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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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CD11b+Gr1int cells induced by LPS administration suppress Th2 cell responses. Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice by incubation under Th2-skewing conditions for 6 days. cDCs and CD11b+Gr1int cells were isolated from the lungs of LPS-treated mice. cDCs and CD11b+Gr1int cells alone (each at 1 × 105 cells/well or at 5 fold more numbers of CD11b+Gr1int cells) or in combination as shown were cultured with Th2 polarized DO11.10 CD4+ T cells (1 × 106 cells/well) and OVA peptide (5 μg/ml). (a) Following cell-surface staining for CD4, intracellular cytokine staining for IL-5 was performed on co-cultured cells after 36 h. Cells were incubated with Golgi Stop (BD Biosciences) for the last 4 h of culture, were fixed with CytoFix/CytoPerm (BD Biosciences), permeabilized with Perm/Wash buffer (BD Biosciences) and labeled with anti-IL-5 mAb. For analysis by flow cytometry, gating on CD4-positive cells revealed a population with lower CD4 expression and blast-like FSC vs SSC properties (not shown), consistent with activated T cells. In each dot plot, the number represents the proportion of IL-5-producing cells (indicated by the box) among activated CD4-positive cells. (b) Cells were stimulated with IL-2 (50 U/ml) for 15 min and STAT5 phosphorylation determined by intracellular staining. (c) Nuclear extracts were analyzed by immunoblotting with antibodies against GATA-3 and β-actin. (d) Reversal of suppression of Th2 (IL-5 and IL-13) cytokine production by Arg 1 inhibitor and anti-IL-10. Culture supernatants were analyzed by multiplex cytokine assay. Data shown are mean ± s.d. * P<0.05, **P<0.01, ***P<0.001 with respect to cDC:CD11b+Gr1int (1:5 ratio).
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Figure 6: CD11b+Gr1int cells induced by LPS administration suppress Th2 cell responses. Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice by incubation under Th2-skewing conditions for 6 days. cDCs and CD11b+Gr1int cells were isolated from the lungs of LPS-treated mice. cDCs and CD11b+Gr1int cells alone (each at 1 × 105 cells/well or at 5 fold more numbers of CD11b+Gr1int cells) or in combination as shown were cultured with Th2 polarized DO11.10 CD4+ T cells (1 × 106 cells/well) and OVA peptide (5 μg/ml). (a) Following cell-surface staining for CD4, intracellular cytokine staining for IL-5 was performed on co-cultured cells after 36 h. Cells were incubated with Golgi Stop (BD Biosciences) for the last 4 h of culture, were fixed with CytoFix/CytoPerm (BD Biosciences), permeabilized with Perm/Wash buffer (BD Biosciences) and labeled with anti-IL-5 mAb. For analysis by flow cytometry, gating on CD4-positive cells revealed a population with lower CD4 expression and blast-like FSC vs SSC properties (not shown), consistent with activated T cells. In each dot plot, the number represents the proportion of IL-5-producing cells (indicated by the box) among activated CD4-positive cells. (b) Cells were stimulated with IL-2 (50 U/ml) for 15 min and STAT5 phosphorylation determined by intracellular staining. (c) Nuclear extracts were analyzed by immunoblotting with antibodies against GATA-3 and β-actin. (d) Reversal of suppression of Th2 (IL-5 and IL-13) cytokine production by Arg 1 inhibitor and anti-IL-10. Culture supernatants were analyzed by multiplex cytokine assay. Data shown are mean ± s.d. * P<0.05, **P<0.01, ***P<0.001 with respect to cDC:CD11b+Gr1int (1:5 ratio).

