Conj532 - Cytokine - Jak/STAT Pathways T-helper cell subsets and cytokine profiles

Th1, Th2 and Th17 cells are a separate lineage of CD4+ T cells, distinct from other T cell subsets. Every specific T helper cells produce its specific cytokines. T-bet, T-box expressed in T cells; FoxP3, forkhead box P3; ROR, retinoid-related orphan receptor. Makoto Kudo, et al. Front Microbiol. 2013;4:263. Cytokines secreted by immune cells “instruct” T-cells

TH1 like Antigen TH2 like response response

Effector B cells ('Be1' and 'Be2' cells) can secrete cytokines, such as interferon-γ (IFNγ), interleukin-12 (IL-12), IL-4 and IL-2, that reinforce and stabilize the cytokine profile of effector T helper 1 (TH1) and TH2 cells. In addition, the effector B cells can recruit additional naive Nature Reviews Immunology 10, 236-247 T cells into the inflammatory response. Cellular Mechanisms in Rheumatoid Arthritis

Pathogenesis of RA: synovial and systemic inflammation. Inflammation in RA is caused by activation of T cells, B cells and macrophages, which releases cytokines such as IL-1, IL-6 and TNF. These cytokines cause local joint damage through increased production of metalloproteinases and activation of osteoclasts. IL-1, IL-6 and TNF also leak out to the blood stream resulting in systemic inflammation: anaemia, thrombocytosis, fatigue, osteoporosis and the acute-phase response. Abbreviations: IL, interleukin; RA, rheumatoid arthritis. Choy, E. H. et al. Nat. Rev. Rheumatol. 9, 154–163 (2013) 3 General types of cytokine receptors

Class I Class II Class III

α α α β (gp130) α γ β

Single chain Unique + Common Multiple unique (dimer) chains (tetramer) chains (2 or 3) Epo, GH, Prl, GM-CSF IL-6, LIF, IL-11, IL-2, Interferons CNTF), CT-1, CLC, OSM, IL-27 and IL-31. Q: why did nature evolve multiple chain receptors? “Canonical” JAK–STAT pathway

Activating cross Binding & PO of STAT Tyr PO4 of JAK PO4 of JAK 4

THM: 3 Tyr-P required; catalyzed by a kinase Dimerization of STATs that is NOT a part of the receptor Three sequential tyrosine phosphorylations triggered by cytokine– receptor interaction. Receptor dimerization allows transphosphorylation and activation of Janus kinases (JAKs). This is followed by phosphorylation of receptor tails and the recruitment of the Signal Transducers and Activators of Transcription (STAT) proteins through their Src-homology-2 domains. STAT tyrosine phosphorylation then occurs. Dimerization of activated (tyrosine phosphorylated) STAT is followed by nuclear entry. Nature Reviews Molecular Cell Biology 3; 651-662 So, lets go into more detail about each of these players Structural organization of STATs Signal Transducers and Activators of Transcription Different regions have different functions or bind different transcriptional regulators YP SP FERM DBD SH3 SH2 TAD

Stat2/p48 Stat2/p300(CBP) Stat1/p300(CBP) Stat1/p300(CBP) Stat1/PIAS1 Stat5/ERK

Stat3/c-Jun Stat1/MCM5 Stat1/p48

Stat5/Nmi At least 6 families of STATS

The domain structure of STAT. The contact regions of STAT-interacting proteins are indicated by red lines. DBD: DNA binding domain; SH3: Src homology 3 domain (poly Pro); SH2: Src homology 2 domain (pY);8 TAD: Transcription activation domain; FERM ( band4.1, ezrin,radixin, & moesin) binds to STATS and other proteins How do SH2 and DNB domains work?

