House dust mite allergen induces asthma via TLR4 triggering of airway structural cells methods
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TLR4 on radioresistant cells determines innate immunity to HDM
We next investigated the importance of TLR4 expression on structural cells in the innate response to complex and relevant allergens such as HDM extracts, also known to contain large amounts of endotoxin,. The HDM extract contained 1.05ng endotoxin/mg extract. HDM administration (100 µg) in the airways of WT->WT and TLR4 -/- ->WT mice resulted in an increase in monocytes (not shown) and DCs ( ) in the airways and increase in CCL2 and CCL3 in the BAL fluid ( online) compared with PBS administration, responses that were severely reduced in TLR4 -/- ->TLR4 -/- and WT->TLR4 -/- mice. There was no induction of KC nor G-CSF, and consequently airway neutrophilia did not develop (data not shown). Because the administration of HDM in the airways is known to induce Th2 responses in the lungs -, we evaluated the production cytokines kn...
Construction of bone marrow chimeras
3-5-wk-old TLR4-deficient or wild-type (WT) recipient mice were sublethally irradiated with 900 rads. On the same day, 5 × 10 4 bone marrow cells from MHCII-EGFP mice (WT for TLR4) or Tlr4 -/- mice were harvested and infused via the tail vein, respectively. For the imaging experiments, MHCII-EGFP mice on a Rag -/- background (no B and T cells) were used as bone marrow donors. These mice were kept in isolators andprovided neomycin-containing water until use 10-12 wk later. To permit chimerism in the lung to be complete, we allowed 10-12 weeks of reconstitution time before experiments were started; the degree of chimerism of CD19 + MHCII + B cells, CD11c + MHCII + DCs, and alveolar macrophages, known to be slowly repopulated following irradiation, was confirmed by measuring GFP positivity after 12 weeks of chimerism, or by immunostaining o...
Immune analysis
Broncho-alveolar lavage (BAL) was performed by injecting 1 ml of PBS containing 0.01mM EDTA. Thoracic draining LNs, lungs, and trachea were collected, and single cell suspensions were prepared as reported. Cell suspensions were stained in PBS supplemented with 2 mM EDTA, 0.5% BSA, and 0.01% sodium azide. Monoclonal antibodies (conjugated to various fluorochromes or biotin) and fluorescence-labeled streptavidin were obtained from BD Biosciences. 2.4G2 (anti-FcγRIII/II) was used to block unspecific antibody binding. Single cell suspensions were stained with FITC anti I-Ad/I-Ed, PE-labeled anti-CD40, anti-CD80, anti-CD86, and APC-labeled anti-CD11c antibodies (all from BD Biosciences). Data were collected on a FACSCalibur (Becton Dickinson) and were analyzed withFlowJo software (Treestar, Inc.).
Immune analysis
To measure cytokine levels, MLN cells were plated in round bottom 96-well plates (10 6 cells/ml) and restimulated with HDM extracts (30 µg/ml) for 5 d, followed by collection of the culture supernantant and assay by commercially available ELISA (R&D systems).
Preparation of explants
Mice were injected intratracheally with LPS or PBS 2 hrs before being euthanised by CO 2 asphyxiation. The tracheal explant was stained with 100 µM SNARF (Invitrogen) for 10 sec. at 37°C in PBS. The trachea was then immobilized on the surface of a Petri dish using Vetbond epoxy glue (3M Animal Care Products) and a mechanical support, and the entire preparation submerged in 20 ml of phenol red free RPMI maintained at 37°C. During microscopy medium was superfused with a gas mixture of 95%O 2 /5% CO 2.
Two-photon microscopy
Two-photon microscopy was conducted using a Bio-Rad Laboratories Radiance 2100MP system equipped with a Nikon 600FN upright microscope, 20 × water immersion lens (N.A. 0.95; Olympus), and either a Mira 900 Sa:Ti femtosecond-pulsed laser driven by a 10-W Verdi pump laser (Coherent) tuned to 880 nm. The typical pixel size of the image field was 1.09 µm, and the x-y dimensions of the scan area were 560 × 560 µm. The typical optical z-step size was 2µm. Serial x-y images were collected over the entire z depth every 30-60 s, and the entire process was repeated for up to 60 min toobtain a four-dimensional dataset.
