Heat Shock Protein 90 Inhibitors Suppress Pyroptosis in THP-1 Cells

Pyroptosis is a recently discovered inflammatory form of programmed cell death which is mostly triggered by infection with intracellular pathogens and critically contributes to inflammation. Mitigating pyroptosis may be a potential therapeutic target in inflammatory diseases. However, small chemicals to reduce pyroptosis is still elusive. In the present study we screened 155 chemicals from a microbial natural product library and found Geldanamycin, an HSP90 inhibitor, profoundly rescued THP-1 cells from pyroptosis induced by LPS plus Nigericin treatment. Consistently, other HSP90 inhibitors, including Radicicol, 17-DMAG and 17-AAG, all ameliorated pyroptosis in THP-1 cells by suppressing the inflammasome/Caspase-1/GSDMD signal pathway in pyroptosis. HSP90 inhibition compromised the protein stability of NLRP3, a critical component of inflammasome. Moreover, upregulated HSP70 may also contribute to this effect. HSP90 inhibition may thus be a potential therapeutic strategy in the treatment of inflammatory diseases in which pyroptosis plays a role.


Introduction
Inflammation is a crucial pathologic process which participates in a vast variety of critical diseases, including atherosclerosis [1], diabetes [2], asthma [3] and inflammatory bower disease [4]. In inflammation, activated immune cells cause tissue damage in multiple mechanisms and the mitigation of inflammation holds the key to the treatment of these diseases. [5] Pyroptosis is a recently discovered inflammatory form of programmed cell death which is triggered by inflammatory stimuli and eventually exacerbates inflammation. [6,7] When an inflammatory signal is received by the cells, it may activateNACHT-, LRR-and pyrin domaincontaining protein 3 (NLRP3), which initiates the assembly of inflammasome. [8] Inflammasome is a potent pro-inflammatory protease complex. Once it is fully assembled, the outermost component, pro-caspase-1 will be cleaved to its active form caspase-1. [8] As an inflammatory protease, caspase-1 has two important substrates in inflammation and pyroptosis, pro-interleukin-1 (pro-IL-1) [8] and gasdermin D (GSDMD) [7].
IL-1 is a potent pro-inflammatory cytokine. It is involved in multiple severe diseases mediated with inflammation. [9] Once synthesized, the full length protein is stored in the cytoplasm as its latent form, pro-IL-1. [9] The outmost component of inflammasome, caspase-1, provides a final cut to pro-IL-1 and converts it into its active form IL-1, which is ready to be released out of the cells to enhance inflammation. [8,9] The other crucial substrate of caspase-1, GSDMD, is also kept in its latent form in the cytoplasm. When inflammasome is assembled, caspase-1 cleaves it into its active N-terminal. The N-terminal GSDMD then forms pores in the plasma membrane to facilitate the release of IL-1. Because the pores formed by N-terminal GSDMD connects the material inside and outside of cells, the cells will eventually swell and break, ending up in pyroptosis. [6,7] Thus, in addition to the release of pro-inflammatory cytokine IL-1, pyroptosis also exacerbates inflammation by releasing damage-associated molecular pattern (DAMP) after cell death. [7] Pyroptosis was first discovered and well-studied in macrophage/monocytes. [10] Many previous studies analyzed pyroptosis pathways in human spontaneously immortalized monocytic cell line THP-1. THP-1 cell line is derived from the peripheral blood of a child with M5 acute monocytic leukemia. It is widely used as a proxy to human monocytes because it mimics many of their crucial functions, including secretion of cytokines upon inflammatory stimulation, assembly of inflammasome, differentiation into macrophages, polarization to M1 and M2 macrophage subtypes, differentiation into mature and immature dendritic cells [11][12][13]. In the present study we also use THP-1 cells to gain some clues about pyroptosis in monocytes.
Given the strong pro-inflammatory effect of pyroptosis, reducing the NLRP3/Caspase-1/GSDMD pyroptotic pathway has become a promising therapeutic strategy. Heat shock protein 90 (HSP90) is a well-known chaperon protein in the heat shock protein family. A previous study suggests that NLRP3 may be a client protein of HSP90. [14] Without HSP90, NLRP3 may misfold before being degraded by proteasome. [15,16] Therefore, it is possible to attenuate pyroptosis by inhibiting HSP90. Indeed, in the present screen for pyroptosis inhibitors, we found multiple HSP90 inhibitors significantly reduced pyroptosis, probably through the misfolding and degradation of NLRP3.
As an important member of HSP chaperons, loss of HSP90 is frequently reported to increase the expression of HSP70, another member of the same chaperon family as compensation. [17] Paradoxically, HSP70 inhibits inflammasome, the key signal complex of pyroptosis. [18] In our current research, we also found the increase of HSP70 expression in the presence of HSP90 inhibitors. Moreover, inhibiting HSP70 would partially compromise the rescuing effect of some HSP90 inhibitors on pyroptosis. The upregulation of HSP70 may be another mechanism that HSP90 inhibitors improve THP-1 cells in pyroptosis.

