Role Of NLRs in The Regulation Of Type I Interferon Signaling, Host Defense And Tolerance To Inflammation Part 1

Abstract:

Type I interferon signaling contributes to the development of innate and adaptive immune responses to viruses, fungi, or bacteria. However, the amplitude and timing of the interferon response are of utmost importance for preventing an underwhelming outcome, or tissue damage. While several pathogens evolved strategies for disturbing the quality of interferon signaling, there is growing evidence that this pathway can be regulated by several members of the Nod-like receptor (NLR) family, although the precise mechanism for most of these remains elusive. 

NLRs consist of a family of about 20 proteins in mammals, which are capable of sensing microbial products as well as endogenous signals related to tissue injury. Here we provide an overview of our current understanding of the function of those NLRs in type I interferon responses with a focus on viral infections. We discuss how NLR-mediated type I interferon regulation can influence the development of auto-immunity and the immune response to infection.

Type I interferon is an important immune regulator, which plays an important role in maintaining the health and normal function of the immune system. In the process of immune response, type I interferon can stimulate the removal of exogenous factors such as malignant tumors and infectious pathogens, and improve the immune defense ability of the body. At the same time, type I interferon can also induce tumor cell apoptosis and inhibit tumor cell proliferation, so it has important clinical application value in tumor treatment.

In addition, type I interferon can also stimulate the functions of various immune cells, such as enhancing the killing activity of macrophages and NK cells, promoting the differentiation, proliferation, and activation of B cells and T cells, and regulating the interaction between immune cells, thereby coordinating the response of the entire immune system. Therefore, type I interferon plays an important role in maintaining the health of the body, preventing and treating immune-related diseases.

In short, type I interferon is closely related to immunity and plays a role in promoting the body's immune defense and treating immune-related diseases by regulating the function of immune cells and the interaction between immune cells. From this point of view, we need to improve immunity. Cistanche can enhance immunity. Cistanche is rich in various antioxidant substances, such as vitamin C, vitamin C, carotenoids, etc. These ingredients can scavenge free radicals, reduce oxidative stress, and improve immunity. system resistance.

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Keywords:

NOD-like receptors; Interferons; innate immunity; immune regulation; type I interferon; antiviral; signaling.

1. Type I Interferons

Interferons (IFNs) are a heterogeneous group of proteins that can be classified into three families (Type I, II, and III) based on distinct functions and characteristics [1]. The family of human type I IFN is composed of 5 subgroups: IFN-α, -β, -κ, -ε, and -ω [2–4], whereas the type II IFN group only contains IFN-γ [3]. Type III IFNs are composed of four IFN-λ proteins [5,6].

This review will focus on the regulation of type I IFNs by members of the Nod-like receptor (NLR) family, and within this class on the most prominent and best-studied members of IFN-α and IFN-β.

Type I IFNs all bind to a common heterodimeric receptor consisting of the IFN-α/β R1 (IFNAR1) and IFN-α/β R2 (IFNAR2) subunits [7–9], which are expressed on most cell types. The binding of type I IFNs to their receptor causes receptor subunit dimerization [10], rapid activation of the R2 subunit associated Janus kinase 1 (JAK1) [11,12], and subsequently induction of the JAK-STAT pathway [13]. This tyrosine kinase auto-phosphorylates and additionally phosphorylates specific residues within the interaction sites of the intracellular domain of the receptor, revealing signal transducer and activator of transcription (STAT) binding pockets [14]. 

After binding the STAT proteins via their Src-homology 2 (SH2) domains, STATs get phosphorylated by activated JAK1, leading to their dissociation from the receptor. IFN-α induces the formation of STAT1/STAT2 heterodimers [15], which can further associate with interferon regulatory factor 9 (IRF9), and subsequently form the IFN-stimulated gene factor 3 (ISGF3) [16]. The ISGF3 translocates into the nucleus to bind interferon-stimulated response elements (ISREs), inducing antiviral response genes [15,17,18]. Furthermore, STAT1 can form homodimers or heterodimers with STAT3. STAT1, STAT3, STAT4, STAT5, and STAT6 form homodimers. 

Dimerization precedes translocation into the nucleus and activation of genes regulated by a gamma interferon activation site (GAS) [19–21], causing a pro-inflammatory response (Figure 1).

