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. 2010 May 25;107(21):9789-94.
doi: 10.1073/pnas.1000779107. Epub 2010 May 5.

Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1beta secretion

Sara Tassi  1 , Sonia Carta, Laura Delfino, Roberta Caorsi, Alberto Martini, Marco Gattorno, Anna Rubartelli
Affiliations

Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1beta secretion

Sara Tassi et al. Proc Natl Acad Sci U S A. .

Abstract

In healthy monocytes, Toll-like receptor (TLR) engagement induces production of reactive oxygen species (ROS), followed by an antioxidant response involved in IL-1beta processing and secretion. Markers of the antioxidant response include intracellular thioredoxin and extracellular release of reduced cysteine. Cryopyrin-associated periodic syndromes (CAPS) are autoinflammatory diseases in which Nod-like receptor family pyrin domain-containing 3 (NLRP3) gene mutations lead to increased IL-1beta secretion. We show in a large cohort of patients that IL-1beta secretion by CAPS monocytes is much faster than that by healthy monocytes. This accelerated kinetics is caused by alterations in the basal redox state, as well as in the redox response to TLR triggering displayed by CAPS monocytes. Indeed, unstimulated CAPS monocytes are under a mild oxidative stress, with elevated levels of both ROS and antioxidants. The redox response to LPS is quickened, with early generation of the reducing conditions favoring IL-1beta processing and secretion, and then rapidly exhausted. Therefore, secretion of IL-1beta is accelerated, but reaches a plateau much earlier than in healthy controls. Pharmacologic inhibition of the redox response hinders IL-1beta release, confirming the functional link between redox impairment and altered kinetics of secretion. Monocytes from patients with juvenile idiopathic arthritis display normal kinetics of redox response and IL-1beta secretion, excluding a role of chronic inflammation in the alterations observed in CAPS. We conclude that preexisting redox alterations distinct from CAPS monocytes anticipate the pathogen-associated molecular pattern molecule-induced generation of the reducing environment favorable to inflammasome activation and IL-1beta secretion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LPS-stimulated CAPS monocytes display accelerated kinetics of IL-1β secretion. (A and B) Monocytes from healthy donors (HD; n = 10) and from the indicated CAPS subjects were cultured for 18 h with 1 μg/mL of LPS. Secreted IL-1β was quantified by ELISA. Data are expressed as ng/mL. (A) Data from single patients. For HDs, mean ± SD is shown. (B) Mean ± SD from untreated patients (n = 5), treated patients (n = 4), and HDs (n = 10). (C) Kinetics of IL-1β secretion by monocytes from 10 HDs and from the indicated CAPS subjects. Monocytes were exposed to LPS for 3, 6, or 18 h, and secreted IL-1β was quantified as in A. (D) Western blots of intracellular pro–IL-1β in monocytes from patients C2 (lanes 1–3), C3 (lanes 7 and 9), and MW1 (lanes 8 and 10) and from one representative HD analyzed in parallel (lanes 4–6), unstimulated (lanes 1, 4, 7, and 8) and stimulated with LPS for 3 h (lanes 2, 5, 9, and 10) or 18 h (lanes 3 and 6). (Lower) The same blots hybridized with anti−β-tubulin. (E) Monocytes from healthy donors and CAPS patients, unstimulated (0) or stimulated for various times with LPS (3, 6, and18 h) were stained with FITC-conjugated anti-annexin V and propidium iodide (PI) and analyzed by flow cytometry (25). Data show the percentage of living cells (annexin V−/PI−), early apoptotic cells (annexin V+/PI−), late apoptotic cells (annexin V+/PI+), and necrotic cells (annexin V−/PI+). Representative data from 2 healthy donors and 2 CINCA patients are shown.
Fig. 2.
Fig. 2.
ROS levels are higher in unstimulated CAPS than in healthy monocytes. Monocytes from four HDs and four CAPS patients (C3, C4, C5, and MW1) were cultured 1 h in the absence (gray columns) or presence (black columns) of LPS, and intracellular ROS levels were quantified by H2DCF-DA fluorometric methods. Data are expressed as mean ± SD relative fluorescence units (RFUs).
Fig. 3.
Fig. 3.
