Chapter1: The Role Of Inflammasomes in Glomerulonephritis

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Abstract: The inflammasome is an immune multiprotein complex that activates pro-caspase 1 in response to inflammation-inducing stimuli and it leads to IL-1β and IL-18 proinflammatory cytokine production. NLRP1 and NLRP3inflammasomes are the best characterized and they have been related to several autoimmune diseases. It is well known that the kidney expresses inflammasome genes, which can influence the development of some glomerulonephritis, such as lupus nephritis, ANCA glomerulonephritis, IgA nephropathy, and anti-GBM nephropathy. Polymorphisms of these genes have also been described to play a role in autoimmune and kidney diseases. In this review, we describe the main characteristics, activation mechanisms, regulation, and functions of the different inflammasomes. Moreover, we discuss the latest findings on the role of the inflammasome in several glomerulonephrites from three different points of view: in vitro, animal, and human studies.

Keywords: inflammasome; NLRP3; glomerulonephritis; innate immunity

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1. The Inflammasome

The immune system is composed of two arms, the innate and adaptive immunity, that are responsible for both immediate and long-term immunity to pathogen-and non-pathogen-derived antigens. Innate immunity detects infections, changes in cellular homeostasis, and tissue damage, subsequently generating inflammation, tissue repair, and homeostatic balance restoration. These effects are promoted by the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs and DAMPs bind to pattern recognition receptors, which include Toll-like receptors (TLRs), cytoplasmic NOD-like receptors (NLRs), and are absent in melanoma 2-like receptors (AIM2). Previous studies have demonstrated the role of several members of the NLR family in the formation of inflammasomes, multiprotein complexes capable of recognizing inflammation-inducing stimuli. These complexes activate pro-caspase-1, which is responsible for the cleavage of multiple substrates, mainly the pro-inflammatory cytokines IL-1β and IL-18. The release of these cytokines by the inflammasome can also be carried out through an inflammatory form of programmed cell death named pyroptosis [4]. Therefore, the activation of the inflammasome develops innate immunity activity in response to tissue infection. The noninfectious stimulus can also activate the inflammasome. Although inflammasomes can be activated by many members of the NLR family, this review will focus mainly on the NLRP3 inflammasome and NLRP1, NLRC4, and proteins absent in melanoma 2(AIM2)(Figure la), also important in many immune diseases. Furthermore, the role of these complexes in different glomerulonephritis will be reviewed.

Figure 1. Inflammasome structure and mechanism of activation. (a) Schematic representation of NLRP3 inflammasome assembly and detailed confirmation of NLRP3 scaffold, adaptor apoptosis speck-like protein(ASC), and the effector procaspase-1.(b)Structure of NLRP1, NLRP3, NLRC4, and AIM2 which participate in the formation of the main inflammasomes. NLR family members (NLRP1, NLRP3, NLRC4) contain leucine-rich repeats(LRR)and a central nucleotide-binding domain (NBD). The N-terminal PYD domain is present in NLRP subfamily members, whereas NLRC4 presents a CARD domain. NLRP1 also contains a C-terminal extension containing a function-to-find domain (FIIND) and a CARD domain. AIM2 is composed of an N-terminal PYD domain and a C-terminal HIN(hematopoietic, interferon-inducible, and nuclear localization) domain. (c) NLRP3 activation pathways and effector functions. NLRP3 inflammasome assembly can be triggered in several ways: PAMPs and DAMPs detection via PRRs, cytokine stimulation via IL-1 receptor(IL-1R), or through TNF link to tumor necrosis factor (TNF)receptors TNFR1 and TNFR2. These elements trigger the transcription of NF-kB, which promotes the transcription of NLRP3 and IL1Bgenes; this is the first signal or priming. The second signal or activation can be produced by ionic flux, K efflux, Ca2+influx, Nat influx, and Cl efflux, reactive oxygen species (ROS), and mitochondrial dysfunction or lysosomal damage. NLRP3 inflammasome assembly provokes IL-1β and IL-18 cytokines'proteolytic maturation, which also participate in autoimmunity development and pyroptosis by the action of gasdermin-D. Protein myeloid differentiation primary response 88, MyD88; apoptosis signal-regulating kinase, ASK; kinases interleukin 1 receptor-associated kinase, IRAK; caspase-8, CASP8; Fas-associated protein with death domain, FADD; P2X purinoceptor 7, P2X7R; transient receptor potential melastatin, TRPM; transient receptor potential vanilloid, TRPV. Figure 1 has been created with BioRender.com.

