Neutrophils play an important role in the early stages of Mycobacterial infection by recruiting to sites of infection and phagocytosing bacteria. However, their overall impact is complex, as they may both help control infection initially but also potentially disseminate bacteria as "Trojan horses" to other organs. While neutrophils have antimicrobial mechanisms, it is unclear if they can directly kill all Mycobacterial species. Their interactions with macrophages in clearance of apoptotic neutrophils and antigen presentation also influence acquired immunity. Overall, neutrophils act as a "double-edged sword" in the pathogenesis of Mycobacterial infections.

1. WELCOME
K.KARTHIK,
MVSc,
VBM, IVRI

2. 4000 A DAY!!!
M.tuberculosis when unleashed, lashes 4000/day

4. 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)

5. 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

6. NEGLECTED NEUTROPHILS
• Poorly ranked in case of TB
Short lived
Easily
activated
Cryopresevation
difficult
Invitro
study
difficult

7. 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

8. 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)

9. BACK TO THE BASICS
Preliminary steps by neutrophils after entry of the
organism:
• Recruitment
• Recognition
• Phagocytosis
• Killing

10. 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

11. 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)

12. STEPS INVOLVED
Initial signal - cytokine release
Activation of endothelium, increase in
adhesion molecules
Influx of neutrophils, initiation of
complement through chemo attractant C5a

13. PHAGOCYTOSIS
• Neutrophils directly interact & internalize
mycobacteria ( Wolf et al., 2007)
2 mechanism mediate interaction
• Direct recognitition
• Opsonisation

14. 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)

15. 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)

16. 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)

17. 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)

18. 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)

19. 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

20. 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

21. NETs & ECTs
NETs ECTs

22. 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)

23. 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

24. Cont..
• Phagocytosis of apoptotic cell- may be anti / pro
inflammatory based on :
Mycobacteria inside neutrophil is alive / dead
• Live: pro inflammatory
• Dead : anti inflammatory

25. AS SIMPLE AS THAT……

26. 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)

27. TOTAL EFFECT OF
NEUTROPHILS

28. 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.