Cell culture and induction of pyroptosis
THP-1 cells were obtained from ATCC (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mM Sodium Pyruvate, 25 mM HEPES (all from Gibco, Grand Island, NY) and 1% penicillin/streptomycin (Corning, Manassas, VA). Pyroptosis was induced according to the previously published procedure.
[19] Briefly, 1 ×10 6 /ml cells were cultured with RPMI 1640 medium containing 2% FBS overnight and then treated with 1 g/ml LPS (Sigma, St. Louis, MO) for 3 hours, followed by 10 g/ml Nigericin (Alfa Aestar, Ward Hill, MA) treatment. Supernatant LDH activity, cell viability and cell protein content were analyzed 1 hour following addition of Nigericin to the culture system unless otherwise indicated.

LDH activity assay
Culture supernatant LDH activity following pyroptosis induction was analyzed with a CytoTox96 Cytotoxicity Assay kit (Promega, Madison, WI) according to the instructions from the manufacturer. Light absorbance was read with Cytation 3 imager (Bio Tek, Winooski, VT).

MTT assay
Cell viability MTT assay was performed after pyroptosis induction by adding 10 l, 5 mg/ml MTT (Sigma) solution into each well of 96-well-plates. After 3 hours of further culture 200 l DMSO (Fisher, Fair Lawn, NJ) was added to each well and incubated for 15 minutes in room temperature protected from light. The absorbance of 570 nm wavelength light was then measured with Cytation 3 imager (Bio Tek, Winooski, VT).

Chemical screening and treatment
To screen for inhibitors of pyroptosis, 155 chemicals from microbial natural product library (Target Molecule, Wellesley Hills, MA) were added into individual wells of 96-well-plates at 50 M 1 hour before the induction of pyroptosis. The effect of chemicals on pyroptosis was measured with supernatant LDH activity and cell viability MTT assays.
To test the effect of HSP90 inhibitors on pyroptosis, Geldanamycin, 17-DMAG, 17-AAG and Radicicol (all from Sigma) were added to culture system 1 hour before the induction of Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200351/883258/bcj-2020-0351.pdf by guest on 09 June 2020 pyroptosis with LPS and Nigericin. MG132 (Sigma) was added to the culture 1 hour before HSP90 inhibitors if indicated. Ver-155008 (Selleckchem, Huston, TX) was added to the cell culture system simultaneously with HSP90 inhibitors. To test protein expression after HSP90 inhibition, cells were collected and lysed without the induction of pyroptosis at indicated time after HSP90 inhibitors were added.

Western Blot
Cell lysis was obtained with Ripa buffer and then mixed with 2×Laemmili loading buffer (65.8 mM Tris-HCI, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue, 5% 2mercaptoethanol) before boiled for 10 minutes. Protein samples and pre-stained protein ruler (Thermo Fisher Scientific, Rockford, IL) were separated by SDS-PAGE in 8%, 10% or 12% resolving gels and then transferred to PVDF membranes (GE, Chicago, IL). After transfer, membranes were blocked with 5% nonfat milk (Millipore, Burlington, MA) in PBS supplemented with 0.1% Tween-20 (Fisher) for 1 hour in room temperature. The membranes were then incubated with the following primary antibodies at 4 C overnight, according to the manufacturers' instruction: Caspase-1, GSDMD, NLRP3, HSP70 (all from Cell Signal Technology, Beverly, MA) HSP90 (Proteintech, Rosemont, IL) and -actin (Santa Cruz, Dallas, TX). After 3 times of wash in PBST, HRP labeled Goat anti-Mouse IgG,(H+L) and Goat anti-Rabbit IgG,(H+L) secondary antibodies (Thermo Fisher Scientific) were used to incubate the membranes. Luminescence was generated after the membranes were exposed to Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and detected with X-ray film.

