Enhanced Inﬂammasome Activity in Patients with Psoriasis Promotes Systemic Inﬂammation

Psoriasis is linked to systemic inﬂammation and cardiovascular comorbidities, but studies of the underlying cellular mechanisms are lacking. The NLRP3 inﬂammasome is genetically associated with psoriasis, and its activation is increasingly linked with cardiovascular disease. In this study, we show that patients with psoriasis exhibited higher plasma levels of inﬂammasome-generated IL-1 b and IL-18, without any correlation to skin lesion severity. Increased constitutive expression of the inﬂammasome sensors NLRP3, NLRP1, and AIM2 was found in peripheral blood cells of the patients and also of those with mild disease, and this was accompanied by an increased caspase-1 reactivity in the myeloid blood subsets. TNF-a was found to activate selectively the NLRP3 inﬂammasome without the requirement for a priming signal. TNF-a was found to signal through the TNFR ‒ caspase-8 ‒ caspase-1 alternative inﬂammasome pathway, which proceeds independently of pyroptosis. Patients who received anti-TNF therapy had normalized plasma IL-1 b and IL-18 levels as well as normalized caspase-1 reactivity. This was in contrast to the patients treated with methotrexate who exhibited persistent, increased caspase-1 reactivity. Thus, we show that the TNF-a e mediated activation of NLRP3 inﬂammasomes in patients with psoriasis may contribute to systemic inﬂammation. Anti-TNF therapy normalized inﬂammasome function, suggesting a mechanism for the cardiovascular risk ‒ reducing effect.


INTRODUCTION
Psoriasis is an immune-mediated systemic disorder that is characterized by inflammatory reactions in the skin and joints (Nestle et al., 2009;Takeshita et al., 2017).The systemic nature of the disease, which entails immune dysregulation beyond the skin, is increasingly being recognized (Boehncke, 2018;Nestle et al., 2009).Psoriasis is associated with increased risks for systemic comorbidities, including obesity, atherosclerosis, and cardiovascular disease (Armstrong et al., 2013;Boehncke, 2018;Gelfand et al., 2006;Mehta et al., 2011), risks that are shared with other chronic inflammatory diseases.A causal link between systemic inflammation and the comorbidities seen in psoriasis remains to be fully elucidated.
In psoriasis, there is a complex interplay between components of the innate and adaptive immune systems (Lowes et al., 2014;Scho ¨n, 2019).Innate immunity seems to be important in the active, severe form that involves systemic and comorbid manifestations (Christophers and van de Kerkhof, 2019;Fanoni et al., 2019).The IL-23/T helper type 17 immune axis plays a critical role in the chronic state, which is supported by the efficacy of immunotherapies directed against TNF-a, IL-17, and IL-23 in the treatment of psoriasis (Harden et al., 2015).
The inflammasomes form the central hub of innate immune regulation.Members of the NLR family of proteins, including NLRP1, NLRP3, NLRC4, and AIM2, assemble into multiprotein complexes (termed inflammasomes) upon sensing microbial or danger signals (Broz and Dixit, 2016).Among the NLRs, NLRP3 is the most extensively studied, and it has been implicated in several chronic inflammatory disorders (Liu et al., 2017;Martinon et al., 2006;Masters et al., 2010;Mathews et al., 2014).The activation of the NLRP3 inflammasome requires an initial priming signal, which triggers the transcriptional upregulation of the NLRP3 and proeIL-1b genes (Bauernfeind et al., 2009).This is followed by a second signal, which results in inflammasome assembly, cleavage of procaspase-1 to active caspase-1, and the production of IL-1b and IL-18 (Latz et al., 2013).
Although there is accumulating evidence for the involvement of inflammasomes in cardiovascular diseases (An et al., 2019), studies elucidating the systemic role of inflammasomes in patients with psoriasis is lacking.In this study, we demonstrate increased inflammasome priming and activity in patients with psoriasis and identify TNF-a as the main regulator of inflammasome expression and function in the peripheral blood of patients with psoriasis.