The binding of IFN-o to its receptor also leads to rapid phosphorylation of receptor subunitR1 associated tyrosine kinase Tyk2 (22-25), which mediates signaling to non-IFN pathways this results in the initiation of the MAP kinase pathway, and activation of p38 and subsequent growth inhibition (26), as well as chromatin remodeling upon translocation of the Crebinding element (CREB)(27). Furthermore, Tyk2 activates phosphoinositide-3-kinase (PI3K), resulting in the activation of the mammalian target of rapamycin (mTOR) pathway and initiation of mRNA translation, as well as activation of the pro-inflammatory nuclear factorkappa-light-chain-enhancer' of activated B-cells (NF-kB) pathway (28].

1.1. Immune Response to Infection and Tissue Tolerance is influenced by the Tiype I interferon Response

Viruses interact with a broad range of proteins in mammalian cells, and their evolution has been driven by antiviral constraints and the adaptation of their host cells. It is hence not surprising that their co-evolution has resulted in highly sophisticated regulatory mechanisms of the timing and amplitude of immune responses to viral challenges. Type I IFN has a central role in controlling viral infections and is also involved in the defense of other pathogens. In 1957, IFNs were discovered by Alick Isaacs and Lean Lindenmannas a soluble factor in the supernatant of the chorioallantoic membrane, challenged with heat-inactivated influenza virus, that interferes with the viral infection in cells, hence the name-interferon”29. type I FNs act both in an autocrine and paracrine manner and prime bystander cells for upcoming viral infection by the latter. Their ability to restrict viral replication is mainly driven by a multitude of interferon-stimulated genes (ISGs). Furthermore, type I IFNs play an important role in the activation of cells that are involved in the development of the adaptive immune response. Here type I IFNs take part in the control of cell expansion and differentiation and determining cytokine and chemokine responses of cells of the lymphoid lineage (30].

Type I IFNs are associated with the rapid induction of a cellular antiviral state, and most cells can produce them in response to an appropriate pattern-recognition receptor (PRR) stimulation. They prime the infected cells, as well as the surrounding cells towards a state of either defense or tolerance [31]. Their importance as protective factors during viral infections was proven by showing the high susceptibility of mice deficient in the IFNAR1 receptor (Ifnar1−/− mice) to vesicular stomatitis virus (VSV), Semliki Forest virus, vaccinia virus (VACV), and lymphocytic choriomeningitis virus (LCMV) [32]. Furthermore, mice with STAT1 deficiency were shown to be highly susceptible to influenza viruses [33], further cementing the importance of type I IFNs in antiviral responses. In humans, several forms of inherited STAT1 deficiencies are associated with a high susceptibility to intracellular bacteria and viruses [34], while some gain-of-function STAT1 mutations are responsible for the development of chronic mucocutaneous candidiasis [35].

In bacterial infections, the functions of type I IFNs are more complex, as they can influence host defense either positively or negatively [30]. Type I IFN treatment of macrophages results in better restriction of bacterial replication during infection with intracellular Legionella pneumophilia or Bacillus anthracis [36–39]. Furthermore, type I IFN appears to protect cells from invasion by Salmonella enterica subsp. enterica ser. Typhimurium (S. Typhimurium) and Shigella flexneri, as mice treated with recombinant type I IFN showed reduced numbers of invasive bacteria in epithelial cells and improved survival [40,41]. Type I IFNs contribute to the activation of macrophages, regarding the production of nitric oxide (NO) and TNFα [42]. However, IFN-α and -β have also been identified as negative regulators of many of the cytokines and chemokines, orchestrating immune responses to bacterial infections, in particular for Listeria monocytogenes [43,44] and S. Typhimurium [44,45] (reviewed in [46]).

Besides bacteria, recognition of fungi, most importantly by the C-type lectin receptor Dectin-1, but also of fungal nucleic acids by Toll-like receptor 7 (TLR7) and TLR9 induces robust type I interferon responses [47,48]. However, as with bacterial infections, type I interferons can also be supportive of pathogen survival [49].