Figures
( a )WT->WT and WT->TLR4 -/- chimeras were injected i.t with LPS or PBS. Tracheal explants were prepared 2hours later and analyzed by two-photon microscopy. Green DCs are located within red epithelial cells. Nuclei were counterstained in blue. ( b ) Levels of GM-CSF measured 24 h after LPS exposure. ( c ) Chimeric mice were injected i.t with fluorescent OVA AF647 together with LPS or PBS. The number of OVA + migrating DCs was enumerated in the MLNs thirty-six hours later. ( d ) On day 0, WT->WT and WT->TLR4 -/- - chimeras received an intravenous injection of CFSE-labelled OVA-specific naive OTII T cells, and were administered with OVA/LPS or OVA/PBS i.t on day 1. T cell proliferation was evaluated in the MLNs at day 5. Proliferation index for each group is shown in the upper right corner of the histograms. ( e ) Cytokine production by MLN cells 5 days after OVA...
Figures
Chimeras were injected i.t with PBS or HDM allergen. The number of MHCII+CD11c+ (a) MHCII+CD11c+CD11b+ (b) DCs in tracheal digests was analyzed 24 hours later. The tracheal counts have been corrected for 1.10 6 CD45 neg structural cells to correct for differently sized explants. ( c ) Production of GM-CSF, (D) TSLP, (E)IL-25 and (F) IL-33 was measured in BAL fluids. *:p<0.05. This is one representative experiment out of 3. 5-6 mice/group were used
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Evidence quotes (8)
We next investigated the importance of TLR4 expression on structural cells in the innate response to complex and relevant allergens such as HDM extracts, also known to contain large amounts of endotoxin,. The HDM extract contained 1.05ng endotoxin/mg extract. HDM administration (100 µg) in the airways of WT->WT and TLR4 -/- ->WT mice resulted in an increase in monocytes (not shown) and DCs ( ) in the airways and increase in CCL2 and CCL3 in the BAL fluid ( online) compared with PBS administration, responses that were severely reduced in TLR4 -/- ->TLR4 -/- and WT->TLR4 -/- mice. There was no induction of KC nor G-CSF, and consequently airway neutrophilia did not develop (data not shown). Because the administration of HDM in the airways is known to induce Th2 responses in the lungs -, we evaluated the production cytokines known to promote eosinophilic inflammation -. The levels of GM-CSF, TSLP, IL-25, and IL-33 ( ) were markedly increased in the BAL fluids of WT->WT animals exposed to a single administration of HDM compared with mice injected with PBS. For comparison, some mice were also injected with a low (100...
3-5-wk-old TLR4-deficient or wild-type (WT) recipient mice were sublethally irradiated with 900 rads. On the same day, 5 × 10 4 bone marrow cells from MHCII-EGFP mice (WT for TLR4) or Tlr4 -/- mice were harvested and infused via the tail vein, respectively. For the imaging experiments, MHCII-EGFP mice on a Rag -/- background (no B and T cells) were used as bone marrow donors. These mice were kept in isolators andprovided neomycin-containing water until use 10-12 wk later. To permit chimerism in the lung to be complete, we allowed 10-12 weeks of reconstitution time before experiments were started; the degree of chimerism of CD19 + MHCII + B cells, CD11c + MHCII + DCs, and alveolar macrophages, known to be slowly repopulated following irradiation, was confirmed by measuring GFP positivity after 12 weeks of chimerism, or by immunostaining of TLR4 expression on cryosections of lungs, followed by confocal imaging.
Broncho-alveolar lavage (BAL) was performed by injecting 1 ml of PBS containing 0.01mM EDTA. Thoracic draining LNs, lungs, and trachea were collected, and single cell suspensions were prepared as reported. Cell suspensions were stained in PBS supplemented with 2 mM EDTA, 0.5% BSA, and 0.01% sodium azide. Monoclonal antibodies (conjugated to various fluorochromes or biotin) and fluorescence-labeled streptavidin were obtained from BD Biosciences. 2.4G2 (anti-FcγRIII/II) was used to block unspecific antibody binding. Single cell suspensions were stained with FITC anti I-Ad/I-Ed, PE-labeled anti-CD40, anti-CD80, anti-CD86, and APC-labeled anti-CD11c antibodies (all from BD Biosciences). Data were collected on a FACSCalibur (Becton Dickinson) and were analyzed withFlowJo software (Treestar, Inc.).