Enhanced plasma levels of IL-1b and IL-18 in untreated patients with psoriasis compared with those in healthy controls
To determine whether there is an increased inflammasome activity in patients with psoriasis, we analyzed the free circulating levels of the inflammasome-generated cytokines IL-1b and IL-18 in the plasma samples of untreated patients with psoriasis (n ¼ 18) and healthy controls (n ¼ 19) using Luminex (R&D Systems, Minneapolis, MN).The PASI score of the included patients was in the range of 0e25, encompassing the entire spectrum of low to severe disease.We found significantly higher plasma levels of IL-1b and IL-18 (Figure 1a and b) in the patients than in the controls, suggesting increased constitutive inflammasome activity, which may have implications for systemic inflammation.The IL-1b levels in the patient group demonstrated a highly significant correlation with the IL-18 levels (r ¼ 0.59, P ¼ 0.009, Pearson's correlation coefficient) (Supplementary Figure S1).We found no correlations between the investigated cytokines and disease severity (PASI), suggesting sources of IL-1b and IL-18 other than the lesional skin.

Enhanced constitutive expression of inflammasome sensors in the peripheral blood subsets of untreated patients with psoriasis
We next investigated the baseline levels of inflammasome sensors in patients with psoriasis (mean age ¼ 49.25 AE 14.2 years, mean PASI ¼ 5.6 AE 6.9) compared with those in healthy controls (mean age ¼ 51.5 AE 15 years).Using a multicolor flow cytometry panel, we determined the The plasma levels of IL-1b and IL-18 were determined in healthy controls (n ¼ 19) and untreated psoriasis patients (n ¼ 18) using Luminex.The Mann-Whitney U test was used for the comparisons.(c) Expression level of NLRP3 is increased in patients with psoriasis.Constitutive expression of inflammasome sensors was determined in the CD14þ, CD16þ, CD4þ, and CD8þ subsets of red blood cell-lysed peripheral blood samples of patients with psoriasis (n ¼ 12) and healthy controls (n ¼ 5) using multicolor flow cytometry.Data are represented percentage gated cells.expression levels of NLRP1, NLRP3, and AIM2 in the CD14þ, CD16þ, CD4þ, and CD8þ subsets of red blood cell-lysed fresh whole blood.Significantly enhanced baseline expression levels of NLRP3 and NLRP1 were noted in the CD14þ monocyte, CD16þ neutrophil, and CD4þ lymphocyte subsets from patients with psoriasis compared with those from the healthy controls (Figure 1c) (Supplementary Figure S2).Similar results were observed with an increased AIM2 expression in the CD14þ and CD16þ subsets in the patients (Supplementary Figure S2).This suggests that the inflammasome sensors exist in a primed state in the immune cell subsets in the peripheral blood of the patients.
Higher caspase-1 reactivity to lipopolysaccharide in the untreated patients with psoriasis than in healthy controls Next, owing to the lack of correlation between inflammasome-generated cytokines and severity, we selected patients with mild PASI and investigated whether the constitutively upregulated peripheral-blood NLRP3 expression results in increased inflammasome function.Using fresh whole-blood samples from patients with mild psoriasis (mean PASI ¼ 3.8 AE 2.6) and healthy controls, we found no detectable differences in spontaneous caspase-1 activity between the two groups (data not shown), suggesting that inflammasome priming in the peripheral blood does not result in its spontaneous activation.Interestingly, in a patient who was suffering an exacerbation of severe psoriasis (PASI ¼ 25) after the sudden withdrawal of systemic treatment, we found increased basal caspase-1 activity in the CD4þ and CD8þ cell subsets.The same patient, who later exhibited chronic-phase psoriasis (PASI ¼ 9), demonstrated normal basal caspase-1 activity in the CD4þ and CD8þ cell subsets (Supplementary Figure S3).These findings suggest that an acute inflammatory state can provoke the constitutive involvement of inflammasomes in the lymphoid subsets, and this is supported by recent data showing NLRP3-dependent IL-1b release in the CD4þ cells from patients with gain-offunction NLRP3 mutations (Arbore et al., 2016).
To elucidate the extent of inflammasome reactivity toward stimuli in the immune cells, we made use of the toll-like receptor 4 agonist lipopolysaccharide (LPS).A total of 1 hour of LPS exposure of fresh whole-blood samples was followed by the determination of caspase-1 activity in the CD14þ, CD16þ, CD4þ, and CD8þ cell subsets.Studies in mice have suggested NLRP3 as an important modulator of age-associated inflammatory changes (Stout-Delgado et al., 2012).Using 18 healthy individuals, we found a distinct negative correlation between LPS-induced caspase-1 reactivity in the CD14þ cells and the age of the individuals (r ¼ À0.5, P ¼ 0.02), suggesting an immune aging of the human monocytes (Supplementary Figure S4).No correlations of caspase-1 reactivity to body mass index and gender were found (data not shown).Using age-matched patients and healthy controls (Table 1), we observed significantly increased caspase-1 reactivity in the CD14þ and CD16þ cell subsets of the patients (Figure 2) compared with those of the controls, which suggests that the priming of NLRP3 in these cells promotes inflammasome reactivity.In contrast, no differences in caspase-1 reactivity were observed in the CD4þ and CD8þ cell subsets (data not shown).We did not detect caspase-3 and/or caspase-7 in the CD14þ and CD16þ blood cells, showing that the fluorescence inhibitor of caspase-1 activity we observed did not arise from the apoptotic caspases but from the inflammasome response (Supplementary Figure S4b).However, the detection of other inflammatory caspases cannot be ruled out.
Exposure to TNF-a selectively primes NLRP3 but not NLRP1 or AIM2 or NLRC4 To investigate the mechanism of inflammasome priming in patients with psoriasis, we investigated the effects of exposing whole-blood samples for 1 hour to the key psoriasis-related cytokines on the gene expression of NLRP1, NLR31, NLRC4, and AIM2.The cytokines IL-17, TNF-a, IL-23, and IFN-g have all been shown to be increased systemically in psoriasis (Enerba ¨ck, 2011).Among the cytokines, exposure to TNF-a alone and in combination with IL-17 induced a substantial 10-fold increase in NLRP3 gene expression compared with unstimulated samples (Figure 3a).Except for the significant decrease in NLRC4 to the combination of IL-17 and TNF-a, no changes in the expression levels of NLRP1, NLRC4, and AIM2 to psoriasis cytokines were observed (Supplementary Figure S5).These results suggest that among the cytokines that are strongly implicated in psoriasis, TNF-a induces substantial and specific priming of NLRP3 but not of any of the other inflammasome-forming NLRs.

