Accumulating evidence suggests that the activation of the innate branch of the immune system plays a pivotal role in the induction and perpetuation of metabolic and aging-related diseases. In this context, the NLRP3 inflammasome pathway has been identified as an important driver of sterile inflammatory processes. De novo protein synthesis of NLRP3 induced by signals such as TLR ligands or TNF is a prerequisite for sustained NLRP3 mediated caspase-1 cleavage and inflammasome activation. Here, we demonstrate in aged mice that spontaneously elevated TNF represents a critical priming signal that functions to control NLRP3 inflammasome activation. Elevated systemic TNF levels were responsible for increased NLRP3 expression and caspase-1 activity in adipose tissues and liver. TNF dependent, spontaneous inflammasome activity in aged mice resulted in impaired glucose tolerance that could be attributed to peripheral insulin resistance. Altogether, these results implicate that TNF-driven NLRP3 expression constitutes an important checkpoint that regulates inflammasome activation, presumably by additional signals such as aging-associated DAMPs.
The NLRP3 protein is a member of the NLR family that detects a large number of exogenous microbe-associated molecular patterns and also endogenous damage-associated molecular pattern (DAMP) structures that are released upon cell or tissue damage. Upon activation, NLRP3 associates with the adaptor protein ASC by homotypic domain interactions and forms an inflammasome to initiate the autocatalytic activation of caspase-1. Active caspase-1 in turn processes the precursors of the proinflammatory cytokines IL-1β and IL-18 to their active and secretable form (1). NLRP3 activation furthermore induces a special type of cell death, known as pyroptosis, which has been shown to induce secondary inflammation in neighboring cells by the release of DAMP molecules (2). Aside from IL-1 and IL-18 secretion, caspase-1 regulates the unconventional release of many other proteins (3) and induces the formation of proinflammatory lipid mediators such as eicosanoids (4).
It is very well accepted that low-grade inflammation plays a causative role in the pathogenesis of metabolic diseases, most prominently in the context of obesity. Metabolic inflammation is orchestrated by an interplay of metabolic cells and immune cells in response to an excess of nutrients. In the long-run, this inflammatory response can result in considerable metabolic changes such as peripheral insulin resistance and the development of type 2 diabetes. The exact origin and initiation mechanisms of this proinflammatory state are not fully understood (5) but increased macrophage infiltration (6) and elevated levels of proinflammatory cytokines in fat tissues, predominantly TNF (7), are commonly observed in obese individuals and mice. More recently it was shown that the cytosolic innate immune sensor NLRP3 pays a pivotal role in this obesity-associated systemic low-grade inflammation resulting in metabolic abnormalities (8–11). In the context of high-fat diet, NLRP3-deficient animals display greatly reduced macrophage activation in fat tissues, reduced signs of systemic inflammation and NLRP3-deficient mice are less prone to develop peripheral insulin resistance with associated glucose intolerance. In these short-term, high-fat diet–based mouse models of obesity, IL-1β is partially required to exert the downstream effects of NLRP3 inflammasome activation leading to glucose intolerance (8, 12). A similar pathophysiology seems to be at play in the context of age-related inflammation that leads to functional decline in multiple organs and the immune system (13). This so-called process of “inflammaging” is observed in aged mice that are fed a normal chow diet. The here-observed low-grade inflammation and associated metabolic changes are also NLRP3 inflammasome mediated, but interestingly independent of the inflammasome effector cytokine IL-1β (13). To this end, while the absence of NLRP3 and caspase-1 protects mice from age-related increases in innate immune activation and improves glycemic control, IL-1R deficient mice are not protected (13). Thus, even though NLRP3 serves as an upstream target that controls obesity induced inflammation and age related inflammation, IL-1β is only involved in mouse models of obesity. The downstream effectors that mediate NLRP3 related metabolic disturbances in old age have not been identified.
