Resumen de Regulación de la activación del inflamasoma nlrp3 por lipina-2 en macrófagos
The innate immune system constitutes the first line of defense against invading microbial pathogens, as it can discriminate self from non-self. It can also sense endogenous danger signals that are released from disrupted host tissue or stressed cells. Macrophages, cells of the innate immune system, are able to recognize PAMPs and DAMPs by different receptors that modulate inflammation through transcriptional (TLRs) and post-transcriptional (NLRs) mechanisms. Key players in those pathways are large intracellular multiprotein complexes called inflammasomes. After exposure to pathogens and danger signals, inflammasomes orchestrate innate immune responses through activation of caspase-1 leading to the maturation of pro-inflammatory cytokines pro-IL-1β and pro-IL-18 and an inflammatory form of death known as pyroptosis. NLRP3 inflammasome is the best characterized to date. It is composed of an NLR receptor, the adaptor protein ASC and pro-caspase-1. In macrophages, its activation and assembly need two signals. The first signal, also known as priming, leads to transcription of genes encoding pro-IL-1β and NLRP3 and it is mediated by activation of the transcription factor NF-κB through various receptors, such as TLRs, IL1R or TNFR. The second signal, provided by DAMPs, activates the inflammasome assembly, resulting in the catalytic processing of pro-caspase-1 into its enzymatically active form. NLRP3 inflammasome is activated by a wide range of stimuli, such as bacterial and viral pathogens, pore-forming toxins, lipids, crystals, vaccine adjuvants and stress cellular signals such as ATP. It is broadly agreed that detection of such a diverse variety of agents cannot bind to NLRP3 directly. Instead, it is thought that NLRP3 monitors a common host-derived factor triggered by all these agents. Several hypotheses on molecular mechanisms leading to NLRP3 activation have been formulated. Three models that may not be exclusive are the most widely accepted. The first model suggests that a decrease in intracellular potassium concentration causes inflammasome activation, the second model points at mitochondrial damage and ROS production, and the third model claims that lysosomal destabilization is the mechanism triggering caspase-1 activation.
Extracellular ATP has been widely used as an inflammasome inducer and exerts its biological action through purinergic P2 receptors such as P2X7R. P2X7R are non-selective cationic channels, abundantly expressed in macrophages, whose activation mediates an influx of Na+ and Ca2+ into the cytosol and the concomitant efflux of K+. ATP binding to P2X7R promotes a decrease in intracellular K+ levels triggering inflammasome assembly, but high concentrations of ATP (in the mM range) are required to elicit NLRP3 activation in a P2X7R dependent manner. P2X7R are located in lipid rafts, membrane microdomains rich in cholesterol and sphingolipids, and their function is tightly regulated. Cholesterol acts as a negative regulator of their activity, and protects cells from P2X7-dependent death.
FFA triggered inflammation has been proposed to be involved in the development of metabolic diseases such as obesity and T2D. Elevated FFA levels in circulation may contribute to these disorders by activating the NLRP3-ASC inflammasome, triggering caspase-1, IL-1β and IL-18 production. The underlying mechanism may be the following: FFAs induce pro-IL-1β production through TLRs and at the same time promote caspase-1 by increasing mtROS through intracellular AMPK inhibition.
Dysregulation of the NLRP3 inflammasome is associated with a wide spectrum of autoinflammatoy diseases. These diseases are characterized by recurrent fevers and unprovoked episodes of inflammation, in which the monocyte-macrophage is the dysfunctional cell and auto-reactive T-cells or auto-antibodies are absent. Furthermore, autoinflammatory disorders are driven by IL-1 oversecretion as IL-1, in particular IL-1β blockade improves clinical symptoms in patients. However, TNF-α neutralization, a highly effective therapy for autoimmune diseases, poorly controls autoinflammatory disorders.
Of all autoinflammatory diseases characterized to date, it is important to highlight the Majeed syndrome, whose molecular mechanism does not lie in mutations of inflammasome proteins or IL-1 biology, but in mutations of lipin-2, a key enzyme in lipid metabolism.
