Seeing inflammation through the innate immune eye

Introduction Inflammation is triggered when innate immune cells detect microbial infection or tissue damage. Surveillance mecha­ nisms involve pattern recognition receptors on the cell surface and in the cytoplasm. Most pattern recognition receptors respond to pathogen­ associated molecular patterns or host­derived damage­associated mole­ cular patterns by triggering activation of various transcription factors. Induction of cytokines promotes the activation and recruitment of leukocytes, which are critical for elim­ inating pathogens and host debris. In order to avoid immunopathology, the system is very tightly regulated by numerous molecules that limit the magnitude and duration of the inflammatory response. In this review we present current knowledge on pathogen recognition through diff­ erent pattern recognition receptors and the complex signalling pathways responsible for activation of inflam­ matory and antimicrobial responses. Conclusion There are still many unresolved questions, such as the exact nature of the molecular events leading to diff­ erent immune receptor activation and also identity of some unknown ligands for the receptors. Unravelling of these will offer insight into what critical components might be target­ ed for better therapeutic benefits in inflammatory disorders. Introduction The role of inflammatory response is to combat infection and tissue injury. The innate immune system constitutes the first line of host defence during infection and therefore plays a very crucial role in the early recognition and subsequent trigger­ ing of a pro-inflammatory response to invading pathogens1. The adaptive immune system, on the other hand, is responsible for elimination of pathogens during the late phase of infection and in the generation of immunological memory. The innate immune response is mediated pri­ marily by phagocytic cells and antigen­presenting cells (APCs), such as macrophages, and dendritic cells (DCs), and has been regarded as relatively non-specific2, whereas the adaptive immune response is charac­ terized by antigen-specific receptors on lymphocytes generated by clonal gene rearrangements. Innate immune cells of tissues such as macrophages, fibroblasts and dendritic cells, as well as circulating leukocytes, recognize pathogen inva­ sion or cellular damage with pattern recognition receptors (PRRs). These receptors detect pathogen­associated molecular patterns (PAMPs), such as pathogen­derived nucleic acids and cell wall components, fungal β-glucan, bacterial flagellin, and lipopolysaccharide (LPS) from Gram­ negative bacteria. PRRs also recog­ nize damage­associated molecular patterns (DAMPs), released from injured cells during apoptosis or necrosis. DAMPs include uric acid, ATP, and the DNA­binding nuclear protein HMGB1 and amyloid β fibrils. Activated PRRs then initiate signal­ ling cascades to trigger the release of factors that promote recruitment of leukocytes to the infected region. In this review, we look into the inflammatory signalling response emanating from the recognition of microbial infection and cellular injury by PRRs. Among PRRs, membrane­bound toll­like receptors (TLRs) play a cen­ tral role in the initiation of immune response against pathogen invasion. However, other PRRs are also involved—including membrane­bound C­type lectin receptors (CLRs), cyto­ solic proteins such as NOD­like recep­ tors (NLRs) and RIG­I­like receptors (RLRs). Among these receptor types, TLRs and RLRs are primarily important for the production of type I interfer­ ons (IFNs), whereas NLRs are known to regulate interleukin-1β (IL-1β) production through activation of caspase­1. Discussion Toll-like receptors TLRs were the first PRRs to be iden­ tified. They are also the most well characterized and recognize a wide range of PAMPs3–5. TLRs are trans­ membrane proteins which comprise an ectodomain, which contains leucine­rich repeats that mediate the recognition of PAMPs, a transmem­ brane region, and cytosolic Toll­IL­1 receptor (TIR) domains that activate downstream signalling pathways. TLRs are expressed either on the cell surface or on intracellular vesicles. To date, 10 and 12 functional TLRs have been identified in human and mice, respectively. Each TLR detects distinct PAMPs from bacteria, viruses, fungi and parasites. Upon recognition of respective PAMPs, TLRs recruit a specific set of adaptor molecules that have TIR doma ins, such as MyD88 and TRIF, and initiate downstream signalling


Introduction
The role of inflammatory response is to combat infection and tissue injury.The innate immune system constitutes the first line of host defence during infection and therefore plays a very crucial role in the early recognition and subsequent trigger ing of a pro-inflammatory response to invading pathogens 1 .The adaptive immune system, on the other hand, is responsible for elimination of pathogens during the late phase of infection and in the generation of immunological memory.The innate immune response is mediated pri marily by phagocytic cells and antigenpresenting cells (APCs), such as macrophages, and dendritic cells (DCs), and has been regarded as relatively non-specific 2 , whereas the adaptive immune response is charac terized by antigen-specific receptors on lymphocytes generated by clonal gene rearrangements.
