Characterisation of toll like receptor 8 as a new innate immune receptor for mycobacterium tuberculosis

Abbreviations ...xiii!. List!of!Publications ...xvii!. Mycobacterium, tuberculosis, MTB.!. Mycobacterium- tuberculosis., Manuscript!. mycobacteria!that!includes!the!M.,avium,complex,!M.,

! ! ! !

! CHARACTERISATION!OF!TOLL.LIKE!RECEPTOR!8!AS!A!NEW!

!

! NATIONAL!UNIVERSITY!OF!SINGAPORE!

2012!

This! work! would! not! have! been! possible! without! the! contribution! of! the!following!people!–!Sonia!Davila!and!Martin!Hibberd!from!the!Genomic!Institute!of! Singapore! for! genomic! information;! Dr! Andres! Floto,! Krisztina! Hegyi,!Catherine! Schaffner,! Karen! Brown,! Lucy! Hepburn,! and! Sarah! Coulter! from! the!Cambridge! Institute! for! Medical! Research! for! pH! data! and! transfection!experiments!on!human!macrophages;!Dr!Sylvie!Alonso,!Lin!Wenwei!and!Vanessa!Koh!for!work!on!MTB;!Dr!Seah!Geok!Teng!for!the!various!mycobacteria!species;!Prof!Ng!Mah!Lee!and!Yap!Han!for!electron!microscopy!work;!!Prof!Mike!Kemeny!and!Christopher!Yang!!for!cloning!the!TLR8.2!receptor;!and!A/Prof!Lu!Jinhua!and!Jason!Goh!for!the!TLR8!WT!plasmid!and!techniques!for!the!dual!luciferase!assay.!!

Life!in!the!laboratory!would!not!have!been!so!enjoyable!without!the!company!of!my! fellow! lab! members,! especially! Voja,! Adrian,! Yanting,! Enwei,! and! Nurhuda.!!Thank! you! for! your! fun! and! laughter,! as! well! as! your! precious! blood.! Special!thanks!go!to!Adrian!for!proofreading!my!thesis.!!I!would!also!like!to!thank!my!family!and!friends!for!their!continuous!support!and!encouragement.!

!

!

Table.of.Contents.

Acknowledgements ii!

Table!of!Contents iii!

Summary viii!

List!of!Figures ix!

List!of!Tables xii!

Abbreviations xiii!

List!of!Publications xvii!

Chapter!1.!Introduction 1!

1.1! Mycobacterium,tuberculosis 2!

1.1.1! Mycobacteria 2!

1.1.2! Human!Exposure!to!Mycobacteria 3!

1.1.3! Epidemiology!of!TB 3!

1.1.4! Pathogenesis!of!TB 4!

1.2! Innate!Immunity!to!Mycobacteria 6!

1.2.1! Host!Genetics!and!Immunity!to!Mycobacteria 6!

1.2.2! Innate!Immunity!to!MTB,Infection 8!

1.2.3! Uptake!and!Recognition!of!Mycobacteria!by!Macrophages 10!

1.2.4! Intracellular!Fate!of!MTB 11!

1.3! The!Role!of!Toll.Like!Receptors!in!Mycobacterium!Infections 12!

1.3.1! Toll.Like!Receptors 12!

1.3.2! TLRs!in!Mycobacterium!Infections 19!

1.4! Toll.Like!Receptor!8 29!

1.4.1! TLR8!Biology 29!

1.4.2! Agonists!of!TLR8 30!

1.4.3!! Expression!and!Localisation!of!TLR8 33!

1.4.4! Immune!Functions!Associated!with!TLR8 34!

1.4.5! TLR8!in!Bacterial!Infections 35!

1.4.6! Genetic!Variation!in!Human!TLR8 36!

1.5! Aims!of!this!Study 37!

Chapter!2.!Materials!and!Methods 39!

2.1! Solutions!and!Buffers 40!

2.1.1! Buffers!for!Cell!Culture!and!Isolation!of!Primary!Cells 40!

2.1.2! Buffers!for!Mycobacteria!Culture!and!Work 40!

2.1.3! Buffers!for!SDS.PAGE!and!Western!Blot 41!

2.1.4! Buffers!for!DNA!Gel!Electrophoresis 42!

2.1.5! Buffers!for!Metabolic!Labeling 42!

2.1.6! Buffers!for!Confocal!Microscopy 43!

2.1.7! Buffers!for!Electron!Microscopy 43!

2.2! Reagents 44!

2.2.1! TLR!Agonists 44!

2.2.2! Antibodies 44!

2.2.3! Plasmids 44!