Lipin-2 is a member of a family of proteins, the lipins, which catalyze the enzymatic conversion of phosphatidic acid to diacylglycerol, a direct precursor of triacylglycerol and some phospholipids. There are three members in this family (lipin-1, lipin-2 and lipin-3) and all of them exhibit Mg2+-dependent PAP activity. Lipins are cytosolic enzymes that translocate onto the endoplasmic reticulum to participate in the glycerolipid biosynthesis pathway or onto the nucleus, to act as transcriptional coactivators. Structurally, these proteins possess two highly conserved domains, N-LIP and C-LIP. The C-LIP domain contains the PAP enzyme active site motif (DXDXT) as well as the LXXIL motif, which has been shown to mediate interaction of lipin with transcriptional activators and coactivators. Members of the lipin family exhibit different tissue expression patterns; lipin-1 is mainly expressed in adipose tissue and skeletal muscle, whereas lipin-2 is mainly expressed in the liver. Lipins have also been found in macrophages, where lipin-1 and lipin-2 play opposite roles in the inflammatory response. It has been described that lipin-1 mediates pro-inflammatory responses through the overactivation of downstream pathways during TLR4 activation. However, lipin-2 has a protective role in macrophages, which depends on the overstimulation of the JNK pathway by saturated fatty acids.
Mutations found in the human LPIN2 gene have been linked to inflammatory based disorders. In particular, four independent LPIN2 mutations are known to cause Majeed syndrome, an autosomal recessive disease first described in 2001 that falls into the category of autoinflammatory disorders. Majeed syndrome starts during infancy and its phenotype includes chronic recurrent multifocal osteomyelitis, congenital dyserythropoietic anaemia, recurrent fevers and inflammation of the bone and skin. Majeed syndrome patients are weakly responsive to treatment with corticosteroids and TNF-α inhibitors but IL-1β blockade is accompanied by symptomatic improvement on clinical and biological parameters. These data revealed that IL-1β plays a key role in the development of the disease.
Studies in humans to elucidate the underlying mechanisms by which mutations in lipin-2 cause this syndrome are limited. Since IL-1β seems to be involved in this process, it was hypothesized that lipin-2 could modulate its production and thereby be involved in the regulation of inflammasome activity.
Taking into consideration all of these previous results, the principal objective in this thesis was to assess the role of lipin-2 in the inflammasome activation in vitro and in vivo.
The purpose of the present study was to explore the role of lipin-2 on inflammasome activation. To accomplish this objective, a classic model of NLRP3 inflammasome activation was used. Priming of macrophages and inflammasome assembly were achieved by LPS and exogenous ATP-induced activation of the P2X7R, respectively.
1. Involvement of lipin-2 in inflammasome priming (first signal) defining its effects on cell signaling, in mouse and human macrophages Inflammasome activation and subsequent IL-1β production has two important checkpoints, priming (signal 1) and assembly (signal 2), but the mechanistic details of those events still remain poorly characterized.
After testing the production of IL-1β in macrophages deficient in lipin-2 treated with LPS (200 ng/ml, 4 h) and ATP (2 mM, 40 min), it was observed that IL-1β release was augmented when lipin-2 was reduced or absent. These data suggest that lipin-2 modulates NLRP3 inflammasome activation, and an important next step would be to determine at which checkpoint lipin-2 was involved in IL-1β production.
To further explore the role of lipin-2 in IL-1β production, experiments were performed to look at whether lipin-2 could be involved in TLR4 signaling pathways during the priming step.
The first goal was to determine whether lipins could be implicated in the up-regulation of pro-IL-1β by LPS. It is well established that the transcription of pro-IL-1β is induced by the activation of the transcription factor NF-κB. To assess whether NF-κB activity was altered when lipin-2 was absent, nuclear expression of this transcription factor was analyzed, and it was found that 30 minutes after LPS stimulation, the induction of nuclear translocation of NF-κB was more marked in cells lacking lipin-2. To determine whether a higher activation of NF-κB in these cells was parallel to a larger induction of pro-IL-1β, protein and mRNA levels were quantified by western blot and RT-q-PCR respectively. As expected, the analysis showed a pronounced increase in pro-IL-1β expression that was higher in the absence of lipin-2. In addition, RT-q-PCR analysis also revealed that the mRNA expression of NLRP3 was increased in cells without lipin-2.