Innate immune cells of tissues such as macrophages, fibroblasts and dendritic cells, as well as circulating leukocytes, recognize pathogen inva sion or cellular damage with pattern recognition receptors (PRRs).These receptors detect pathogenassociated molecular patterns (PAMPs), such as pathogenderived nucleic acids and cell wall components, fungal β-glucan, bacterial flagellin, and lipopolysaccharide (LPS) from Gram negative bacteria.PRRs also recog nize damageassociated molecular patterns (DAMPs), released from injured cells during apoptosis or necrosis.DAMPs include uric acid, ATP, and the DNAbinding nuclear protein HMGB1 and amyloid β fibrils.Activated PRRs then initiate signal ling cascades to trigger the release of factors that promote recruitment of leukocytes to the infected region.
In this review, we look into the inflammatory signalling response emanating from the recognition of microbial infection and cellular injury by PRRs.
Among these receptor types, TLRs and RLRs are primarily important for the production of type I interfer ons (IFNs), whereas NLRs are known to regulate interleukin-1β (IL-1β) production through activation of caspase1.

Toll-like receptors
TLRs were the first PRRs to be iden tified.They are also the most well characterized and recognize a wide range of PAMPs [3][4][5] .TLRs are trans membrane proteins which comprise an ectodomain, which contains leucinerich repeats that mediate the recognition of PAMPs, a transmem brane region, and cytosolic TollIL1 receptor (TIR) domains that activate downstream signalling pathways.TLRs are expressed either on the cell surface or on intracellular vesicles.To date, 10 and 12 functional TLRs have been identified in human and mice, respectively.Each TLR detects distinct PAMPs from bacteria, viruses, fungi and parasites.
Upon recognition of respective PAMPs, TLRs recruit a specific set of adaptor molecules that have TIR doma ins, such as MyD88 and TRIF, and initiate downstream signalling events that lead to the secretion of inflammatory cytokines, type I IFNs, chemokines and antimicrobial pep tides 6 .These responses cause neu trophil recruitment, activation of macrophages and induction of IFN stimulated genes, resulting in clear ance of pathogens.
TLR1, TLR2, TLR4, TLR5 and TLR6 are localized on the cell surface and recognize microbial membrane com ponents, whereas TLR3, TLR7, TLR8 and TLR9 are expressed in intracel lular vesicles and recognize nucleic acids 7 .However, TLR11 is expressed both on the cell surface and in intracellular compartments 8 .TLR13 is also expressed in intracellular vesicles 7 .Intracellular vesicles in immune cells, in which TLR3, TLR7, TLR8 and TLR9 are include the endoplasmic reticulum (ER), endosomes and lysosomes 7 .The intracellular localization helps TLRs to recognize nucleic acids delivered to the intracellular compartments after the uptake of pathogens by infected cells.Different PAMPs are being sensed by different TLRs (Table 1).