TNF-a treatment upregulates the expression of proeIL-1b and proeIL-18
We next examined the role of TNF-a in the transcriptional regulation of proeIL-1b and proeIL-18 in whole blood.We found that the 1-hour exposure of whole blood to TNF-a resulted in a remarkable (>160-fold) increase in proeIL-1b expression (Figure 3b).A comparatively smaller but significant increase in proeIL-18 was observed (Figure 3c).These findings indicate that TNF-a induces the transcriptional upregulation of both proeIL-1b and proeIL-18.The combination of IL-17 with TNF-a resulted in a small but significant increment in proeIL-1b gene expression, which was not observed when IL-17 was used alone.Thus, TNF-a primes not only NLRP3 but also IL-1b and IL-18.

TNF-a activates caspase-1 without the requirement for a second stimulus
To determine whether any of the psoriasis-associated cytokines induce a fully activated inflammasome, thereby activating caspase-1, we exposed fresh whole blood for 1 hour to IL-17, TNF-a, IL-23, or IFN-g, followed by flow cytometry.TNF-a exposure resulted in a distinct increase in caspase-1 activity, which was present in the innate immune CD14þ and CD16þ populations but was absent in the lymphoid CD4þ and CD8þ cell subsets (Figure 4a and b).A significant increase in caspase-1 activity was also observed when using the combination of IL-17 and TNF-a but not when IL-17 alone was used, primarily indicating a TNF-a effect.IL-23 addition gave rise to a very small but significant increase in caspase-1 activity.Our data suggest that TNF-a both primes and activates the NLRP3 inflammasome, thereby bypassing the requirement for a second signal.