In line with the previous results, the role of lipin-2 in LPS activation was further confirmed by quantitative analysis of proinflammatory cytokines. It was observed that the inhibition of lipin-2 expression led to a significant induction of Tnfa and Il6 gene expression, and ELISA measurements verified that Tnfa increased mRNA expression by LPS in cells lacking lipin-2 resulted in an increased production of its protein levels. Taking these data into consideration, TNF-α overproduction seen in cells without lipin-2 could act as a priming step through TNFR1 and TNFR2, and participate in the higher IL-1β generation reported in the absence of lipin-2.
Collectively, these data suggest that lipin-2 depletion increases pro-IL-1β and NLRP3 production, possibly through the overactivation of NF-κB as well as modulates proinflammatory cytokine levels. However, the fact that lipin-2 plays an important role in transcriptional regulation of NLRP3, does not discard its involvement in NLRP3 deubiquitination, a very important process for inflammasome activation.
MAPKs are also activated through TLR4 receptors and they also seem to play a role in inflammasome activation. For this reason, the effect of lipin-2 depletion on the level of phosphorylation of members of the MAPKs pathway was studied by western blot. It was found that treatment of macrophages lacking lipin-2 with LPS significantly increased the phosphorylation of JNK, ERK and p38 compared to wt, suggesting that lipin-2 may negatively regulate MAPKs activation by LPS. The fact that the absence of lipin-2 increases MAPKs activation may be related to an enhanced inflammasome activation in those cells, probably due to an increase in speck formation through ASC phosphorylation by JNK.
2. Involvement of lipin-2 in inflammasome activation (second signal), defining its effect on P2X7 receptor activity, potassium efflux, ASC oligomerization, caspase-1 activation and pyroptosis Once it was determined that lipin-2 has a key role during the first step, experiments were performed to look at its involvement in the second signal. Analysis of IL-18 production, a constitutively expressed cytokine and whose maturation depends on inflammasome activation, showed an important increase in LPS primed cells lacking lipin-2 after stimulation with ATP, which suggested that lipin-2 could be implicated in caspase-1 activation (second signal).
Macrophage treatment with ATP causes activation of purinergic receptor P2X7. To determine whether lipin-2 interferes with that process, patch-clamp whole-cell configuration studies were carried out. Application of ATP dramatically enhanced the amplitude of the inward and outward currents through P2X7R in cells lacking lipin-2. These data suggest that ATP promotes the P2X7R opening to a pore dilated state that is larger when lipin-2 is absent. In addition, these data were confirmed by the use of NMDG+ as the extracellular charge-carrying ion.
As cellular ion gradients control inflammasome activation, intracellular K+ concentration was measured. Application of ATP led to an important drop in the intracellular content of K+, which was found to be more marked when lipin-2 was absent. Moreover, the significant decrease in the intracellular amounts of this ion elicited by ATP were analyzed at different time points and remained lower overtime if lipin-2 was not present. These data show that lower intracellular concentration of K+ may be responsible for the enhanced NLRP3 inflammasome activation and concomitant IL-1β overproduction observed in primed macrophages lacking lipin-2 after ATP treatment. Moreover, other cellular events triggered during the second step of inflammasome activation were investigated and higher ASC oligomerization, greater activation of caspase-1 and enhanced membrane permeabilization, a process that precedes pyroptosis, were detected in lipin-2 deficient macrophages. Collectively, these data suggest that absence of lipin-2 leads to overstimulation of the NLRP3 pathway, triggering IL-1β and IL-18 production in an ASC and caspase-1 dependent manner, effects that may be due to the fact that absence of lipin-2 leads to an increase of ATP receptor activity, triggering a higher K+ efflux from the cell and in turn, increasing the assembly of the inflammasome and its activity.