2.3! Primary!Cells!and!Cell!Culture 47!

2.3.1! Cell!Culture 47!

2.3.2! Differentiation!of!THP1!and!THP1!Blue!cell!lines 47!

2.3.3! Isolation!and!Culture!of!Primary!Human!Macrophages 48!

2.4! Mycobacteria!Culture 49!

2.4.1! Mycobacteria!Culture 49!

2.4.2! Alexa!Fluor®!647!Labelling!of!BCG 49!

2.4.3! Generation!of!Mycobacterial!Homogenates 50!

2.4.4! Treatment!of!BCG!Homogenates!for!Ligand!Identification 50!

2.4.5! Isolation!of!BCG!RNA 51!

2.4.6! Isolation!of!BCG!DNA 51!

2.4.7!! Isolation!of!BCG!Proteins 52!

2.4.8! 16S!derived!phylogeny!of!the!Mycobacterium!genus 53!

2.5! Genotyping!TLR8!M1V!Frequencies 53!

2.6! Transfection 54!

2.6.1! Lipofectamine!Transfection!of!HEK293T!cells 54!

2.6.2! Accell!TLR8!small!interfering!RNA!transfection!in!THP1 54!

2.6.3! Transfection!in!primary!macrophages 55!

2.7! Analysis!of!NFκB!activation 55!

2.7.1! Quantification!of!NFκB!activation!in!THP1!Blue!cells 55!

2.7.2! Dual.Luciferase!Reporter!Assay 55!

2.8! Analysis!of!TLR8!Transcripts!and!Proteins 56!

2.8.1! Signal!Peptide!Prediction 56!

2.8.2! RT.PCR 57!

2.8.3! SDS.PAGE!and!Western!Blot 58!

2.8.4! Analysis!of!TLR8!Glycosylation 59!

2.9! Imaging 60!

2.9.1! Confocal!Microscopy 60!

2.9.2! Transmission!Electron!Microscopy 60!

2.10! Infection!Studies 61!

2.10.1! Mycobacteria!Killing!Assay!By!Quantification!of!CFU 61!

2.10.3! Analysis!of!BCG!Phagolysosome!Maturation 62!

2.10.4! Measurement!of!Phagosomal!pH 63!

2.10.5! Bioplex!Cytokine!Array 63!

2.10.6! Bioplex!Phosphoprotein!Array 64!

2.10.7! Prostaglandin!E 2! Analysis 65!

2.11! Statistical!Analysis 65!

Chapter!3.!Results!I 66!

3.1! Introduction 67!

3.2! In,Silico,Analysis!of!5’!Polymorphisms 70!

3.3! TLR8!M1V!is!the!Predominant!Variant!in!the!Singaporean!Population 70!

3.4! The! Regulation! of! TLR8! Expression! is! Unaffected! by! the! 5’! Regulatory! and! M1V!Polymorphisms 75!

3.5! The!TLR8!M1V!Variant!Uses!An!Alternative!Start!Codon 78!

3.6! TLR8!WT!and!M1V!Differ!in!the!N.Region!of!Their!Signal!Peptide 81!

3.7! TLR8!WT!and!M1V!Variants!Undergo!Similar!Processing!in!the!ER 85!

3.8! TLR8!WT!and!M1V!Both!Traffic!Via!the!Common!Secretory!Pathway 90!

3.9! TLR8!WT!and!M1V!Both!Contain!an!Intracellular!Targeting!Motif 92!

3.10! TLR8!Variants!Have!Differential!Preferences!for!Endosomal!Compartments94! 3.11! TLR8!M1V!Shows!a!Stronger!NFκB!Response 99!

Chapter!4.!Results!II 107!

4.1! Introduction 108!

4.2! Establishing! THP1! as! an! Experimental! Model! for! Studying! Mycobacteria! Recognition!by!TLR8 108!

4.3! TLR8!is!Recruited!to!the!Mycobacterial!Phagosome 112!

4.4! TLR8!is!Involved!in!Mycobacterial!Sensing 114!

4.5! TLR8!is!a!Mycobacteria!Sensor 117!

4.6! The!Generation!of!the!TLR8.2!Fusion!Receptor 119!

4.7! The!Expression!of!the!TLR8.2!Fusion!Receptor 121!

4.8! Ligand!Screening!with!the!TLR8.2!Fusion!Receptor 123!

4.9! The!Identification!of!the!Mycobacteria!Derived!Ligand!for!TLR8 125!

4.10! Reduced! TLR8! Levels! Lead! to! Impaired! Mycobactericidal! Activity! in! Monocytic!Cells 129!

