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TLR4/MyD88-induced CD11b+Gr-1 int F4/80+ non-migratory myeloid cells suppress Th2 effector function in the lung.

Arora M, Poe SL, Oriss TB, Krishnamoorthy N, Yarlagadda M, Wenzel SE, Billiar TR, Ray A, Ray P - Mucosal Immunol (2010)

Bottom Line: LPS promoted the development of a CD11b(+)Gr1(int)F4/80(+) lung-resident cell resembling myeloid-derived suppressor cells in a Toll-like receptor 4 and myeloid differentiation factor 88 (MyD88)-dependent manner that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells.Suppression of Th2 effector function was reversed by anti-interleukin-10 (IL-10) or inhibition of arginase 1.These data suggest that CD11b(+)Gr1(int)F4/80(+) cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT
In humans, environmental exposure to a high dose of lipopolysaccharide (LPS) protects from allergic asthma, the immunological underpinnings of which are not well understood. In mice, exposure to a high LPS dose blunted house dust mite-induced airway eosinophilia and T-helper 2 (Th2) cytokine production. Although adoptively transferred Th2 cells induced allergic airway inflammation in control mice, they were unable to do so in LPS-exposed mice. LPS promoted the development of a CD11b(+)Gr1(int)F4/80(+) lung-resident cell resembling myeloid-derived suppressor cells in a Toll-like receptor 4 and myeloid differentiation factor 88 (MyD88)-dependent manner that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells. LPS effects switched from suppressive to stimulatory in MyD88(-/-) mice. Suppression of Th2 effector function was reversed by anti-interleukin-10 (IL-10) or inhibition of arginase 1. Lineage(neg) bone marrow progenitor cells could be induced by LPS to develop into CD11b(+)Gr1(int)F4/80(+)cells both in vivo and in vitro that when adoptively transferred suppressed allergen-induced airway inflammation in recipient mice. These data suggest that CD11b(+)Gr1(int)F4/80(+) cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.

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Suppression of HDM- and TH2 cell-induced eosinophilic inflammation in the airways by LPS. (a) Diagram of the treatment protocol. HDM (100 μg per mouse) ± LPS (10 μg LPS per mouse) was administered intratracheally (i.t.) at the indicated time points. Lung-draining LNs were harvested from one group 7 days after one HDM instillation ± LPS to assess CD4+ T cell priming. The rest of the animals were challenged with HDM ± LPS and at 72 h after the final instillation, BAL fluid and lung tissue samples were obtained. (b) ELISPOT assay of cells harvested from lung-draining LNs. Naive mice were used as control. For all the groups, the total cell population was used for ELISPOT assay and the number of cytokine-expressing Th cells was estimated based upon the percentage of CD4+ T cells determined by flow cytometry. For the HDM and HDM+LPS groups, CD4+ T cells were also purified by magnetic bead selection prior to being subjected to the assay to confirm that the cytokines were being produced by CD4+ T cells. In all cases, cells were stimulated for 8-10 h with PMA (25 ng/ml) plus ionomycin (500 ng/ml). Results are expressed as the number of cytokine-producing cells per sample. Data shown are mean ± SD **, P <0.01. The data shown are representative of two independent experiments. (c) Counts of cells recovered in the BAL fluid. Values are mean + SEM ***, P<0.005. (d) The concentrations of IL-5, IL-13 and IFN-g in lung homogenates were measured by multiplex assay and are presented as mean ± SEM *, P<0.05 and **, P<0.01. Histological examination of lung sections stained with PAS for assessment of inflammation and mucus production. Lung infiltrates around bronchovascular bundles where eosinophilic response is most pronounced in disease were of +4 grade in animals that received HDM and +1/2 in those that received LPS together with HDM. Arrows indicate mucus staining. The data shown are representative of three independent experiments. (e) Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice and were injected i.v. into either LPS-treated mice or naïve recipient mice (5 × 106 cells/mouse). The mice were then exposed to aerosolized OVA daily for 7 days and were analyzed 24 h after the last exposure. Total and differential counts of cells recovered in the BAL fluid (upper panel) and H&E staining of lung sections (lower panels) of both group of mice were performed. Values are mean ± SEM *, P<0.01 and**, P<0.001. The data shown are representative of two independent experiments.
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Figure 4: Suppression of HDM- and TH2 cell-induced eosinophilic inflammation in the airways by LPS. (a) Diagram of the treatment protocol. HDM (100 μg per mouse) ± LPS (10 μg LPS per mouse) was administered intratracheally (i.t.) at the indicated time points. Lung-draining LNs were harvested from one group 7 days after one HDM instillation ± LPS to assess CD4+ T cell priming. The rest of the animals were challenged with HDM ± LPS and at 72 h after the final instillation, BAL fluid and lung tissue samples were obtained. (b) ELISPOT assay of cells harvested from lung-draining LNs. Naive mice were used as control. For all the groups, the total cell population was used for ELISPOT assay and the number of cytokine-expressing Th cells was estimated based upon the percentage of CD4+ T cells determined by flow cytometry. For the HDM and HDM+LPS groups, CD4+ T cells were also purified by magnetic bead selection prior to being subjected to the assay to confirm that the cytokines were being produced by CD4+ T cells. In all cases, cells were stimulated for 8-10 h with PMA (25 ng/ml) plus ionomycin (500 ng/ml). Results are expressed as the number of cytokine-producing cells per sample. Data shown are mean ± SD **, P <0.01. The data shown are representative of two independent experiments. (c) Counts of cells recovered in the BAL fluid. Values are mean + SEM ***, P<0.005. (d) The concentrations of IL-5, IL-13 and IFN-g in lung homogenates were measured by multiplex assay and are presented as mean ± SEM *, P<0.05 and **, P<0.01. Histological examination of lung sections stained with PAS for assessment of inflammation and mucus production. Lung infiltrates around bronchovascular bundles where eosinophilic response is most pronounced in disease were of +4 grade in animals that received HDM and +1/2 in those that received LPS together with HDM. Arrows indicate mucus staining. The data shown are representative of three independent experiments. (e) Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice and were injected i.v. into either LPS-treated mice or naïve recipient mice (5 × 106 cells/mouse). The mice were then exposed to aerosolized OVA daily for 7 days and were analyzed 24 h after the last exposure. Total and differential counts of cells recovered in the BAL fluid (upper panel) and H&E staining of lung sections (lower panels) of both group of mice were performed. Values are mean ± SEM *, P<0.01 and**, P<0.001. The data shown are representative of two independent experiments.

