Inhibitory effect of PPIs on PAMP-induced IL-1β and TNF-α secretion in vitro

Secretion of IL-1β by primary human monocytes activated with LPS was increased at low pH (Figure 1a), in agreement with the previous data.17, 18, 19, 20 Interestingly, IL-1β secretion was strongly inhibited by the PPI omeprazole (OME) both at acidic and neutral pH (Figure 1a). OME displayed an IC 50 of 100 μ M, and inhibited IL-1β secretion up to 80% at 300 μ M (Figure 1c, left panel). Although LPS-induced TNF-α is not increased by low pH, OME also inhibited TNF-α secretion (Figures 1b and c, right panel). Dose–response experiments with other PPIs21, 22 provided data similar to OME both for IL-1β and TNF-α (Figures 1d–g). Toxicity, evaluated by trypan blue staining and lactate dehydrogenase release, was virtually absent at doses lower than 400 μ M with all PPIs tested (not shown). The inhibitory effect of PPIs on cytokine production is not restricted to TLR4 stimulation: also R848- (TLR7/8 ligand) and zymosan- (TLR2 ligand) induced secretion of IL-1β and TNF-α was impaired (Figures 1h and i). Similarly, the marked secretion of IL-1β and TNF-α that follows the simultaneous stimulation of monocytes with the three TLR ligands7 was inhibited (Figures 1h and i; LRZ).

Figure 1 OME inhibits IL-1β and TNF-α secretion induced by different PAMPs in human healthy monocytes. (a and b) Healthy monocytes were incubated in the medium at neutral pH (pH 7.4) or acidic pH (pH 6.5) with LPS (100 ng/ml) in the absence or presence of OME (300 μ M). Secreted IL-1β (a) and TNF-α (b) were quantified after 18 and 6 h, respectively. Data are expressed as ng/ml (N=5, mean±S.E.M.). (c–g) Dose–response experiments with 10–300 μ M of OME (c), ESO (d), lansoprazole (e), pantoprazole (f) and rabeprazole (g) were performed. Supernatants were collected after 18 or 6 h to quantify IL-1β (left panels) and TNF-α (right panels). Data are expressed as the percentage of secretion of PPI versus PPI-untreated cells; mean±S.E.M. of four experiments. (h and i) Monocytes were stimulated for 18 and 6 h with LPS (100 ng/ml), R848 (5 μg/ml) and zymosan (ZYM, 20 μg/ml), alone or in combination (LRZ), in the presence or absence of OME. Secreted IL-1β (h) and TNF-α (i) were quantified as above. Data are expressed as ng/ml (N=5, mean±S.E.M.). *P<0.05; **P<0.01; ***P<0.001 Full size image

The effect of OME on IL-1β secretion was investigated on monocytes from patients affected by cryopyrin-associated periodic syndrome (CAPS), a very rare autoinflammatory disease where gain-of-function mutations in the inflammasome gene NLRP3 cause huge secretion of IL-1β.27 As shown in Figure 2, OME inhibited IL-1β secretion by >80% in all the four patients examined.

Figure 2 OME prevents secretion by monocytes from patients affected by CAPS. Monocytes from CAPS patients (N=4) and healthy donors (N=4) were stimulated with 100 ng/ml of LPS alone or in combination with OME (300 μ M). Secreted IL-1β was quantified by enzyme-linked immunosorbent assay (ELISA) in 18 h supernatants. Data are expressed as ng/ml. **P<0.01; ***P<0.001 Full size image

PPIs inhibit TNF-α and IL-1β secretion at different levels

The amount of TNF-α mRNA in monocytes stimulated with LPS in the presence of OME was found to be ~50% less than that detected in monocytes exposed to LPS alone (Figure 3a), a decrease consistent with the decreased TNF-α secretion (Figure 3b). However, no difference in the activation of inflammation-related transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), was observed in untreated or OME-treated monocytes (Supplementary Figure S1). Moreover, in spite of the marked inhibition of secretion (Figure 3b), OME affected neither IL-1β gene expression (Figure 3a) nor intracellular accumulation of the precursor pro-IL-1β protein in LPS-stimulated monocytes (Figure 3c), suggesting that the inhibitory effect of the drug is located post-translationally, at the level of inflammasome activation. Accordingly, both on LPS-primed monocytes (Figure 3d) and peritoneal murine macrophages (Figure 3e), OME inhibited IL-1β secretion induced by extracellular adenosine triphosphate (ATP) or nigericin, a toxin that strongly activates the inflammasome,28 and prevented ATP- and nigericin-induced generation of active caspase-1 (Figure 3f). Both ATP and nigericin trigger a marked efflux of K+ from cells, resulting in a drop in [K+] i , which is a crucial step in NLRP3 inflammasome activation and IL-1β secretion.29, 30 Remarkably, nigericin-induced K+ release from monocytes was prevented by OME (Figures 3g and h). Thus, OME directly or indirectly interferes with K+ efflux, resulting in hindrance of inflammasome assembly and caspase-1 activation, and consequently of IL-1β secretion.