4.11! Discussion 132!

Chapter!5.!Results!III 136!

5.1! Introduction 137!

5.2! The!M1V!Polymorphism!Affects!the!Kinetics!of!Phagosome!Maturation 137!

5.3! The!Mycobacteria!Phagosomes!of!TLR8!M1V!Macrophages!Are!Characteristic! of!Bacteriocytic!Phagosomes 143!

5.4! TLR8! M1V! Expressing! Macrophages! Have! Enhanced! Intracellular! Mycobactericidal!Activity 147!

5.5! Molecular! Mechanisms! for! Functional! Differences! Between! TLR8! WT! and! TLR8!M1V 151!

5.6! Effector!Mechanisms!for!Functional!Differences!Between!TLR8!WT!and!TLR8! M1V 153!

5.7! An!Analysis!of!the!Cyclooxygenase!2!Pathway 156!

5.8! TLR8!M1V!Transfection!‘Rescues’!WT!Primary!Macrophages 160!

5.10! Discussion 163!

Chapter.6:.Discussion 169!

Chapter.7 References 181!

!

Summary.

Previous! reports! have! implicated! the! chemokine! receptor! CCR5! and! Toll.like!

receptors! TLR2! and! TLR4! in! the! innate! immune! recognition! of! Mycobacterium,

tuberculosis, (MTB).! In! this! study,! we! provide! comprehensive! genetic,!

biochemical,! cellular! and! mechanistic! data! linking! intracellular! TLR8! to! the!detection! and! host! response! to! pathogenic! mycobacteria.! Natural!polymorphisms! in! TLR8! have! been! implicated! in! susceptibility! or! protection!from! pulmonary! MTB! infections! in! Indonesian! and! Russian! cohorts.! Here! we!demonstrate! that! the! protective! polymorphism! termed! rs3764880! (M1V)!generates! a! functional! TLR8! receptor! variant! with! an! altered! signal! peptide,!causing! the! trafficking! of! TLR8! to! early! rather! than! late! endosomes.! The!protective! TLR8! polymorphism! results! in! increased! inflammatory! cytokine!release,!promotes!phagosomal!acidification!and!enhances!intracellular!killing!of!MTB.! These! results! indicate! that! the! TLR8! receptor! pathway! may! represent! a!

new!target!for!pharmacological!or!immunological!manipulation!in!MTB,infection.

!

Figure!3.3! Allelic! frequencies! of! SNP! rs3764880! in! various!

Figure!5.3! Quantification! of! phagosome! acidification! with!

Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5.!Science!314(5798):!454.458.!

!

Oh!SHO,!Hegyi!K,!Davila!S,!Mustafa!F,!Schaffner!C,!Brown!K,!Hepburn!L,!Coulter!S,!Koh!HQV,!Lin!W,!Alonso!S,!Ng!ML,!Dass!RH,!Yang!MLC,!Kemeny!DM,!Seah!GT,!Lu!

JH,! Hibberd! ML,! Floto! RA! and! MacAry! PA.! Polymorphisms in TLR8 control.

phagosome responses to Mycobacterium- tuberculosis., (Manuscript!

submitted) .

Aziz! NH,! Oh! SHO,! Gan! SU,! Shui! GH,! Noor! Shahid,! Lim! YT,! Chan! CE,! Hanson! BJ,!

Wenk! MR,! MacAry! PA.! Dissecting the role of lipids in FcKmediated.

.Chapter.1 Introduction.

and! treatment! ! the! M., tuberculosis! complex,! causative! agents! of! tuberculosis! (TB);! M., leprea, which! causes! leprosy;! and! the! group! of! typically! opportunistic!

non.tuberculous! mycobacteria! (also! known! as! atypical! or! environmental!

mycobacteria)!that!includes!the!M.,avium,complex,!M.,scrofulaceum,,M.,marinum, and!the,M.,fortuitum,complex!(Brooks!et!al.,!2010).!!!

MTB!along!with!M.,bovis,!M.,africanum,!M.,canettii,!M.,microti,!M.,caprae,!and!M., pinnipedii,are!members!of!the!MTB!complex!(Bouakaze!et!al.,!2010;!Brooks!et!al.,!

2010).! Despite! being! genetically! similar,! having! more! than! 85%! DNA.DNA!relatedness,!they!exhibit!differences!in!host!tropism!(Imaeda,!1985;!Sreevatsan!

et!al.,!1997).!For!instance,!while!MTB!and!M.,africanum,are!human!pathogens,!M.,

bovis!is!a!pathogen!mainly!of!cattle!whereas!M.,mircoti,causes!disease!in!small!

wild!mammals,!namely!voles,!wood!mice!and!shrews!(Brooks!et!al.,!2010;!Wells!and!Oxon,!1937).!!!