3. Effect of cholesterol on inflammasome overactivation in lipin-2 deficient macrophages Lipin-2 deficient cells were found to have lower cholesterol levels than control cells. In order to determine whether these decreased levels were related to the finding that absence of lipin-2 triggers NLRP3 inflammasome hyperactivation, cellular cholesterol levels were increased by preincubating cells with 100 µg/ml cholesterol (complexed with MCD to make it water-soluble). Once it was determined that cholesterol levels were similar in cells with and without lipin-2, different events triggered by inflammasome activation were studied: K+ efflux, ASC oligomerization, caspase-1 activity and IL-1β production. Responses mediated by P2X7 receptors after ATP application, such as ionic currents, were diminished by cholesterol loading. Furthermore, it was also observed that ATP treatment of LPS primed macrophages after incubation with cholesterol-loaded MCD produced a profound inhibition of ASC oligomerization, caspase-1 activity and IL-1β production. Taken together these results suggest that restored cholesterol levels in cells lacking lipin-2 is essential to reduce inflammatory responses associated to NLRP3, as decreased levels of this lipid seem to play a key role during the activation of P2X7R by ATP, participating in NLRP3 inflammasome activation.
In this scenario it is likely that lipin-2 is necessary for maintaining cholesterol homeostasis. Lipin-2 may modulate cholesterol levels through HMGCR, the key enzyme in cholesterol synthesis or through cholesterol efflux by regulating ABCA1 and ABCG1. However, since lipin-2 is a lipid metabolic enzyme, it can also be speculated that lipin-2 deficiency leads to perturbations in the bilayer structure in the immediate lipid environment of the P2X7 receptor, enhancing its activity through modifications in its gating properties. Lipin-2 absence could alter ceramide and lysolipid levels and thereby alter membrane structure and fluidity. In the same line of thinking, modification in DAG and PA levels, phospholipids precursors which are control by lipins, might also contribute to stabilize the dilated state of P2X7R pore.
In recent years, the lipid environment has been considered to be crucial for the maintenance of the structure and function of membrane proteins, a fact that has led to the development of new methods to understand lipid interactions. Among these methods, high resolution mass spectrometry stands out, which is emerging with the full potential to monitor different modes of lipid binding to membrane proteins. Studying the interaction between P2X7R and surrounding lipids in the presence and absence of lipin-2 could reveal new data about how this enzyme regulates cholesterol homeostasis and the inflammatory response.
4. Role of lipin-2 during in vivo treatment with LPS One of the differences between humans and mice with mutations in lipin-2 is that mice do not develop the Majeed Syndrome. Although lipin-2 knock-out mice did exhibit features of the disease such as anemia, they failed to show evidences of osteomyelitis. These data suggest that other components in addition to lipin-2 deficiency may contribute to the appearance of the Majeed syndrome.
Majeed syndrome patients have elevated pro-inflammatory cytokines such as IL-1β in serum infering that inflammasome activity may be altered. As intraperitoneal injection of LPS triggers IL-1β production in a NLRP3 inflammasome dependent manner, a model of septic shock induced by LPS was used to determine whether infection, as an additional factor in mice may contribute to develop this autoinflammatory condition. Mice were injected intraperitoneally with LPS (10 mg/kg) and after 3 hours they were euthanized, and blood was collected to analyze cytokine production in serum. It was observed that mice lacking lipin-2 showed an increased production of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α when compared to wt mice. These data suggest that lipin-2 absence promotes inflammasome overactivation in vivo in a mice model of septic shock. Moreover, the mRNA expression of pro-inflammatory genes in livers and spleens from mice treated with LPS was also analyzed. In lipin-2 deficient livers, LPS promotes a significant increase of pro-inflammatory genes such as Il1b, Il6, Tnfa, Il12b and Nos2 in comparison with wt livers. Additionally, genes encoding inflammasome proteins and IL-1 receptor were also evaluated, and Nalp3 and Il1r gene expression were found to be up-regulated when lipin-2 was absent. In the same way, to test whether lipin-2 contributed to a marked inflammation in spleens following LPS injection, RT-q-PCR analyses were performed, and it was found that Il1b, Tnfa and Nalp3 gene expression was up-regulated in inflamed spleens from Lpin2-/- mice. Moreover, LPS treatment showed that spleens from mice lacking lipin-2 were bigger in size, which could be due to a higher oxidative response in those animals. Since LPS treatment promotes an enhanced pro-inflammatory status in lipin-2 deficient mice, it may also be speculated that infectious agents, such as bacteria or viruses, may constitute an additional environmental factor triggering the Majeed syndrome. This hypothesis was formulated because children are highly exposed to these agents during the first year of life, and if they have mutations in lipin-2, they might develop worse episodes of inflammation leading to the medical profile observed in Majeed syndrome patients. In the same way, the vaccination schedule for infants could also contribute to Majeed syndrome development, as alum salts, one of the main components in vaccines is able to increase IL-1β production and neutrophil recruitment to peritoneal cavity in mice. It would be of great interest to subject mice to the same vaccine schedule as children to evaluate whether vaccines triggers the disease or not. Moreover, children but not mice may also be exposed to UV and other skin irritants, another two NLRP3 inducers that trigger IL-1β production in a NLRP3 dependent manner and could also participate in the development of the clinical symptoms observed in Majeed syndrome patients.