Mentions: We next sought to determine whether the CD11b+Gr1int cells could block activation of transcription factors such as STAT5 and GATA-3 in freshly primed Th2 cells. GATA-3 is the key regulator of Th2 differentiation as previously described by us and others 25-29 and a role for STAT5 in Th2 differentiation has also been shown 30,31. The protocol of priming involving 6 days of culture under Th2-skewing conditions does induce GATA-3, albeit in at low levels in a small fraction of the CD4+ T cells, as judged by expression of GATA-3-dependent genes such as T1ST2 31. Administration of anti-T1ST2 into mice blocks the effector function of adoptively transferred OVA-specific Th2 cells including Th2 cytokine secretion and eosinophilic airway inflammation. The expression of T1ST2 has been recently shown to require GATA-3 and activated STAT5 31. We reasoned that if both STAT5 and GATA-3 are required for expression of Th2 cytokine genes and other Th2 expressed molecules such as T1ST2, the efficient Th2 suppression observed in vivo (Figure 4) might be due to the inability of primed Th2 cells migrating into tissue to be reactivated by tissue DCs resulting in GATA-3 upregulation and STAT5 activation. We, therefore, asked whether stimulatory effects of lung cDCs on the primed Th2 cells would be compromised by the LPS-induced lung CD11b+Gr1int cells. As measures of Th2 activation, we examined Th2 cytokine production, STAT5 phosphorylation and GATA-3 expression (Figure 6). The frequency of IL-5-secreting Th2 cells was ascertained by intracellular cytokine staining (ICS) after coculture of the primed Th2-skewed cells with either lung cDCs, CD11b+Gr1int cells, or their mixture for 36 h as shown in Figure 6a. The ratio of DC to CD11b+Gr1int cells used was 1:5 based on our quantitation of these cells in the lungs after LPS administration over a 4 day time period which ranges from 5-10-fold more than cDCs. As expected with specific antigen-induced stimulation, as opposed to PMA plus ionomycin which would activate all primed T cells, a fraction of CD4 cells displayed blast-like (activated) morphology (by FSC vs SSC, data not shown) as well as typically lower CD4 expression32,33. Among reactivated CD4 cells (reduced CD4 expression), the frequency of IL-5-secreting cells was greater with stimulation by lung cDCs as compared to lung CD11b+Gr1int cells (Figure 6a). 5-fold more CD11b+Gr1int cells reduced cytokine production by 55.0 ± 3.5%. However, as expected, the addition of 5-fold more lung cDCs slightly increased cytokine production (data not shown). Furthermore, IL-5 production was inhibited when the cDCs were mixed with the CD11b+Gr1int cells in a 1:5 ratio by 60.7 ± 2.4 % (Figure 6a). The inhibition was not reversed when the NOS2 inhibitor L-NIL was used. We next cocultured the primed Th2 cells with cDCs or CD11b+Gr1int cells as above, removed the culture supernatant and exposed the cells briefly to IL-2 to assess STAT5 phosphorylation. IL-2 was able to induce STAT5 phosphorylation in cells cocultured with lung DCs and same numbers of CD11b+Gr1int cells but not when 5 fold more CD11b+Gr1int cells were used (Figure 6b). When added to cDCs, the cells partially inhibited STAT5 phosphorylation which was reversed by L-NIL. The reversal of suppression by L-NIL was expected since NO blocks Jak3-mediated STAT5 activation34,35. With respect to GATA-3 expression, lung cDCs induced an increase in GATA-3 expression in the primed Th2 cells. Similar to results obtained for STAT5 phosphorylation, equal numbers of CD11b+Gr1int cells as cDCs also caused GATA-3 upregulation. However, at 5-fold higher numbers and when mixed with DCs, GATA-3 upregulation was not achieved (Figure 6c). Of note, however, the cells maintained a low level of GATA-3 expression. Addition of L-NIL only slightly reversed GATA-3 expression (Figure 6c).


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)

CD11b+Gr1int cells induced by LPS administration suppress Th2 cell responses. Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice by incubation under Th2-skewing conditions for 6 days. cDCs and CD11b+Gr1int cells were isolated from the lungs of LPS-treated mice. cDCs and CD11b+Gr1int cells alone (each at 1 × 105 cells/well or at 5 fold more numbers of CD11b+Gr1int cells) or in combination as shown were cultured with Th2 polarized DO11.10 CD4+ T cells (1 × 106 cells/well) and OVA peptide (5 μg/ml). (a) Following cell-surface staining for CD4, intracellular cytokine staining for IL-5 was performed on co-cultured cells after 36 h. Cells were incubated with Golgi Stop (BD Biosciences) for the last 4 h of culture, were fixed with CytoFix/CytoPerm (BD Biosciences), permeabilized with Perm/Wash buffer (BD Biosciences) and labeled with anti-IL-5 mAb. For analysis by flow cytometry, gating on CD4-positive cells revealed a population with lower CD4 expression and blast-like FSC vs SSC properties (not shown), consistent with activated T cells. In each dot plot, the number represents the proportion of IL-5-producing cells (indicated by the box) among activated CD4-positive cells. (b) Cells were stimulated with IL-2 (50 U/ml) for 15 min and STAT5 phosphorylation determined by intracellular staining. (c) Nuclear extracts were analyzed by immunoblotting with antibodies against GATA-3 and β-actin. (d) Reversal of suppression of Th2 (IL-5 and IL-13) cytokine production by Arg 1 inhibitor and anti-IL-10. Culture supernatants were analyzed by multiplex cytokine assay. Data shown are mean ± s.d. * P<0.05, **P<0.01, ***P<0.001 with respect to cDC:CD11b+Gr1int (1:5 ratio).