In general, NLRP3 can be activated by a wide array of endogenous DAMPs, which include cholesterol crystals, uric acid crystals, extracellular ATP, reactive oxygen species, and a number of lipids (14). The wide and structurally diverse array of agents inducing NLRP3 activation implies that NLRP3 cannot function as a direct receptor for these substances, yet that rather a shared downstream pathway engaged by NLRP3 activating DAMPs may enable NLRP3 activation. Several of the NLRP3 activating DAMPs accumulate during the aging process (15, 16) and thus could potentially trigger NLRP3 in vivo, yet a clearly defined structural ligand that would trigger NLRP3 activation in the context of aging has not been identified and it is unlikely that a single aging-associated NLRP3 activator exists. Apart from that, prior to its activation, NLRP3 requires an additional proinflammatory licensing step. To this end, resting mouse macrophages are critically dependent on a proinflammatory priming signal to upregulate NLRP3 expression to sufficient protein levels (17). TNF was shown to mediate sensitization to the endogenous danger signal ATP in the absence of microbial stimulation (18). Additionally, it was recently shown, that rapid priming by LPS can license the NLRP3 inflammasome independently of NLRP3 protein levels, a process that was attributed to MyD88 and IRAK1/4 signaling (19, 20). Deubiquitination of resting NLRP3 induced by this rapid TLR priming cascade was suggested to constitute the molecular mechanism of this response (19, 20). Interestingly, the requirement for priming is only observed for the NLRP3 inflammasome pathway, whereas other inflammasome sensors are not dependent on such a signal. This additional safeguard mechanism of NLRP3 activation might have evolved to protect the host from inflammatory damage in the context of cell or tissue damage.
To what extent NLRP3 inflammasome priming signals play a role in NLRP3-associated sterile inflammatory diseases causing metabolic changes has not been addressed. In this work, we present evidence that priming of the NLRP3 inflammasome by the proinflammatory cytokine TNF critically regulates NLRP3 expression to trigger caspase-1 activity in metabolic tissues and inflammasome mediated insulin resistance in old age.
Materials and Methods
Macrophage culture and stimulation
Primary macrophages were obtained by culturing bone marrow cells from C57BL/6 mice with 30% L929 supernatant conditioned DMEM medium containing 10% FCS for 7 d. A total of 7 × 105 cells per condition in 500 μl serum free medium was used for stimulation experiments. Cells were primed for the indicated time with 200 ng/ml ultra-pure LPS from Escherichia coli, 400 ng/ml Pam3CSK4, 1 μg/ml poly(I:C) (Invivogen) or 100 ng/ml recombinant TNF (Peprotech). Cells were stimulated for 60 min with 6.5 μM Nigericin, 5 mM ATP (Sigma-Aldrich) or 3 h with Lipofectamine 2000 (Invitrogen) transfected dsDNA (pCI plasmid DNA). Cyclohexemide (CHX) was added 30 min before addition of the priming agent.
ASC pyroptosome formation assay
Immortalized and lentivirally transduced ASC-mYFP macrophages were kindly provided by Prof. Latz. ASC pyroptosomes were quantified by epifluorescence microscopy and imageJ software. Confocal laser microscopy was used for high-resolution images of choleratoxin subunit B and Hoechst dye stained cells.
Female C57BL/6JRccHsd and C57BL/6JRccHsd aged mice were purchased from Harlan Laboratories. Mice were fed ad libitum with a normal chow diet. We performed all experiments in compliance with the local authorities (protocol number 84-02.04.2014.A1111). Infliximab was dosed 3.5 mg/kg body weight and injected i.p. with a total volume of 100 μl PBS daily for a period of 4 wk. Hepatocytes were isolated as described (21) and liver CD11b+ cells were isolated by MACS technology (Miltenyi). Adipocytes and the stroma vascular fraction (SVF) were isolated as previously described (22).