In summary, the activators to which children but not mice are exposed that could be involved in the development of Majeed syndrome should be outlined. Moreover, it will also be useful to elucidate whether NLRP3 alone or in combination with other inflammasomes is responsible for IL-1β overproduction when lipin-2 in absent. Understanding the actual mechanisms involved in the development of this autoinflammatory syndrome could bring new insights into the pathophysiology of the disease and provide highly effective therapies for these patients.
5. Involvement of lipin-2 in metabolic inflammasome activation with palmitic acid in mouse and human macrophages It is important to highlight that palmitic acid has also been described as an NLRP3 inflammasome inducer but differs from other inflammasome activators because it can act as signal 1 and signal 2. Since lipin-2 attenuates the pro-inflammatory response in human macrophages and RAW cells activated by palmitic acid, it was analyzed whether lipin-2 was involved in IL-1β production by this fatty acid. First of all, NF-κB activation was studied by western blot and it was shown that inhibition of lipin-2 expression led to an overactivation of p65 and p50 subunits of NF-κB. In addition, it is worth noting that NF-κB overactivation was accompanied by an increase in Il1b mRNA expression by palmitic acid. Thus, it can also be concluded that macrophage stimulation by palmitic acid promotes IL-1β maturation in a caspase-1 dependent manner, and inhibition of lipin-2 exacerbates such production.
Activation of LPS primed macrophages with palmitic acid led to stronger caspase-1 activation, and IL-1β generation was markedly augmented when lipin-2 was absent. To elucidate the cellular mechanism by which palmitic acid triggers the NLRP3 inflammasome, IL-1β production was assessed after MAPKs inhibition. Palmitic acid enhanced IL-1β secretion in lipin-2 deficient macrophages was effectively blocked after MAPKs inhibition, suggesting that MAPKs might be key players in inflammasome overactivation caused by lipin-2 depletion.
Surprisingly, lipin-2 seemed to play a different role in BMDM during palmitic acid induced inflammasome activation. IL-1β production remained largely unaffected under palmitic acid treatment in lipin-2 deficient BMDM when compared to wt macrophages and was even diminished when LPS primed BMDM were stimulated with palmitic acid. The pathophysiological relevance of these findings may reside in the differential behavior of mouse and human lipin-2 deficient macrophages after palmitic acid treatment, what led to the hypothesis of breastfeeding as a trigger for Majeed syndrome, due to breastmilk is rich in this fatty acid. Taken together, the differences found between human macrophages and BMDMs in IL-1β production in a metabolic context might explain why mice lacking lipin-2 do not develop the Majeed syndrome and humans do.
To further understand the IL-1β generation in mouse and human macrophages by palmitic acid, it would be of great interest to explore non-canonical inflammasome activation by intracellular fatty acids. Non-canonical inflammasome also appears to require two signals: a priming signal that increases the expression of inflammasome components, such as pro-IL-1β and caspase-11 and a second signal to trigger caspase activation. The data obtained in the present work could be explained if palmitic acid fails to produce non-canonical inflammasome activity in LPS primed lipin-2 deficient macrophages but not in wt macrophages or if pro-caspase-11 induction is lower in primed macrophages lacking lipin-2. If that hypothesis were to be confirmed, non-canonical inflammasome could be considered as the underlying mechanism by which palmitic acid exhibits distinct properties in the presence or absence of lipin-2.
To conclude, the results obtained in this work show that lipin-2 is a key regulator during classic activation (LPS and ATP) and metabolic activation (LPS and palmitic acid) of the NLRP3 inflammasome in macrophages. These findings could open new avenues for developing new drugs for the treatment of pathological conditions associated with processes in which IL-1β production is altered.