2. NLR Family Inflammasomes

The NLR family comprises 23 human genes. Members of this family show common structural elements: C-terminal series of leucine-rich repeats (LRRs) and central nucleotide-binding domains (NBD), a component of the larger NACHT domain. Furthermore, NLR family members can be divided into different subfamilies depending on their N-terminal effector domain: caspase-activation and recruitment domain (CARD), baculovirus inhibitor of apoptosis protein repeat (BIR), or pyrin domain (PYD). The NLRP and NLRC subfamilies are the most important, the former being the best-characterized subfamily of NLRs. The NLRP subfamily members have PYD domains at their N-terminal while the NLRC proteins have one or more CARD domains [8-10]. NLR family members NLRP1, NLRP3, and NLRC4 have been the best studied in inflammasome formation.

2.1.NLRP Subfamily

The NLRP subfamily is composed of 14 members in the human genome, plus 3 paralogs in mice being NLRP1(NALP1/CARD7)the first to be described in forming inflammasomes. Its structure consists of an N-terminal PYD followed by a NACHT domain and LRRs. This is also contributed by a C-terminal extension containing a function-to-find domain (FIIND), which auto processes NLRP1 into two polypeptide chains, and a CARD domain, that leads to caspase-1 activation and the consequent pro-inflammatory cytokine release. It has been reported that NLRP1 mutations can play a role in inflammatory diseases such as psoriasis [15], rheumatoid arthritis (RA)[16], or in systemic lupus erythematosus (SLE).

NLRP3 inflammasome(Cryopyrin/Nalp3/Cias1/Pypaf1)is the most widely studied and is the only known member to be activated by numerous pathogenic and sterile inflammatory signals. Furthermore, NLRP3 plays a role in the regulation of IL-1β production in macrophages [18,19]. NLRP3 is composed of the NLRP3 scaffold, an adaptor apoptosis speck-like protein (ASC), and the effector procaspase-1. It interacts with ASC via PYD-PYD homotypic interactions to promote the formation of the inflammasome by recruiting and activating procaspase-1 to generate active caspase-1(Figure lb). This effector protein leads to the conversion of the cytokine precursors pro-IL-1β and pro-IL-18 into mature and biologically active IL-1β and IL-18. The main attention given to the NLRP3 inflammasome has been due especially to its implication in the pathogenesis of several human inflammatory diseases, particularly the cryopyrin-associated periodic syndromes(CAPS)[21]. Focusing on its critical role in regulating inflammation, the NLRP3 inflammasome could be of great importance to therapies targeting inflammation.

2.2. IPAF-NAIP Subfamily

Its most well-studied element, NLRC4(IPAF/CARD12), was previously characterized as an ICE-protease activating factor (IPAF)regarding its capacity for activating caspase-1. Nevertheless, posterior studies clearly placed its domain structure in the NLR family, and as it possessed a CARD domain, it was renamed NLRC4. The CARD domain allows it to directly bind to the CARD of caspase-1 without the participation of ASC [24]. However, NLRC4 is able to bind to ASC and efficiently activate caspase-1, as well as caspase-8, and apoptotic caspase.

NLRC5 is a less well-known inflammasome that links both innate and adaptive immune responses by regulating major histocompatibility complex(MHC) I class expression. It is expressed in macrophages, dendritic cells, T cells, B cells, and fibroblasts [27]. Moreover, an observed interaction with the NLRP3 inflammasome seemed to have a synergistic effect on IL-1β cleavage, thus it may positively modulate NLRP3 inflammasome activation [28]. Therefore, NLRC5 could form a functional inflammasome, but more studies are needed to know its physiological function more accurately.