After recognizing the microbial PAMPs, the TLRs activate the signal ling pathways that provide specific immunological responses.The res ponses initiated by individual TLRs depend on the recruitment of TIR domaincontaining adaptor protein (e.g.MyD88, TRIF, TIRAP or TRAM) 6 .MyD88 relays signals culminating in NFkB and MAP kinase activation and the induction of inflammatory cytokines.It is engaged by all TLRs (with the exception of TLR3).TLR3 and TLR4 use TRIF to activate an alternative pathway leading to the activation of NFkB and IRF3 and the induction of type I IFN and inflam matory cytokine productions.TLR2 and TLR4 use TIRAP as an additional adaptor to recruit MyD88.TRAM acts as a bridge between TLR4 and TRIF.TLR4 is the only TLR that recruits four adaptor proteins and activates two distinct signalling pathways: the 'TRIFdependent' and 'MyD88 dependent' pathways 6 .TLR4 initially recruits TIRAP and MyD88.TIRAP localizes to the plasma membrane, where it serves to bridge the inter action between MyD88 and TLR4 upon LPS engagement 9 .MyD88 then recruits IRAKs, TRAF6 and the TAK1 complex, leading to activation of NFkB and MAP kinases 6 .TLR4 is then endocytosed and delivered to intracellular vesicles to form a com plex with TRAM and TRIF, which then recruits TRAF3 and the protein kinases TBK1 and IKK, which phos phorylates IRF3, leading to type I IFN expression.
TLR5 also signals through MyD88 to induce inflammatory cytokine production.However, TLR5 also recruits TRIF, in addition to MyD88, which leads to the activation of NFkB rather than IRF3 11 .
TLR7 and TLR9 are expressed pri marily in plasmacytoid DCs (pDCs), but signal also through MyD88.MyD88 forms a complex with TRAF3, TRAF6, IRAK1, IKKα and IRF7.Then, IRF7 is phosphorylated by IRAK1 and IKKα, which then translocates into the nucleus to regulate the expression of type I IFN.
Although TLRs play a central role in the initiation of immune responses against a number of pathogens, it has become apparent that PRRs other than TLRs are also involved in PAMP recognition and the control of innate immunity.These include membrane bound CLRs, cytosolic proteins such as NLRs and RLRs.

C-type lectin receptors
CLRs are a large superfamily of membrane proteins comprising one or more Ctype lectinlike domains, which largely elicit inflammatory responses by recognizing fungal and bacterial PAMPs.The term 'Ctype lectin' was introduced to distinguish between Ca 2+ dependent and Ca 2+ independent carbohydratebinding lectins.CLRs have at least one carbo hydrate recognition domain, which is a compact structural module contain ing conserved residues and deter mines the carbohydrate specificity of the CLR 12 .CLRs exist as both soluble and transmembrane proteins.The transmembrane CLRs function as PRRs, and can be divided into two groups: (1) CLRs belonging to the mannose receptor family and (2) CLRs including the DCassociated Ctype lectin 1 and DC immunorecep tor subfamilies.CLRs are primarily expressed by DCs and interact with pathogens through the recognition of carbohydrate (mannose, fucose and glucan) structures.Mannose structures allows the recognition of viruses and fungi.Fucose structures more specifically recognize some bacteria and helminths, while glucan structures recognize some fungi and mycobacteria 13,14 .Recognition by CLRs leads to the internalization of the pathogen, its degradation and subsequent antigen presentation 14 .
CLR triggering by different patho gens and the ensuing under lying signalling cascade depend on carbohydrate-specific signalling pathways and the DC subset.There are two ways by which CLRs induce sig nalling pathways.(1) CLRs such as macrophage inducible Ctype lectin (CLeC4e), dectin 2 (CLeC6A) and blood DC antigen 2 protein (BDCA2) induce signalling pathways through immunoreceptor tyrosinebased acti vation motif (ITAM) containing adap tor molecules, such as Fc receptor γ-chain (FcRγ) or DAP12 15,16 .(2) Other CLRs, such as dectin 1 and DC-specific ICAM3 grabbing nonintegrin, induce signalling pathways through the activation of protein kinases or phatases that interact with their cytoplasmic domains 17,18 .Many CLRs are known to induce signalling path ways that modulate TLR-induced gene expression at the transcription level in an NFkBdependent fashion.

