Secretion of IL-1β From Monocytes in Gout Is Redox Independent

Abstract

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays important roles in immunity but is also implicated in autoimmune disease. The most well-established mechanism of IL-1β secretion is via activation of the NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome which requires an initial priming signal followed by an activating signal. However, the precise mechanism by which the inflammasome is activated remains unclear. The role of reactive oxygen species (ROS) in this process is contradictory, with some studies suggesting that ROS are crucial while others describe opposite effects. In this study, we evaluated the effects of oxidative stress on IL-1β secretion. Gout is a disease driven solely by IL-1β secretion in response to monosodium urate (MSU) crystals which form during periods of hyperuricemia and thus presents an opportunity to study factors contributing to IL-1β secretion. Sera and monocytes were isolated from patients with gout to determine whether differences in antioxidant status could explain the susceptibility of these individuals to gout attacks. In addition, sera and monocytes were collected from patients with chronic kidney disease (CKD) for comparison as this condition is associated with high levels of oxidative stress and disturbances in serum uric acid levels. There were differences in some aspects of antioxidant defenses in gout patients and these were mainly due to higher serum uric acid. Monocytes from gout patients were more responsive to priming, but not activation, of the NLRP3 inflammasome. However, expression of the components of the NLRP3 inflammasome were unaffected by priming or activation of the inflammasome, nor were these expression levels differentially regulated in gout patients. Inhibition of ROS by N-Acetyl Cysteine inhibited TLR2-induced priming of the NLRP3 inflammasome, but had no effect on MSU-induced activation. Together these findings demonstrate that oxidative stress only affects priming of the NLRP3 inflammasome but does not influence activation.

Keywords: IL-1β; NLRP3 inflammasome; antioxidant capacity; chronic kidney disease; gout; reactive oxygen species; redox regulation.

Figure 1

The priming but not activating signal is ROS-dependant in human monocytes. (A) IL-1β secretion from primary human monocytes. Cells were stimulated for 18 h with Pam3 and MSU crystals ± NLRP3 inhibitor MCC950. Bars represent means ± standard deviation and are representative of at least three independent experiments. (B) Primary human monocytes were stimulated for 2 h with Pam3, MSU crystals, or Pam3 + MSU at the concentrations specified and ROS measured by ROS-Glo™ assay. (C,D) Primary human monocytes were stimulated for 6 h with Pam3 (100 ng/mL) ± MSU crystals (10 mg/dL) alongside the ROS inhibitor NAC (5–10 mM). ROS levels were measured by ROS-Glo™ assay (C) and IL-1β secretion was determined by ELISA (D). Cell viability was determined at the end of the experiments shown in (C,D) by cell-titer Glo assay (E,F, respectively). Data represent means from 6 individual donors + SEM (n = 6). Significance was determined by Kruskall-Wallis analysis comparing treated vs. untreated controls (^*P < 0.05, ^**P < 0.01).

Figure 2

Gene expression of IL1B and SOD2 in human monocytes is increased by TLR2 activation but unaffected by soluble uric acid. Primary human monocytes were stimulated for 6 h with Pam3 (100 ng/mL) following an initial exposure to culture media (RPMI) or soluble uric acid (30 mg/dL). Gene expression of IL1B (A), SOD2 (B), and TXNRD1 (C) were quantified by qPCR. (D) Human monocytes were stimulated as above for measurement of intracellular total antioxidant capacity (TAC) and expressed per mg of protein. Data represents means from 3 to 4 individual donors + SEM. Statistical analyses were performed by Mann-Whitney tests between control and uric acid treated samples in the presence of Pam3 (ns = not significant).

Figure 3

Serum antioxidant capacity in gout, CKD and RA patients. Serum was isolated from whole blood of healthy controls (n = 52), gout (n = 50), CKD (n = 42), or RA (n = 36) patients. (A) Uric acid concentration was determined using amplex-red based uric acid assays. (B) GPx and (C) SOD activities were measured using kinetic enzyme assays. (D) TAC was measured by TAC assay kit and quantified by reference to a Trolox standard curve. Serum enzyme activities and TAC ware expressed as activity per mg protein. Individual points represent means for 2–3 replicate measurements for each donor. Error bars represent means ± SEM of the entire population. Significance was determined by Kruskall–Wallis analysis comparing disease vs. healthy controls (^*P < 0.05, ^**P < 0.01, ^****P < 0.0001).

Figure 4

Monocytes from gout patients secrete higher levels of IL-1β in response to Pam3, but there is no correlation between IL-1β production and serum uric acid concentration. Monocytes were isolated from whole blood from healthy (n = 40), gout (n = 40), or CKD (n = 37) donors and stimulated for 18 h with cell culture media (A), Pam3 (100 ng/mL) (B), MSU (10 mg/dL) (C), or a combination of MSU and Pam3 (D). IL-1β secretion was determined by ELISA. (E) Correlation of IL-1β secretion from monocytes and serum uric acid concentration in gout patients. Each point represents means from 3 to 6 replicate measurements. (F) IL-1β secretion from monocytes from gout patients was analyzed by grouping samples according to serum uric acid concentration. Hyperuricemia is represented by a serum uric acid of ≥6.8 mg/dL and normouricemia by serum uric acid of ≤ 6.8 mg/dL. Error bars represent mean ± SEM. Individual points represent means from 3 to 6 replicate measurements for each donor. Error bars represent means ± SEM of the entire population. Significance was determined by Kruskall-Wallis analysis comparing disease vs. healthy controls (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

Figure 5

Expression of genes coding for components of the NLRP3 inflammasome are unchanged in monocytes from gout patients. cDNA was prepared from human monocytes from healthy (n = 37), gout (n = 43), or CKD (n = 35–37) donors. Expression of CASP1 (A), IL1B (B), NLRP3 (C), and PYCARD (D) was measured by absolute qPCR. Copy numbers were calculated using standard curves for each gene of interest and normalized to expression of the reference genes B2M and RPL32. Individual points represent means from 2 to 3 replicate measurements for each donor and error bars represent means ± SEM. Significance was determined by Kruskall-Wallis (^*P < 0.05). Correlation of IL1B gene expression in monocytes from gout patients with IL-1β secretion in response to (E) Pam3 or (F) Pam3 + MSU crystals. Each point represent means from 3 to 6 replicate measurements for each donor. Error bars represent mean of entire population ± SEM. Correlation significance was determined by Spearman correlation test.

Figure 6

Monocytes from gout patients express higher levels of TXNRD1 but overall total intracellular antioxidant capacity is unchanged. cDNA was prepared from monocytes isolated from healthy (n = 37), gout (n = 43), or CKD (n = 37) donors. Expression of TXNRD1 (A) and SOD2 (B) was calculated by absolute qPCR. Copy numbers were calculated using standard curves for each gene of interest and normalized to expression of reference genes B2M and RPL32. (C) Monocytes from healthy (n = 9), gout (n = 13), or CKD (n = 5) donors were lysed in NP40 cell lysis buffer and intracellular TAC and protein content were measured. Individual points represent means from 2 to 3 replicate measurements for each donor. Error bars represent means ± SEM of the entire population. Significance was determined by Kruskall-Wallis comparing disease vs. healthy controls (^*P < 0.05).