Statistics
Statistical analysis was performed with GraphPad Prism software (GraphPad, San Diego, CA). Data were expressed as mean±SEM. For comparison between two groups, the unpaired Student's t-test was used. For multiple comparison, one way ANOVA followed by Turkey's post hoc analysis was used. A value of p<0.05 was considered significant.

Geldanamycin inhibits pyroptosis
To search for a pyroptosis inhibitor, we screened of 155 chemicals in the microorganism component library according to the effect on LPS plus Nigericin induced pyroptosis in THP-1 cells. Among them, Geldanamycin was the most remarkable to reduce the supernatant LDH activity ( Figure 1A) and increase cell viability in MTT assay ( Figure 1B). Because Geldanamycin is an inhibitor of HSP90, we further analyzed the expression of HSP90 in THP-1 cells after the stimulation of LPS and Nigericin. We found HSP90 is abundantly expressed in THP-1 cells and is not influenced by LPS or Nigericin. In contrast, HSP70, another important heat shock protein, was not detectible and not induced by LPS or Nigericin. ( Figure 1C).
We further found that Geldanamycin dose dependently ameliorates pyroptosis in THP-1 cells with the maximum activity at 30 M as shown in supernatant LDH activity and cell viability Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200351/883258/bcj-2020-0351.pdf by guest on 09 June 2020 ( Figure 2A). Moreover, we examined the classical NLRP3/caspase-1/GSDMD pyroptosis signal pathway and found the protein expression of NLRP3 as well as the activation of caspase-1 and GSDMD through cleavage were reduced by Geldanamycin in a dose dependent manner both 30 minutes and 60 minutes after the treatment of Nigericin ( Figure 2B and C). We thus confirm that Geldanamycin ameliorates the pyroptosis induced by LPS plus Nigericin in THP-1 cells.

HSP90 inhibitors reduce pyroptosis
Given Geldanamycin is an HSP90 inhibitor and its potency in reducing pyroptosis, we wonder whether other HSP90 inhibitors would have similar function. Indeed, Radicicol, another HSP90 inhibitor also dose dependently reduced LDH release and increased cell viability. On the other hand, 17-DMAG and 17-AAG significantly improved cell viability but did not ameliorate LDH release ( Figure 3). All these three HSP90 inhibitors dose dependently blocked the classical NLRP3/caspase-1/GSDMD pyroptosis signal pathway ( Figure 4). These results all indicate that HSP90 inhibition suppress pyroptosis.

HSP90 inhibitors induce NLRP3 degradation through proteasome
NLRP3 is the central molecule in inflammasome which initiates pyroptosis. In addition, HSP90 has been found to possibly assist the folding of HSP90 protein into correct structure and prevent its degradation. [14] Since we have already found that HSP90 inhibitors reduce the protein expression of NLRP3, we wonder if its degradation in the absence of HSP90 is the reason. We thus tested the protein expression of NLRP3 in the presence of HSP90 inhibitors without pyroptosis induction and found that all four HSP90 inhibitors time dependently reduced the protein level of NLRP3 ( Figure 5A and B). Moreover, this effect was hindered by MG132, a proteasome inhibitor ( Figure 5C and D). HSP90 may thus be critical for the protein structure and stability of NLRP3 while its inhibitors may reduce pyroptosis by increasing its misfolding and subsequent degradation through proteasome.