TNF-a-induced inflammasome activity is dependent upon NLRP3 and ROS
To elucidate whether TNF-aeinduced inflammasome activity is dependent upon NLRP3, we added the specific NLRP3 inhibitor MCC950 at 1 hour before exposing the whole blood to the combination of TNF-a and IL-17.This resulted in a substantially decreased proeIL-1b expression (from w160-fold to w30-fold) (Figure 5a).In contrast, there was no decrease in proeIL-18 on preincubation with MCC950 (data not shown).ROS generation is a well-known trigger for inflammasome activation (Tschopp and Schroder, 2010).The addition of the ROS scavenger N-acetyl-L-cysteine resulted in a significant decrease in the TNF-a þ IL-17einduced proeIL-1b gene expression, suggesting that ROS are involved in TNF-a þ IL-17einduced proeIL-1b activation.(Figure 5a).
Similarly, 1 hour of preincubation with MCC950 before the exposure of the whole blood to TNF-a resulted in significantly decreased caspase-1 activity, demonstrating that TNF-aeinduced inflammasome activity is largely dependent on NLRP3 (Figure 5b).

TNF-aeinduced inflammasome activity involves caspase-8
Caspase-8 signaling has been suggested to act upstream of NLRP3 in the LPS-mediated alternative inflammasome signaling in human monocytes (Gaidt et al., 2016).To determine whether the TNF-aeinduced inflammasome activity in blood cells involves caspase-8, we added the caspase-8 inhibitor (Z-IETD-FMK) at 1 hour before stimulation with TNF-a, followed by the determination of caspase-1 activity.We found a significant decrease in caspase-1 activity on the inhibition of caspase-8 (Figure 5c), thereby confirming that TNF-a signaling in monocytes proceeds through the TNF receptor-caspase-8-caspase-1 pathway.

TNF-a activates the alternative inflammasome pathway in monocytes
The recently described alternative inflammasome pathway in human monocytes has been shown to bypass the need for a second stimulus and proceed independently of pyroptosis (Gaidt et al., 2016).On the basis of our observation that TNF-a did not require a second stimulus for inflammasome activation, we investigated whether the TNF-a exposure resulted in pyroptosis by determining the release of lactate dehydrogenase (LDH).Because potassium ion efflux is sufficient to activate NLRP3, we made use of nigericin as a positive control (Mun ˜oz-Planillo et al., 2013).We found that just as in the case with LPS, TNF-a did not result in LDH release, whereas robust LDH release was observed when nigericin was used.Our data suggest that TNF-a activates the alternative inflammasome pathway (Figure 5d).

Patients with psoriasis treated with TNF-a inhibitors display normalized inflammasome function
Our data demonstrated an important role for TNF-a in inflammasome priming and activation.As anticipated, we found that the TNF-a levels in the plasma were elevated in the patients with psoriasis (n ¼ 18) compared with those in the healthy individuals (n ¼ 23) (Supplementary Figure S6), thereby confirming the finding that TNF-a is present in the circulation and may act as a trigger for inflammasome activation (Bai et al., 2017).
TNF-a inhibition using neutralizing antibodies is highly effective in treating psoriasis (Harden et al., 2015).In patients with psoriasis who were treated with antieTNF-a antibodies for at least 8 months, we found an attenuated LPS-induced upregulation of proeIL-1b gene expression in the whole blood compared with that in the whole blood of the untreated patients, suggesting that the antieTNF-a treatment reduces the priming of the inflammasome (Figure 6a).These patients also exhibited lower IL-1b and IL-18 plasma levels than the untreated patients (although not reaching statistical significance) but without a difference in plasma levels compared with those in the healthy controls (Supplementary Figure S7).
Interestingly, compared with the untreated patients (mean PASI ¼ 3.8 AE 2.6), the patients treated with TNF-a inhibitors (mean PASI ¼ 2.1 AE 1.98), despite having comparable PASI levels with the untreated groups, displayed significantly reduced caspase-1 reactivity in the CD14þ and CD16þ cells (Figure 6b).In fact, the caspase-1 reactivity displayed by these patients was similar to the levels displayed by healthy controls.
Methotrexate is a folate antagonist that at a standard dose of 15e20 mg per week is a well-established therapy for inflammatory conditions such as psoriasis.Intriguingly, an increased caspase-1 reactivity in the CD14þ and CD16þ subsets persisted in the patients with psoriasis who were treated with methotrexate (mean PASI ¼ 2.4 AE 1.53) compared with the reactivity in the patients treated with antieTNF-a (Figure 6c).
The above data strongly support the assumption that TNF-a plays a key role in the increased inflammasome reactivity in patients with psoriasis.The marked difference in caspase-1 reactivity between the patients treated with methotrexate and those treated with antieTNF-a antibodies suggests that targeting TNF-a is superior in treating the inflammasomeinduced systemic inflammation in psoriasis.