Type I IFNs are of equal importance in orchestrating adaptive immune responses to infection by transcriptional regulation of a broad range of target genes. Notably, type I IFNs induce and support the production of type II IFNs, mainly IFN-γ in NK cells directly [50,51], and support the production of IL-12 in dendritic cells (DCs) [52]. They can further enhance responses of myeloid cells, B cells, and T cells upon viral infection, leading to improved clearance of viruses and the establishment of a robust adaptive T and B cell memory repertoire. In antigen presentation, IFN-γ induces the transcription of MHC class I and class II by inducing the expression of two NLR family members, caspase activation and recruitment domain (CARD) containing 5 (NLRC5) and MHC class II transcriptional activator (CIITA), respectively [53,54]. Meanwhile, it was found that the expression of many other NLRs is regulated by both type I and type II IFNs. In the following section, we describe in detail how NLRs are regulated by type I IFNs and how they modulate the outcome of type I IFN responses. We discuss how deregulation of NLRs can result in susceptibility to either infection or auto-inflammatory disease as a consequence of pathogen dissemination or a lowered tissue tolerance to stress damage

1.2. Induction of Type I Interferon Response by Nucleic Acid Sensing

Recognition of pathogen-associated molecular patterns (PAMPs) by evolutionary conserved PRRs is the initial step for mounting a rapid innate immune response. After sensing potentially noxious non-self molecules, PRRs activate a defined set of signaling cascades, culminating in the induction of a state of tolerance or defense in the host cell. This allows the production and release of cytokines, which signal to neighboring cells for recruiting immune cells for the initiation of a specific adaptive immune response.

PRRs are localized in different subcellular compartments. Toll-like receptors (TLRs), C-type lectins, and scavenger receptors cover the cell surface, as well as, in the case of TLRs, membranes of the endosomal compartment. NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and cyclic GMP-AMP synthase (cGAS) monitor the cytoplasm for cell damage or the presence of invasive pathogens. Activation of these receptors results in the induction or repression of type I IFNs secretion, which will be discussed in the following chapters and is summarized in Figure 1.

Detection of cytosolic DNA is mainly mediated by the ubiquitously expressed cGAS and the absence of melanoma 2 (AIM2) protein. This not only includes foreign DNA derived from pathogens but also cytosolic chromatin resulting from genotoxic stress. While cGAS activation induces type I IFNs, detection of cytosolic DNA by AIM2 results in pyroptotic cell death as a consequence of the activation of caspase-1 and the subsequent processing and release of mature IL-1β and IL-18 [55]. 

Binding to cytosolic DNA renders cGAS in an active state, leading to the synthesis of the second messenger cyclic GMP-AMP (cGAMP) with a mixed-linkage backbone (c[G(20,50 )pA(30,50 )p]), which in turn is sensed by the protein referred as a stimulator of interferon genes (STING) [56–59], located at the membrane of the endoplasmic reticulum [60]. Activation of STING leads to its translocation into the Golgi network and activates the TRAF family member associated NF-κB activator-binding kinase 1 (TBK1). After auto-phosphorylation, TBK1 subsequently activates IRF3 through direct binding [61]. 

This enables its dimerization, translocation into the nucleus, and initiation of transcription of type I IFNs. IRF3 activation results in an initial wave of transcription with IFN-β and IFN-α4 as central transcription targets. Transcription of IRF7 is also induced for allowing a positive feedback loop leading to a second wave of type I IFNs secretion [62]. STING is the essential mediator of this response as its deficiency abolishes cGAS-induced IRF3 activation and IFN-β induction [63]. cGAS deficiency in mouse bone marrow-derived macrophages (BMDMs) is detrimental to the induction of antiviral type I IFN responses towards DNA viruses such as herpes simplex virus (HSV) 1, VACV, and murine gammaherpesvirus 68, but does not influence the response towards the RNA virus Sendai virus (SeV) [64,65]. Besides the activation of IRF3, STING also functions as an activator of NF-κB. For an extensive review of the functions of cGAS-STING activation, the reader is referred to [66].

Studies in cells from cGAS−/− mice have proven that cGAS is the main DNA sensor in antigen-presenting cells, such as plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs). Depletion of cGAS in those cells rendered them unresponsive to DNA transfection and infection with DNA viruses [67]. The type I IFN response towards these nucleic acids is also essential as a priming signal for the function of the DNA-induced AIM2 inflammasome assembly [55].

Besides nucleic acids from several DNA viruses like cytomegalovirus [68,69], HSV 1 [67], VACV [67], and retroviruses [70], cGAS is also the sensor for microbial DNA from invasive bacteria and protozoans such as L. monocytogenes [71–73], Chlamydia trachomatis [74], Mycobacterium tuberculosis [75–77], Toxoplasma gondii [78], and Leishmania major [79].