HSP70 upregulation may contribute to the rescuing effect of HSP90 inhibitors
HSP70 is another important chaperon in the family of HSPs. Previous reports suggest that the expression of HSP70 may be upregulated when HSP90 is inhibited. [17] Because HSP70 is also known to interfere with the signal of inflammasome, [18] we further examined whether HSP70 is involved in the effects of HSP90 inhibitors on pyroptosis. Consistent with the previous reports, in THP-1 cells the expression of HSP70 is time dependently upregulated by the inhibitors of HSP90 ( Figure 6A and B). We thus further tested whether Ver-155008, an HSP70 inhibitor, would interfere with the function of HSP90 inhibitors. In LPS plus Nigericin induced pyroptosis model of THP-1 cells, we found Ver-155008 did not change the LDH reduction of Geldanamycin or Radicicol. However, the cell viability improvement of Radicicol was partially reduced by Ver-155008, while the Geldanamycin effect was also reduced but not reaching a statistical significance ( Figure 6C and D). The HSP70 upregulation is thus possibly involved in the HSP90 inhibiting effects on pyroptosis.

Discussion
Pyroptosis is a recently discovered type of programmed cell death. It is triggered by inflammation and further enhance inflammation. Given its potent pro-inflammatory function, hampering pyroptosis may be a potential therapeutic strategy for inflammatory diseases. However, the current search for pyroptosis inhibitors is still elusive. In the present study we screened 155 natural chemicals from microorganisms and found Geldanamycin inhibits pyroptosis in THP-1 cells induced by LPS and Nigericin. Moreover, other HSP90 inhibitors, Radicicol, 17-DMAG and 17-AAG all exhibited similar function as Geldanamycin. Our results indicate that HSP90 is a potential therapeutic target and HSP90 inhibitors may provide therapeutic options to inflammatory diseases where pyroptosis is involved.
Pyroptosis is a type of necrosis like programmed cell death which is triggered by inflammatory stimuli and results in amplifying inflammation. [7,20] First of all, the assembled and activated inflammasome processes nonfunctional pro-IL-1 into bioactive IL-1, which is a potent proinflammatory cytokine. [7,21] Meanwhile, the pores on the plasma membrane formed with activated GSDMD, another substrate of inflammasome, help to release IL- out of the cells and stimulate inflammatory signal of other cells. This process simultaneously would release other content of cells, many of which are categorized as damage-associated molecular patterns because of their capacity to stimulate inflammation. [7,21] As pyroptosis is abundantly found in monocytes and macrophages, it may be considered a self-expanding cellular mechanism to exacerbate inflammation. [22] Blocking the signal cascade of pyroptosis is thus probably the key to alleviate inflammatory diseases.
The NLRP3/Caspase-1/GSDMD pathway lies in the center of pyroptosis, and the assembly of NLRP3 inflammasome is an early critical step. [23,24] Therefore, modulating the expression or function of NLRP3 is a potential method to stop pyroptosis. Previous reports show that NLRP3 is a client of chaperon protein HSP90, which helps to form the correct three dimensional structure. [25] Without HSP90, NLRP3 is probably misfolded and then degraded. [15,16] Consistently, our current study found that HSP90 inhibitors time and dose dependently reduce the protein expression of HSP90, and it is reversed by MG132, a proteasome inhibitor. More importantly, HSP90 inhibitors significantly ameliorate cell death and improve cell viability in LPS plus Nigericin induced pyroptosis, suggesting a promising therapeutic target of HSP90 in pyroptosis and inflammation.
In the present study we found that MG132 rescued the protein degradation of NLRP3 after HSP90 inhibition. However, because HSP90 is essential for the correct NLRP3 protein folding, it is very likely that NLRP3 goes through misfolding before degradation by proteasome in the presence of HSP90 inhibitors. [15,16] In such situation, even though MG132 rescues the protein amount of NLRP3, the misfolded protein may not be efficient to assemble inflammasome and mediate pyroptosis. Moreover, MG132 is a well-known inhibitor of NF-B, which has a complicated regulatory mechanism on inflammasome by both increasing and decreasing its activation. [26,27] The interplay between MG132 and HSP90 inhibitors in pyroptosis warrants a more detailed separate study.