Mycobacteria!cannot!be!classified!as!gram!positive!or!negative.!Instead,!they!are!characterised!as!acid.fast!bacteria!for!their!resistance!to!decolorisation!by!acid!alcohol! during! staining! procedures.! Mycobacteria! have! characteristic! thick! cell!walls! which! are! extremely! rich! in! complex! lipids! such! as! mycolic! acids,! waxes!

1.1.2 Human.Exposure.to.Mycobacteria.

Exposure! to! environmental! mycobacteria! is! common! and! highly! prevalent.!Environmental! mycobacteria! can! be! found! in! a! large! variety! of! environments!including!air,!soil,!water,!treated!water,!and!food;!and!thus!frequently!come!into!contact! with! the! skin,! and! digestive! and! respiratory! epithelial! (Casanova! and!Abel,! 2002;! Primm! et! al.,! 2004).! The! bacille! Calmette.Guérin! (BCG)! vaccine,!

containing! an! attenuated! strain! of! M., bovis,! has! been! in! use! since! 1921.! It! is!

estimated!that!more!than!one!billion!people!have!received!BCG,!administered!to!neonates! and! infants! in! many! countries! as! part! of! childhood! immunisation!programmes! (WHO,! 2011).! TB! and! leprosy! are! endemic! in! most! developing!

countries!of!the!world,!exposing!large!populations!to!MTB,and!M.,leprae,(WHO,! 2011).,!

!

1.1.3 Epidemiology.of.TB.

Present!since!antiquity,!TB!is!one!of!the!oldest!diseases!known!to!mankind!and!continues!to!have!a!devastating!impact!on!public!health!in!the!developing!world,!and!on!immuno.compromised!individuals!in!the!developed!world.!!The!ability!of!MTB!to!efficiently!establish!latency!in!the!human!host!has!enabled!the!tubercle!

1.1.4 Pathogenesis.of.TB.

The! initial! phase! of! a! MTB! infection! occurs! with! inhaled! mycobacterium! being!taken! up! by! lung! resident! alveolar! macrophages.! The! bacterium! may! be!destroyed! by! the! macrophage,! eliminating! infection! completely.! Alternatively,!the! mycobacterium! may! escape! this! initial! destruction,! multiplies! to! large!numbers,!and!kills!the!macrophage.!Mycobacteria!are!consequently!released!into!the! extracellular! environment! where! they! are! taken! up! by! other! macrophages,!thus!contributing!to!the!establishment!of!an!active!infection!of!the!lung.!Infected!macrophages!secrete!cytokines!and!chemokines!that!attract!dendritic!cells!and!

additional!macrophages.!These!cells!accumulate!to!form!a!granulomatous!lesion,!a!characteristic!feature!of!TB!(Iseman,!2003; van!Crevel!et!al.,!2002).!!!

T!cell!immunity!develops!two!to!three!weeks!post!infection.!On!recruitment!to!the! infection! site,! antigen! specific! CD4! and! CD8! T! cells! activate! infected!macrophages! via! the! production! of! cytokines,! primarily! interferon! gamma!(IFNγ).! CD8! T! cells! also! contribute! to! the! control! of! infection! by! lysing!chronically!infected!macrophages,!serving!to!release!intracellular!mycobacteria!for! uptake! by! activated! macrophages! that! have! a! cytokine! driven! improved!capacity!for!phagocytosis!and!killing!(Raviglione!and!O'Brien,!2008;!van!Crevel!et!al.,!2002).!!!

The!granuloma!physically!contains!the!infection,!preventing!dissemination,!with!caseation!of!the!centre!occuring!as!macrophages!die.!The!anaerobic!conditions,!as! well! as! low! pH! within! the! caseous! tissue! inhibit! the! growth! of! extracellular!MTB,! and! the! mycobacteria! may! remain! dormant! there! for! decades.! ! However,!under! conditions! of! failing! immune! surveillance,! disease! may! progress! via! the!liquefaction! of! the! caseous! center,! forming! an! air.filled! cavity! in! which!extracellular! bacilli! multiply.! The! cavity! may! rupture! nearby! bronchi,! allowing!the! bacilli! to! be! disseminated! through! the! airways,! to! the! circulatory! and!lymphatic!system!and!thus,!causing!a!systemic!infection!(van!Crevel!et!al.,!2002).!!