Additionally, NOD1 is the founding member of the NLR family, and together with NOD2, they were the first NLRs identified as sensors for PAMPs [29]. NOD1(NLRC1)and NOD2(NLRC2)receptors can activate NF-kB and lead to the production of inflammatory cytokines. Nevertheless, they have not been described to form an inflammasome complex.

3. Non-NLR Family Inflammasomes

Recently, other inflammasomes not belonging to the NLR family have been widely described, such as the proteins absent in melanoma 2(AIM2) and pyrin inflammasomes. AIM2 was described as a sensor able to trigger inflammasome activation, pyroptosis, and release of IL-1β and IL-18 in response to intracellularly delivered double-stranded DNA (dsDNA) detection [31].AIM2 is a member of the ALR family of proteins, composed of an N-terminal PYD domain and a C-terminal HIN(hematopoietic, interferon-inducible, and nuclear localization) domain. Moreover, it negatively regulates inflammation and type I interferon (IFN) responses independent of its inflammasome function [33]. Different studies have elucidated a link between increased AIM2 expression and several human diseases, such as atherosclerosis, skin disease, and chronic kidney disease.

4. Mechanisms of NLRP3 Inflammasome Activation

The inflammasome can be understood as a two-sided element and it regulates pathogen infection, but when the immune response triggered is not tightly regulated, it can be involved in pathologies such as CAPS and autoinflammatory disorders [21]. Inflammasomes can recognize a wide variety of endogenous or exogenous, sterile or infectious stimuli within the cell (PAMPs and DAMPs), which trigger its assembly and activation. This process can be explained by considering the upstream sensors recognizing activating signals, the adapters and the downstream effectors ]35. The unfeasibility of direct interaction between NLRP3 and this diversity of stimuli led to a cellular event producing a conformational change in NLRP3, converting it into an active form. Nevertheless, there is no unique mechanism for the activation of the NLRP3 inflammasome 36]. NLRP3 activation can be triggered by PAMPs and DAMPs detection via PRRs, such as TLRs and NLRs, by cytokine stimulation via IL-1 receptor (IL-1R), or through TNF link to tumor necrosis factor (TNF)receptors TNFR1 and TNFR2 [37]. Moreover, there are mediators that facilitate signal transduction of these receptors: the adaptor protein myeloid differentiation primary response 88(MyD88), the apoptosis signal-regulating kinase(ASK)1 and ASK2, interleukin 1receptor-associated kinase (IRAK)1 and IRAK4, caspase-8(CASP8), Fas-associated protein with death domain (FADD),ubiquitin-binding protein SHARPIN and TRAF-interacting protein with forkhead-associated domain (TIFA). All these elements trigger the transcription of NF-kB, which promotes the transcription of NLRP3 and IL1Bgenes, habilitating the cell for responding to NLRP3 activators.

NLRP3 inflammasome activation in macrophages is a two-step process, thus it requires a priming signal. In the priming process, a non-activating stimulus causes the transcriptional expression of the main components of the inflammasome, this being the first hit'. Second stimuli or 'second hit' aggravates the functional activity of the NLRP3 inflammasome [39]. Activation of the NLRP3inflammasome can be produced by different stimuli, including ionic flux, K+efflux, Ca2+influx, Na+influx and Cl- efflux, reactive oxygen species (ROS), and mitochondrial dysfunction or lysosomal damage. K+efflux channels P2X purinoceptor 7(P2X7R) participate in this type of inflammasome activation. Other plasma-membrane-resident Ca2+ channels, namely transient receptor potential melastatin 2 (TRPM2), TRPM7, and transient receptor potential vanilloid 2 (TRPV2), can lead to Ca2+influx to the cytosol [21](Figure lc). Mitochondria regulate homeostasis and respond to changes in intracellular K+ and ROS, resulting in mitochondrial dysfunction and apoptosis. Additionally, mitochondrial apoptotic signaling stimulated by NF-kB causes the production of IL-1β. Oxidized mitochondrial DNA is released as a consequence of mitochondrial dysfunction and apoptosis, and it directly activates the NLRP3 inflammasome [40-42].