YP SP FERM DBD SH3 SH2 TAD

Tyr Tyr P

SH2

Phosphorylation and SH2-phosphotyrosine binding

STAT monomers STAT dimer (binds DNA) Structure of STAT bound to DNA

SH2 domain

Linker domain

DNA-binding domain

Coiled coil domain (a) Crystal structure of an N and C-terminally truncated Stat1 Nutcracker Model molecule bound to DNA. The structure of truncated Stat3 is virtually superimposible with that of Stat1 (Chen et al., 1998). STAT domain structure and protein binding sites

The core structure (amino acids 130–712) shows binding of a STAT1 dimer to DNA and the location of binding sites of various proteins in various domains. The amino- terminal structure, the placement of which in the intact structure is undefined, also interacts with various partners, as does the carboxy-terminal transactivation domain, the structure of which is unknown. CBP, CREB binding protein; IRF, interferon regulatory factor; Mcm, mini chromosome maintenance; Nmi, N- Myc interactor; PIAS, protein inhibitor of 11 activated STAT. Nature Reviews Molecular Cell Biology 3; 651-662 Structure & number of Jaks

JH7 JH6 JH5 JH4 JH3 JH2 JH1

N FERM C

Pseudokinase Kinase Domain JANUS KINASES Domain

Size Identity Chromosome Expression

Tyk2 140 kDa 36% 19p13.2 Ubquitous Jak1 135 kDa 36% 1p31.2 Ubiquitous Jak2 130 kDa 47% 10p23 Ubiquitous Jak3 120 kDa - 4q31 Myeloid/Lymphoid

Seven domains, termed Jak homology (JH) domains 1-7 are shared among Jaks. The JH1 domain is the kinase domain and the JH2 domain is a pseudokinase domain whose precise function has not yet been determined. 12 Q: What does acronym, JAK stand for? Janus - the “two-faced” god, keeper of the gate

The Janus kinases, were thought to contain 2 types of phosphate-transferring domains. Thus, it is named after “Janus”, the Roman two-faced gatekeeper13 of the heavens. Different cytokine receptors bind different combinations of Jaks

TYPE HORMONE PHOSPHORYLATION Single specificity Growth Hormone Homo-phosphorylation Promiscuous IL-6 Multi- phosphorylation (all JAKs) Obligate Hetero INFα, INFγ Hetero-phosphorylation (2 different JAKs) Jak1 How determined? INFα Tyk2 (Mutate Jak1 ---> no Tyk2 PO4 Jak1 Why important? INFγ (Mutate Jak1 ---> no Jak2 PO Jak2 4 PO O 14 Different4 Jaks PO4 different STATS Interferon receptors and activation of classical JAK–STAT pathways by type I and type II interferons AllExample type I interferons of how (IFNs) one bind can a common get specificity receptor which is known as the type I IFN receptor. The type I IFNof functionreceptor is composedby different of two interferonssubunits, IFNAR1 by and IFNAR2,expressing which are and associated utilizing with different the Janus activated kinasescombinations (JAKs), tyrosine of cytokine kinase 2 (TYK2) receptors, and JAK1, respectively. A single type II IFN, IFN-g, binds a distinct cell-surfaceJaks and receptor, STATS which is known as the type II IFN receptor. This receptor is also composed of 2 subunits, IFNGR1 and IFNGR2, which are associated with JAK1 and JAK2, respectively. Activation of the JAKs that are associated with the type I IFN receptor results in tyrosine phosphorylation of STAT2 (signal transducer and activator of transcription 2) and STAT1; this leads to the formation of STAT1–STAT2–IRF9 (IFN-regulatory factor 9) complexes, which are known as ISGF3 (IFN-stimulated gene (ISG) factor 3) complexes. These complexes translocate to the nucleus and bind IFN-stimulated response elements (ISREs) to initiate gene transcription. Both type I and II IFNs also induce formation of STAT1– STAT1 homodimers that translocate to the nucleus and bind GAS elements in the promoter of some ISGs, thereby initiating transcription of these genes. The GAS 15 element and ISRE sequences are shown. From Nat Rev Immunol 376 | MAY 2005 How is Cytokine Function Regulated?