Furthermore,!clinical!symptoms!of!TB!can!be!extremely!diverse,!representing!the!outcome! of! complex! interactions! between! the! host! and! mycobacterium.! TB!primarily!presents!as!a!pulmonary!disease,!but!it!may!spread!to!any!part!of!the!body,! ranging! from! the! central! nervous! system,! lymphatic! system,! circulatory!system,!genitourninary!systerm,!bones,!joint!and!skin.!!Disease!localisation!and!severity! also! varies! upon! the! onset! of! active! TB! (van! Crevel! et! al.,! 2002).!Tuberculous! pleuritis! is! usually! self.limiting! whereas! the! most! serious! disease!manifestation!is!miliary!TB,!which!is!characterised!by!the!hematogenous!spread!of! mycobacteria! throughout! the! body! (van! Crevel! et! al.,! 2002).! The! immune!mechanisms!that!distinguish!individuals!who!develop!clinical!manifestations!of!TB!and!those!who!do!not!are!largely!unknown,!although!a!predictive!IFN!driven!transcriptional! signature! for! active! TB! that! correlates! with! disease! extent! has!

In! particular,! genetically! influenced! variation! in! macrophage! function! has! been!demonstrated! in! several! studies! to! influence! susceptibility! to! mycobacterium!infection! (Berrington! and! Hawn,! 2007).! Black! populations! in! the! United! States!are!reported!to!be!more!susceptible!to!TB!than!Caucasians,!and!the!basis!for!this!racial! difference! has! been! attributed! to! the! differential! permissiveness! of! host!macrophages! to! contain! internalised! mycobacteria! (Crowle! and! Elkins,! 1990).!!!Also,!individuals!with!chronic!granulomatous!disease,!a!genetic!disorder!wherein!mutations! in! the! NADPH! oxidase! complex! result! in! defectiveness! of! oxidative!defence!in!phagocytes,!are!vulnerable!to!mycobacterial!diseases!(Casanova!and!Abel,! 2002).! Mutations! in! natural! resistance! associated! macrophage! protein! 1!(Nramp1)! have! been! shown! in! mice! to! result! in! impaired! early! resistance! to!

mycobacteria!infections,!in!particular!to!M.,bovis,BCG!(Gros!et!al.,!1981;!Vidal!et!

al.,! 1993).! Nramp1! is! recruited! to! the! mycobacteria! phagosome! where! it!functions!as!a!membrane!transporter,!depleting!phagosomes!of!divalent!cations!required!for!mycobacterial!survival,!and!mice!with!defective!Nramp1!are!unable!

to!contain!intracellular!M.,bovis,BCG!replication!(Gros!et!al.,!1981;!Jabado!et!al.,!

2000).!In!humans,!several!polymorphisms!in!the!human!NRAMP1!gene!have!also!been!linked!to!increased!risk!of!pulmonary!TB!(Casanova!and!Abel,!2002).!!

Reactivation) (5910%))

Latent)Infection)(90%))

Elimination?) Innate)immunity)

Adaptive)immunity)

Similar! observations! have! been! made! in! humans.! In! 1929,! a! tragic! incident!occurred! whereby! 249! infants! up! to! 10! days! old! in! Lübeck,! Germany! were!administered!a!BCG!vaccine!contaminated!with!live,!virulent!MTB.!Seventy!nine!of!the!infants!died!of!TB,!however,!the!one!hundred!and!seventy!three!survivors!show!that!innate!resistance!in!young!infants,!known!to!have!little!or!no!adaptive!immunity,!can!be!effective!against!mycobacteria!infections!(Schluger,!2005).!!

In! a! meta.analysis! of! the! efficacy! of! the! BCG! vaccine! in! preventing! TB,! it! was!found! that! vaccination! efficacy! was! variable,! averaging! at! 50%! protection.!Protective! effect! appears! to! vary! according! to! geography! ! clinical! trials!conducted!in!the!United!Kingdom!have!consistently!shown!a!protective!effect!of!60! to! 80%,! but! trials! conducted! elsewhere! have! shown! little! or! no! protective!effect.! A! detrimental! effect! was! even! reported! in! Madras,! South! India,! and!efficacy! appears! to! fall! as! one! gets! closer! to! the! equator! (Colditz! et! al.,! 1994).!Vaccination! was! more! protective! against! disseminated! disease! rather! than!

who!have!previously!recovered!could!be!caused!by!new!exogenous!reinfections!(van!Rie!et!al.,!1999).!These!studies!demonstrate!that!in!spite!of!existing!T.cell!immunity,! whether! acquired! via! vaccination! or! natural! infection,! there! is!insufficient!protection!against!the!initial!pulmonary!infection!(van!Crevel!et!al.,!2002).! ! This! implies! a! critical! role! for! the! host! innate! immune! system! in! the!