Another intriguing discovery is the involvement of HSP70 in the effect of HSP90 inhibitors on pyroptosis. As another important member of HSP chaperon family, HSP70 is frequently found to be upregulated when HSP90 function is compromised via inhibitors or genetic modulation, Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200351/883258/bcj-2020-0351.pdf by guest on 09 June 2020 probably as a compensatory response. [17] However, HSP70 was found to regulate NLRP3 in a sharp different way compared to HSP90. Instead of helping folding and stabilizing NLRP3, HSP70 directly binds to NLRP3 and renders it incapable of forming inflammasome in response to inflammatory stimuli. [18] The upregulation of HSP70 thus makes it more difficult to initiate the NLRP3/Caspase-1/GSDMD pyroptotic pathway. In the present study we found extremely low expression of HSP70 in THP-1 cells with or without the pyroptotic stimulation. However, all HSP90 inhibitors remarkably increased its expression, providing a possibility to further block pyroptosis via HSP70. Indeed, HSP70 inhibitor Ver-155008 partially reduced the function of Radicicol, suggesting an involvement of HSP70 upregulation in its effect.
Four HSP90 inhibitors from two distinct structural classes were analyzed in the present study. Among them Geldanamycin, 17-DMAG and 17-AAG belong to class benzoquinone ansamycin polyketide. 17-DMAG and 17-AAG are derived from Geldanamycin by replacing chemical groups on the 17 position. Radicicol, on the other hand, belongs to class resorcylic acid lactone. The two classes bind to HSP90 ATP/ADP pocket in different ways because of their distinct chemical structure. [28] Albeit their high specificity to HSP90, they also have some off-target effects. For example, 17-DMAG and 17-AAG are found to generate reactive oxygen species (ROS).
[29] In the present study 17-DMAG and 17-AAG drastically reduced the cleavage of Caspase-1 and GSDMD, while the upstream signal NLRP3 was mildly inhibited. This may be a result of some unknown off-target effects. More specific HSP90 inhibitors are to be developed to address the exact mechanisms. HSP90 inhibitors have been reported to alleviate inflammation in vivo in other studies. 17-AAG was found to attenuate LPS induced sepsis and lung injury by inhibiting systemic inflammation. [30] Ganetespib, another HSP90 inhibitor, also improves LPS induced lung injury by suppressing myeloid cell response. [31] In an alcoholic liver disease model, 17-DMAG ameliorates the liver injury by reducing monocyte infiltration and desensitizing liver macrophage to LPS. [32] 17-DMAG also protects streptozotocin-induced diabetic apolipoprotein E-deficient mice from nephropathy and atherosclerosis via suppressing inflammation. [33] Moreover, HSP90 inhibitors also show a protective role in several autoimmunity animal models. [34] Our present study made a connection between HSP90 inhibitors and pyroptosis, an important and powerful contributor of inflammation. Pyroptosis suppression by HSP90 inhibitors may be a mechanism in these in vivo inflammatory studies.
In recent years multiple novel pathways of programmed cell death were discovered. In contrast to apoptosis, some of these pathways manifest necrotic features and enhance inflammation via releasing cell content through permeabilized cell membrane. Necroptosis, for example, is a type of programmed cell death triggered by death receptors, including Fas, tumor necrotic factor receptors, interferon receptors or toll-like receptors. These receptors go through the caspase-8 signal pathway and activate the RIPK1/3 to eventually phosphorylate and activate the plasma membrane pore forming molecule MLKL. [20,35] Upon the pore formation in necroptosis, inflammation will be further propagated by the released DAMPs, including HMGB1, ATP and mitochondrial DNA. [20,35] Interestingly, HSP90 is also found indispensable in the signal cascade of necroptosis. Inhibiting HSP90 skews the cell fate from pro-inflammatory necroptosis to anti-inflammatory apoptosis. [36] Added with the current discovery about the role of HSP90 in pyroptosis, HSP90 inhibition may be a potent therapeutic strategy in protecting from the tissue damage raised from overwhelmingly pro-inflammatory cell death.

Conclusion
In the current study we found HSP90 inhibitors rescue pyroptosis in THP-1 cells induced by LPS plus Nigericin stimulation. HSP90 inhibition compromises protein stability of NLRP3, a critical component in inflammasome. Upregulated HSP70 may also be involved in the suppression of pyroptosis.        Figure 5A and the cell protein content of HSP70 was examined with Western Blot (A) and statistically analyzed (B). HSP70 inhibitor VER-155008 (30 M) was added together with HSP90 inhibitors one hour before the induction of pyroptosis with LPS plus Nigericin. Cell culture supernatant LDH (C) and cell viability (D) was analyzed one hour after Nigericin treatment. Images represent one of three independent experiments. N=3, *P<0.05 versus 0 in B and versus DMSO in D