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Macrophages monitor tissue osmolarity and induce inflammatory response through NLRP3 and NLRC4 inflammasome activation.

Ip WK, Medzhitov R - Nat Commun (2015)

Bottom Line: Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses.Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice.Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Interstitial osmolality is a key homeostatic variable that varies depending on the tissue microenvironment. Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses. Hyperosmolality due to high dietary salt intake has been linked to pathological inflammatory conditions. Little is known about the mechanisms of sensing the hyperosmotic stress by the innate immune system. Here we report that caspase-1 is activated in macrophages under hypertonic conditions. Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice. Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.

No MeSH data available.


Related in: MedlinePlus

Hyperosmotic stress induces caspase-1 activation and caspase-1-dependent IL-1β secretion. (a–d) BMDMs primed with LPS were maintained in isotonic conditions (control) or switched to hyperosmotic conditions (+ 200 mOsm (a, b) or as indicated (c, d)) by adding NaCl, glucose, or sorbitol for 3 h (a, c, d), overnight (b), or for the indicated times (c). LPS-primed BMDM stimulated with ATP (5 mM) for 30 min (a), 2 h (b), or the indicated times (c), or with alum (250 μg/ml) for 4 h (a) or 6 h (b), were used as positive controls. The cleavage of caspase-1 to its active p10 subunit in BMDMs was detected by immunoblot analysis in cell lysates (a, c) or in media supernatants (b; see also Supplementary Fig. 2a), or the caspase-1 activation was visualized by incubation with a fluorescent cell-permeable probe that binds only to activated caspase-1 (FLICA)(d). Scale bar, 10 μm. (e) Wild-type (C57BL/6) or caspase-1-deficient (Casp1−/−) BMDMs primed with LPS were incubated overnight in isotonic conditions (+ 0 mOsm) or hypertonic conditions (+ 100 or 200 mOsm). IL-1β in media supernatants was measured by ELISA. Data are representative of three independent experiments. Data in e are the mean ± s.d. of quadruplicates.
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Figure 3: Hyperosmotic stress induces caspase-1 activation and caspase-1-dependent IL-1β secretion. (a–d) BMDMs primed with LPS were maintained in isotonic conditions (control) or switched to hyperosmotic conditions (+ 200 mOsm (a, b) or as indicated (c, d)) by adding NaCl, glucose, or sorbitol for 3 h (a, c, d), overnight (b), or for the indicated times (c). LPS-primed BMDM stimulated with ATP (5 mM) for 30 min (a), 2 h (b), or the indicated times (c), or with alum (250 μg/ml) for 4 h (a) or 6 h (b), were used as positive controls. The cleavage of caspase-1 to its active p10 subunit in BMDMs was detected by immunoblot analysis in cell lysates (a, c) or in media supernatants (b; see also Supplementary Fig. 2a), or the caspase-1 activation was visualized by incubation with a fluorescent cell-permeable probe that binds only to activated caspase-1 (FLICA)(d). Scale bar, 10 μm. (e) Wild-type (C57BL/6) or caspase-1-deficient (Casp1−/−) BMDMs primed with LPS were incubated overnight in isotonic conditions (+ 0 mOsm) or hypertonic conditions (+ 100 or 200 mOsm). IL-1β in media supernatants was measured by ELISA. Data are representative of three independent experiments. Data in e are the mean ± s.d. of quadruplicates.

Mentions: We next tested whether hyperosmotic stress activates caspase-1, which is necessary for processing and secretion of IL-1β. Immunoblot analysis of cell lysates from BMDMs showed that addition of NaCl, glucose or sorbitol (i.e., + 200 mOsm) into the culture medium induced the cleavage of caspase-1 into the active subunit p10 (Fig. 3a) and its release into the culture medium (Fig. 3b). This caspase-1 cleavage was also demonstrated in macrophage cell line J774 but not in other cell types such as mouse embryonic fibroblasts, L929 cells and Hepa cells (Supplementary Fig. 3), suggesting that hyperosmotic stress-induced activation of caspase-1 is not ubiquitous and may be macrophage specific. Consistently, dose- and time-dependent activation of caspase-1 in BMDMs was observed specifically upon hyperosmotic challenge to BMDMs (Fig. 3c). The activation of caspase-1 was also demonstrated by using FLICA, a fluorescent inhibitor that binds covalently to the active caspase-1 (Fig. 3d). To confirm that the IL-1β secretion by hyperosmotic stress was caspase-1-dependent, caspase-1-deficient BMDMs were used. Only minimal release of IL-1β was observed as compared with BMDMs from wild-type mice (Fig. 3e). We also measured lactate dehydrogenase in the supernatants as an indicator of cell viability, which confirmed that there was no obvious cytotoxicity under hypertonic conditions (Supplementary Fig. 4). Collectively, these results suggest that caspase-1 is activated in the inflammasome complex upon hyperosmotic challenge, leading to the processing and secretion of IL-1β.