We! have! previously! identified! the! CCR5! chemokine! receptor! as! an! immune!

receptor! for! MTB, (Floto! et! al.,! 2006).! It! is! known! that! constitutive!

1.2.4 Intracellular.Fate.of.MTB.

The!extraordinary!success!of!MTB!lies!in!its!ability!to!create!a!niche!in!host!cells,!namely! macrophages,! by! manipulating! the! intracellular! environment! to! avoid!

destruction.!!The!arrest!of!MTB,phagosome!maturation!and!prevention!of!fusion!

to! lysosomes! is! key! to! its! propensity! to! survive! and! multiply! within! infected!macrophages.! In! resting! macrophages,! MTB! establishes! a! niche! in! a! privileged!compartment,!characterised!by!the!persistence!of!early!endosomal!markers,!that!resists! acidification! thereby! evading! destruction! (Clemens! and! Horwitz,! 1995;!

1.3 The.Role.of.TollKLike.Receptors.in.Mycobacterium.Infections.

1.3.1 TollKLike.Receptors!

The!innate!immune!system!is!the!first!line!of!defence!against!invading!pathogens,!and!is!based!on!the!recognition!of!molecular!signatures!that!are!broadly!shared!across!pathogens!and!thus!discriminate!against!self.cells!and!tissues.!!Detection!of!these!pathogen!associated!molecular!patterns!(PAMPs)!is!mediated!by!PRRs!that! initiate! a! host! defence! response! against! the! invading! pathogen! (Janeway,!1989).!PRR!signalling!also!leads!to!dendritic!cell!maturation,!thereby!regulating!the!initiation!of!the!adaptive!immune!response!(Janeway!and!Medzhitov,!2002;!Kawai!and!Akira,!2011).!!!!

TLRs! are! the! key! PRRs! that! regulate! both! innate! and! adaptive! immune!responses.!These!type!I!transmembrane!glyco.proteins!recognise!a!wide!range!of!PAMPs! including! lipids,! lipoproteins,! proteins! and! nucleic! acids! derived! from!viruses,!bacteria,!fungi!and!parasites!(Table!1.1).!Structurally,!TLRs!are!defined!by! the! presence! of! a! leucine! rich! repeat! in! the! extracellular! domain! (or! the!topologically! equivalent! lumen! of! membrane! enclosed! intracellular!compartments)! for! the! recognition! of! PAMPs! and! a! Toll.IL1R! (TIR)! domain!(homologous!to!the!cytoplasmic!region!of!the!IL1!receptor)!at!the!intracellular!domain,!responsible!for!downstream!signalling!(Bowie!and!O'Neill,!2000;!Meylan!et!al.,!2006).!!

To!date,!there!are!10!and!12!functional!TLRs!characterised!in!humans!and!mouse!respectively.! TLR10! is! not! functional! in! mice,! while! TLR11,! TLR12! and! TLR13!have!been!evolutionarily!lost!in!humans!(Kawai!and!Akira,!2010).!!!While!most!cell! types! express! at! least! one! TLR,! the! greatest! panel! of! TLRs! is! expressed! in!

Cellular!localisation!is!important!for!TLR!function.!TLRs!that!sense!nucleic!acids!(TLR3,!TLR7,!TLR8,!TLR9)!belong!to!the!intracellular!sub.family!of!TLRs!and!are!expressed! within! the! endoplasmic! reticulum! (ER),! endosomes,! multivesicular!bodies! and! lysosomes.! ! The! intracellular! localisation! or! compartmentalisation!physically! prevents! recognition! of! host! nucleic! acids,! while! enabling! access! to!foreign! nucleic! acids! derived! from! phagocytosed! viruses,! bacteria! or! infected!cells!(Blasius!and!Beutler,!2010).!The!other!TLRs!are!expressed!primarily!on!the!cell! surface! and! therefore! mediate! recognition! of! microbial! membrane!components,! but! can! also! be! internalised! into! endosomal! compartments! after!ligand!recognition!(Beutler,!2009;!Kawai!and!Akira,!2011).!!

!