Glucose tolerance test and insulin tolerance test
Glucose tolerance test (GTT) and insulin tolerance test was performed on 4 h fasted mice in the morning hours. 1.8 mg Glucose per gram body weight (Braun) or 0.83 mU/g body weight insulin (Sanofi) was injected i.p. in a total volume of 70 μl of NaCl 0.9%. Glucose concentration was measured with a blood glucose meter (Accu-Chek; Roche) in tail vein blood at the indicated time points.
Protein isolation and Western blot analysis
Nitrogen frozen tissue was grinded and lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% Sodiumdesoxycholate, 1% TritonX100, 0.1% SDS, protease inhibitor) before the protein concentration was determined using a BCA protein assay (Pierce) and equal amounts were loaded for SDS-page. Cell culture supernatants were precipitated with methanol/chloroform before caspase-1 was detected. Western blot analysis was performed as described (23) with minor modifications. Abs for β-actin and ubiquitin (SantaCruz), caspase-1 (Adipogen and SantaCruz), ASC and NLRP3 (Adipogen), and IL-1β (RnD) were used. Immunoprecipitation was performed as described (19).
Total cellular RNA from 50 mg grinded tissue or 1 × 106 cells was isolated with TRIzol reagent (Invitrogen). Total RNA was treated with DNase I (Fermentas) and reverse transcribed with M-MuLV reverse transcriptase (Fermentas) using a oligodT adapter. Quantitative PCR was performed with SybrGreen according to the manufacturer’s protocol (Fermentas). Following primers were used: Nlrp3 5′-GATGCTGGAATTAGACAACTGC-3′ and 5′-GATCATTGTTGCCCAGGTTC-3′; Hprt 5′-CTGGTGAAAAGGACCTCTCG-3′ and 5′-TGAAGTACTCATTATAGTCAAGGGCA-3′; Emr1 (F4/80) 5′-CTTTGGCTATGGGCTTCCAGTC-3′ and 5′-GCAAGGAGGACAGAGTTTATCGTG-3′; Casp1 5′-CCAGGCAAGCCAAATCTTTA-3′ and 5′-TCAGCTGATGGAGCTGATTG-3′; Actb 5′-AGCCATGTACGTAGCCATCC-3′ and 5′-CTCTCAGCTGTGGTGGTGAA-3′; Il1b 5′-CTGCAGCTGGAGAGTGTGG-3′ and 5′-GGGGAACTCTGCAGACTCAA-3′. Expression of target genes was normalized to Hprt expression and plotted as arbitrary units on a linear scale if not otherwise indicated.
TNF, IL-1β, IL-6 (BD OptEIA), IL-18 (MBL) and insulin (Millipore) were measured in cell culture supernatants and serum by ELISA according to the manufacturer’s instructions.
We used a two-tailed Student t test to determine significance in differences between treatments; * indicates p < 0.05 or p < 0.01; ** indicates p < 0.005 or p < 0.001. Quantifiable results are expressed as mean + SEM.
TNF is a TLR-independent priming signal of the NLRP3 inflammasome
Since microbe-associated molecular patterns are not necessarily present at a systemic level in the context of metabolic or aging related inflammation, we turned to the role of TNF as a potential priming signal for NLRP3 inflammasome activation in this setting. TNF is a proinflammatory cytokine that engages the NF-κB pathway and it is also upregulated in aged individuals (24, 25). It was previously shown that TNF can promote ATP and silica mediated NLRP3 activation (18). Interestingly, also long term exposure to TNF leads to sustained ATP-induced caspase-1 activation by the NLRP3 inflammasome, while long term exposure to LPS led to tolerization of macrophages (18). Thus, a constantly elevated level of TNF could mediate NLRP3 licensing in vivo. Here we show, that priming of murine bone marrow–derived macrophages (BMMΦs) with TNF for 2 h did not affect the mRNA levels of IL-1β, yet resulted in a considerable induction of NLRP3 expression (Fig. 1A). IL-18, on the other hand, was constitutively expressed and remained unchanged under these conditions. In line with these data, TNF incubation consequently led to dose dependent NLRP3 protein expression, whereas these TNF induced NLRP3 protein levels were marginally lower compared with LPS priming (Fig. 1B). As expected from the mRNA data, TNF priming did not result in relevant pro–IL-1β protein expression after short periods (4 h), whereas LPS readily induced IL-1β expression. To study whether TNF-primed macrophages were capable of NLRP3 inflammasome activation, we stimulated TNF-primed macrophages with the canonical NLRP3 stimulus Nigericin and studied ASC pyroptosome formation as an IL-1β independent readout of inflammasome activation (Fig. 1C). These studies revealed that Nigericin only triggered ASC pyroptosome formation in the presence of a priming signal, with both LPS and TNF being equally potent. Analogous results were obtained when we studied caspase-1 cleavage (Fig. 1D). Here, a dose dependent increase in NLRP3 protein expression correlated with the total NLRP3 inflammasome activity represented by caspase-1 cleavage (Fig. 1B, 1D). Since the caspase-1 substrate pro–IL-18 was constitutively expressed in MΦs and not regulated by TNF, secreted IL-18 mirrored the levels of activated caspase-1 (Fig. 1E). On the other hand, as expected, matured IL-1β was not secreted in TNF pretreated and activated MΦs (Fig. 1D). Altogether, these findings confirmed and substantiated the important role of the induction of NLRP3 protein expression as a critical denominator of inflammasome output activity. Moreover, these results showed that IL-1β maturation and secretion could be uncoupled from inflammasome activation in the context of TNF priming. Unlike IL-1β, IL-18 is constitutively expressed in monocytic cells at the mRNA and protein level, while this expression is not or only minimally altered by proinflammatory signaling cascades. As such, NLRP3 activation in TNF primed cells results only in caspase-1 cleavage and IL-18 secretion, but not in secretion of IL-1β.
NLRP3 licensing by TNF requires de novo protein synthesis
Others and we have previously shown that NLRP3 induction by proinflammatory stimuli is a critical step in NLRP3 activation (17, 18). This has led to the widely accepted two-step model of NLRP3 inflammasome activation: NLRP3 protein induction by proinflammatory stimuli that induce NF-κB signaling and the presence of a NLRP3 activation signal (17, 26). Additionally, it was recently shown, that rapid priming by LPS can license the NLRP3 inflammasome independently of increased NLRP3 protein levels (19). To address the role of NLRP3 de novo protein expression in the context of LPS and TNF priming, we conducted a series of experiments, in which we added the translation inhibitor CHX as illustrated (Fig. 2A). In accordance with the above-described study, we detected caspase-1 cleavage in LPS-primed macrophages stimulated with Nigericin already after short priming periods independent of de novo protein synthesis. However, caspase-1 cleavage in response to NLRP3 activation after prolonged LPS pretreatment (3 h and longer) or TNF pretreatment was completely blocked by CHX and thus critically dependent on de novo protein synthesis (Fig. 2A, 2B). Of note, CHX did not compromise the activation of the AIM2 inflammasome by transfected DNA at any time point tested (Fig. 2A). Furthermore, TNF priming did in contrast to rapid LPS priming (20 min) not result in comparable deubiquination (Fig. 2C). In summary, these data indicated that NLRP3 priming by proinflammatory signals can be distinguished in two phases: A first, rapid priming phase, as exerted by LPS, facilitates NLRP3 activation independently of de novo gene expression but requires deubiquitination of NLRP3. On the other hand, a second, delayed priming phase is critically dependent on de novo protein expression of NLRP3. TNF, in contrast to LPS, only engages the delayed priming phase and as such requires de novo NLRP3 expression for its activation.