Macrophages monitor tissue osmolarity and induce inflammatory response through NLRP3 and NLRC4 inflammasome activation.

Ip WK, Medzhitov R - Nat Commun (2015)

Hyperosmotic stress induces caspase-1 activation and caspase-1-dependent IL-1β secretion. (a–d) BMDMs primed with LPS were maintained in isotonic conditions (control) or switched to hyperosmotic conditions (+ 200 mOsm (a, b) or as indicated (c, d)) by adding NaCl, glucose, or sorbitol for 3 h (a, c, d), overnight (b), or for the indicated times (c). LPS-primed BMDM stimulated with ATP (5 mM) for 30 min (a), 2 h (b), or the indicated times (c), or with alum (250 μg/ml) for 4 h (a) or 6 h (b), were used as positive controls. The cleavage of caspase-1 to its active p10 subunit in BMDMs was detected by immunoblot analysis in cell lysates (a, c) or in media supernatants (b; see also Supplementary Fig. 2a), or the caspase-1 activation was visualized by incubation with a fluorescent cell-permeable probe that binds only to activated caspase-1 (FLICA)(d). Scale bar, 10 μm. (e) Wild-type (C57BL/6) or caspase-1-deficient (Casp1−/−) BMDMs primed with LPS were incubated overnight in isotonic conditions (+ 0 mOsm) or hypertonic conditions (+ 100 or 200 mOsm). IL-1β in media supernatants was measured by ELISA. Data are representative of three independent experiments. Data in e are the mean ± s.d. of quadruplicates.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4430126&req=5

Figure 3: Hyperosmotic stress induces caspase-1 activation and caspase-1-dependent IL-1β secretion. (a–d) BMDMs primed with LPS were maintained in isotonic conditions (control) or switched to hyperosmotic conditions (+ 200 mOsm (a, b) or as indicated (c, d)) by adding NaCl, glucose, or sorbitol for 3 h (a, c, d), overnight (b), or for the indicated times (c). LPS-primed BMDM stimulated with ATP (5 mM) for 30 min (a), 2 h (b), or the indicated times (c), or with alum (250 μg/ml) for 4 h (a) or 6 h (b), were used as positive controls. The cleavage of caspase-1 to its active p10 subunit in BMDMs was detected by immunoblot analysis in cell lysates (a, c) or in media supernatants (b; see also Supplementary Fig. 2a), or the caspase-1 activation was visualized by incubation with a fluorescent cell-permeable probe that binds only to activated caspase-1 (FLICA)(d). Scale bar, 10 μm. (e) Wild-type (C57BL/6) or caspase-1-deficient (Casp1−/−) BMDMs primed with LPS were incubated overnight in isotonic conditions (+ 0 mOsm) or hypertonic conditions (+ 100 or 200 mOsm). IL-1β in media supernatants was measured by ELISA. Data are representative of three independent experiments. Data in e are the mean ± s.d. of quadruplicates.
Mentions: We next tested whether hyperosmotic stress activates caspase-1, which is necessary for processing and secretion of IL-1β. Immunoblot analysis of cell lysates from BMDMs showed that addition of NaCl, glucose or sorbitol (i.e., + 200 mOsm) into the culture medium induced the cleavage of caspase-1 into the active subunit p10 (Fig. 3a) and its release into the culture medium (Fig. 3b). This caspase-1 cleavage was also demonstrated in macrophage cell line J774 but not in other cell types such as mouse embryonic fibroblasts, L929 cells and Hepa cells (Supplementary Fig. 3), suggesting that hyperosmotic stress-induced activation of caspase-1 is not ubiquitous and may be macrophage specific. Consistently, dose- and time-dependent activation of caspase-1 in BMDMs was observed specifically upon hyperosmotic challenge to BMDMs (Fig. 3c). The activation of caspase-1 was also demonstrated by using FLICA, a fluorescent inhibitor that binds covalently to the active caspase-1 (Fig. 3d). To confirm that the IL-1β secretion by hyperosmotic stress was caspase-1-dependent, caspase-1-deficient BMDMs were used. Only minimal release of IL-1β was observed as compared with BMDMs from wild-type mice (Fig. 3e). We also measured lactate dehydrogenase in the supernatants as an indicator of cell viability, which confirmed that there was no obvious cytotoxicity under hypertonic conditions (Supplementary Fig. 4). Collectively, these results suggest that caspase-1 is activated in the inflammasome complex upon hyperosmotic challenge, leading to the processing and secretion of IL-1β.

Bottom Line: Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses.Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice.Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Interstitial osmolality is a key homeostatic variable that varies depending on the tissue microenvironment. Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses. Hyperosmolality due to high dietary salt intake has been linked to pathological inflammatory conditions. Little is known about the mechanisms of sensing the hyperosmotic stress by the innate immune system. Here we report that caspase-1 is activated in macrophages under hypertonic conditions. Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice. Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.

No MeSH data available.


Related in: MedlinePlus