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Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

Bourdonnay E, Zasłona Z, Penke LR, Speth JM, Schneider DJ, Przybranowski S, Swanson JA, Mancuso P, Freeman CM, Curtis JL, Peters-Golden M - J. Exp. Med. (2015)

Bottom Line: Secretion is tunable and occurs both in vitro and in vivo.SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice.Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School; and Department of Environmental Health Sciences, School of Public Health; University of Michigan, Ann Arbor, MI 48109.

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AM-derived SOCS attenuates pulmonary STAT activation in vivo. (A–D) Mouse lungs were pretreated oropharyngeally with 50 µl saline alone or saline containing ∼3 × 106 MPs isolated from CM from AMs (A–D) or PMs (A and C). 2 h later, mice received an oropharyngeal dose of 50 µl saline alone or saline containing 0.1 µg IFNγ. 1 h thereafter, their AMs were removed by lavage, and lung homogenates were prepared from the middle right lung for analysis of p-STAT1 (A) and p-STAT3 (B) by WB and from the inferior right lung for analysis of MCP-1 mRNA by qRT-PCR (C). p-STAT1 levels in lysates of lavaged AMs were analyzed by WB (D). (E) Mice were treated with intrapulmonary saline alone or saline containing AM MPs before IFNγ, as in A, and lung sections prepared from the left lung were incubated with hematoxylin to stain nuclei blue and anti-pSTAT1, followed by DAB to stain p-STAT1 red; photographs were taken using an Eclipse E600 microscope (40 magnification), and insets represent enlarged images (top); p-STAT1 staining was quantified by first separating the colors using a color deconvolution plugin (ImageJ software) and performing densitometric analysis of red staining (bottom) in 10 randomly selected fields, which were expressed relative to the area of the whole field. Bars, 500 µm. (A–E) Bar graphs represent the mean ± SE from a minimum of three mice per group in one experiment, which was representative of at least three independent experiments (A, C, and D) or the mean ± SE from 10 randomly selected fields from one representative experiment (E). In B, the blot shown is representative of two independent experiments. *, P < 0.05 versus untreated mice (A, C, and D); **, P < 0.05 versus IFNγ-treated mice not pretreated with AM-derived MPs (A, C, and E).
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fig7: AM-derived SOCS attenuates pulmonary STAT activation in vivo. (A–D) Mouse lungs were pretreated oropharyngeally with 50 µl saline alone or saline containing ∼3 × 106 MPs isolated from CM from AMs (A–D) or PMs (A and C). 2 h later, mice received an oropharyngeal dose of 50 µl saline alone or saline containing 0.1 µg IFNγ. 1 h thereafter, their AMs were removed by lavage, and lung homogenates were prepared from the middle right lung for analysis of p-STAT1 (A) and p-STAT3 (B) by WB and from the inferior right lung for analysis of MCP-1 mRNA by qRT-PCR (C). p-STAT1 levels in lysates of lavaged AMs were analyzed by WB (D). (E) Mice were treated with intrapulmonary saline alone or saline containing AM MPs before IFNγ, as in A, and lung sections prepared from the left lung were incubated with hematoxylin to stain nuclei blue and anti-pSTAT1, followed by DAB to stain p-STAT1 red; photographs were taken using an Eclipse E600 microscope (40 magnification), and insets represent enlarged images (top); p-STAT1 staining was quantified by first separating the colors using a color deconvolution plugin (ImageJ software) and performing densitometric analysis of red staining (bottom) in 10 randomly selected fields, which were expressed relative to the area of the whole field. Bars, 500 µm. (A–E) Bar graphs represent the mean ± SE from a minimum of three mice per group in one experiment, which was representative of at least three independent experiments (A, C, and D) or the mean ± SE from 10 randomly selected fields from one representative experiment (E). In B, the blot shown is representative of two independent experiments. *, P < 0.05 versus untreated mice (A, C, and D); **, P < 0.05 versus IFNγ-treated mice not pretreated with AM-derived MPs (A, C, and E).

