polyI:C for 48 h. Cell-free supernatants were removed and analyzed by ELISA. Data shown are mean +/− SE from triplicate cultures. These data are representative of three separate experiments. One-way ANOVA and Newman-Keuls post-test was used to analyze significance (* P ≤ 0.05, *** P ≤ 0.001).
Figure 6
Gene expression analysis of M1 (A) and M2 (B) macrophage marker expression by bone marrow derived macrophages from _S. mansoni_-sensitized TLR3+/+ and TLR3−/− mice. Bone marrow derived macrophages were treated with SEA for 24 h before RNA extraction and analysis. Transcript level of each gene, under each condition, is expressed as fold over transcript levels in macrophages from sensitized TLR3+/+ mice, incubated in media alone. ELISA analysis of cell-free supernatants collected after 24 h stimulation of macrophages with SEA or media alone is shown in C. Data are representative of 2 independent experiments and expressed as mean +/− SE from triplicate wells. One-way ANOVA and Newman-Keuls post-test was used to analyze significance. (* P ≤ 0.05, *** P ≤ 0.001).
Figure 7
Phenotype of lymphocytes derived from whole lung samples of TLR3+/+ and TLR3−/− mice day 8 after egg embolization in _S. mansoni_-sensitized mice. Lung cells were dispersed and stained with anti-CD4, anti-CD8, anti-CD25 and anti-foxP3 as described in Materials and Methods. A representative dot plot (B) shows gating of CD4+ cells as an inset. The percentage of CD25+and foxP3 + cells in the CD4+ gate is shown. The bar graph (A) shows CD4+ and CD8+ cells based on lymphocyte gate. The events shown for CD25+ and CD25+foxP3+ cells are based on CD4+ lymphocyte gate. Data shown are representative of three separate experiments using three to five mice per group. Significance was determined by using one-way ANOVA and Newman-Keuls post-test. Significant differences are shown as * P ≤ 0.05, ** P ≤ 0.01
Figure 8
IL-17 levels in ex vivo cultures from lungs (A) and draining lymph node (B) cells from 5–6 _S. mansoni_-sensitized mice per group at day 8 after egg embolization. Cells were cultured with media alone or with SEA antigen for 48 h prior to Bioplex analysis for IL-17. Data shown are mean ± SEM from triplicate wells. One-way ANOVA and Newman-Keuls post-test was used to analyze significance. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with media alone conditions. Flow cytometric analysis of dispersed lung cells showing elevated levels of Th17 cells is shown in C. The top panel indicates the number of IL-17+ cells in CD4+ lymphocyte gate. Significance was determined using Student’s t-test. Significant differences are shown as ** P ≤ 0.01. Representative dot plot is shown in bottom panel of Fig 8C. Boxed region shows percentage of IL-17+ cells in CD4+ lymphocyte gate.
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