To measure cytokine levels, MLN cells were plated in round bottom 96-well plates (10 6 cells/ml) and restimulated with HDM extracts (30 µg/ml) for 5 d, followed by collection of the culture supernantant and assay by commercially available ELISA (R&D systems).
Mice were injected intratracheally with LPS or PBS 2 hrs before being euthanised by CO 2 asphyxiation. The tracheal explant was stained with 100 µM SNARF (Invitrogen) for 10 sec. at 37°C in PBS. The trachea was then immobilized on the surface of a Petri dish using Vetbond epoxy glue (3M Animal Care Products) and a mechanical support, and the entire preparation submerged in 20 ml of phenol red free RPMI maintained at 37°C. During microscopy medium was superfused with a gas mixture of 95%O 2 /5% CO 2.
Two-photon microscopy was conducted using a Bio-Rad Laboratories Radiance 2100MP system equipped with a Nikon 600FN upright microscope, 20 × water immersion lens (N.A. 0.95; Olympus), and either a Mira 900 Sa:Ti femtosecond-pulsed laser driven by a 10-W Verdi pump laser (Coherent) tuned to 880 nm. The typical pixel size of the image field was 1.09 µm, and the x-y dimensions of the scan area were 560 × 560 µm. The typical optical z-step size was 2µm. Serial x-y images were collected over the entire z depth every 30-60 s, and the entire process was repeated for up to 60 min toobtain a four-dimensional dataset.
( a )WT->WT and WT->TLR4 -/- chimeras were injected i.t with LPS or PBS. Tracheal explants were prepared 2hours later and analyzed by two-photon microscopy. Green DCs are located within red epithelial cells. Nuclei were counterstained in blue. ( b ) Levels of GM-CSF measured 24 h after LPS exposure. ( c ) Chimeric mice were injected i.t with fluorescent OVA AF647 together with LPS or PBS. The number of OVA + migrating DCs was enumerated in the MLNs thirty-six hours later. ( d ) On day 0, WT->WT and WT->TLR4 -/- - chimeras received an intravenous injection of CFSE-labelled OVA-specific naive OTII T cells, and were administered with OVA/LPS or OVA/PBS i.t on day 1. T cell proliferation was evaluated in the MLNs at day 5. Proliferation index for each group is shown in the upper right corner of the histograms. ( e ) Cytokine production by MLN cells 5 days after OVA/LPS administration. *:p<0.05. These panels show one representative out of 2-4 experiments. 4-6 mice/group were used.
Chimeras were injected i.t with PBS or HDM allergen. The number of MHCII+CD11c+ (a) MHCII+CD11c+CD11b+ (b) DCs in tracheal digests was analyzed 24 hours later. The tracheal counts have been corrected for 1.10 6 CD45 neg structural cells to correct for differently sized explants. ( c ) Production of GM-CSF, (D) TSLP, (E)IL-25 and (F) IL-33 was measured in BAL fluids. *:p<0.05. This is one representative experiment out of 3. 5-6 mice/group were used
Machine-readable layer
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"text": "( a )WT->WT and WT->TLR4 -/- chimeras were injected i.t with LPS or PBS. Tracheal explants were prepared 2hours later and analyzed by two-photon microscopy. Green DCs are located within red epithelial cells. Nuclei were counterstained in blue. ( b ) Levels of GM-CSF measured 24 h after LPS exposure. ( c ) Chimeric mice were injected i.t with fluorescent OVA AF647 together with LPS or PBS. The number of OVA + migrating DCs was enumerated in the MLNs thirty-six hours later. ( d ) On day 0, WT->WT and WT->TLR4 -/- - chimeras received an intravenous injection of CFSE-labelled OVA-specific naive OTII T cells, and were administered with OVA/LPS or OVA/PBS i.t on day 1. T cell proliferation was evaluated in the MLNs at day 5. Proliferation index for each group is shown in the upper right corner of the histograms. ( e ) Cytokine production by MLN cells 5 days after OVA..."
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