Figure 3 OME downmodulates IL-1β and TNF-α secretion with different mechanisms. (a) Real-time PCR of IL-1β and TNF-α mRNA levels, 3 h from exposure to LPS or LPS+OME. Data are expressed as fold change of mRNA levels in cells stimulated with LPS or LPS+OME versus untreated cells (mean of normalized expression±S.E.M.; N=4). (b) IL-1β (18 h) and TNF-α (6 h) secreted by monocytes from the same subjects analyzed in (a). Data are expressed as the percent of secretion by LPS+OME versus LPS (mean±S.E.M.). (c) Western blot analysis of intracellular pro-IL-1β in monocytes unstimulated or at different time points from LPS stimulation, with or without OME. α-Tubulin is used as the loading control. One representative experiment out of five is shown. (d and e) IL-1β secreted by human monocytes (d) or murine peritoneal macrophages (e) primed 3 h (d) or 18 h (e) with LPS and then exposed 30 min to ATP, at 1 mM (d) or 5 mM (e) or to 20 μM nigericin (Nig, 20 min) with or without OME (mean±S.E.M.; N=4). (f) Western blot of p10 caspase-1 in cell lysates from murine macrophages. α-Tubulin is shown as the loading control (one representative experiment out of three). (g) Monocytes stimulated 3 h with LPS were loaded with PBFI and incubated in medium alone (LPS) or with 20 μ M nigericin without (LPS+Nig) or with 300 μ M OME (LPS+Nig+OME). Data are expressed as 340/380 ratio of the PBFI fluorescence intensity measured every 60 s for 20 min (mean±S.E.M.; N=3). (h) PBFI fluorescence intensity after 20 min in the different culture conditions depicted in (g) is expressed as percent versus time 0. *P<0.05, **P<0.01; ***P<0.001 Full size image

The molecular target of PPIs on gastric cells is the hydrogen potassium ATPase (H+/K+ ATPase) proton pump.21 However, macrophages do not express this proton pump (Figure 4a). Among the other proton pumps, we focused on vacuolar ATPases (v-ATPases). These pumps, usually restricted to intracellular acidic organelles,31 are expressed on the surface of certain cell types including tumor cells where they were proposed to be a target of PPIs.32 Interestingly, CD14+ monocytes, unlike CD3+ lymphocytes, are positive for surface v-ATPases and positivity increases following LPS stimulation (Figures 4b and c), suggesting a possible role of surface-bound v-ATPases as PPI receptors on activated monocytes.

Figure 4 Macrophages do not express the gastric H+/K+ ATPase, but display surface v-ATPases. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA coding for the β-subunit of the gastric H+/K+ proton pump on peritoneal mouse macrophages (Mφ) untreated or exposed 3 h to LPS (Mφ+LPS). Data are expressed as fold changes of normalized expression versus murine stomach (mean±S.E.M. of three experiments). (b and c) PBMCs from healthy donors were double stained with anti-v-ATPase and anti-CD14 antibody (Ab) or anti-CD3 Ab time 0 or 3 or 6 h after exposure to LPS and analyzed by FACS. In (b), data are expressed as the relative fluorescence intensity (RFI) of v-ATPase in CD14+ (monocytes) and CD3+ (T-lymphocytes) cells (mean±S.E.M. of three independent experiments). Statistical analysis is referred to monocytes and evaluated versus t0. *P<0.05; **P<0.01 versus (c) a representative experiment of costaining (out of 3) is shown: 41% of CD14+ cells (upper plot) and 1.88% of CD3+ cells are positive for surface v-ATPases at 3 h from LPS exposure. *P<0.05; **P<0.01 Full size image