The most important family of cytosolic RNA-sensors is the RIG-I-like receptor family (RLRs), consisting of the retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). These proteins can sense the 5-prime di- and tri-phosphates of short, blunt-end double-stranded (ds)RNA by RIG-I, or long dsRNA by MDA5 [80]. All three proteins contain DExD/H box domains with ATPase function, which are crucial for RNA binding. RIG-I and MDA5 further contain two CARD. These N-terminal domains are responsible for further downstream signaling by binding to the CARD domain of the mitochondrial antiviral signaling protein (MAVS). The C-terminal domain of RIG-I serves as an inhibitory domain, keeping the protein in an inactive state until it binds RNA and conformational changes are induced [81].

After the binding of different cytosolic RNA species, both MDA5 and RIG-I are subject to K63-linked ubiquitination, both by covalent, and non-covalent attachment [82]. Either RIG-I, tripartite motif-containing protein 25 (TRIM25) [82] or Riplet [83,84] can function as E3 ubiquitin ligases. This process enables RIG-I to homotetrameric [85] and localizes to MAVS at the outer mitochondrial membrane initiating its oligomerization [86]. This multimerization of MAVS results in its activation and enables the recruitment of additional downstream adaptor proteins TRAF2, TRAF6, and TRADD [87,88]. Subsequently, TRAF3 [89] and TANK [90] are recruited to facilitate the activation of TBK1 and IKKε, which then phosphorylate the transcription factors IRF3 and IRF7. Activation of those two factors enables their homodimerization and translocation into the nucleus where they initiate transcription of type I and type III IFNs [91–94]. LGP2 does not contain a CARD domain and hence was proposed not to function in signaling, but rather as a regulator of RIG-I or MDA5 function [95].

1.3. Induction of Type I Interferon Responses by Membrane-Bound TLRs

While most of the members of the TLR family of TLRs may activate the NF-κB signaling cascade by MyD88, type I IFNs are induced by TLRs via activation of TRIF [96]. Among those TLRs, TLR4 has proven to be the most important inducer of type I IFNs. Recognition of LPS, or several viral proteins, leads to the activation of TRIF. TRIF can then directly associate with TBK1, inducing IRF3 activation and translocation into the nucleus as described above [97,98]. Further, TLR3, which also signals via TRIF, and TLR7 and TLR9 are inducers of IFN responses [98]. TLR7 and TLR9, are mainly expressed in pDCs where they induce type I IFN expression in a MyD88-dependent manner. pDCs constitutively express IRF7, and it has been shown that MyD88 can form a complex with IRF7 to trigger its activation and transcriptional activity [99,100]. For a more comprehensive review of TLR-induced immune signaling, see [101,102].

1.4. Induction of Interferon Responses by NLRs

Besides membrane-bound TLRs and cytosolic RLRs, the NOD-like receptor (NLR) protein family is another group of cytosolic PRRs. In mammals, a total of 22 human NLRs have been described [103]. NLRs are characterized by a common tripartite motif, consisting of a central nucleotide binding and oligomerization (NACHT) domain, C-terminal leucine-rich repeats (LRRs), and a variable N-terminal effector domain. According to their effector domain, NLRs are categorized into different subgroups: CARD-transcription and activation domain (CARD-AD) containing NLRA, baculovirus inhibitor of apoptosis (BIR) domain carrying NLRB, caspase activation and recruitment domain (CARD) containing NLRC and pyrin domain (PYD) containing NLRP [104]. NLRX1 contains an unconventional N-terminal domain, which shares no homology with the N-terminal domains of the other protein-family members. It is further unique as it contains a mitochondrial localization sequence (MLS) [105].

NOD1 and NOD2 were the founding and name-giving members of this protein family [106–108]. NOD1 and NOD2 function as intracellular sensors of peptidoglycan (PGN) components from the bacterial cell wall to initiate an appropriate immune response [106,107,109–111]. However, not all proteins of this subfamily function as bona fide PRRs. This is indicated by the fact that no direct ligand binding, or even direct activator, has been discovered for most members of the NLR protein family. Furthermore, some of the NLRs with known activators, like NLRC4 [112], do not bind to their activators directly, but rather need accessory proteins. Besides the function of NLRs as PRRs with direct induction of pro-inflammatory signaling pathways (NOD1, NOD2, NLR family apoptosis inhibitory protein NAIP), some NLRs form a specialized multiprotein complex, the inflammasome. 

