SHANK-associated RH domain interacting protein (SHARPIN) is required for optimal NLRP3 inflammasome activation

Abstract

The NLRP3 inflammasome is a multimeric protein complex assembled in response to a wide array of pathogens and danger-associated molecular patterns. Despite the ability of NLRP3 to respond to diverse cues, the mechanisms controlling assembly of this complex are contested. Recently published studies show that HOIL-1, a member of the linear ubiquitin chain assembly complex (LUBAC), contributes to activation of the NLRP3 inflammasome. SHARPIN, along with HOIP and HOIL-1 assembles the LUBAC complex. Herein, we examined whether SHARPIN is required for the activation of NLRP3 inflammasome. Utilizing Sharpin^cpdm macrophages (deficient in SHARPIN expression), we demonstrate that SHARPIN is required for optimal activation of the NLRP3 inflammasome by both canonical and non-canonical stimuli. Furthermore, Sharpin^cpdm macrophages had dramatic defects in both NFκB and MAP kinase pathways, suggesting a role in transcriptional priming of the NLRP3 inflammasome. In conclusion, our study identified SHARPIN as a novel regulator of the NLRP3 inflammasome.

INTRODUCTION

Nod-like receptors (NLRs) are cytoplasmic sensors that recognize and respond to several inflammatory triggers. Some NLRs, including NLRP1b, NLRP3 and NLRC4 recruit bipartite adaptor protein ASC and caspase-1 to assemble multi-protein complexes known as inflammasomes (1). In particular, NLRP3 responds to a wide variety of triggers such as ATP, the microbial toxin nigericin, uric acid crystals and enteric pathogens such as Vibrio cholerae, Escherichia coli and Citrobacter rodentium (2). A recent study identified HOIL-1 as an integral upstream regulator of the NLRP3 inflammasome (3). In this study, the authors suggested linear ubiquitination of ASC by HOIL-1 as the molecular mechanism for activation of the NLRP3 inflammasome (3). HOIL-1 along with HOIL-1 interacting protein (HOIP) and SHANK-associated RH interacting protein (SHARPIN) constitutes the linear ubiquitin chain assembly complex (LUBAC) (4). While it could be posited that the LUBAC complex might be involved in the regulation of the NLRP3 inflammasome, the roles of other proteins in the LUBAC complex have not been characterized.

The multiprotein LUBAC complex consists of HOIL-1, HOIP and the recently identified component SHARPIN (4). Even though HOIL-1 is required for optimal activation of NFκB in mouse embryonic fibroblasts (MEFs), Hoil1^−/− mice are phenotypically normal (5). However, mutations causing defective SHARPIN expression in mice (Sharpin^cpdm) result in severe hyperinflammation (6). Sharpin^cpdm mice develop chronic inflammatory dermatitis and inflammation of the gut and lungs as early as 4 weeks of age (6). In addition, Sharpin^cpdm mice display underdeveloped secondary lymphoid organs suggesting an important role for SHARPIN in the development of these organs. Similar to Hoil1^−/− MEFs, Sharpin^cpdm MEFs also had significantly reduced NFκB activation in response to TNF due to SHARPIN’s involvement in the LUBAC complex (7–9). Thus, it is clear from these studies that while both SHARPIN and HOIL-1 are components of the LUBAC complex, they exert different functions. Furthermore, the importance of SHARPIN in inflammasome activation has not been studied.

Herein, we used Sharpin^cpdm macrophages to study the role of SHARPIN during inflammasome activation. We find that SHARPIN is critical for both canonical and non-canonical NLRP3 inflammasome activation, but not for activation of the NLRC4 and AIM2 inflammasomes. We further show that Sharpin^cpdm BMDMs have defective activation of NFκB, ERK1/2 and p38 MAP kinases that regulate expression of components of the NLRP3 inflammasome. In conclusion, we demonstrate for the first time that SHARPIN regulates NFκB and MAP kinase activation in response to TLR stimulation and controls NLRP3 inflammasome activation. This study highlights the complexity of regulatory mechanisms that are in place to control the NLRP3 inflammasome and adds SHARPIN as additional upstream regulator that can potentially be targeted for controlling aberrant NLRP3 inflammasome activation.