Impaired antioxidant response in CAPS monocytes. Real-time PCR analysis of xCT mRNA in monocytes from the indicated CAPS patients and from two HDs, untreated (gray columns) or after 6 h of exposure to LPS (black columns). (A) Data are expressed as fold changes (mean ± SD) in untreated CAPS monocytes compared with untreated HD monocytes. (B) Data are expressed as fold changes (mean ± SD) in LPS-stimulated (black columns) compared with unstimulated monocytes (gray columns) of the same subject. NS, not significant. (C) Extracellular cysteine was quantified in 18 h supernatants of monocytes from HDs (n = 6) or CAPS patients (n = 6) cultured without LPS (−; gray columns) or with LPS (18 h LPS; black columns). Data are expressed as mean ± SD. (D) Intracellular Trx in monocytes from a CAPS patient (lanes 1 and 2) and an HD (lanes 3 and 4), unstimulated (lanes 1 and 3) and stimulated for 18 h with LPS (lanes 2 and 4). Results from one representative HD out of the six analyzed are shown. (Lower) The same blot hybridized with anti–β-tubulin.
Fig. 4.
Fig. 4.
LPS accelerates xCT expression and cysteine release in CAPS monocytes. (A) Real- time PCR analysis of xCT mRNA in monocytes from patient MW1 and from three HDs, untreated (gray columns) or treated with LPS for 2 h (black columns). Data are expressed as fold change (mean ± SD) compared with untreated HD monocytes. (B and C) Kinetics of cysteine release (B) and IL-1β secretion (C) after LPS stimulation by cells from MW1 and MW2 and from one HD analyzed in parallel. Results are representative of three healthy donors analyzed.
Fig. 5.
Fig. 5.
Redox active drugs block IL-1β secretion by CAPS monocytes. IL-1β secretion by monocytes from HDs (n = 10) or from the indicated CAPS patients, stimulated for 18 h with LPS in the absence (black columns) or presence of DPI (gray columns; HD, C4, C5, and MW4) or BCNU (white columns; HD, C5, and MW4). Monocytes from patient C4 was analyzed twice, before (C4 pre) and 1 week after (C4 post) the start of anti–IL-1 therapy.
Fig. 6.
Fig. 6.
Redox remodeling and IL-1β secretion in monocytes from SoJIA patients stimulated with LPS. (A) IL-1β secretion by monocytes from HDs (n = 5) and SoJIA patients exposed to LPS for 3, 6, or 18 h. (B) ROS production by monocytes from HDs (n = 5) and the indicated SoJIA patients cultured for 1 h in the absence (gray columns) or presence (black columns) of LPS. Data are expressed as RFUs. (C) Released cysteine in the 18-h supernatants of unstimulated (gray columns) and stimulated (black columns) monocytes from HDs (n = 5) and the indicated SoJIA patients. (D) Western blot analysis of Trx in monocyte lysates from a representative HD (out of the six performed; lanes 1 and 2), and three SoJIA patients (lanes 3–8), unstimulated (lanes 1, 3, 5, and 7) or stimulated for 18 h with LPS (lanes 2, 4, 6, and 8). (Lower) Same blot hybridized with anti–β-tubulin. (E) IL-1β secreted by monocytes from SoJIA patients (n = 4) stimulated for 18 h with LPS in the absence (black columns) or presence of DPI (gray columns) or BCNU (white columns). Data are expressed as mean ± SD.
Fig. 7.
Fig. 7.
How do the different redox states and redox responses to LPS in healthy and CAPS monocytes influence the rate of IL-1β secretion? (A) Redox is balanced in resting healthy monocytes (with low basal levels of ROS and of the antioxidant systems Trx and cystine/cysteine cycle). (B) LPS triggering induces pro–IL-1β synthesis and rapid ROS production (first step). To counteract this oxidative hit, an antioxidant response with up-regulation of Trx and increased cysteine release occurs (second step). This redox remodeling, required for pro–IL-1β processing (8), takes several hours. Therefore, IL-1β secretion is slow and peaks at 18 h (third step). (C) In contrast, in resting mutated CAPS monocytes (asterisks), antioxidant systems are up-regulated due to chronic oxidative stress (first and second steps already occurring in resting monocytes). (D) Therefore, on LPS stimulation, IL-1β processing and secretion is anticipated due to the preexisting or rapidly generated antioxidant conditions that promote inflammasome activation and processing (third step).
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