Mentions: We tested the in vivo ability of AM-derived SOCS3 to influence pulmonary inflammatory signaling by the direct intrapulmonary administration of MPs, using as negative controls MPs that lacked SOCS3. We took advantage of the fact that SOCS3 protein exhibits 100% similarity between rat and mouse by using rat AMs as a source of MPs and normal C57BL/6 mice as recipients. IFNγ activates not only STAT1 but also STAT3 (Qing and Stark, 2004). Intrapulmonary pretreatment with ∼3 × 106 MPs/mouse inhibited IFNγ-induced STAT1 activation (Fig. 7 A), STAT3 activation (Fig. 7 B), and mRNA expression of the STAT-dependent chemokine monocyte chemotactic protein 1 (MCP-1, or CCL2; Fig. 7 C) in lung homogenates depleted of AMs by lavage just before harvest. Interestingly, no corresponding inhibition of STAT1 activation was noted in the lavaged AMs themselves (Fig. 7 D), suggesting that AECs were the target cells responsible for the inhibition noted in lung homogenates. That phosphorylated STAT1 was found mainly in AECs of the IFNγ-challenged lung, and that this AEC STAT1 activation was attenuated by prior intrapulmonary administration of AM-derived MPs, was verified by immunohistochemical staining of lung sections for phospho-STAT1 (Fig. 7 E). In contrast to the effects of AM-derived MPs, administration of the same number of rat PM-derived MPs, isolated from CM which lacks SOCS3 (Fig. 6 C), failed to attenuate lung STAT1 activation (Fig. 7 A) and MCP-1 mRNA expression (Fig. 7 C). These negative data for PS-positive but SOCS3-negative, PM-derived MPs exclude the possibility that the antiinflammatory effects of AM-derived MPs can be explained by potential nonspecific antiinflammatory effects attributable to the PS on their surface.


Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

Bourdonnay E, Zasłona Z, Penke LR, Speth JM, Schneider DJ, Przybranowski S, Swanson JA, Mancuso P, Freeman CM, Curtis JL, Peters-Golden M - J. Exp. Med. (2015)