A: Several types of negative feedback Diagram of domains in STAT-induced STAT Inhibitors, SOCS & CIS proteins

At least eight proteins belong P-tyrosine binding (STAT competitor Elongin B/C to the SOCS family of Kinase domain binding binding proteins are shown (upper (Kinase Inhibitor) (ubiquitination) panel). They are characterized by the presence of an SH2 central domain and the SOCS box domain at the C-terminus. A small domain called kinase inhibitory region (KIR), only found in SOCS1 and SOCS3, is shown as a small Other SOCS Box containing proteins box at the N-terminal region. SOCS proteins can interact with phosphotyrosine phosphorylated proteins through their SH2 domain and with Elongin BC through their SOCS box domain. Other proteins containing a SOCS box domain but CIS = Cytokine-Induced SH2 protein; lacking a SH2 domain are SOCS = Suppressor of Cytokine Signaling also shown (lower panel). SSI = STAT-induced STAT Inhibitor Rico-Bautista et al 2006 Negative regulation of cytokine signaling: STAT-

induced STAT inhibitor Naka et al.,TiBs, 24:394-398

= SOCS1

SOCS bind to and inhibit JAKs CIS inhibits STAT binding

SSI-1 type inhibition CIS-1 type inhibition THM - 2 sites of inhibition (Jaks or STAT binding to Receptor) (a) Binding of JAK to cytokine receptors and activation of STAT. (b) SSI-type inhibition of cytokine signaling. The gene encoding SSI-1 is induced by STAT dimers, resulting in the production of SSI-1 and inhibition of cytokine signaling by binding of SSI-1 to the kinase domain of the JAK family. (c) CIS1-type inhibition of cytokine signaling. The gene encoding CIS1 is induced by STAT5 dimers, resulting in the production of CIS1 and inhibition of cytokine signaling by binding of CIS1 to the STAT of cytokine receptors. 18 Abbreviations used: CIS1, cytokine-inducible SH2 protein 1; GAS motif, -Stat activated site; JAK, Janus , SSI, STAT-induced STAT inhibitor; STAT, signal transducers and activators of transcription. Other negative regulators of STAT proteins

Phosphatases (a) and suppressors of cytokine signalling (SOCS proteins) (b) block further STAT activation in the cell cytoplasm. In the nucleus, nuclear phosphatases (c) can mediate STAT dephosphorylation, and interactions with proteins that inhibit activated STAT proteins (PIAS) (d) can also occur. In addition, naturally occurring short forms of STATs can potentially act as dominant- negative proteins by occupying DNA as non- functional protein or by binding to a wild-type STAT protein (e). JAK, ; STAT, signal transducers and activators of transcription. Note, also shown in green is a likely regulation of JAKs by ubiquitination/phosphorylation

Why so many different mechanisms for controlling STATS?

Nature Reviews Molecular Cell Biology 3; 651-662 (2002); 19 STATS: Transcriptional control and biological impact Proposed mechanisms for inhibiting the JAK– STAT pathway by PIAS proteins

Fig 4 | Different PIAS (protein inhibitor of activated STAT) Block DNA binding proteins can inhibit the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway through distinct mechanisms. a | PIAS1 and PIAS3 block the DNA-binding activity of STAT Act as co- dimers. repressors b | PIASX and PIASY might act as transcriptional co-repressors of STAT by recruiting other co- repressor proteins such as histone deacetylase (HDAC). c | PIAS proteins can promote the conjugation of small ubiquitin- related modifier (SUMO) to STAT1. The significance of STAT1 sumoylation in regulating STAT1 activity is controversial and needs to be clarified Promote sumoylation