Mentions: Since our experiments suggested that the LPS-induced CD11b+Gr1int cells did not traffic to the lung-draining LNs, as was also observed in the prior study 7, our next question was whether these cells would temper effector T cell responses in the tissue. We chose a mouse model of allergic airway inflammation induced by the common household allergen, house dust mite (HDM) (Figure 4a). Mice were sensitized and challenged with HDM 17. After one instillation of HDM and allowing time for priming, we assessed IL-5+ CD4+ T cells by ELISPOT assay in the lung-draining LNs. As shown in Figure 4b, significantly greater numbers of IL-5+ CD4+ T cells were discernible in the LNs of both HDM and HDM+LPS groups as compared to that in the LNs of naive mice again demonstrating that a high dose of LPS had minimal effects at the LN level. The slightly lower numbers of IL-5+ cells under conditions of HDM+LPS should be considered in light of the fact that activated T cells can enter LNs from tissues via afferent lymphatics 18. Thus, the number of effector T cells in the draining LNs after immunization is governed by two criteria- cells that remain in the LN and those that migrate to the antigen-delivery site and reenter the lymph node via the afferent lymph. Since all of our evidence indicated that the CD11b+Gr1int cells develop in the lung without detectable presence in the LNs, we did not expect to see any influence on effector response in the LNs soon after priming which is what we observed. However, when the lungs were examined after 3 rounds of challenges with the allergen alone or in the presence of LPS, a significant reduction in airway inflammation as well as Th2 cytokine levels in the lung was observed when LPS was mixed with HDM (Figure 4, panels c and d). Thus, LPS treatment induced a condition in the lung that did not favor effector Th2 responses. Most importantly, LPS did not cause more IFN-γ production in the lung suggesting that the inhibition was not due cross-regulation by this cytokine. If our inference was correct that the LPS-induced tissue-dwelling myeloid cells would suppress Th2 effector function in the lung, we expected in vitro- generated Th2 cells to be unable to induce inflammation in the airways of LPS-treated mice. To test this, Th2 cells were generated in vitro by repetitive stimulation of CD4+ (DO11.10) T cells under Th2-skewing conditions. The cells were then adoptively transferred into control and LPS-treated mice and the cells were recruited to the lung by challenge with aerosolized OVA 19,20}. There was a remarkable difference in the level of airway inflammation between the LPS-treated and untreated groups with severe inhibition observed when cells were transferred into LPS-treated mice (Figure 4e). This observation was in line with a previous report in which intravenous LPS administration was shown to suppress experimental asthma in a NOS2-dependent fashion although the mechanism for this observation was not identified 9.