Two! main!TLR! signalling! pathways! have! been! characterised! to! date.! The! first!pathway,! common!to! all! TLRs,! is! through! MyD88,! leading! to! the! activation! of!nuclear! factor! kappa! B! (NFκB)! and! mitogen! activated! protein! kinase! (MAPK),!and!inflammatory!cytokine!production.!The!second,!only!described!for!TLR3!and!TLR4,!is!through!the!activation!of!TRIF!–!an!alternative!pathway!leading!to!NFκB!and!interferon!regulatory!factor!3!(IRF3)!activation,!and!the!induction!of!type!I!IFN!and!inflammatory!cytokines!(Kawai!and!Akira,!2011;!Yamamoto!et!al.,!2002)!(Figure!1.2).!!

TLR!signalling!is!complex!and!allows!for!individual!TLRs!to!initiate!overlapping!and! distinct! signalling! pathways,! depending! on! the! stimulus! (Beutler,! 2009).!How! the! different! ligand.TLR! interactions! lead! to! differential! activation! of!

specific!TLRs.!For!instance,!in!the!case!of!Candida!infection,!TLR2./.!mice!showed!increased! resistance! as! TLR2! activation! favours! the! induction! of!immunosuppressive! interleukin! 10! (IL10),! while! TLR4!defective! mice! showed!increased! susceptibility! to! disseminated! infection,! as! a! result! of! impaired!chemokine!expression!and!neutrophil!recruitment!(Netea!et!al.,!2006;!Netea!et!al.,!2002).!!!

TLRs! in! our! innate! defences! are! indispensible.! Blocking! all! TLR! signalling! via!inactivation! of! both! MyD88! and! TRIF! results! in! severe! immunocompromise,!which! is! evident! in! MyD88! and! TRIF! deficient! mice! that! succumb! to!opportunistic! infections! before! weaning! age! (Beutler,! 2009).! Autosomal.recessive! MyD88! deficiency! predisposes! young! patients! to! recurrent! life.threatening!pyogenic!bacterial!infections!(von!Bernuth!et!al.,!2008).!Also,!studies!on! polymorphic! variants! of! TLRs! demonstrate! that! even! mutations! affecting!individual!TLR!function!can!affect!the!incidences!and/or!progression!of!several!inflammation! related! disease! including! sepsis,! immunodeficiencies,!atherosclerosis!and!asthma;!or!cause!susceptibility!to!specific!infections!such!as!TB!(Misch!and!Hawn,!2008;!Schwartz!and!Cook,!2005).!!

!

Several! mycobacterial! components! have! been! reported! as! agonists! for! TLRs!(Table!1.2)!(Jo!et!al.,!2007).!!Unique!to!the!mycobacterium!family!is!the!proline.glutamic!acid.polymorphic!guanine.cytosine.rich!sequence!(PE_PGRS)!family!of!proteins,! located! on! the! extracellular! surface! of! mycobacteria.! Functions! of!PE_PGRS! are! largely! unknown,! although! it! has! been! widely! proposed! to! be!involved! in! immune! evasion! (Tian! and! Jian.Ping,! 2010).! Several! PE_PGRS!antigens!have!been!found!to!activate!TLR2,!inducing!TNFα!production!in!human!macrophages,! and! the! maturation! and! activation! of! human! dendritic! cells!(Bansal!et!al.,!2010;!Basu!et!al.,!2007).!!

The!19kDa!lipoprotein,!a!cell!wall!component!of!MTB,!is!a!secreted!antigen!that!also!binds!TLR2.!It!is!a!major!stimulator!of!IL12!production!in!human!monocytic!cell!lines!via!TLR2!activation;!and!can!induce!killing!of!intracellular!MTB!in!both!

mouse!and!human!macrophages!by!distinct!mechanisms!(Brightbill!et!al.,!1999;!Thoma.Uszynski!et!al.,!2001).!

'

Table'1.2'|'TLR'recognition'of'mycobacterial'components!

Mycobacterial Component TLR usage Species

19 kDa lipoprotein TLR2 MTB, M bovis

27 kDa lipoprotein TLR2 MTB

33 kDa lipoprotein TLR2 M leprae

38 kDa glycolipoprotein TLR2 and TLR4 MTB

Ligands! have! also! been! identified! for! TLR4.! The! mycobacterial! 38kDa!glycolipoprotein!induces!TNFα!and!IL6!production!in!human!monocytes!through!both! TLR2! and! TLR4! engagement! (Jung! et! al.,! 2006).! Mycobacterial! HSP65!signals!exclusively!via!TLR4!(Bulut!et!al.,!2005).!!

intracellular!MTB,(Liu!et!al.,!2006).!