DISCUSSION
Psoriasis entails a low-grade systemic inflammation, as evidenced by the increased C-reactive protein levels in patients compared with those in healthy subjects (Dowlatshahi et al., 2013).The systemic manifestations of psoriasis, including obesity and atherosclerosis, lead to cardiovascular disease and an increased risk of major adverse cardiovascular events ( Armstrong et al., 2013;Boehncke, 2018;Gelfand et al., 2006;Mehta et al., 2011).Insulin resistance and endothelial dysfunction, both of which are associated with atherosclerosis, have been suggested as the pathogenic link between psoriasis and cardiovascular comorbidity (Boehncke, 2018).In line with this concept, TNF-a, which is a major insulin antagonist and a known inducer of endothelial dysfunction, has been found to be systemically enhanced in cases of psoriasis (Hotamisligil et al., 1993;Picchi et al., 2006).Although this provides a solid conceptual basis for the linkage between the systemic inflammation in psoriasis and cardiovascular comorbidity, there remains a lack of mechanistic evidence (Boehncke et al., 2011;Yamanaka and Mizutani, 2015).
In this study, we have investigated the inflammasomes and provide mechanistic data about their role in triggering the systemic inflammation in psoriasis.We demonstrate that patients with psoriasis have increased systemic levels of IL-1b and IL-18 and possess primed inflammasome components.No correlations of the above cytokines with PASI were observed in the patients with mild-to-severe PASI, suggesting that the inflammasome-dependent systemic inflammation in these patients is independent of their cutaneous manifestations.Using the blood from patients with mild-to-moderate psoriasis (PASI < 7.5) in all the caspase-1 reactivity experiments, we could show that the myeloid cells from untreated patients with psoriasis exhibit enhanced caspase-1 production in response to LPS.Importantly, we show that TNF-a selectively activates the NLRP3 inflammasome without the requirement for a priming signal, resulting in the activation of caspase-1.In accordance with this, we show that patients with psoriasis who are treated with antieTNF-a antibodies do not have increased plasma levels of IL-1b and IL-18 and do not exhibit increased caspase-1 reactivity compared with untreated patients.
IL-1b signaling plays a well-documented role in the pathogenesis of psoriasis (Lowes et al., 2014).IL-1b has been shown to be critical for the differentiation and activation of IL-17eproducing T cells (Sutton et al., 2009), and IL-1b protein levels in the psoriatic skin have been shown to correlate with disease severity and treatment response (Cai et al., 2019).It is increasingly recognized that activation of the NLRP3 inflammasome contributes to the pathogenesis of cardiovascular diseases (An et al., 2019).NLRP3 expression in adipose tissue has been shown to be associated with coronary atherosclerosis (Bando et al., 2015).The NLRP3-specific inhibitor MCC950 prevents diet-induced cardiovascular disease in mice through autophagy induction (Pavillard et al., 2017).Data from the Canakinumab Anti-Inflammatory Thrombosis Outcome Study trial (Ridker et al., 2017) reveal a statistically significant lower rate of recurrent cardiovascular events in patients treated with the antieIL-1b antibody canakinumab, suggesting a mechanistic role for the inflammasome in linking systemic inflammation with cardiovascular diseases (Ridker et al., 2017).Moreover, the antithrombogenic effect of statins has been attributed to reduced NLRP3 and IL-1b expression (Satoh et al., 2014).
We performed our experiments using unmanipulated whole blood in order to reflect accurately the in vivo conditions (Mallone et al., 2011).The NLRP3 inflammasome is mainly described in myeloid cells, with CD14þ monocytes being the main source of IL-1b.Neutrophils have recently been shown to possess functional inflammasome components (Bakele et al., 2014), and the involvement of CD4þ cells in inflammasome hyperactivity has recently been demonstrated in patients with gain-of-function mutations in NLRP3 (Arbore et al., 2016).Consistent with the above studies, we find increased expression of NLRP3 not only in the CD14þ subset but also in the CD16þ and CD4þ subsets