20 Nature Reviews Immunology 3; 900-911 Pias Proteins act as E3 for SUMO

Left - Ubiquitin is coupled to E-1 ubiquitin-activating and in turn transferred to E-2 ubiquitin- conjugating enzyme. E3 ubiquitin combines with the charged E2 and forms an isopeptide bond between ubiquitin and the target protein. PIAS proteins act as E3 ligases for SUMO. SUMO shares 18% homology with ubiquitin. Right - PIAS1, PIAS3 and PIASx sumoylate STAT1 at Lys-703- close to Tyr-701 where JAK is phosphorylated. STAT1 can be modified by SUMO at lysine residue 703. Direct interactions between PIAS1 and STAT121 may interfere with the STAT1 ability to bind DNA. Biochemical Pharmacology 70 (2005) 649–657 Example of Feedback Regulation by SOCS The role of SOCS-3 in THM - induction of SOCS-3 causes decreased cytokine coupling Leptin signaling and resistance

O hr 1hr 2 hr 4hr stimulation with leptin CHO-OBRI CIS mRNA

SOCS-1 mRNA

SOCS-2 mRNA

SOCS-3 mRNA

LeptinTHM induces - specificity SOCS-3, butof inductionnot CIS, SOCS-1, or SOCS-2, mRNA in CHO cells expressing 23 theand long different form of the time leptin coursesreceptor. J Flier lab JBC 274:30059 ‘99 How important are SSI proteins?

Studies with SOCS1 KO mice In SOCS1 knockout mice, negative regulation of cytokine signaling is SOCS-1 KO ---> Post-Embryonic diminished Lethal

Lack of binding of Rescued by cross SSI-1 to JAK leads to of heterozygotes to prolonged activation INF-γ KO of the JAK/STAT pathway and prolonged action of cytokines. Abbreviations: JAK, Janus tyrosine kinase, SSI, STAT-induced STAT inhibitor; STAT, signal transducers and activators of transcription. 25 Naka et al.,Trends in Biochemical Sciences, 24:394-398 “Cross talk” between Jak/STAT and other signaling pathways

THM1: Other pathways can be activated by ligand binding to "cytokine" receptors

THM2: Jaks can bind & activate other tyrosine kinase pathways

THM3: Other serine kinase pathways can modulate Jak/STAT function

THM4: SOCS proteins can modulate other pathways Activation of CRKL by the type I INF receptor, and role of CRKL in type-I INF-mediated signaling FigJak2 2 CRKLcan alsois present phosphorylate as a latent cytoplasmic CRKL form that allowingconstitutively it to associates be active with itself the guanine-nucleotide- just like a exchange factor (GEF) C3G. A member of the STAT STAT and form a heterodimer (signal transducer and activator of transcription) family Itof can proteins, also STAT5, scaffold is associated and activate with tyrosine C3G, kinase 2 (TYK2) that is bound to the type I interferon (IFN) (GEF)receptor leading subunit IFNAR1. to increased After engagement RAP1 activity. of the type I IFN receptor by an IFN, CRKL associates with TYK2 and undergoes rapid tyrosine phosphorylation. The activated form of CRKL forms a signaling complex with STAT5, which also undergoes TYK2-dependent tyrosine phosphorylation. The CRKL–STAT5 complex translocates to the nucleus and binds specific GAS (IFN-activated site) elements that are present in the promoters of certain IFN-stimulated genes (ISGs), which initiates transcription of these genes. The specific GAS sequence bound by CRKL–STAT5 is shown. The IFN-dependent phosphorylation (activation) of CRKL also results in induction of the GEF activity of C3G. C3G subsequently regulates the small G-protein RAP1, resulting in activation of this GTPase, which may then promote growth-inhibitory responses JAK, Janus activated kinase. Nat Rev Immunol 376 | MAY 2005 | Mechanisms of activation of MAP kinase, p38 and its downstream effectors by type I interferons THM: Tyk2 and Jak1 can also Interferon (IFN)-activated JAKs regulate the phosphorylationdirectly activate (activation) GEFs of VAV or other guanine-nucleotide-exchange factors (GEFs), resulting in downstream activation of RAC1 and, possibly, other small G proteins (SGPs) that can regulate the signaling pathway of the mitogenactivated (MAPK) p38. A MAPK kinase kinase (MAPKKK) is subsequently activated and regulates downstream activation of the MAPK kinases MAPKK3 and MAPKK6, which directly phosphorylate p38, resulting in its activation. Activated p38 subsequently regulates activation of multiple downstream effectors, including MAPK-activated protein kinase 2 (MAPKAPK2), MAPKAPK3, mitogen- and stress-activated kinase 1 (MSK1) and MAPK-interacting protein kinase 1 (MNK1). IFNAR1, type I IFN receptor subunit 1; IFNAR2, type I IFN receptor subunit 28 2; TYK2, . Nat Rev Immunol 376 MAY 2005 JAK2-mediated activation of STATs and ERK/MAPK by GH or a growth factor (GF)