TLR9! has! also! reported! roles! in! the! immune! response! to! MTB.! ! Mycobacterial!DNA! isolates,! a! potential! ligand! for! bacterial! DNA! sensing! TLR9,! were! able! to!activate! human! and! mouse! macrophages! although! DNA! from! virulent!mycobateria! showed! attenuated! activation! as! compared! to! the! response! from!avirulent! strains! (Kiemer! et! al.,! 2008).! Also,! macrophages! isolated! from! TLR9!knockout! mice! were! more! permissive! for! intracellular! replication! of! avirulent!

forms!of!MTB,!however,!the!absence!of!TLR9!had!no!effect!on!more!virulent!MTB!strains.!

Although! TLR7! is! not! normally! implicated! in! mycobacterium! infections,!stimulation!of!TLR7!by!ligands!imiquimod!or!single!stranded!RNA!(ssRNA)!can!induce! autophagy! in! RAW! 264.7! macrophages! and! subsequently! enhance!

bactericidal!activity!in!M.,bovis,BGC!infected!macrophages!(Delgado!et!al.,!2008).!!!

Interestingly,!MTB!can!also!exploit!TLR!signalling!to!its!advantage.!Excessive!TLR!signalling! can! downregulate! the! immune! response! as! part! of! a! homeostatic!feedback! to! limit! excessive! activation! that! could! lead! to! tissue! damage!(Manicassamy! and! Pulendran,! 2009).! Studies! have! shown! that! in! infected!macrophages,!prolonged!TLR2!signalling!induced!by!mycobacterial!lipoproteins!resulted! in! the! inhibition! of! major! histocompatibility! complex! (MHC)! class! II!expression,!thereby!limiting!antigen!presentation!and!the!subsequent!induction!of!effector!T!cell!reponses!(Harding!and!Boom,!2010).!!

The! persistence! of! MTB, within! the! phagosomes! of! host! macrophages! provides!

opportunities! for! long.term! stimulation! of! TLRs! recruited! to! these!compartments! (Harding! and! Boom,! 2010).! Also,! even! within! the! macrophage!phagosome,! live! MTB! are! able! to! shed! surface! proteins! and! lipids! such! as! the!19kDa! lipoprotein! and! actively! export! these! TLR! agonists! out! of! the! host! cell,!allowing!the!chronic!stimulation!of!surface!TLRs!(Beatty!et!al.,!2000;!Beatty!and!Russell,! 2000;! Neyrolles! et! al.,! 2001).! Therefore,! although! a! negative! feedback!mechanism!during!chronic!infection!may!be!beneficial!to!the!host!by!limiting!the!harmful! effects! of! excessive! inflammation,! an! overwhelming! stimulation! of! the!

Whether!individual!TLRs!by!themselves!are!of!fundamental!significance!in!innate!resistance!to!mycobacteria!is!still!unclear.!TLR2!knockout!mice!exhibited!no!or!

minor! susceptibility! to! low! dose! MTB, infection,! but! succumb! only! at! high!

infection! levels! (Bafica! et! al.,! 2005;! Drennan! et! al.,! 2004;! Reiling! et! al.,! 2002;!Sugawara!et!al.,!2003).!There!is!also!some!suggestion!that!TLR2!functions!as!a!regulator! of! inflammation,! and! in! its! absence,! long.term! mortality! in! MTB!infected!mice!result!from!uncontrolled!inflammation!and!lung!injury!(Drennan!et!al.,!2004).!!

Susceptibility! of! TLR4! deficient! mice! to! MTB! infection! varies! between! various!studies.! Some! studies! show! that! TLR4! deficient! mice! are! equally! resistant,!regardless! of! infection! dose,! while! others! demonstrated! susceptibility! to! TB!infection! reflected! by! an! enhanced! mycobacterial! outgrowth! and! an! increased!mortality!(Abel!et!al.,!2002;!Branger!et!al.,!2004;!Chackerian!et!al.,!2001;!Reiling!et!al.,!2002).!TLR9!has!also!been!demonstrated!in!the!TLR9!knockout!mouse!to!be! required! for! the! regulation! of! mycobacterial.induced! granulomatous!response! (Ito! et! al.,! 2009).! ! Analogous! to! TLR2,! more! profound! effects! and!susceptibility! were! observed! only! at! higher! MTB! infection! doses! (Bafica! et! al.,!2005).!!!!

In! contrast! to! MyD88! deficient! mice,! mice! lacking! individual! TLRs! are! not!

dramatically! susceptible! to! MTB, infections.! This! indicates! some! form! of!

redundancy!in!the!role!of!individual!TLRs!or!that!co.operation!among!multiple!TLRs! is! required! in! mediating! the! immune! response! towards! mycobacteria!infections.! TLR2! and! 9! double! knockouts! showed! high! susceptibility! to! MTB!