Inflammasome formation has in common, with that apoptosis-associated speck protein (ASC), which is recruited by PYD of the activated NLR. Consequently, a highly organized multiprotein signaling platform is built, to which pro-caspase-1 is recruited, resulting in the maturation of pro-IL-1β and pro-IL-18 [113]. Non-PRR functions have also been described for two other NLR proteins, namely MHC class II transactivator (CIITA) and NLRC5, which are transcriptional regulators, that have been described to shuttle into the nucleus, where they can interact with a multiprotein transcription complex, termed MHC enhanceosome, to induce the transcription of MHC class II and MHC class I genes, respectively [114–117]. Nuclear translocation and direct transcriptional regulation have further been described for NLRP3 [118] and NOD2 [119]. Several other NLRs have been recently described as modulators of innate immune responses. For details about the functions of NLR proteins, the reader is referred to recent review articles [120–122]. However, to this date, there are still several NLR proteins whose functions have not been studied.

In the following sections, we provide an overview of our current understanding of the functions of NLRs in IFN responses. For a summary see Table 1 and Figure 2.

2. Negative Regulatory Feedback on Type I Interferon Responses by NLRs

2.1. NLRX1

NLRX1 has been associated with diverse signaling pathways. It attenuates NF-κB activation upon TLR activation [138,139,176] and can enhance ROS production, thereby enhancing the JNK pathway [177–180]. Furthermore, NLRX1 also promotes autophagy through association with Tu translation elongation factor (TUFM) [140] and enhances IRF1 protein levels upon viral infection by attenuating the inhibition of mRNA translation by protein kinase R (PKR) [181]. NLRX1 has also been implicated in the induction of apoptosis [182] and regulation of the NLRP3 inflammasome [183,184].

Besides those functions, NLRX1 is one of the best-described NLRs that regulates type I IFN responses. NLRX1 does not seem to be a sensor of viral or bacterial infection, but rather a negative regulator of type I IFNs [105,138]. Its unusual function is underscored by the fact that it contains an MLS in its N-terminus [105,178,185]. Although, the exact localization at the mitochondria is still a matter of debate, as both localization to the mitochondrial matrix and the outer mitochondrial membrane [105] have been reported.

Through interaction with MAVS, NLRX1 negatively regulates RIG-I-MAVS-dependent IFN-β induction by disruption of the interaction of MAVS and RIG-I [105,138]. Hence, overexpression of NLRX1 results in impaired RIG-I-dependent antiviral signaling and thereby enhanced viral replication [141,142]. NLRX1 might target MAVS for proteasomal degradation through the recruitment of poly (rC) binding protein 2 (PCBP2) which is recruited by the NACHT domain of NLRX1 [142]. Silencing of NLRX1 in pDCs, where NLRX1 is constitutively expressed, and in monocyte-derived DCs (moDCs), in which basal levels of NLRX1 are increased during differentiation, also leads to higher RLR-induced levels of type I IFN [143], supporting a negative regulation of RIG-I-induced signaling.

Knockdown of NLRX1 leads to enhanced transcription levels of IFNb1, STAT2, and the 20 -50 -oligoadenylate synthetase 1 gene (OAS1) after viral infection, suggesting a negative regulatory role of NLRX1 on the IFN-β/STAT2/OAS1 axis [138]. Accordingly, virus infection causes higher expression of IFNa2, IFNb1, OAS1, and STAT2 in Nlrx1−/− mice when compared to wild-type mice. However, such heightened antiviral response lowered tissue tolerance towards lung damage [138]. Fas-associated factor 1 (FAF1) on the other hand, was identified as an inhibitor of NLRX1-mediated reduction of type I IFN expression. FAF1 competes with MAVS for binding to NLRX1 and therefore positively regulates virus-induced type I IFN secretion. It is proposed that upon FAF1 binding, NLRX1 dissociates from MAVS, which is then able to interact with RIG-I and enhance type I IFN induction [144]. Another mechanism through which NLRX1 can inhibit the induction of type I IFNs is by binding to STING. This interaction is enhanced upon viral infection and dissociates TBK1 from the protein complex [145]. Furthermore, NLRX1 is involved in the regulation of autophagy. Interaction of mitochondrial TUFM with NLRX1 was suggested to enhance autophagy and by that to inhibit type I IFN signaling [140].