RIG-I and MDA5 consist of two
Nterminal caspaserecruitment doma ins (CARDs), a DExD⁄H box RNA helicase domain and a Cterminal repressor domain (RD), whereas LGP2 lacks a CARD and appears to be a positive regulator of signalling by MDA5 and RIGI 22 .
RLRs recognize viral RNAs in the cytoplasm.RNA virus infection leads to the generation of dsRNA and RNAs with 5'-triphosphate ends in infected cells.The helicase domain and RD are important for the recognition of these RNAs, while the CARDs are essential for triggering intracellu lar signalling cascades 21 .dsRNA is present in cells infected with dsRNA viruses as well as generated during the course of ssRNA virus replica tion.As host cells do not produce dsRNA, the innate immune system discriminates between host and viral RNAs by the presence of dsRNA.MDA5 is responsible for IFN produc tion to dsRNA stimulation 23 .Reci procally, RIGI is essential for IFN production in response to ssRNA with 5'-triphosphate ends 24 .
RIGI and MDA5 proteins discrimi nate the lengths of dsRNA.Small dsRNAs of up to 1 kb are recognized by RIGI but not by MDA5.On the other hand, dsRNAs bigger than 2 kb can be recognized by MDA5.RIGImediated signalling is posi tively and negatively controlled by ubiquitination of RIGI.The CARDs of RIGI undergo Lys63linked ubi quitination by TRIM25, a ubiquitin E3 ligase 25 .This ubiquitination is necessary for efficient activation of the RIG-I signalling pathway.RIG-I also undergoes ubiquitination by the ubiquitin ligase RNF125, which leads to its proteasomal degradation 26 .Ubiquitination by RNF125 is consid ered to inhibit aberrant activation of RIGI signalling.
In response to viral RNAs, RIGI and MDA5 associate with an adapter protein designated as mitochondrial antiviral signalling (MAVS), also known as virus-induced signalling adapter (VISA) or CARD adapter inducing IFNb (CARDIF) 27,28 .MAVS contains a CARD in its Nterminus and shares homology with the first CARDs of RIGI and MDA5 for homo typic CARD-CARD interaction.The interaction is followed by MAPK activation and transcription induc tion by IRF3, IRF7 and NF-kB.Many of the components found down stream from TRIF in TLR signal ling also are engaged by MAVS.For example, the kinases TBK1 and IKKε mediate IRF3/7 activation and induction of type I IFN genes 29 .

NOD-like receptors
NLRs are the family of cytosolic immune receptors characterized by the presence of two shared features, Cterminal leucinerich repeats (LRRs) and a NACHT nucleotide binding domain (NBD).The LRR domains are responsible for the recognition of PAMPs and proteinprotein interactions, whereas the NBD domain binds ribonucleotides and regulates selfoligomerization 30 .
Though the NLRs have com mon features, they differ in their Nterminal domains.These dif ferences are used to classify the NLR protein members.The largest group has an Nterminal pyrin domain (PYD) and is therefore called 'NLRP'.Another group, which has an Nterminal CARD, contains the proteins nucleotidebinding oligomer ization domaincontaining 1 (NOD1, also called NLRC1), NOD2 (NLRC2) as well as the NLR family, CARD domaincontaining 4 (NLRC4, also known as IPAF).Other NLR family members also have an acidic trans activation domain or a baculoviral inhibitory repeatlike domain, such as the NLR family, apoptosis inhibi tory protein 5 (NAIP5).
NLRP1, NLRP3 and NLRC4 assemble multimolecular protein complexes called 'inflammasomes' in response to various activators, leading to the activation of inflammatory caspases.Activated caspase1 regulates the maturation of IL-1β and IL-18 cyto kines.AIM2, also known as PYHIN4, not belonging to the NLRP family, but to a different protein family (PYHIN), also assembles an inflammasome 31 .Different inflammasomes are acti vated by different stimulus (Table 2).
