WELCOME

K.KARTHIK,

MVSc,

VBM, IVRI

4000 A DAY!!!

M.tuberculosis when unleashed, lashes 4000/day

NEUTROPHILS IN TB

INTRODUCTION

• TB is transmitted through aerosol – macrophages and dendritic cells first to encounter bacilli

• Participation of neutrophils in monocyte recruitment, granuloma formation and lung repair (Antony et al., 1983)

FACTS ABOUT NEUTROPHILS

• Kills organism by both oxidative (phagocytic) & non oxidative ( degranulation )

( Kumar et al., 2010)

GRANULE TYPE PROTEIN

azurophilic granules (or "primary granules")

myeloperoxidase, bactericidal/permeability-increasing protein (BPI), Defensins, and the serine proteases neutrophil elastase

specific granules (or "secondary granules"

Lactoferrin and Cathelicidin

tertiary granules cathepsin and gelatinase

NEGLECTED NEUTROPHILS

• Poorly ranked in case of TB

Short lived

Easily activated

Cryopresevation

difficult

Invitro study

difficult

NEED OF THIS TOPIC?

• Commonly affected phagocyte in human

Eum et al., 2010

• Contribute to control of TB in bloodi

Mirtineau et al., 2007

• Neutrophil driven interferon- disease pathogenesis

Berry et al., 2010

NEUTROPHILS IN TB – 2 WAY TRACK

TRACK 1• Neutrophils causes decrease

in downstream CFU in lung – with 200 M.tuberculosis infection

( Sugarwara et al., 2004)• Depleting murine

granulocyte before challenge with infection , increase CFU

( Barrios et al., 2006)

TRACK 2• No effect on CFU in case of

granuloctye depletion

( Seiler et al., 2000)• RB6-8C5 monoclonal

antibody – to deplete granulocyte receptor , also targets dendritic and monocytes

( Wojtasiak et al., 2010)

BACK TO THE BASICS

Preliminary steps by neutrophils after entry of the organism:

• Recruitment• Recognition• Phagocytosis• Killing

NEUTROPHIL RECRUITMENT

PACE OF RECRUITMENT (HOURS)

PLACE ORGANISM REFERENCE

1 Multiple perivascular sites

M.tuberculosis Long et al., 1931

2 Hepatic infiltration M.avium Feng et al., 2003

3 Skin infiltration BCG , rabbits Shigenaga et al., 2001

4 Dermal infiltration BCG , mice Abadie et al., 2005

MECHANISM OF RECRUITMENT

• Sensitized animals- powerful immune response to mycobacterial challenge ( Long et al., 1931)

• IL 17 & IL 23 from Th 17 – masters the orchestera ( Cruz et al., 2010)

• IL8 from macrophage also joins the party

(Lyons et al., 2002)

STEPS INVOLVED

Initial signal - cytokine release

Activation of endothelium, increase in adhesion molecules

Influx of neutrophils, initiation of complement through chemo attractant C5a

PHAGOCYTOSIS

• Neutrophils directly interact & internalize mycobacteria ( Wolf et al., 2007)

2 mechanism mediate interaction• Direct recognitition• Opsonisation

DIRECT RECOGNITION• Pattern recognition receptor mediate interaction

(May et al., 1987)• TLR2 also involved• Impaired control of M.tuberculosis & M.avium in TLR2

deficient mice (Feng et al., 2003)• TLR2 – mycobacterial ligand – lipoarabinomannan / 19 Kda

lipoprotein

(Neufert et al., 2001)• TLR4 also involved – blocking – reduce IL8 production

( Godaly et al., 2005)

• Complement receptor 3&4 also bind directly

(Aleman et al., 2004)

OPSONISATION

• Opsonisation also plays important role in regulating phagocytosis

• Reduction in ficoll isolated neutrophils to phagocytose after heat inactivation of serum

( Majeed et al., 1998)

DOES IT KILL MYCOBACTERIA?

• Controversial stuff• Theoretically – it kills & halt during early infection

MECHANISM:• Human neutrophil peptides – α defensin – cationic –

azurophil granules bind to anionic molecules

(Fu et al., 2003)• M.avium, M.kansasii, M.smegmatis, M.tuberculosis

fail to bind ( Perskrist et al., 2002)

Cont..

• M.tuberculosis gene lysX – similar to S.aureus gene mprF – increases lysine content – decreases negative charge- decrease suseptibility to HNP

( Maloney et al., 2009)• HNP also be taken by macrophages – ability to kill

organism ( Sharma et al., 2000)• Phagocytosis of apoptotic neutrophils by

macrophages – restriction of mycobacterial growth

( Tan et al., 2006)

TROJAN HORSE

• In the absence of killing the bacteria, neutrophils traffics the infection to other organs – GRANULOCYTE TROJAN HORSE

( Eruslanow et al., 2005)• Mice treated with anti IL17 during infection shows

100 fold low organism in spleen

(Redford et al., 2010)

NEUTROPHIL EXTRACELLULAR TRAPS (NETs)

• NETs composed of nuclear chromatin / mitochondrial DNA associated with histones & granular antimicrobial proteins

( Yousefi et al., 2009)• Formed in respone to pro inflammatory stimuli

( Brinkmann et al., 2004)• It traps Mycobacteria ( Ramos et al., 2009) – unable to

kill – instaed it kill Listeria – confirming anti microbial property

• Hence it causes localization

– basis of granuloma

ECTOSOMES (ECTs)

• Is the vesicles from cell membrane in respone to stimuli ( Gasser et al., 2003)

• Ranges from 50-200 nm – have chemo attractant & pro inflammatory property

• It is cholesterol enriched , express CD35 ( CR1)

(Gasser et al., 2003)• ECTs from PMN bind to endothelial & macrophages

but not to red cells – play role in immune response

NETs & ECTs

NETs ECTs

NEUTROPHILS & MACROPHAGES CO-OPERATION

• Clearance of short lived neutrophils is carried out by macrophages

• Neutrophil derived chemokines attract monocyte from blood (Mantovani et al., 2011)

• Mycobacterial Lipoarabinomannan – stimulate macrophage chemo taxis (Fietta et al., 2000)

• Increase in apoptosis in neutrophils after mycobacterial internalization – oxidative process (Persson et al., 2008)

APOPTOSIS- ANTI / PROINFLAMMATORY

ANTI INFLAMMATORY• Apoptosis – anti

inflammatory results in induction of TGF ß, PGE2

• Inhibits IL6 ,IL8, IL12 & TNF from macrophages

( Krysko et al., 2006)

PRO INFLAMMATORY• Pro inflammatory due to

expression of heat shock proteins

(Perrson et al., 2008)• Activation of macrophage

by neutrophil proteases

Cont..

• Phagocytosis of apoptotic cell- may be anti / pro inflammatory based on :

Mycobacteria inside neutrophil is alive / dead

• Live: pro inflammatory• Dead : anti inflammatory

AS SIMPLE AS THAT……

NEUTROPHILS ON ACQUIRED IMMUNITY

• Neutrophils produce IL12, interferon gamma, macrophage inflammatory protein – attracts T lymphocte ( Seiler et al., 2003)

• Produce IL10 limit acquired immunity

(Dorhoi et al., 2010)• Cross present antigen to dendritic cells

( Morel et al., 2008)

TOTAL EFFECT OF NEUTROPHILS

CONCLUSION• Neutrophils are seen in the early stages of the

Mycobacterial infection.• In chronic cases the same neutrophils may act in the

pathology of Granuloma formation• Thus neutrophil act as a “Double edged Sword”.• Whether it kills Mycobacteria ??• It may disseminate the organism to various organs.

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