It should be noted that the inhibitory effect of NLRX1 on MAVS-dependent type I IFN induction is somewhat controversial, as several groups could not validate the above-described effects [146–148]. As it was shown that NLRX1 differentially affects IRF3- and IRF1- mediated responses, this might explain, at least in part, these contradictory findings [181].

2.2. NLRC3

NLRC3 can negatively regulate several signaling pathways such as NF-κB [186,187], mTOR [188], and the assembly and activity of the NLRP3 inflammasome [189]. It was further shown to attenuate auto-immune and virus-specific CD4+ T cell responses by inhibition of TNF and IFN-γ production [187,190] and by that reducing proliferation of Th1 and Th17 cells [187].

NLRC3 also restricts type I IFN production in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and HSV1 infection by directly impeding the interaction between STING and TBK1 [149]. Mechanistically, NLRC3 blocks STING trafficking from the ER to a perinuclear/golgi location and endoplasmic-associated puncta after DNA sensing [149]. This negative regulation of STING by NLRC3 prevents TBK1-dependent phosphorylation of IRF3 through its binding to Ras GTPase-activating-like protein IQGAP1 [191]. NLRC3 deficiency in murine BMDMs and MEFs results in higher DNA- and HSV1-induced type I IFN, IL-6, and TNF production. Consequently, Nlrc3−/− mice infected with HSV1 show reduced morbidity and viral load [149]. NLRC3 might also play a role in RIG-I-induced IFN response [192], however, the predominant effect is on the cGAS-induced pathway [149].

Negative regulation of TLR signaling by NLRC3 is mediated by the formation of a complex with TRAF6, and it was proposed that cellular complexes of TRAFs with regulatory NLRs, dubbed “TRAFasomes”, exist that act as regulatory platforms [186]. It remains to be established if such a scenario might also contribute to the regulation of interferon pathways by NLRC3.

Besides its function as a negative regulator, NLRC3 can bind double-stranded viral DNA by its LRR with high affinity, which leads to an increase of its ATPase activity of the NBD by 10-fold. The binding of ATP diminishes the interaction of the NBD with STING, leading to the activation of the type I IFN pathway [193].

2.3. NLRC5

NLRC5 is part of a distinct set of NLRs that function as transcriptional regulators of MHC class I and class II genes [114,151,194]. Both NLRC5 and CIITA bind to their respective transcriptional targets in MHC promotor regions via the same multiprotein DNA binding complex [115,117,195,196]. NLRC5 is constitutively expressed in a broad range of lymphoid organs and barrier tissues, such as the lung and the gastrointestinal tract, that are a gateway for several pathogens [114,151,194]. Expression of NLRC5 and subsequent MHC class I gene expression can be enhanced by stimulation with IFN-γ [114,151,194,197].

In the first characterization of NLRC5, it was reported that it influences transcription from ISRE and GAS reporter elements, while overexpression of NLRC5 resulted in elevated levels of IFN-α mRNA in HeLaS3 cells. Those results were confirmed by siRNA-mediated knockdown [114], and we showed that in THP-1 cells and primary dermal fibroblasts siRNA knockdown of NLRC5 reduces IFN-β and CXCL10 induction upon SeV infection [151]. NLRC5 was shown to inhibit influenza A virus (IAV) replication in the lung epithelial cell line A549 and to increase RIG-I and type I IFN transcription [152]. 

Interaction between NLRC5 and RIG-I was confirmed independently by Cui et al. However, these authors reported a negative effect of NLRC5 overexpression on type I IFN luciferase reporter activation by poly(I: C) [153]. Meanwhile, the knockdown of NLRC5 in several different cell lines was shown to lead to increased IFN-β responses towards either poly(I: C) treatment or VSV infection [153]. However, the regulation of IFN-β activation by NLRC5 remains a matter of debate [151]. Noteworthy, Nlrc5−/− mice in which exon 4 was targeted neither showed altered basal nor poly(I: C)-induced IFN-β serum levels compared to wildtype animals [154]. This is contrasted by studies using another NLRC5 knockout mouse model, in which exon 8 was targeted. Ex vivo stimulation with VSV or poly(I: C), as well as a systemic challenge with VSV, resulted in higher levels of IFN-β and stronger phosphorylation of IRF3 [155].