Apart from the NLR-ASC-caspase-1 canonical inflammasome activation, there is also a non-canonical inflammasome characterized by its activation via caspase-1l in mice with the human orthologs caspase-4/5. Caspase-11 recognizes lipopolysaccharide(LPS) transfected into the cytosol from Gram-negative bacteria, directly binding to its CARD domain. It initiates proteolytic maturation of IL-1β as well as pyroptotic cell death in a GSDMD-dependent manner [35,43]. Non-canonical inflammasome activation by a component of LPS was shown in a previous study where mice lacking caspase-1l were resistant to LPS-induced lethality, even in the presence of TLR4 [44]. However, from the canonical and non-canonical inflammasomes, an alternative pathway of inflammasome activation was observed. It does not require K+ efflux, induction of ASC speck formation, or leading to subsequent pyroptosis, and was spread by TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3.

NLRP3 inflammasome can be regulated at a post-transcriptional and post-translational level. At the post-transcriptional level, epigenetic factors such as DNA methylation and histone acetylation can regulate NLRP3 mRNA expression in response to Mycobacterium tuberculosis infection. Dysregulation of epigenetic mechanisms could contribute to the pathological development of autoinflammatory syndromes by upregulating the expression of inflammasome components. MicroRNAs are also studied as post-transcriptional regulators of NLRP3 inflammasomes (miR-223, miR-133a, miR-7, miR-30e..)[35,47]. NLRP3 inflammasome activation can also be regulated by post-translational modifications, mainly phosphorylation, and ubiquitination. These modifications are often linked. They can provoke different fates on the NLRP3 protein, including the modification of interacting protein networks, trafficking, change in subcellular localization, activation/inhibition of enzymatic activity, and proteasomal, lysosomal, or autophagic degradation [48]. In fact, a recent study showed that NLRP3 phosphorylation in its LRR domain can regulate inflammasome assembly.

5. Inflammasome Effector Functions

As previously stated, inflammasomes play a crucial role in the innate immune system by their ability to control the activation of the proteolytic enzyme caspase-1, which leads to proteolytic maturation of the proinflammatory cytokines IL-1β and IL-18, as well as pyroptosis cell death [50]. Mature IL-1β binds to IL-1R, promoting the heterodimerization of the receptor and the subsequent recruitment of components such as MyD88. IL-1βleads to the release of other cytokines such as IL-lα, IL-6, and TNF-α as well as other factors that control the growth and differentiation of immune cells. IL-18 participates in many physiological pathways. A higher level of IL-18 can cause metabolic syndromes. For instance, chronic inflammation generated in adipose tissues can lead to insulin resistance and type 2 diabetes mellitus.

Another important process carried out by inflammasomes is a lytic form of programmed cell death named pyroptosis. Both canonical inflammasome signaling, recruiting caspase-1, and noncanonical inflammasome, via caspase-4, caspase-5(in humans), and caspase-11 (in mice), can trigger pyroptosis. It is characterized by cell swelling, mem-brane lysis, and the release of inflammatory compounds into the extracellular space, such as IL-1β, IL-6, and IL-18. Previous studies have shown that masterminds, a group of pore-forming effector proteins, can play inflammatory caspase-induced pyroptosis, being the N-terminal domain of gasdermin-Dsufficient to trigger the process. Additionally, caspase-8-dependent apoptosis is an additional pathway resulting from inflammasome activation. AIM2 and NLRP3 inflammasomes showed cleaved forms of apical caspase-8 and executioner caspase-3, in response to cytosolic DNA and nigericin, respectively. The process occurred independently from caspase-1 but depended on the inflammasome adapter ASC. Interestingly, a recent study described the capacity of the Z-DNA binding protein 1(ZBP1), an innate immune sensor capable of activating cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis) together with the NLRP3 inflammasome.