Aging enhanced NLRP3 expression and inflammasome activation in the adipose tissue
Rapid priming may be more relevant for the immediate inflammasome activation in response to microbial pathogens. Continuous exposure to TNF and other NF-κB activating stimuli may lead to elevated NLRP3 levels in macrophages and license NLRP3 activation in chronic diseases. In this regard we hypothesized that long-term elevated proinflammatory TNF levels, as observed in aged individuals (27), might modulate the systemic inflammasome activity by priming NLRP3 tissue expression. To address this possibility, we turned to the well-established model of aging-associated inflammation, in which a critical role has been ascribed to the NLRP3 inflammasome (13). To this end, we compared 7 wk to 20 mo old C57BL/6 mice that were fed on a normal chow diet and cohoused under SPF conditions. 20 mo old animals showed an increase in body weight that was accompanied by increased serum levels of TNF, compared with 7 wk old wild type mice (Fig. 3A). Moreover, in line with previous reports (13), aged mice showed a dramatic increase in the caspase-1 dependent cytokine IL-18 within serum, serving as a systemic and measurable surrogate parameter of inflammasome activation (Fig. 3A). Serum levels of IL-1β were below the ELISA detection limit (Fig. 3A). Indeed, analyzing the activation status of caspase-1 by Western blot, we detected substantially increased levels of active and cleaved caspase-1 in the adipose and hepatic tissues of old mice, whereas total tissue caspase-1 was not substantially different in young and old individuals (Fig. 3B). Cleaved IL-1β was not detectable by Western blot in the tissue lysates (Fig. 3B), likely attributable to the detection limit of the method. Consequently, these data do not fully exclude the presence of elevated IL-1β levels. To investigate whether bone marrow macrophages from aged mice display a changed inflammasome activity that could be responsible for enhanced activity of caspase-1 in old age, we analyzed the NLRP3 and AIM2 response in bone marrow–derived macrophages of young and aged mice (Fig. 4A). Bone marrow–derived macrophages from 7 wk to 20 mo old animals did not show a difference in caspase-1 cleavage after triggering the inflammasome with Nigericin or DNA in vitro. Furthermore, we could not detect caspase-1 cleavage in stimulated isolated adipocytes from epididymal adipose tissue or isolated hepatocytes. In contrast, caspase-1 activation could be seen in the myeloid cell containing SVF of epididymal adipose tissue and CD11b+ cells sorted from mouse livers (Fig. 4B, 4C). We thus conclude that the main part of caspase-1 cleavage contributing to the elevated inflammasome activity in the liver and adipose tissues of aged mice is confined to myeloid cells. In line with this notion, isolated adipocytes and hepatocytes also failed to display an inducibility of NLRP3 mRNA expression upon TNF exposure comparable with myeloid liver cells or the myeloid cell containing SVF of the adipose tissue (Fig. 4D, 4E). However, the spontaneous inflammasome activation in old age was accompanied by upregulated NLRP3 mRNA levels in the liver, s.c. adipose tissue, epididymal adipose tissue, and mesenterial adipose tissue of 20 mo versus 7 wk old wild type mice (Fig. 3C), whereby the mRNA levels of IL-1β and IL-18 were not altered in a relevant fashion (Supplemental Fig. 1A, 1B). We further observed that old mice displayed a significantly impaired glucose tolerance when i.p. challenged with glucose [Fig. 3D and (13)]. Of note, this limited glucose tolerance could be attributed to an impaired peripheral insulin tolerance and not a failure of pancreatic insulin secretion, given the fact that blood insulin levels were even increased in aged mice (Fig. 3E). In summary, these data indicated, that increased NLRP3 expression in the liver and adipose tissues in the context of aging were associated with enhanced inflammasome activation and subsequent impaired glucose tolerance.