Treatment with esomeprazole protects mice from LPS-induced sepsis

The above results prompted us to investigate the therapeutic potentials of PPIs in systemic inflammation in vivo. Esomeprazole (ESO) was preferred to OME, as it was found superior in reducing gastric acid secretion when parenterally administered.33 A murine model of acute endotoxic shock was used.26 Forty mice were injected intravenously with a lethal dose of LPS (12.5 mg/kg); a second group of 40 mice received ESO intraperitoneally 30 min before LPS. In the first 24 h after LPS injection, mice from both groups exhibited signs of disease, including alterations in weight, temperature and mobility. Sixty percent of mice treated with ESO improved from the second day and fully recovered, whereas only two mice in the control group survived LPS injection (Figure 5a). The systemic production of TNF-α and IL-1β was strongly inhibited in mice treated with ESO (Figure 5b). At variance, the serum levels of other inflammatory and anti-inflammatory mediators including IL-10, IL-6 and IL-1 receptor antagonist (IL-1Ra) were not significantly changed in ESO-treated mice (Supplementary Figure S2). When ESO was injected 30 min after LPS injection, an important therapeutic effect was still evident, with 40% of survival (Figure 5a). Increase of survival time, with 14% of mice that fully recovered, was observed even when ESO treatment was performed after 1.5 h from LPS, when serum TNF-α had already reached a high level (Figure 5b).

Figure 5 ESO protects mice from LPS shock, suppresses the systemic production of TNF-α and IL-1β and induces resistance to LPS rechallenge. (a) Mice were injected intravenously with LPS (12.5 mg/kg) alone (N=40), or received intraperitoneally ESO (12.5 mg/kg) 30 min before (N=40), 30 min after (N=10) or 90 min after (N=7) the LPS injection. Mice were monitored for survival. (b) TNF-α (left panel) and IL-1β (right panel) in sera from LPS- and LPS+ESO-treated mice were quantified (ng/ml) 90 min (TNF-α) or 4 h (IL-1β) after LPS injection, respectively (mean±S.E.M., N=11). (c) Fifteen mice ESO treated and that survived the first LPS shock (survived LPS+ESO) were rechallenged with LPS, without any treatment, 3 weeks after the first LPS injection. As control, 10 naive mice were injected with LPS. Mice were monitored for survival. (d) TNF-α (left) and IL-1β (right) levels (ng/ml) were detected in the serum of naive and rechallenged mice by enzyme-linked immunosorbent assay (ELISA) (mean±S.E.M.; N=8 for TNF-α, N=7 for IL-1β). (e) Twelve mice receive a single injection of ESO 15 days before LPS challenge (ESO pre-treated). A control group of 10 naive mice received LPS only. Mice were monitored for 21 days for survival. (f) Serum levels of TNF-α (left) or IL-1β (right) from ESO pre-treated and naive mice were quantified as above. Data are expressed as ng/ml (mean±S.E.M.; n=6). *P<0.05, **P<0.01 and ***P<0.001 Full size image

ESO-treated mice that survived shock are resistant to rechallenge with LPS

A group of 15 LPS+ESO-treated mice that recovered were rechallenged with LPS, without other treatments, at 3 weeks after the first injection. A control group of naive mice (N=10) received the same dose of LPS. Remarkably, 80% of mice that had recovered from the first shock (Figure 5c, survived LPS+ESO) also survived the rechallenge (Figure 5c) without exhibiting overt signs of disease. In contrast, all naive mice died. The systemic production of TNF-α and IL-1β after the second challenge of LPS was much lower in ESO-treated mice that survived the first LPS injection than in control mice (Figure 5d).

As a control, the potential suppressive effect of ESO per se was studied. Two groups of mice were injected with a single dose of ESO alone or with an equal volume of saline solution. After 2 weeks, both groups received a lethal dose of LPS (12.5 mg/kg): while all control mice died within 48 h, 25% of ESO-pre-treated mice survived (Figure 5e). Systemic production of TNF-α by ESO-pre-treated mice after challenge with LPS was decreased with respect to controls, whereas IL-1β decrease was not significant (Figure 5f).

Macrophages from mice that survived LPS injection display tolerance to different TLR agonists in vitro

Peritoneal macrophages from ESO-treated mice that survived the first or the second LPS challenge were collected at 3 weeks after the LPS injection and stimulated in vitro with agonists of different TLRs (Figure 6). Significantly less TNF-α and IL-1β were secreted by macrophages from mice that survived compared with macrophages from naive mice in response not only to LPS (Figure 6a) but also to other TLR ligands (Figures 6d and e). Also, the burst of IL-1β secretion induced in primed macrophages by short exposure to ATP was strongly suppressed both in macrophages from mice that survived the first (Figure 6b) and the second LPS challenge (Figure 6e), in spite of an expression of the purinergic P2X (purinergic P2X, ligand-gated ion channel 7), ligand-gated ion channel 7 (P2X7) receptor similar to naive macrophages (Figure 6c).34 Suppression was long lasting: decreased cytokine production was still observed in macrophages collected from ESO-cured mice at 60 days after the first LPS injection (not shown).