MATERIALS AND METHODS

Mice

C57BL/6 WT and Sharpin^cpdm mice were both purchased from Jackson laboratory (Bar Harbour, Maine) bred at St. Jude Children’s Research Hospital. Animal studies were conducted under protocols approved by St. Jude Children’s Research Hospital and Ghent University Committee on Use and Care of Animals.

Western blotting and cytokine analysis

Bone marrow-derived macrophages (BMDMs) were prepared and stimulated as described before (10). Samples for immunoblotting were prepared by combining cell lysates with culture supernatants as described previously (11). Cytokine and chemokine concentrations were determined using multiplex ELISA (Millipore. IL-1β (eBioscience) and IL-18 (MBL international) concentrations were determined by classical ELISA. LDH release in the supernatants was determined by LDH release assay kit (Promega).

Real-time PCR

Transcript level of Nlrp3, pro-Il1b and Tnfa was quantified as described before (10). β-actin expression was used for normalization, and results are presented as fold induction over levels untreated control cells.

Statistics

GraphPad Prism 5.0 software was used for data analysis. Data are represented as mean ± standard errors of mean (SEM).

RESULTS AND DISCUSSIONS

Chronic proliferative dermatitis (cpdm) mice were first described as C57BL/6 mice that acquired a spontaneous mutation that resulted in severe inflammation of skin and other epithelial tissues (12). In 2007, these mice were re-named “Sharpin^cpdm” after the discovery that the phenotype was caused by a nonsense mutation in the Sharpin gene that resulted in full SHARPIN-deficiency (6). To study the role of SHARPIN in NLRP3 inflammasome activation, we generated macrophages from the bone marrow of WT and Sharpin^cpdm mice and stimulated these macrophages with LPS (TLR4 agonist) or Pam3CSK4 (TLR2 agonist) for 3.5 hours followed by ATP for 30 minutes. TLR priming followed by ATP addition induces canonical NLRP3 inflammasome activation (13). Surprisingly, we discovered that both LPS/ATP and Pam3CSK4/ATP- induced caspase-1 activation and IL-1β cleavage were dramatically reduced in Sharpin^cpdm BMDMs (Fig. 1A). In agreement with these Western blotting observations, IL-1β and IL-18 levels in the supernatants of LPS+ATP- and Pam3CSK4+ATP-stimulated Sharpin^cpdm BMDMs were considerably lower than the levels secreted by WT macrophages (Fig. 1B-C). Moreover, pyroptotic cell death as measured by LDH release in the supernatants was at background levels in stimulated Sharpin^cpdm macrophages (Fig. 1D). To further examine whether the observed results were specific to macrophages, we stimulated WT and Sharpin^cpdm bone marrow derived dendritic cells (BMDCs) with LPS and ATP. Canonical NLRP3 inflammasome activation was similarly abrogated in Sharpin^cpdm dendritic cells (Supplemental Fig. 1A). However, whether SHARPIN is also involved in modulating NLRP3 inflammasome in other cells such as Neutrophils, T cells and B cells are not known and will be studied in the future. Regardless, these observations collectively establish SHARPIN as a key regulator of canonical NLRP3 inflammasome activation.

Figure 1. Canonical NLRP3 inflammasome activation is abrogated in Sharpin^cpdm BMDMs.

WT and Sharpin^cpdm BMDMs were stimulated with LPS (500ng/ml) or Pam3CSK4 (1μg/ml) for 4 hours with ATP for the last 30 minutes. A) Cell lysates probed for caspase-1, IL-1β, NLRP3 and β-actin. B and C) IL-1β and IL-18 levels in the cell culture supernatant. D) Cell death determined by measuring LDH released in the supernatants. Data are presented as mean ± SEM from four independent experiments. Student t-test was performed for statistical analysis. **=p<0.01, ****=p<0.0001.