While the role of NLRC5 as a key regulator of MHC class I gene regulation is well established, the role of NLRC5 in type I IFN responses seems to be highly dependent on the cell type and the organismic context [156]. This is well illustrated by the observation, that knockdown of NLRC5 increases RIG-I-induced antiviral IFN response in pDCs, while it does not affect the same pathway in moDCs. Interestingly, those two cell types differ in their basal expression level of NLRC5 [143], suggesting a differential contribution of NLRC5 to IFN control in these cell types. Besides its role in antigen presentation, it is plausible that NLRC5 has further roles in antiviral immunity linked to the innate detection of viruses besides its role in antigen presentation, as suggested by some of the studies discussed above.

2.4. NLRP2

In humans, NLRP2 is predominantly expressed in the brain, pancreas, kidney, and reproductive tissues such as the testis and placenta [157,198,199]. In immune cells, NLRP2 is upregulated in macrophages in response to the B-DNA analog dAdT, as well as in T cells upon activation of RIG-I [198]. Differences exist between mouse and human cell populations. In contrast to human cells, NLRP2 is not upregulated in mouse CD3+ T cells upon RNA and DNA sensing, while in mouse CD14+ myeloid cells, RNA sensing leads to increased expression of NLRP2 [198]. NLRP2 protein levels were shown to be upregulated upon IFN-β, IFN-γ, and LPS treatment in macrophage-like differentiated human THP-1 cells, whereas CpG treatment did not affect NLRP2 protein levels [157]. Among the cytokines that are regulating NLRP2 expression, co-treatment with IFN-γ and TNF-α enables non-canonical inflammasome activation by intracellular LPS in brain pericytes [158].

In terms of type I IFN regulation, NLRP2 can bind TBK1 leading to perturbed interaction with IRF3, resulting in reduced IFN-β production [159], albeit this is a singular observation at present.

2.5. NLRP4

NLRP4 has only been studied recently. Although containing a PYD, NLRP4 does not interact with the inflammasome adaptor protein ASC [200] and does not affect IL1β secretion [201]. It was described to regulate the formation of the autophagosome and autophagic processes [201,202], and to negatively regulate the NF-κB response [163,203]. Furthermore, NLRP4 has been described to play a role in embryonic development [204]. It is expressed in human oocytes and early embryos [205], and seven gene copies of Nlrp4 are expressed in murine oocytes [206–208]. Knockdown of Nlrp4e in murine oocytes causes developmental arrest between the 2- and the 8-cell stage [204].

NLRP4 represses type I IFN responses by targeting TBK1 for degradation. This is mediated by the recruitment of the deleted E3 ubiquitin ligase 4 (DTX4). NLRP4 interacts with the kinase domain of phosphorylated TBK1 which facilitates K48-linked polyubiquitination of TBK1 at lysine residue 670 by DTX4 [164]. This degradation might be mediated by a signalosome complex including NLRP4, ubiquitin-specific peptidase 38 (USP38), DTX4, TRAF interacting protein (TRIP), and potentially some phosphatases that remain to be identified. Upon viral infection, TBK1 gets activated, resulting in its K63- and K33-linked ubiquitination. The formation of this complex leads to the editing of K33-linked ubiquitination at lysine residue 670 at TBK1 and its replacement by K48-linked polyubiquitination [165]. 

However, this might not be the only pathway as the dual specificity tyrosine phosphorylation regulated kinase 2 (DYRK2) was found to contribute to NLRP4-mediated degradation of TBK1 [166]. DYRK2 phosphorylates TBK1 on serine residue 527, which is essential for the recruitment of NLRP4 and enhances the interaction of the two proteins. This promotes the K48-linked polyubiquitination of TBK1. The authors suggest that DYRK2 enhances the degradation of TBK1 through the NLRP4-DTX4 nexus [166]. In rat cardiac muscle cells, reduced TBK1 and IRF3 levels were reported upon overexpression of NLRP4 in a dose-dependent manner [163].

We still know very little about the physiological function of NLRP4. The association of the knockdown of NLRP4 isoforms with a developmental defect in oocytes might result from missing tolerance towards paternal DNA as has been shown for NLRP14 (see below). Data cumulating from the studies summarized above suggest that a key mechanism of NLRP4 is the control of the half-life of TBK1 through proteasomal degradation.

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