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Blood, Vol. 113, Issue 2, 377-388, January 8, 2009
Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology
Blood Baenziger et al.
113: 377
Supplemental materials for: Baenziger et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 58.6 KB)
- Figure S1. R848-induced lymphopenia and increased neutrophil and monocyte numbers are TLR7 dependent (JPG, 77.6 KB)
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Mice were treated with 1 mg/kg/d R848 for 7 days. Blood lymphocytes, monocytes, neutrophils, and thrombocytes numbers were determined with a hemocounter one hour after the last injection. Effects are TLR7 dependent but IFNAR and IRF 7 signaling seems not to be critically involved because effects in R848-treated mice lacking IFNAR or IRF7 were similar as in wild-type mice. Data of at least 3 individually measured animals are shown but IFNAR−∕− thrombocyte counts (#) where only 2 animals are shown.
- Figure S2. Disruption of the lymphoid structure is not further compromised after 21 days of chronic TLR7 stimulation (JPG, 228 KB)
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(A) Spleen cryosections of C57BL/6 mice showing a dose dependent malformation of lymphoid follicles with enlarged T and B-cell zones, and reduced marginal zone B lymphocytes. Lymphoid disruption was not further compromised after 21 days of treatment as compared to the 7 day-treatment (see also Fig. 3). (B) MOMA-1+ and F4/80+ macrophages were not affected. N ≥ 3.
- Figure S3. Disruption of the lymphoid structure is not further compromised even after 42 days of chronic TLR7 stimulation (JPG, 245 KB)
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(A–B) See legend Fig. S2 for details.
- Figure S4. Disruption of the lymphoid structure is TLR7 dependent (JPG, 413 KB)
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(A) Spleen cryosections of C57BL/6, TLR7−∕−, IFNAR−∕− and IRF7−∕− mice after 7 days of treatment with 1 mg/kg/d R848, showing a TLR7 dependent malformation of splenic lymphoid follicles since TLR7−∕− mice showed normal T- and B-cell zones as well as unaltered marginal zone B-lymphocyte rings. IRF7 knockout mice showed no obvious symptoms of lymphoid follicle malformation. In contrast, IFNAR−∕− mice showed somewhat inflated follicles with loosened rings of marginal zone B cells. (B) MOMA-1+ and F4/80+ macrophages as well as FDC-M1 networks and PNA+ germinal centres (data not shown) were not affected. N ≥ 3.
- Figure S5. Germinal center B cells and MFG-E8 mRNA are decreased at day 7 and increased or unaffected at day 21/42 of repetitive TLR7 triggering (JPG, 52.2 KB)
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(A) C57BL/6 mice were treated as indicated. Splenic germinal center B-cell frequencies were evaluated by flow cytometry by staining for B220 in conjunction with FITC-labeled PNA (upper panel) or GL7 antibody (lower panel). Percentage of double positive cells was normalized to that of corresponding mock treated animals. Error bars indicate 95% confidence intervals. N ≥ 3. (B) RNA was extracted from spleen (N = 3), pooled for reverse transcription and analyzed for MFG-E8 transcripts (see Supplementary Methods). Mean normalized MFG-E8 expression was calibrated to the matching mock control. Error bars indicate SEM of mean normalized expression relative to calibrator.
- Figure S6. Immune activation and lymphoid system disruption is MyD88 dependent (JPG, 163 KB)
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MyD88−∕− mice were treated with 1mg/kg/d R848 for 7 days. (A) Blood was analyzed for the presence of lymphocytes, monocytes, and neutrophiles 1 h after the last injection. There was a tendency towards a slight lymphopenia and increased neutrophile numbers in MyD88−∕− mice, although this was not statistically significant. Monocyte numbers were increased. This suggests some MyD88/TLR independent mechanisms. However, white blood cell counts were not altered in TLR7−∕− animals (Fig. 1 and Fig. S1). (B) Increase in the percentage of lymphoid subsets expressing CD69 is MyD88 dependent. Splenic lymphoid subpopulations from R848-treated MyD88−∕− animals displayed no activated phenotype. CD69 expression was analyzed by flow cytometry. (C) Disruption of the lymphoid structure is MyD88 dependent. There was no malformation of splenic lymphoid follicles with enlarged T- (CD4) and B-cell zones (not shown), and reduced marginal zone B lymphocytes (CD21/35; arrowhead) in MyD88−∕− mice. (D) Relative contraction of lymphoid subsets in spleen is MyD88 dependent. Splenocytes were stained with indicated antibodies and relative numbers were assessed by flow cytometry. (E) Splenomegaly is MyD88 dependent. Spleen weight was first normalized to total body weight and, second, normalized to the spleen weight of mock-treated animals. Error bars indicate 95% confidence intervals. (F) Cytokine deregulation is MyD88 dependent. Cytokine levels at day 7 are shown. TNF-α, IL-6, and IL-1β levels in MyD88−∕− were below detection limit (0.8, 0.8, and 0.3 pg/ml, respectively). Error bars indicate standard error. N ≥ 3.
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