Figure 6 Macrophages from mice survived LPS rechallenge secrete less TNF-α and IL-1β in response to different TLR agonists. (a and b) Peritoneal macrophages from ESO-treated mice survived the first LPS shock (N=4) and from naive mice (N=6) were stimulated with LPS (a) or primed 18 h with LPS and then exposed 30 min to ATP (b), and TNF-α (a) and IL-1β (a and b) were quantified in supernatants after 4 or 18 h. (c) Real-time PCR analysis of P2X7 receptor mRNA from macrophages from naive mice or from mice survived the first LPS shock (survived LPS+ESO) after 3 h exposure to LPS. (d–f) Peritoneal macrophages from ESO-treated mice survived the second LPS shock (N=4) and from naive mice (N=6) were stimulated with LPS, zymosan (ZYM), R848, Pam(3)CSK(4) (PAM3), poly(I:C) and flagellin (Flag) for 4 (d) or 18 h (e) or with LPS, Pam(3)CSK(4) or poly(I:C) for 18 h followed by 30 min with 5 mM ATP (f). Secreted TNF-α (d) and IL-1β (e and f) were quantified (ng/ml; mean±S.E.M.; N=4). *P<0.05; **P<0.01; ***P<0.001 Full size image

The surface expression of v-ATPases was analyzed on macrophages from naive and ESO-cured mice at 3 weeks after LPS challenge. The expression was highly variable in the different mice examined, and similar in macrophages from naive and LPS+ESO-treated mice, either before or after LPS stimulation. However, a slightly less relative fluorescence intensity was observed in the macrophages from ESO-treated mice that survived LPS injection (Supplementary Figure S3).

Mice that survived LPS-ESO display resistance to zymosan-induced generalized inflammation model

To understand whether the long-lasting suppressed cytokine production in vitro in response to different TLR agonists by macrophages from ESO-cured mice corresponds to a cross-resistance in vivo, an inducer of systemic inflammation other than LPS, namely, zymosan,35 was injected intraperitoneally in five ESO-treated mice that recovered from the first LPS challenge and in seven naive mice as control. Although this dose of zymosan was not lethal, resulting in 57% of mice that fully recovered, survival was higher in the group of mice that had survived LPS+ESO (80%; Figure 7a). Moreover, among survivors, naive mice exhibited a significantly stronger weight loss in the first 2 days (Figure 7b), and more intense signs of disease. In mice that recovered from zymosan-induced inflammation, the serum level of TNF-α (but not of IL-1β) was significantly lower in the group of mice survived LPS+ESO than in naive mice (Figure 7c). Thus, ESO-treated mice that survived LPS shock acquire resistance also to zymosan.

Figure 7 ESO-treated mice that survived LPS shock display resistance to zymosan-induced generalized inflammation. ESO-treated mice that recovered from the first LPS challenge (survived LPS+ESO, N=5) and naive mice (N=7) were injected with zymosan (Zym, 1 g/kg). Mice were monitored for survival (a) and for loss of body weight (b; N=4). (c) Serum levels of TNF-α (left; N=4 naive+Zym and N=5 survived Zym-treated mice) and of IL-1β (right; N=3 naive+Zym and N=4 survived Zym-treated mice) were quantified (ng/ml; mean±S.E.M.). *P<0.05 Full size image

ESO is effective on thioglycollate-induced peritonitis

To address the efficacy of PPIs in a non-infectious setting independent of TLR stimulation, we used the sodium thioglycollate peritoneal inflammation mouse model.36, 37 Two groups of mice were injected intraperitoneally with thioglycollate and one of these received ESO intraperitoneally 30 min earlier. A third group received saline only. The number of leukocytes infiltrating the peritoneum of mice treated with ESO and thioglycollate was similar to that of control mice, and markedly lower compared with that of untreated, thioglycollate-injected mice, both after 4 (Figure 8a) and 72 h (Figure 8c). The percentage of infiltrating cells was instead similar, although, as expected,37 after 4 h neutrophils prevailed (Figure 8b), whereas after 72 h macrophages were the most represented cell type (Figure 8d). In keeping with these results, the levels of the neutrophil chemokines MIP-2 (macrophage inflammatory protein 2) and KC and of the monocyte chemoattractant protein-1 (MCP-1)37 were lower in peritoneal lavage and serum of ESO-treated mice compared with untreated, thioglycollate-injected mice (Figures 8e–g).