Upon activation, the inflamma some oligomerizes and recruits procaspase1 directly by CARD interaction (e.g.NLRP1 or NLRC4 inflammasomes) or indirectly via the adaptor protein apoptosis associated specklike protein (ASC).In the latter case, NLRP3 or AIM2, for example, interact with ASC via the homotypic interaction of their PYDs.ASC, in turn, interacts with procaspase1 via their CARDs.Caspase1 in resting stage is pres ent in a catalytically inactive proform.The formation of the inflamm asome initiates autocatalytic activation of caspase1, and then cleaves the zymo gens (proIL1β and proIL18) to active IL1β and IL18.
NLRP1 differs the other mem bers of the NLR family in its dom ain organization.Like other members of the NLRP subgroup, it has an N-terminal PYD, followed by an NBD and an LRR region.However, in con trast to all other members, NLRP1 has a Cterminal extension consist ing of a FIIND motif and a CARD.NLRP3 (cryopyrin), the most studied of all inflammasomes, contains the NLRtypical elements LRR and NBD and an Nterminal PYD.It can also recruit the adaptor protein ASC by PYD interactions 32 .Oligomerization of NLRP3 in response to a stimulus and subsequent recruitment of ASC can activate caspase1.In order to oligomerize, NLRP3 further requires binding of ATP to its NBD element 30 .Interestingly, only one large NLRP3 inflammasome is formed per cell primarily consisting of adaptor ASC.NLRP3 activation is a two-step process.The first signal leads to NF-KB activation, which upregulates pro-IL-1β and NLRP3 expression.The second signal is generated by the stimulus itself in the form of crystals, aggregated protein, ATP or bacterial toxins [33][34][35] .The second signal causes either lyso somal damage or potassium efflux or ROS production, which eventu ally leads to the inflammasome activation.In Gramnegative bacteria, NLRP3 activation requires a third sig nal in which TRIF activates caspase-11 via type I IFN signalling 36 .Caspase11 subsequently synergizes with NLRP3 activation to regulate caspase1 acti vation.
NLRC4 is the only member of the NLRC family known to activate an inflammasome.With its CARD, NLRC4 directly interacts with caspase-1 without the ASC adaptor protein.However, interaction with the adaptor protein ASC is required for enhanced IL-1β production 37 .
AIM2 contains an HIN200 domain, which recognizes double-stranded DNA, and a PYD domain, which is required for the interaction of ASC and the formation of a caspase1 activating inflammasome 31 .
New members of the NLR family (NLRP6 and 12) also induce inflamma some activation in an ASC-caspase1 dependent fashion 38,39 .

Pathologic implication
More than any other cytokine family, the IL1 family of ligands and recep tors is gaining importance due to its association with various acute and chronic inflammation.Of the IL-1 family, IL-1β is of prime importance these days.Several autoinflammatory diseases such as the Muckle-Wells syndrome (MWS), familial cold auto inflammatory syndrome (FCAS) and neonatal onset multisystem inflamma tory disease (NOMID) characterized by episodes of fever, localized inflam mation and skin rashes are attributed to mutations in the CIAS1 gene encoding NLRP3 or in the NLRP3 promoter.The classic autoinflamma tory diseases-familial Mediterra nean fever (FMF), pyogenic arthritis (PAPA syndrome) and cryopyrin associated periodic syndromes (CAPS) are also caused by aberrant IL-1β production.More common diseases such as cancer, gout, type II diabetes, atherosclerosis and rheumatoid arthr itis (RA) are responsive to IL-1β neu tralization.Anakinra, an IL1 receptor antagonist, is the first known drug for treating RA.Anti-IL-1β antibodies are also being used for the treatment of these diseases 40 .

Conclusion
There are still many unresolved questions, such as the exact nature of the molecular events leading to dif ferent immune receptor activation and also identity of some unknown ligands for the receptors.Unravelling of these will offer insight into what critical components might be targeted for better therapeutic benefits in inflammatory disorders.As more tools become available, the future of inflammation research will be more exciting.