TLR4/MyD88-induced CD11b+Gr-1 int F4/80+ non-migratory myeloid cells suppress Th2 effector function in the lung.

Arora M, Poe SL, Oriss TB, Krishnamoorthy N, Yarlagadda M, Wenzel SE, Billiar TR, Ray A, Ray P - Mucosal Immunol (2010)

Suppression of HDM- and TH2 cell-induced eosinophilic inflammation in the airways by LPS. (a) Diagram of the treatment protocol. HDM (100 μg per mouse) ± LPS (10 μg LPS per mouse) was administered intratracheally (i.t.) at the indicated time points. Lung-draining LNs were harvested from one group 7 days after one HDM instillation ± LPS to assess CD4+ T cell priming. The rest of the animals were challenged with HDM ± LPS and at 72 h after the final instillation, BAL fluid and lung tissue samples were obtained. (b) ELISPOT assay of cells harvested from lung-draining LNs. Naive mice were used as control. For all the groups, the total cell population was used for ELISPOT assay and the number of cytokine-expressing Th cells was estimated based upon the percentage of CD4+ T cells determined by flow cytometry. For the HDM and HDM+LPS groups, CD4+ T cells were also purified by magnetic bead selection prior to being subjected to the assay to confirm that the cytokines were being produced by CD4+ T cells. In all cases, cells were stimulated for 8-10 h with PMA (25 ng/ml) plus ionomycin (500 ng/ml). Results are expressed as the number of cytokine-producing cells per sample. Data shown are mean ± SD **, P <0.01. The data shown are representative of two independent experiments. (c) Counts of cells recovered in the BAL fluid. Values are mean + SEM ***, P<0.005. (d) The concentrations of IL-5, IL-13 and IFN-g in lung homogenates were measured by multiplex assay and are presented as mean ± SEM *, P<0.05 and **, P<0.01. Histological examination of lung sections stained with PAS for assessment of inflammation and mucus production. Lung infiltrates around bronchovascular bundles where eosinophilic response is most pronounced in disease were of +4 grade in animals that received HDM and +1/2 in those that received LPS together with HDM. Arrows indicate mucus staining. The data shown are representative of three independent experiments. (e) Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice and were injected i.v. into either LPS-treated mice or naïve recipient mice (5 × 106 cells/mouse). The mice were then exposed to aerosolized OVA daily for 7 days and were analyzed 24 h after the last exposure. Total and differential counts of cells recovered in the BAL fluid (upper panel) and H&E staining of lung sections (lower panels) of both group of mice were performed. Values are mean ± SEM *, P<0.01 and**, P<0.001. The data shown are representative of two independent experiments.
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Figure 4: Suppression of HDM- and TH2 cell-induced eosinophilic inflammation in the airways by LPS. (a) Diagram of the treatment protocol. HDM (100 μg per mouse) ± LPS (10 μg LPS per mouse) was administered intratracheally (i.t.) at the indicated time points. Lung-draining LNs were harvested from one group 7 days after one HDM instillation ± LPS to assess CD4+ T cell priming. The rest of the animals were challenged with HDM ± LPS and at 72 h after the final instillation, BAL fluid and lung tissue samples were obtained. (b) ELISPOT assay of cells harvested from lung-draining LNs. Naive mice were used as control. For all the groups, the total cell population was used for ELISPOT assay and the number of cytokine-expressing Th cells was estimated based upon the percentage of CD4+ T cells determined by flow cytometry. For the HDM and HDM+LPS groups, CD4+ T cells were also purified by magnetic bead selection prior to being subjected to the assay to confirm that the cytokines were being produced by CD4+ T cells. In all cases, cells were stimulated for 8-10 h with PMA (25 ng/ml) plus ionomycin (500 ng/ml). Results are expressed as the number of cytokine-producing cells per sample. Data shown are mean ± SD **, P <0.01. The data shown are representative of two independent experiments. (c) Counts of cells recovered in the BAL fluid. Values are mean + SEM ***, P<0.005. (d) The concentrations of IL-5, IL-13 and IFN-g in lung homogenates were measured by multiplex assay and are presented as mean ± SEM *, P<0.05 and **, P<0.01. Histological examination of lung sections stained with PAS for assessment of inflammation and mucus production. Lung infiltrates around bronchovascular bundles where eosinophilic response is most pronounced in disease were of +4 grade in animals that received HDM and +1/2 in those that received LPS together with HDM. Arrows indicate mucus staining. The data shown are representative of three independent experiments. (e) Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice and were injected i.v. into either LPS-treated mice or naïve recipient mice (5 × 106 cells/mouse). The mice were then exposed to aerosolized OVA daily for 7 days and were analyzed 24 h after the last exposure. Total and differential counts of cells recovered in the BAL fluid (upper panel) and H&E staining of lung sections (lower panels) of both group of mice were performed. Values are mean ± SEM *, P<0.01 and**, P<0.001. The data shown are representative of two independent experiments.
Mentions: Since our experiments suggested that the LPS-induced CD11b+Gr1int cells did not traffic to the lung-draining LNs, as was also observed in the prior study 7, our next question was whether these cells would temper effector T cell responses in the tissue. We chose a mouse model of allergic airway inflammation induced by the common household allergen, house dust mite (HDM) (Figure 4a). Mice were sensitized and challenged with HDM 17. After one instillation of HDM and allowing time for priming, we assessed IL-5+ CD4+ T cells by ELISPOT assay in the lung-draining LNs. As shown in Figure 4b, significantly greater numbers of IL-5+ CD4+ T cells were discernible in the LNs of both HDM and HDM+LPS groups as compared to that in the LNs of naive mice again demonstrating that a high dose of LPS had minimal effects at the LN level. The slightly lower numbers of IL-5+ cells under conditions of HDM+LPS should be considered in light of the fact that activated T cells can enter LNs from tissues via afferent lymphatics 18. Thus, the number of effector T cells in the draining LNs after immunization is governed by two criteria- cells that remain in the LN and those that migrate to the antigen-delivery site and reenter the lymph node via the afferent lymph. Since all of our evidence indicated that the CD11b+Gr1int cells develop in the lung without detectable presence in the LNs, we did not expect to see any influence on effector response in the LNs soon after priming which is what we observed. However, when the lungs were examined after 3 rounds of challenges with the allergen alone or in the presence of LPS, a significant reduction in airway inflammation as well as Th2 cytokine levels in the lung was observed when LPS was mixed with HDM (Figure 4, panels c and d). Thus, LPS treatment induced a condition in the lung that did not favor effector Th2 responses. Most importantly, LPS did not cause more IFN-γ production in the lung suggesting that the inhibition was not due cross-regulation by this cytokine. If our inference was correct that the LPS-induced tissue-dwelling myeloid cells would suppress Th2 effector function in the lung, we expected in vitro- generated Th2 cells to be unable to induce inflammation in the airways of LPS-treated mice. To test this, Th2 cells were generated in vitro by repetitive stimulation of CD4+ (DO11.10) T cells under Th2-skewing conditions. The cells were then adoptively transferred into control and LPS-treated mice and the cells were recruited to the lung by challenge with aerosolized OVA 19,20}. There was a remarkable difference in the level of airway inflammation between the LPS-treated and untreated groups with severe inhibition observed when cells were transferred into LPS-treated mice (Figure 4e). This observation was in line with a previous report in which intravenous LPS administration was shown to suppress experimental asthma in a NOS2-dependent fashion although the mechanism for this observation was not identified 9.