PI3K

AKT

THM: Just because effect is due to a cytokine, doesn’t mean that it has to be STAT pathway

29 Current Biology Linda A. Winston, Tony Hunter 1996, 6:668-671 transcription in such pathological settings is needed. It is also important to elucidate whether the JAK-STAT pathway partici- pates in Ang II regulation of the renal microcirculation, glomerular pressure and sodium tubular transport.

RAS Components Regulation by the JAK-STAT Pathway

There has been substantial evidence of interactions between the RAS and pro-inflammatory factors. Ang II increases cytokine production via activation of nuclear factor-kB. In addition, Ang II increases infiltration of immune cells in tissues which in turn intensifies local cytokine production. Indeed, Ang II-infused animals and Ang II-treated tissues exhibit enhanced expression of plasma and tissue pro-inflammatory cytokines such as tumor necrosis factor a (TNF-a), interleukin 1β (IL-1β), IL-6 and Figure 1. Scheme illustrating the activation of the JAK-STAT pathway by Ang II through AT1R. Ang II activates JAK2 via the G protein-dependent IFN-c. This is best characterized in Ang II-dependent hyperten- 53-58 and -independent mechanisms leading to gene transcription and sion. In the following paragraphs we will address the impact of vasoconstriction. Various second messengers including PKC, Pyk2, these interactions on the RAS expressed along the nephron Arhgef1 and SHP2 are involved in these pathways. because intrarenal RAS regulation as well as other local RAS is a key factor in the development of hypertension.8 JAK2 induces phosphorylation of a RhoA guanine nucleotide Renin. Juxtaglomerular apparatus (JGA) cells in the kidney are exchange factor, Arhgef1 in vascular smooth muscle cells.47,48 The the primary source of circulating renin. Although IL-6 is known activated Arhgef1 then stimulates the RhoA-Rho kinase axis to be a strong activator of STAT3, IL-6 decreases renin expression resulting in augmentation of blood pressure, which was delineated in As4.1 cells, an immortalized renin-producing renal tumor cell by showing that Arhgef1 knockout mitigates Ang II-induced high line.59 Moreover, in the same cell line, IL-1β attenuates renin blood pressure accompanied by attenuation of Rho kinase expression mediated via the p44/42 MAPK-STAT3 pathway.60 activation.47 In line with this finding, recent studies show that Oncostatin M is an endotoxin-responsive pro-inflammatory JAK2 pharmacological inhibition attenuates the hypertensive cytokine that also downregulates renin expression via activation response of Ang II-infused animals. Specifically, JAK2 knock- of STAT5 in As4.1 cells.59 These results suggest that activation of down in smooth muscle cells49 and administration of a JAK2 the JAK-STAT pathway reduces renin expression in the kidney. inhibitor50 prevent the development of hypertension by Ang II However, other reports contradict this concept. Despite that IL- infusion supporting JAK2 crucial role in the development of Ang 1β suppresses renin expression via STAT3 activation in As4.1 II-dependent hypertension. cells, it has been demonstrated that in vivo infusion of IL-1β In terms of renal injury, it is known that hyperglycemia, via increases plasma renin levels and blood pressure.61 It is also known Ang II, induces the JAK-STAT pathway in glomerular mesangial what while chronic Ang II infusion suppresses renin expression cells.40 This in vitro finding goes in line with the in vivo and secretion in JGA cells,62 renin expression in principal cells of observation that both AT1R blockage and JAK2 inhibition renal connecting tubules and collecting ducts is increased. This is prevent the progression of proteinuria and hypertension in an important observation, because the presence of all RAS streptozotocin-induced diabetic nephropathy.51 As another evid- components in the tubular fluid indicates that local intratubular ence of critical roles of the JAK-STAT pathway in the Ang II contributes to the blood pressure elevation.63 Thus, our development of renal injury, administration of a JAK2 inhibitor knowledge regarding renin regulation via the JAK-STAT pathway ameliorated development of renal injury accompanied by is far from complete, but it is possible that the contradicting suppression of JAK-STAT activity in renal ischemia/reperfusion results may be explained on the bases of cell specificity. model.52 Further, SOCS1 and SOCS3 overexpression, that Angiotensinogen. From the point of view of Ang production, Activationsuppresses of the theJAK-STAT intrarenal pathway JAK-STAT via pathway, AT1R abrogates the renin is the rate-limiting processing enzyme in the RAS cascade.64 development of renal injury.43,44 Importantly, when SOCS3 is However, because plasma angiotensinogen concentration is close knockdown, the unopposed Ang II induction of STAT activation to Km for the reaction with renin,65 angiotensinogen level is also and c-Fos/c-Jun expression results in more severe renal damage. regarded as an important factor influencing RAS activity.66,67 The Thus, these observations suggest that the JAK-STAT pathway major source of plasma angiotensinogen is the liver. In addition, activation by Ang II plays a crucial role and SOCS proteins serve angiotensinogen can also be synthesized in the kidneys.68-70 MSH as limiting factors in the progression of renal damage in the RAS- Intrarenal angiotensinogen is mainly produced in renal proximal 5HT induced renal injury. On the basis of the evidence, new questions tubular cells.71-73 The importance of this observation is supported arise with regards to the potential contribution of the JAK-STAT by experiments showing that renal proximal tubule-specific pathway to the development of hypertension and renal injury. In overexpression of angiotensinogen in transgenic animals causes particular, dissecting the contribution of STAT-stimulated gene the development of hypertension and renal injury.74 Importantly,