AM-derived SOCS attenuates pulmonary STAT activation in vivo. (A–D) Mouse lungs were pretreated oropharyngeally with 50 µl saline alone or saline containing ∼3 × 106 MPs isolated from CM from AMs (A–D) or PMs (A and C). 2 h later, mice received an oropharyngeal dose of 50 µl saline alone or saline containing 0.1 µg IFNγ. 1 h thereafter, their AMs were removed by lavage, and lung homogenates were prepared from the middle right lung for analysis of p-STAT1 (A) and p-STAT3 (B) by WB and from the inferior right lung for analysis of MCP-1 mRNA by qRT-PCR (C). p-STAT1 levels in lysates of lavaged AMs were analyzed by WB (D). (E) Mice were treated with intrapulmonary saline alone or saline containing AM MPs before IFNγ, as in A, and lung sections prepared from the left lung were incubated with hematoxylin to stain nuclei blue and anti-pSTAT1, followed by DAB to stain p-STAT1 red; photographs were taken using an Eclipse E600 microscope (40 magnification), and insets represent enlarged images (top); p-STAT1 staining was quantified by first separating the colors using a color deconvolution plugin (ImageJ software) and performing densitometric analysis of red staining (bottom) in 10 randomly selected fields, which were expressed relative to the area of the whole field. Bars, 500 µm. (A–E) Bar graphs represent the mean ± SE from a minimum of three mice per group in one experiment, which was representative of at least three independent experiments (A, C, and D) or the mean ± SE from 10 randomly selected fields from one representative experiment (E). In B, the blot shown is representative of two independent experiments. *, P < 0.05 versus untreated mice (A, C, and D); **, P < 0.05 versus IFNγ-treated mice not pretreated with AM-derived MPs (A, C, and E).
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fig7: AM-derived SOCS attenuates pulmonary STAT activation in vivo. (A–D) Mouse lungs were pretreated oropharyngeally with 50 µl saline alone or saline containing ∼3 × 106 MPs isolated from CM from AMs (A–D) or PMs (A and C). 2 h later, mice received an oropharyngeal dose of 50 µl saline alone or saline containing 0.1 µg IFNγ. 1 h thereafter, their AMs were removed by lavage, and lung homogenates were prepared from the middle right lung for analysis of p-STAT1 (A) and p-STAT3 (B) by WB and from the inferior right lung for analysis of MCP-1 mRNA by qRT-PCR (C). p-STAT1 levels in lysates of lavaged AMs were analyzed by WB (D). (E) Mice were treated with intrapulmonary saline alone or saline containing AM MPs before IFNγ, as in A, and lung sections prepared from the left lung were incubated with hematoxylin to stain nuclei blue and anti-pSTAT1, followed by DAB to stain p-STAT1 red; photographs were taken using an Eclipse E600 microscope (40 magnification), and insets represent enlarged images (top); p-STAT1 staining was quantified by first separating the colors using a color deconvolution plugin (ImageJ software) and performing densitometric analysis of red staining (bottom) in 10 randomly selected fields, which were expressed relative to the area of the whole field. Bars, 500 µm. (A–E) Bar graphs represent the mean ± SE from a minimum of three mice per group in one experiment, which was representative of at least three independent experiments (A, C, and D) or the mean ± SE from 10 randomly selected fields from one representative experiment (E). In B, the blot shown is representative of two independent experiments. *, P < 0.05 versus untreated mice (A, C, and D); **, P < 0.05 versus IFNγ-treated mice not pretreated with AM-derived MPs (A, C, and E).
Mentions: We tested the in vivo ability of AM-derived SOCS3 to influence pulmonary inflammatory signaling by the direct intrapulmonary administration of MPs, using as negative controls MPs that lacked SOCS3. We took advantage of the fact that SOCS3 protein exhibits 100% similarity between rat and mouse by using rat AMs as a source of MPs and normal C57BL/6 mice as recipients. IFNγ activates not only STAT1 but also STAT3 (Qing and Stark, 2004). Intrapulmonary pretreatment with ∼3 × 106 MPs/mouse inhibited IFNγ-induced STAT1 activation (Fig. 7 A), STAT3 activation (Fig. 7 B), and mRNA expression of the STAT-dependent chemokine monocyte chemotactic protein 1 (MCP-1, or CCL2; Fig. 7 C) in lung homogenates depleted of AMs by lavage just before harvest. Interestingly, no corresponding inhibition of STAT1 activation was noted in the lavaged AMs themselves (Fig. 7 D), suggesting that AECs were the target cells responsible for the inhibition noted in lung homogenates. That phosphorylated STAT1 was found mainly in AECs of the IFNγ-challenged lung, and that this AEC STAT1 activation was attenuated by prior intrapulmonary administration of AM-derived MPs, was verified by immunohistochemical staining of lung sections for phospho-STAT1 (Fig. 7 E). In contrast to the effects of AM-derived MPs, administration of the same number of rat PM-derived MPs, isolated from CM which lacks SOCS3 (Fig. 6 C), failed to attenuate lung STAT1 activation (Fig. 7 A) and MCP-1 mRNA expression (Fig. 7 C). These negative data for PS-positive but SOCS3-negative, PM-derived MPs exclude the possibility that the antiinflammatory effects of AM-derived MPs can be explained by potential nonspecific antiinflammatory effects attributable to the PS on their surface.

Bottom Line: Secretion is tunable and occurs both in vitro and in vivo.SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice.Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School; and Department of Environmental Health Sciences, School of Public Health; University of Michigan, Ann Arbor, MI 48109.

Show MeSH