Whereas the effector functions have been widely studied, there are several additional roles of the inflammasome complexes that have been less characterized. IL-1β is a leader-less cytoplasmic protein whose secretion mechanisms are poorly defined. An endoplasmic reticulum(ER)/Golgi-independent mechanism termed 'unconventional protein secretion was shown, and it was dependent on caspase-1 activation. However, the specific mechanisms and molecular components involved in this process are unclear. Another emerging role of inflammasomes is the activation of eicosanoids, bioactive molecules derived from membrane lipids that play a role in homeostatic and pathological processes. Furthermore, a link between inflammasome activation and autophagy as well as regulation of phagosome maturation has been observed.

6. The Role of the Inflammasome in Adaptive Immunity and Autoimmunity

The production of proinflammatory cytokines is critical for an effective innate response, as well as a mechanism by which the innate immune system influences the subsequent development of an adaptive immune response. As it is well known, inflammasomes are components of the innate immune system that produce the pro-inflammatory cytokines IL-1β and IL-18, and they drive the differentiation of specific lineages of helper T cells (Th1, Th2, Th17, and regulatory T cells), which are the main players in adaptive immunity [60]. On the other side, an aberrant inflammasome activation is responsible for the development of CAPS, as well as other common diseases such as metabolic disorders, crystal-related diseases, and autoimmune diseases. Inflammation is also crucial in many renal diseases, including acute kidney injury (AKI) and chronic kidney disease(CKD). Although the innate immune system is always involved, in these conditions, adaptive immunity plays the main role.

Concerning autoimmune diseases, they are characterized by self-reactive cells and the overproduction of autoantibodies, produced because of a lack of immunological tolerance and aberrant autoreactive immune responses. The pathogenesis of autoimmune diseases remains to be clarified, but it has been demonstrated that aberration in innate and adaptive immunity is involved. NLRP3 inflammasome has been recently linked with innate immune signal recognition and induction of autoreactive immune responses, probably being a checkpoint in innate immunity to cause distorted adaptive immunity [62,63]. Therefore, how can the NLRP3 inflammasome function affect the development of autoimmune diseases? Cytokines released by the inflammasome, especially IL-1β, produce an inflammatory effect that promotes the development of most autoimmune diseases, including RA and inflammatory bowel disease. Furthermore, the NLRP3 inflammasome is also responsible for autoimmune diseases due to an adaptive immune dysfunction. IL-1 mediates T cell proliferation, thus it can promote autoreactive T cells to cause b-cell death [63,66]. NLRP3 inflammasome promotes Th1 differentiation in RA, induced by IL-1β in a caspase-1-dependent manner, and it can also induce differentiation and polarization of Th2, Th17, and dendritic cells in other autoimmune diseases [63,67]. Th17 cells can produce proinflammatory cytokines namely IL-17A， IL-17F， IL-21, and IL-22， while Th1 cells secrete IFN-γ， induced by IL-18, and all of these factors contribute to autoimmunity development. Additionally, autoimmune diseases can be promoted by pyroptosis, leading to the release of cellular debris and its reaction with immune cells, triggering inflammation.

Indeed, multiple polymorphisms in inflammasome genes have been associated with the susceptibility and development of autoimmune diseases. For instance, rare gain-of-function variants can be implicated in hereditary inflammatory diseases, characterized by uncontrolled production of IL-1β and/or IL-18, named inflammasomopathies. Mutations in NLRP3 are the prototypic inflammasomopathy, but they have also been described as autoinflammatory diseases associated with mutations that activate the NLRP1, NLRC4, and pyrin inflammasomes [69,70]. Moreover, single nucleotide polymorphisms (SNPs) play a crucial role in autoimmune diseases, and they can affect the priming of inflammasomes, some of their components, or end products (IL-1β, IL-18).