TNF regulates NLRP3 expression and inflammasome activity in aged mice
To address the role of TNF in regulating NLRP3 activity during the aging process, we blocked TNF using infliximab, which has been successfully used to neutralize TNF in mice (28). As expected, i.p. application of infliximab to 22 mo old mice led to reduced serum concentration of TNF without affecting the body weight (Fig. 5A). Notably, infliximab treatment had no impact on serum levels of IL-6 (Fig. 5A), and thus not generally affected proinflammatory cytokine production. As expected, serum IL-1β was not detectable by ELISA. Consistent with our hypothesis, NLRP3 mRNA expression was significantly decreased in the liver and adipose tissues (s.c. adipose tissue, epididymal adipose tissue, and mesenterial adipose tissue) of aged infliximab-treated mice compared with the control-treated group (Fig. 5B). Of note, inhibition of NLRP3 expression was not due to a decrease in macrophage infiltration in these tissues (Fig. 5C). In fact, normalizing NLRP3 expression to the macrophage-specific marker Emr1 (F4/80) still displayed a significant decrease in NLRP3 expression in these tissues in the presence of TNF blockade (Fig. 5D). Thus, antagonizing TNF reduces the expression of the inflammasome sensor NLRP3 in the liver and adipose tissues preferentially by reduction of macrophage-intrinsic NLRP3 expression within tissues. In contrast, the mRNA levels of IL-1β and IL-18 were not relevantly changed by infliximab treatment (Supplemental Fig. 1C, 1D).
TNF regulates caspase-1 activity and glucose tolerance in aging
To test, whether the reduction in NLRP3 expression also impacts on tissue specific inflammasome activity, we next analyzed caspase-1 cleavage in untreated and infliximab-treated mice. Application of infliximab did not considerably influence the tissue expression of the inflammasome adaptor protein ASC and procaspase-1 (Fig. 6A–D). However, while active caspase-1 was present in lysates of liver, s.c.-, epididymal- and mesenterial adipose tissue of aged mice, infliximab treatment led to a dramatic inhibition of this enzyme (Fig. 6A–D). In accordance with the reduced presence of activated caspase-1 in adipose tissues and liver, the serum concentration of the caspase-1-processed cytokine IL-18 was also markedly reduced in infliximab-treated mice (Fig. 6E). These results indicated a generally reduced systemic inflammasome activity upon TNF blockade. To address the functional consequences of decreased inflammasome activity in the presence of infliximab, we went on to study glycemic control in the two groups of aged mice. Antagonizing TNF led to a greatly improved glucose tolerance profile that was comparable to young mice (Fig. 6F). Of note, this improved glucose tolerance in infliximab-treated mice could be attributed to an enhanced peripheral insulin activity. Indeed, basal levels of serum insulin were reduced in infliximab-treated mice (Fig. 6G) and blood glucose levels were lower in the infliximab group after an i.p. insulin challenge (Fig. 6G). In summary, these results indicated that downregulation of NLRP3 also translated in reduced NLRP3 inflammasome activation in aged mice, normalizing their glycemic control.
Understanding the pathways initiating and perpetuating sterile inflammation in old age is important to define therapeutic strategies to either inhibit age related inflammation or to impair inflammation dependent metabolic and degenerative disorders in the elderly. In this regard, the NLRP3 inflammasome was recently identified as a key innate sensing pathway that controls age-related systemic low-grade inflammation. To this end, NLRP3 deficiency was shown to protect aged mice from spontaneous caspase-1 activation, peripheral insulin resistance, bone loss, thymic demise and cognitive decline (13). It was speculated, that a variety of DAMPs such as uric acid (29), ATP (30), reactive oxygen species (31), lipotoxic fatty acids (10), cholesterol (32) and ceramides (9) that accumulate during aging might be responsible for triggering NLRP3 in vivo (13). While these considerations provide valid models of NLRP3 activation, it will be difficult to experimentally prove their involvement in vivo. On the other hand, little is known about the endogenous priming signals that are at play in the context of NLRP3-driven sterile inflammatory conditions. Many in vitro models focus on TLR ligands, most prominently LPS, to prime NLRP3 activation. While TLR signaling, e.g., mediated by DAMPs, might be at play in the context of sterile inflammatory conditions in vivo, the role of alternative priming signals has not been explored. Here, we complement previous work (18) and define TNF as a key regulator of NLRP3 expression and inflammasome activity in murine macrophages. In vitro, we observed that TNF was able