The non-canonical NLRP3 inflammasome requires caspase-11 for the activation of caspase-1 and downstream IL-1β production (14). Specifically, it was shown that cytoplasmic LPS are directly recognized by caspase-11 to activate caspase-1 and promote pyroptotic cell death (15). Citrobacter rodentium infection of macrophages activates the non-canonical NLRP3 inflammasome (14). To ascertain whether SHARPIN is also required for non-canonical NLRP3 inflammasome activation, WT and Sharpin^cpdm BMDMs were infected with C. rodentium. Caspase-1 activation and subsequent IL-1β and IL-18 production were all substantially reduced in the absence of SHARPIN, demonstrating the importance of SHARPIN in the regulation of the non-canonical NLRP3 inflammasome (Supplemental Fig. 1B). Interestingly, caspase-11 expression was slightly reduced in Sharpin^cpdm BMDMs compared to the levels of WT BMDMs. Altogether, these results establish SHARPIN as a central regulator of canonical and non-canonical NLRP3 inflammasome activation.

To examine whether SHARPIN is specifically required for NLRP3 inflammasome activation, we analyzed the levels of Salmonella enterica Typhimurium-induced NLRC4 and poly(dA:dT)-induced AIM2 inflammasome activation in WT and Sharpin^cpdm macrophages (16–18). S. Typhimurium induced similar levels of caspase-1 activation and IL-18 production in WT and Sharpin^cpdm BMDMs (Fig. 2A and 2B). Similarly, poly(dA:dT) stimulated similar levels of caspase-1 activation and IL-18 production in WT and Sharpin^cdpm BMDMs (Fig. 2A and 2B). These results clearly demonstrate that SHARPIN is dispensable for NLRC4 and AIM2 inflammasome activation. In accordance with caspase-1 and IL-18 data, pyroptotic cell death as measured by LDH release following S. Typhimurium or poly(dA:dT) stimulation was similar in WT and Sharpin^cpdm BMDMs (Fig. 2C).

Figure 2. SHARPIN is dispensable for inflammasome activation during S. Typhimurim infection and poly(dA:dT) transfection.

WT and Sharpin^cpdm BMDMs were infected with S. Typhimurium for 2 hours or transfected with 1μg poly(dA:dT) for 4 hours. A) Cell lysates blotted for caspase-1 and β-actin. B and C) IL-18 levels in the cell culture supernatants. D) Cell death determined by LDH released in the supernatants from S. Typhimurium and poly(dA:dT) stimulated samples. Data were analyzed for statistical significance using Student t-test and presented as mean ± SEM. All data are representative of four independent experiments. *=p<0.05.

Activation of the NLRP3 inflammasome requires two signals, 1) a priming signal that upregulates expression of NLRP3 and pro-IL-1β and 2) activation signals provided by NLRP3 agonists such as ATP and nigericin that trigger NLRP3 oligomerization and caspase-1 maturation. Previous studies have shown that SHARPIN is required for efficient NFκB signaling during TNFα, IL-1, CD40 and LPS induced signaling in MEFs and B cells, but its role in macrophages is not clear (8, 9, 14). We first evaluated whether LPS and Pam3CSK4 induced production of IL-6, KC and TNFα was dependent on SHARPIN. Production of IL-6, KC and TNFα were considerably reduced in Sharpin^cpdm BMDMs, suggesting a central role for SHARPIN in TLR signaling (Supplemental Fig. 2). Remarkably, LPS-induced expression of NLRP3 and ASC was similar in WT and Sharpin^cpdm BMDMs (Fig. 3A). However, the expression of pro-IL-1β in Sharpin^cpdm BMDMs was dramatically blunted (Fig. 1A and 3A). Messenger RNA analysis of LPS-stimulated BMDMs showed that while Nlrp3 expression was not affected, pro-Il1b expression was reduced in Sharpin^cpdm BMDMs (Fig. 3B and 3C). Furthermore, Tnfa expression was also reduced in Sharpin^cpdm BMDMs (Fig. 3D). HOIL-1 was recently shown to be involved in linear ubiquitination of ASC and to regulate NLRP3 inflammasome activation (3). While a possible role for SHARPIN in linear ubiquitination of ASC cannot be excluded, our study clearly demonstrates an critical role for SHARPIN in LPS-induced priming events.

