ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical, Non-Canonical, and Alternative Routes of Inflammasome Activation in Human THP1 Cells

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  Figure 1. Cyclic Guanossine monophosphate (cGMP)/Protein kinase G—I (PKG-I) axis drives Atrial natriuretic peptide (ANP)/ B-type natriuretic peptide (BNP) inhibitory effect on Interleukin (IL)-1β secretion. THP-1 cells were treated with ANP or BNP (10−8 M) (A,B) or 8-Br-cGMP (100 µM) (C) for 1 h (A,C) or 30 min (B). (A,C) NPR-1 protein expression by Western blot. Protein loading was assessed by re-probing the blots with an anti-β-actin antibody. Western blots are representative of n = 3 independent experiments. (B) Cells were lysed, and cGMP intracellular levels were measured by ELISA assay (basal cGMP: 64 fmol/106 cells). (D) THP-1 cells, either pretreated for 10 min with ANP or BNP (10−8 M) or 8-Br-cGMP (100 µM) or left untreated, were exposed to LPS (10 µg/mL) for 20 min and treated with ATP (5 mM) for 48 h. When used, KT-5823 (1μM) was added 1 h before ANP, BNP, or cGMP. The supernatants were collected and IL-1β release was measured by ELISA. All histograms indicate mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated control cells; ° p < 0.05, °° p < 0.01, versus LPS + ATP-treated cells; # p <…

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  Figure 2. cGMP/PKG-I axis Is Involved in the priming mechanism controlling IL-1β production. THP-1 cells were pre-treated for 10 min with ANP (10−8 M) or BNP (10−8 M) or cGMP (100 µM 8-Br-cGMP) in the absence or presence of LPS (10 µg/mL for 20 min) + ATP (5 mM for 40 min). When KT-5823 (1 μM) was used, it was added 1 h before ANP, BNP, or 8-Br-cGMP treatment. Cell lysates were immunoblotted for p-p65-NF-κB and total p65-NF-κB, p-ERK 1/2 and total ERK1/2 (A) or pro- and mature-IL-1β (B). Protein loading was assessed by re-probing the blots with an anti-β-actin antibody. Representative western blots images are shown. (C–F) Histograms of densitometric quantification. Bars represent the ratio between phosphorylated and total protein band intensity. Untreated cells were used as control and assumed as 1. When no band appears in control cells, LPS + ATP was assumed as 1. All histograms indicate the mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated cells; ° p < 0.05, °° p < 0.01, °°° p < 0.001 versus LPS + ATP treated cells; ## p < 0.01, ### p < 0.001 versus KT-5823 + LPS + ATP treated cells.

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  Figure 3. cGMP/PKG-I axis is involved in the activation step of IL-1β maturation. THP-1 cells were pre-treated for 10 min with ANP (10−8 M) or BNP (10−8 M) or cGMP (100 µM 8-Br-cGMP) in the absence or presence of LPS (10 µg/mL for 20 min) + ATP (5 mM for 40 min). When KT-5823 (1 μM) was used, it was added 1 h before ANP, BNP, or 8-Br-cGMP treatment. Cell lysates were immunoblotted for NLRP3, NLRP1, ASC, AIM2 (A) or Caspase-1 (C). The blots were stripped and re-probed with mouse anti-β-actin to confirm equal loading. Representative western blots images are shown. (B,D) Histograms of densitometric quantification. Bars represent the ratio between respective protein and β-actin band intensity. Untreated cells were used as control and assumed as 1. The histogram indicates mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated cells; °° p < 0.01, °°° p < 0.001 versus LPS + ATP treated cells; # p < 0.05 and ## p < 0.01 versus KT-5823 + LPS + ATP treated cells.

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  Figure 4. cGMP/PKG-I axis induces NLRP3 phosphorylation. THP-1 cells were pre-treated for 10 min with ANP (10−8 M) or BNP (10−8 M) or cGMP (100 µM 8-Br-cGMP) in the absence or presence of LPS (10 µg/mL for 20 min) + ATP (5 mM for 40 min). When KT-5823 (1 μM) was used, it was added 1 h before ANP, BNP, or 8-Br-cGMP treatment. (A) ASC oligomerization assessed by crosslinking assay followed by Western blot analysis. (C) Cell lysates were immunoblotted for p-NLRP3 (Ser295) or total NLRP3. The blots were stripped and re-probed with mouse anti-β-actin, to confirm equal loading. Representative western blots images are shown. (B,D) Histograms represent densitometric quantification. Bars represent the ratio between active p-NLRP3 and NLRP3 or dimeric and trimeric ASC and β-actin band intensity. Untreated cells were used as control and assumed as 1. The histogram indicates mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, *** p < 0.001 versus untreated cells; °° p < 0.01 and °°° p < 0.001 versus LPS + ATP treated cells; # p < 0.05 and ## p < 0.01 versus KT-5823 + LPS + ATP treated cells.

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  Figure 5. cGMP but not PKG-I is involved in non-canonical and alternative inflammasome activation. THP-1 cells were pre-treated for 10 min with ANP (10−8 M) or BNP (10−8 M) or cGMP (100 µM 8-Br-cGMP) in the absence or presence of LPS (10 µg/mL for 20 min) + ATP (5 mM for 40 min). When KT-5823 (1 μM) or 3-isobutyl-1-methylxanthine (IBMX) (10 μM) were used, they were added 1 h before ANP, BNP, or 8-Br-cGMP treatment. Cell lysates were immunoblotted for active Cathepsin B (A), Gasdermin D (B,E), RIPK1 (C), or Caspase-8 (D,F). Protein loading was assessed by re-probing the blots with an anti-β-actin antibody. One representative image is shown. Representative western blots images are shown. Histograms of densitometric quantification. Bars represent the ratio between respective protein and β-actin band intensity. Untreated cells were used as control and assumed as 1. All histograms indicate mean ± SD of at least n = three independent experiments, each one tested in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated cells; ° p < 0.05, °° p < 0.01 and °°° p < 0.001 versus LPS + ATP treated cells; # p < 0.05 versus KT-5823 + LPS + ATP treated cells.

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  Scheme 1. Scheme of the experimental protocol.

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  Figure 6. NPs interfere with canonical, non-canonical and alternative routes of inflammasome activation. Canonical inflammasome activation requires two signals: the first, represented by TLR-ligand engagement, involves NF-κB and ERK1/2 activation and culminates in the increased expression of pro-IL-1β and PRRs, such as NLRP3; the second, provided by stress signals, results in Caspase-1 activation and pro-IL-1β cleavage. NPs, by binding to NPR-1 induce an intracellular increase in cGMP which activates PKG-I. This latter is responsible for NF-κB and ERK1/2 inhibition and possibly for the phosphorylation of NLRP3, thus impeding the assembly of the inflammasome platform. Non-canonical inflammasome signaling depends on the activation of Caspase-4 and -5 in human by a direct binding to cytosolic LPS. Caspase-4/5 process Gasdermin D which translocates to the plasma membrane forming pores that may cause K+ efflux responsible for inflammasome platform assembly. Moreover, Gasdermin D may serve as a conduit for the release of IL-1β. NPs-induced increase in intracellular cGMP levels inhibits Gasdermin D cleavage. Alternative inflammasome activation pathway culminates, upon TLR4 engagement, TRIF and…