of patients with psoriasis.Increased LPS-induced caspase-1 reactivity in the CD14þ and CD16þ cells of the untreated patients was observed, which is in line with our data showing NLRP3 priming in the patients' cells.The observed lack of correlations between IL-1b and IL-18 and the severity of psoriasis as well as with the increased caspase-1 reactivity in patients with mild PASI suggests an ongoing, low-grade inflammatory process in the blood that is acting independently of the skin manifestations.Moreover, our data support that psoriasis may be a part of the autoinflammatory keratinization diseases (Akiyama, 2020;Akiyama et al., 2017).The CD4þ T cells, despite the primed NLRP3, did not display enhanced caspase-1 reactivity in response to short-term exposure to LPS, which may reflect a requirement for additional T-cell activation, as shown by Arbore et al (2016).
TNF-a expression is increased in adipose tissues, including adipocytes and macrophages in obese patients, and this potentially contributes to its circulating levels while also leading to the amplification of pre-existing inflammatory processes (Yost and Gudjonsson, 2009).Inhibitors of TNF-a have previously been demonstrated to reduce cardiovascular risk in patients with psoriasis as well as reducing the risk for diabetes, which is in line with the insulin-antagonist effect of TNF-a (Hotamisligil et al., 1993).Interestingly, the riskreducing effect of TNF-a was demonstrated to be superior to that of methotrexate (Wu et al., 2017).Furthermore, data from a recent Cardiovascular Inflammation Reduction Trial show that among patients with stable atherosclerosis, treatment with methotrexate does not reduce the levels of IL-1b, IL-6, and C-reactive protein and does not result in fewer cardiovascular events compared with placebo (Ridker et al., 2019).Intriguingly, we show that the patients treated with methotrexate, like the untreated patients, have significantly increased caspase-1 reactivity, which is not seen in the patients treated with inhibitors of TNF-a.
TNF-a has been shown to be a key transcriptional regulator of NLRP3 inflammasome components in murine models (McGeough et al., 2017).Previous studies using isolated human monocytes and polymorphonuclear neutrophils have shown that exposure to TNF-a results in significantly increased expression of NLRP3 (O'Connor et al., 2003) and proeIL-1b (Marucha et al., 1990), respectively.Surprisingly, we found that exposing whole blood to TNF-a resulted in the production of NLRP3-dependent active caspase-1, without the need for a second signal.The recently described alternative inflammasome signaling utilized by toll-like receptor 4-LPS in human monocytes has been shown to bypass the requirement for a second signal, leading to caspase-1 activation through the receptor-interacting serine/threonine-protein kinase 1 and caspase-8 pathways (Gaidt et al., 2016) and is independent of pyroptosis.Accordingly, we show that TNF-aeinduced caspase-1 activation in monocytes is dependent on caspase-8 and proceeds without pyroptotic cell death.TNF-a signaling takes place through the TNFreceptor 1 and engages the receptor-interacting serine/threonine-protein kinase 1-TRADD-caspase-8 pathway, leading to regulated necrosis (Vanden Berghe et al., 2014).Pyroptotic cell death characterizes the classical but not the alternative inflammasome pathway, thereby allowing the cells in the latter case to respond with IL-1b production without committing to cell death, which may be instrumental in mediating a low-grade, chronic inflammation.Our study demonstrates that TNF-a in the blood of patients with psoriasis stimulates the NLRP3 inflammasome.However, additional data are required to validate the contribution of TNF-aemediated NLRP3 activation in the systemic inflammation and comorbidities of psoriasis.
In summary, we show that there is an enhanced NLRP3 inflammasome function in blood samples from patients with psoriasis.Our data support a role for innate immune system and inflammasomes in systemic inflammation in psoriasis, with TNF-a being the main mediator of inflammasome activation.The cardiovascular risk-reducing effect of TNF-a inhibitors may be mechanistically explained by attenuated inflammasome activity.