to induce NLRP3 expression and thus license the NLRP3 inflammasome for subsequent activation. Interestingly, we found this activity to be decoupled from IL-1β secretion, a classical hallmark of inflammasome activation. Moreover, in contrast to TLR4 engagement, TNF was not able to provide a de novo protein expression-independent, rapid priming signal, yet required prolonged activity to facilitate NLRP3 activation. Given the fact that aging-associated inflammasome activation exerts its effects independently of IL-1 (13), we turned our attention to “inflammaging,” a condition that has already been associated with increased TNF production and inflammasome activation, yet independence of IL-1. In fact, systemic TNF levels steadily increase during lifespan (27), reaching high concentrations in aged individuals (24, 25). In line with these data, we detected augmented TNF levels in the serum of aged mice that were fed on a normal chow diet. In aged mice, elevated serum TNF concentrations were associated with increased mRNA levels of NLRP3 and processed caspase-1 in the liver and s.c. and visceral adipose tissues. These results suggested that TNF governs the expression and thus activity of NLRP3 in tissues that are central for metabolic inflammation in vivo. Corroborating this assumption, we found that neutralizing TNF resulted in a decrease in macrophage intrinsic NLRP3 expression in metabolic tissues, attenuated inflammasome activation and improved glycemic control.
Adipose tissue macrophages (ATMs) represent the greatest fraction of leukocytes in adipose tissue (33) and they are responsible for the majority of TNF expression within adipose tissue of obese mice (6). Indeed, they might also be responsible for elevated systemic TNF levels in aged individuals since the adipose tissue fraction increases during the lifespan (33). The elevated local and systemic proinflammatory adipokine profile during age-induced obesity is determined by a quantitative increase and phenotypic change in ATMs. Progressive lipid accumulation and lipotoxic effects within ATMs heralds switching of macrophages from an anti-inflammatory M2 to a proinflammatory M1 phenotype (34), which furthermore promotes TNF and the expression of other proinflammatory cytokines. In line with this, NLRP3 mRNA expression also correlates with body weight in experimental models of calorie-restricted mice (9).
Treatment with TNF inhibiting agents has already been shown to block metabolic and aging-associated sterile inflammation leading to metabolic syndrome. In patients with severe and chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, anti TNF treatment significantly reduces the incidence of type 2 diabetes (35). Furthermore, long-term anti-TNF treatment improved insulin resistance in patients with rheumatoid arthritis (36, 37). However, short term TNF blockade in patients with metabolic syndrome or overt type 2 diabetes could not demonstrate beneficial effects on glucose metabolism (38). This indicates that the severity of inflammation and the duration of treatment might play a pivotal role for therapeutic efficacy. Given the critical role of the NLRP3 inflammasome in this disease entity, we consider it quite likely that anti-TNF therapy exerts its effects, at least in part, by blocking inflammasome activation in these patients. Yet it cannot be excluded that inhibition of TNF exerts additional beneficial effects that go beyond the inhibition of inflammasome activation and species specific differences in the metabolic rate, environmental factors and alterations at every level of glucose regulation complicate translation into human biology (39).
In summary, the results presented here implicate that TNF-driven NLRP3 expression constitutes an important regulator of inflammasome activity in metabolic tissues in a murine model of aging. Of note, downstream effects of inflammasome activation not only include IL-1β and IL-18 secretion but also eicosanoid production and pyroptosis and its associated proinflammatory effects. Given these pleiotropic signals, it appears plausible that metabolic derangement encountered in the context of inflammasome activation is mediated by multiple, redundant pathways, rather than a single signaling cascade. As such, targeting upstream events such as proinflammatory signals that license inflammasome activation could be an attractive therapeutic strategy in these diseases next to inhibiting inflammasome activation itself.
We thank Silke Hegenbarth for technical support.
This work was supported by grants from the German Research Foundation (SFB704 and SFB670) and the European Research Council (ERC-2009-Stg 243046) to V.H. V.H. is a member of the Excellence cluster ImmunoSensation. This work is part of the doctoral thesis of S.N.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.