252 JAK-STAT Volume 1 Issue 4

Ang II activates JAK2 via the G protein-dependent and -independent mechanisms leading to gene transcription and vasoconstriction. Various second messengers including PKC, Pyk2, Arhgef1 and SHP2 are involved in these pathways. JAK-STAT 1:4, 250–256; Please - Don’t get behind on reading. You can be sure that some of exam questions will come from the readings.

For example, in the research paper I assigned that came out just last week, Do you think that the authors are correct when they say that these inflamasomes that contain NLRP3 are direct binders and effectors for cAMP action on immune system?? 31 SOCS can regulate insulin pathway at several points

Flier 06 SOCS proteins inhibit signaling by binding to the insulin receptor, thereby blocking access of signaling intermediates and inhibiting insulin activity, leading to a reduction in insulin-receptor directed phosphorylation of IRS-1 and its downstream events, and by targeting IRS-1 and IRS-2 for proteosomal degradation. Abbreviations: PKB, protein kinase B (also known as Akt); PDK1 and 2, phosphoinositide-dependent kinase 1 and 2; PI(4,5)P2, phosphatidylinositol (4,5)- bisphosphate; PI(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate); Shr, C-terminal SH2 domain-32 containing adaptor protein. Termination of STAT1 signaling via acetylation

IFNs induce STAT1 signaling. The nuclear HAT CBP catalyzes acetylation of phosphorylated nuclear STAT1. Subsequently, TCP45 is recruited and STAT1 becomes dephosphorylated, exits the nucleus, and acquires latency.