MATERIALS AND METHODS
The detailed protocols are described in the Supplementary Materials.

Study subjects and samples
All the patients included in the study were examined, and the diagnosis of psoriasis was verified by a dermatologist at the Department of Dermatology at Linko ¨ping University Hospital, Linko ¨ping, Sweden.PASI was used to appraise disease severity.The recruited patients had PASI between 0 and 25.The study was approved by the Local Ethics Committee and conducted according to the Declaration of Helsinki principles.Written informed consent was obtained from all patients and healthy controls.Peripheral blood samples from the study subjects were collected in and immediately processed for flow cytometry or RNA isolation.Plasma was collected using standard procedures.
All cytokines were purchased from R&D Systems.

RNA extraction and quantitative real-time PCR
RNA was extracted from whole blood using the QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.Quantitative real-time PCR was performed to detect the genes of interest using SYBR green (Applied Biosystems, Foster City, CA) or predesigned TaqMan assays in the 7500HT PCR apparatus (Applied Biosystems).GAPDH or RPLPO were used as reference genes.
Flow cytometric analysis NLRP1, NLRP3, and AIM2 detection in white blood cell subsets.
Active caspase 1 and caspase-3 and/or caspase-7 detection using fluorescence inhibitor of caspase-1 flow cytometry.Peripheral blood samples were left untreated or treated with LPS for 1 hour at 37 C.The red blood cells were lysed and stained with CD4-pacific blue, CD14-APC-Cy7, CD8-phycoerythrin, and CD16-PE-Cy7 (all from BD Biosciences).Caspase-1 activity was determined using a caspase-1 fluorescence inhibitor of caspase-1 kit and caspase-3 and/ or caspase-7 activity was detected using FLICA 660 (ImmunoChemistry Technologies, Bloomington, MN) according to the manufacturer's instructions.All flow cytometric analyses were performed on the Gallios flow cytometer (Beckman Coulter LS, Brea, CA) and analyzed using Kaluza Analysis, version 1.3 (Beckman Coulter).The gating strategy is shown in Supplementary Figure S8.

Cytokine determinations in plasma samples using Luminex
The plasma levels of IL-1b, IL-18, and TNF-a were determined using Luminex kits (R&D Systems) following the manufacturer's instructions.

LDH assay
Monocytes were isolated from PBMCs using EasySep Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL Technologies, Vancouver, Canada).The Pierce LDH cytotoxicity kit (Life Technologies, Carlsbad, CA) was used to determine pyroptotic cell death according to the manufacturer's instructions.

Statistical analysis
Data analysis was performed in GraphPad Prism,version 8.3 (GraphPad Software,San Diego,CA).Differences between the patients and the healthy individuals were determined using either the Mann-Whitney U test or the unpaired t-test with Welch's correction.The pairwise comparisons in the in vitro experiments were determined using a paired t-test.A P-value 0.05 was considered statistically significant.Data are represented as mean AE SEM.Correlations were determined using Pearson's correlation coefficients.

Figure 1 .
Figure 1.Systemic levels of inflammasome cytokines and NLRP3 expression are increased in patients with psoriasis.(a) Systemic levels of IL-1b and (b) IL-18 levels are increased in patients with psoriasis.The plasma levels of IL-1b and IL-18 were determined in healthy controls (n ¼ 19) and untreated psoriasis patients (n ¼ 18) using Luminex.The Mann-Whitney U test was used for the comparisons.(c) Expression level of NLRP3 is increased in patients with psoriasis.Constitutive expression of inflammasome sensors was determined in the CD14þ, CD16þ, CD4þ, and CD8þ subsets of red blood cell-lysed peripheral blood samples of patients with psoriasis (n ¼ 12) and healthy controls (n ¼ 5) using multicolor flow cytometry.Data are represented percentage gated cells.An unpaired t-test with Welch's correction was performed.*P < 0.05, ****P < 0.0001.Error bars represent mean AE SEM.ns, not significant.