Bottom Line: LPS promoted the development of a CD11b(+)Gr1(int)F4/80(+) lung-resident cell resembling myeloid-derived suppressor cells in a Toll-like receptor 4 and myeloid differentiation factor 88 (MyD88)-dependent manner that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells.Suppression of Th2 effector function was reversed by anti-interleukin-10 (IL-10) or inhibition of arginase 1.These data suggest that CD11b(+)Gr1(int)F4/80(+) cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Pittsburgh, Pennsylvania, USA.

ABSTRACT
In humans, environmental exposure to a high dose of lipopolysaccharide (LPS) protects from allergic asthma, the immunological underpinnings of which are not well understood. In mice, exposure to a high LPS dose blunted house dust mite-induced airway eosinophilia and T-helper 2 (Th2) cytokine production. Although adoptively transferred Th2 cells induced allergic airway inflammation in control mice, they were unable to do so in LPS-exposed mice. LPS promoted the development of a CD11b(+)Gr1(int)F4/80(+) lung-resident cell resembling myeloid-derived suppressor cells in a Toll-like receptor 4 and myeloid differentiation factor 88 (MyD88)-dependent manner that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells. LPS effects switched from suppressive to stimulatory in MyD88(-/-) mice. Suppression of Th2 effector function was reversed by anti-interleukin-10 (IL-10) or inhibition of arginase 1. Lineage(neg) bone marrow progenitor cells could be induced by LPS to develop into CD11b(+)Gr1(int)F4/80(+)cells both in vivo and in vitro that when adoptively transferred suppressed allergen-induced airway inflammation in recipient mice. These data suggest that CD11b(+)Gr1(int)F4/80(+) cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue.

Show MeSH
Related in: MedlinePlus