Figure 3. SHARPIN is required for pro-Il1b and Tnfa mRNA expression, but not Nlrp3 mRNA expression during LPS stimulation.

A) WT and Sharpin^cpdm BMDMs were stimulated with LPS for the indicated periods of time. Cells were washed and cell lysates were analyzed for the protein expression of NLRP3, ASC and pro-IL-1β by Western blot. β-actin was used as a loading control. B-D) WT and Sharpin^cpdm BMDMs were stimulated with LPS for 1, 2 and 4 hours. RNA in the stimulated cells was harvested by the Trizol method and the expression of Nlrp3 (B), pro-Il1b (C) and Tnfa (D) mRNA were determined. Data are presented as mean ± SEM and representative of at least three independent experiments.

To ascertain the signaling pathways that are affected in the absence of SHARPIN, WT and Sharpin^cpdm BMDMs were stimulated with LPS (priming signal 1 required for NLRP3 inflammasome activation) and the activation of several signaling pathways was determined by Western blotting. These studies demonstrated that IκB phosphorylation and degradation were markedly reduced in Sharpin^cpdm BMDMs when compared to WT controls (Fig. 4A). Similarly, phosphorylation of ERK1 and ERK2 MAP kinases were reduced in the absence of SHARPIN (Fig. 4B). p38 MAP kinase phosphorylation was similarly inhibited in Sharpin^cpdm BMDMs (Fig. 4C). Together, these studies establish an important role of SHARPIN in regulating NFκB, ERK and p38 MAP kinase activation during LPS stimulation in BMDMs and provide a mechanistic link for regulation of the NLRP3 inflammasome.

Figure 4. Activation of NFκB, ERK and p38 MAP kinases is blunted in Sharpin^cpdm BMDMs.

A-B) WT and Sharpin^cpdm BMDMs were stimulated with LPS for the indicated time. Cells were washed, lysed with RIPA buffer and total protein concentration was quantified. Equal amount of protein samples were loaded and the activation of IκB (A), ERK1 and ERK2 (B) and p38 MAP kinase (C) was determined by blotting samples for total and phospho- antibodies. Normalized phospho-IκB was determined by taking normalized ratio of phospho-IκB over β-actin. IκB degradation was determined by normalizing the IκB levels to 0 min IκB. Total activation of ERK1/2 and p38 were analyzed by taking normalized band intensity ratios of phospho- over total protein levels. Data are representative of at least three independent experiments.

To our knowledge, this is the first report to demonstrate a central role for SHARPIN in activation of both canonical and non-canonical NLRP3 inflammasome in macrophages. This regulation of the NLRP3 inflammasome by SHARPIN could be at two levels - priming and activation. Our study demonstrates that SHARPIN is required for efficient activation of NFκB, ERK and p38 MAP kinases during the initial priming step of the NLRP3 inflammasome. Previous studies with HOIL-1 (a member of LUBAC complex along with HOIP and SHARPIN) showed a rather direct role for HOIL-1 in linear ubiquitination of ASC and activation of NLRP3 inflammasome. Our study shows that SHARPIN, although present in the same LUBAC complex, controls NLRP3 inflammasome activation by regulating its transcriptional priming. In conclusion, our results establish SHARPIN as a novel and central regulator of NLRP3 inflammasome signaling. Our results underscore the complexity of the NLRP3 inflammasome and uncover another molecule that could potentially be targeted to modulate and control NLRP3-associated inflammatory disorders.

Abbreviations

SHARPIN

SHANK-associated RH interacting protein

NLR

NOD-like receptor

LUBAC

Linear ubiquitin chain assembly complex

HOIP

HOIL-1L interacting protein

RIP

receptor-interacting protein

TLR

Toll-like receptor

BMDM

bone marrow derived macrophages

PAM

Pam3CSK4

WT

wild-type