Figure 3 .
Figure 3.Effect of psoriasis-related cytokines on mRNA levels of NLRP3, proeIL-1b, and proeIL-18.(a) The effect of 1 hour of exposure of whole blood at 37 C to IL-17, TNF-a, IL-23, and IFN-g on the expression of NLRP3 was determined.The effects of 1 hour of exposure of whole blood to psoriasis-related cytokines on the expression levels of (b) proeIL-1b and (c) proeIL-18 were determined.The samples were analyzed using qRT-PCR and the results are shown as fold-changes relative to the unstimulated sample (n ¼ 3e5).A paired t-test was used for the comparisons.Error bars represent mean AE SEM.*P < 0.05, **P < 0.001.ns, not significant.

Figure 4 .
Figure 4. TNF-a induces caspase-1 activation in the absence of a priming signal.The effects of 1 hour of exposure of whole blood to psoriasis-related cytokines on the caspase-1 activity in (a) CD14þ and (b) CD16þ cells were determined.The samples were analyzed by determining caspase-1 activity by flow cytometry and the results are shown as fold-changes in MFI relative to an unstimulated sample (n ¼ 7).A paired t-test was performed for comparisons.Error bars represent mean AE SEM.*P < 0.05.MFI, mean fluorescence intensity.

Figure 5 .
Figure 5. TNF-a-primes and activates the inflammasome.(a) Whole blood was preincubated for 1 hour with NLRP3 inhibitor MCC950 or ROS inhibitor NAC followed by 1 hour of stimulation with IL-17 or TNF-a or both.proeIL-1b expression was determined using qRT-PCR.Data are represented as fold-changes relative to the unstimulated control (n ¼ 3e5).(b) Whole blood was preincubated for 1 hour with a MCC950, followed by 1 hour of stimulation with TNF-a, after which the caspase-1 activity was determined using flow cytometry.Data are represented as MFI (n ¼ 6).(c) Whole blood was preincubated for 1 hour with caspase-8 inhibitor Z-IETD-FMK followed by 1-hour stimulation with TNF-a.Caspase-1 activity was determined using flow cytometry.Data are shown as MFI (n ¼ 3e5).Error bars represent mean AE SEM.*P < 0.05, **P < 0.001.(d) Monocytes from peripheral blood were treated with LPS or TNF (30 minutes) followed by nigericin treatment (1.5 hours) or left untreated.LDH release is shown as mean of six donors.FLICA, fluorescence inhibitor of caspase-1 activity; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; NAC, N-acetyl-L-cysteine.

Figure 6 .
Figure 6.Psoriasis patients treated with antieTNF-a antibodies have reduced inflammasome activity.(a) Peripheral blood samples from patients treated with TNF-a-antibody (n ¼ 7), untreated (n ¼ 7), and treated with methotrexate (n ¼ 6) were exposed to lipopolysaccharide (100 ng/ml) for 1 hour at 37 C. proeIL-1b expression was determined using qRT-PCR.(b) Peripheral blood samples from patients treated with antieTNF-a (n ¼ 9), untreated (n ¼ 7), and healthy controls (n ¼ 14) were exposed to lipopolysaccharide for 1 hour at 37 C and caspase-1 reactivity was determined using flow cytometry.(c)Peripheral blood samples from patients treated with methotrexate (n ¼ 7), antieTNF-a (n ¼ 9), and untreated (n ¼ 7) were exposed to lipopolysaccharide for 1 hour at 37 C and caspase-1 reactivity was determined using flow cytometry.Data in (a-c) are represented as fold-changes of stimulated and/or unstimulated samples.The Mann-Whitney U test was performed for comparisons.Error bars represent mean AE SEM.*P < 0.05.MFI, mean fluorescence intensity.

Table 1 .
Description of Study Subjects are represented as fold-changes relative to stimulated and/or unstimulated samples for all the individuals.*P < 0.05, **P < 0.001.The Mann-Whitney U test was used for the comparisons.Error bars represent mean AE SEM.MFI, mean fluorescence intensity.