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Figure 6: CD11b+Gr1int cells induced by LPS administration suppress Th2 cell responses. Th2 cells were generated in vitro using CD4+ T cells from DO11.10 mice by incubation under Th2-skewing conditions for 6 days. cDCs and CD11b+Gr1int cells were isolated from the lungs of LPS-treated mice. cDCs and CD11b+Gr1int cells alone (each at 1 × 105 cells/well or at 5 fold more numbers of CD11b+Gr1int cells) or in combination as shown were cultured with Th2 polarized DO11.10 CD4+ T cells (1 × 106 cells/well) and OVA peptide (5 μg/ml). (a) Following cell-surface staining for CD4, intracellular cytokine staining for IL-5 was performed on co-cultured cells after 36 h. Cells were incubated with Golgi Stop (BD Biosciences) for the last 4 h of culture, were fixed with CytoFix/CytoPerm (BD Biosciences), permeabilized with Perm/Wash buffer (BD Biosciences) and labeled with anti-IL-5 mAb. For analysis by flow cytometry, gating on CD4-positive cells revealed a population with lower CD4 expression and blast-like FSC vs SSC properties (not shown), consistent with activated T cells. In each dot plot, the number represents the proportion of IL-5-producing cells (indicated by the box) among activated CD4-positive cells. (b) Cells were stimulated with IL-2 (50 U/ml) for 15 min and STAT5 phosphorylation determined by intracellular staining. (c) Nuclear extracts were analyzed by immunoblotting with antibodies against GATA-3 and β-actin. (d) Reversal of suppression of Th2 (IL-5 and IL-13) cytokine production by Arg 1 inhibitor and anti-IL-10. Culture supernatants were analyzed by multiplex cytokine assay. Data shown are mean ± s.d. * P<0.05, **P<0.01, ***P<0.001 with respect to cDC:CD11b+Gr1int (1:5 ratio).
Mentions: We next sought to determine whether the CD11b+Gr1int cells could block activation of transcription factors such as STAT5 and GATA-3 in freshly primed Th2 cells. GATA-3 is the key regulator of Th2 differentiation as previously described by us and others 25-29 and a role for STAT5 in Th2 differentiation has also been shown 30,31. The protocol of priming involving 6 days of culture under Th2-skewing conditions does induce GATA-3, albeit in at low levels in a small fraction of the CD4+ T cells, as judged by expression of GATA-3-dependent genes such as T1ST2 31. Administration of anti-T1ST2 into mice blocks the effector function of adoptively transferred OVA-specific Th2 cells including Th2 cytokine secretion and eosinophilic airway inflammation. The expression of T1ST2 has been recently shown to require GATA-3 and activated STAT5 31. We reasoned that if both STAT5 and GATA-3 are required for expression of Th2 cytokine genes and other Th2 expressed molecules such as T1ST2, the efficient Th2 suppression observed in vivo (Figure 4) might be due to the inability of primed Th2 cells migrating into tissue to be reactivated by tissue DCs resulting in GATA-3 upregulation and STAT5 activation. We, therefore, asked whether stimulatory effects of lung cDCs on the primed Th2 cells would be compromised by the LPS-induced lung CD11b+Gr1int cells. As measures of Th2 activation, we examined Th2 cytokine production, STAT5 phosphorylation and GATA-3 expression (Figure 6). The frequency of IL-5-secreting Th2 cells was ascertained by intracellular cytokine staining (ICS) after coculture of the primed Th2-skewed cells with either lung cDCs, CD11b+Gr1int cells, or their mixture for 36 h as shown in Figure 6a. The ratio of DC to CD11b+Gr1int cells used was 1:5 based on our quantitation of these cells in the lungs after LPS administration over a 4 day time period which ranges from 5-10-fold more than cDCs. As expected with specific antigen-induced stimulation, as opposed to PMA plus ionomycin which would activate all primed T cells, a fraction of CD4 cells displayed blast-like (activated) morphology (by FSC vs SSC, data not shown) as well as typically lower CD4 expression32,33. Among reactivated CD4 cells (reduced CD4 expression), the frequency of IL-5-secreting cells was greater with stimulation by lung cDCs as compared to lung CD11b+Gr1int cells (Figure 6a). 5-fold more CD11b+Gr1int cells reduced cytokine production by 55.0 ± 3.5%. However, as expected, the addition of 5-fold more lung cDCs slightly increased cytokine production (data not shown). Furthermore, IL-5 production was inhibited when the cDCs were mixed with the CD11b+Gr1int cells in a 1:5 ratio by 60.7 ± 2.4 % (Figure 6a). The inhibition was not reversed when the NOS2 inhibitor L-NIL was used. We next cocultured the primed Th2 cells with cDCs or CD11b+Gr1int cells as above, removed the culture supernatant and exposed the cells briefly to IL-2 to assess STAT5 phosphorylation. IL-2 was able to induce STAT5 phosphorylation in cells cocultured with lung DCs and same numbers of CD11b+Gr1int cells but not when 5 fold more CD11b+Gr1int cells were used (Figure 6b). When added to cDCs, the cells partially inhibited STAT5 phosphorylation which was reversed by L-NIL. The reversal of suppression by L-NIL was expected since NO blocks Jak3-mediated STAT5 activation34,35. With respect to GATA-3 expression, lung cDCs induced an increase in GATA-3 expression in the primed Th2 cells. Similar to results obtained for STAT5 phosphorylation, equal numbers of CD11b+Gr1int cells as cDCs also caused GATA-3 upregulation. However, at 5-fold higher numbers and when mixed with DCs, GATA-3 upregulation was not achieved (Figure 6c). Of note, however, the cells maintained a low level of GATA-3 expression. Addition of L-NIL only slightly reversed GATA-3 expression (Figure 6c).

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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Related in: MedlinePlus