THM: One more mechanism HAT of regulation

TCP45 is a tyrosine phosphatase

CBP is a HAT and Tyr Ptase CBP is a CREB binding protein

Kramer & Heinzel, Molecular and Cellular 33 Endocrinology (2009) A phospho-acetyl switch controls STAT1 signaling A phospho-acetyl switch controls STAT1 signaling. Modifications of STAT1 are dynamically regulated. A phospho-acetyl switch controls STAT1 upon activation by IFN. Serine phosphorylation of STAT1 regulates repressive sumoylation of STAT1 (pY, tyrosine phosphorylation; Ac, lysine acetylation; pS, serine phosphorylation; Su,sumoylation; MAPK,MAPkinases). STAT1/STAT2 heterodimers serve as example.

Acetylation of STAT1 antagonizes its IFN-induced phosphorylation. The balance between STAT1 acetylation and phosphorylation determines STAT1 activity and IFN 34 Kramer & Heinzel, Molecular and Cellular signaling. STAT1 homodimers serve as an example. Endocrinology (2009) Intracellular sensors in innate immunity to viruses: a mechanism for control of cytokine synthesis

After induction many cytokines need to be activated by proteolytic clipping of the prohormone PRRs = pattern recognition receptors TLRs ( Toll-like), RLRs (RIG-1-like), CLRs (C-type lectin), & NLRs (nucleotide binding domain leucine rich repeats CARD = caspase recruitment domain Figure 1 | Intracellular sensors in innate immunity to viruses. Viral pathogen-associated molecular patterns (PAMPs) activate nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and inflammasomes to initiate signalling cascades that lead to the production of pro-inflammatory cytokines, thereby amplifying antiviral innate immune responses. In the presence of viral PAMPs, NLR family PYD-containing protein 3 (NLRP3) and absent in melanoma 2 (AIM2) oligomerize and recruit the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) through homotypic pyrin domain (PYD) interactions. The caspase-recruitment domain (CARD) of ASC binds the CARD of pro-caspase‑1, leading to caspase‑1 activation and the production of interleukin‑1β (IL‑1β) and IL‑18 through cleavage of pro-IL‑1β and pro-IL‑18. Retinoic acid inducible gene‑I (RIG‑I) contains an RNA helicase domain and an amino-terminal CARD. The helicase domain of RIG‑I senses the 5ʹ-triphosphate moiety of single-stranded (ss)RNA virus genomes and then signals through CARD–CARD interactions with the adaptor molecule mitochondrial antiviral signalling protein (MAVS). This results in the phosphorylation and activation of interferon (IFN) response factor 3 (IRF3) and IRF7 to turn on the transcription of type I IFN (IFNα/β) genes. RIG‑I also regulates IL‑1β production transcriptionally and post-translationally following recognition of 5ʹ-triphosphate double-stranded (ds)RNA. Whereas RIG‑I-triggered transcription of pro-IL‑1β depends on nuclear factor-κB (NF- κB) activation and is mediated by MAVS, inflammasome formation, caspase‑1 activation, and IL‑1β and IL‑18 production in response to RIG‑I activation involve ASC. The NLRs NOD2, NLR family member X1 (NLRX1) and NLR family CARD-containing protein 5 (NLRC5) associate with MAVS. Whereas NOD2 mediates the induction of type I IFNs, NLRX1 and NLRC5 inhibit RIG‑I– MAVS interactions and thereby negatively regulate type I IFN production. LRR, leucine-rich repeat; MAPK, mitogen-activated protein kinase; MYD88, myeloid differentiation primary- response protein 88; RIPK2, receptor-interacting serine-threonine protein kinase 2; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRIF, TIR-domain- containing adaptor protein inducing IFNβ. 36