Mycobacteria and phagocytes: Neutrophils make a lot of

difference

Alexander Apt

Professor and Head

Laboratory for Immunogenetics

Central Institute for Tuberculosis

Moscow, Russia

Email: asapt@aha.ru

Neutrophil infiltration of the lungs in TB-infected susceptible

and resistant mice

0 1 2 3 4 50

5

10

15

20

25

30

**

*

*

*

I/St

A/Sn

weeks post infection

percen

t/l

un

g

0 1 2 3 4 50

1

2

3

4

5

6

7

8

**

*

*

*

weeks post infection

10

6/l

un

g

E. Eruslanov et al., Infection & Immunity, 2005

B

Mouse

strain

Neutrophil yield (106/mouse) % Phagocytosis

b Phagocytic

numberc

I/St 25.3 2.2 39.5 4.0 66.5 5.7

A/Sn 9.3 2.5 7.8 0.9 27.1 3.5

A

E. Eruslanov et al., Infection & Immunity, 2005

0

30

60

90

120

150

CBA CBA/N

Su

rviv

al tim

e, d

ays

0

5000

10000

15000

20000

25000

CBA CBA BCG CBA/N CBA/NBCG

IFN

-g-p

rod

ucin

g c

ells p

er

lun

g

0

20

40

60

80

100

20 30 40 50

Per

cen

t su

rviv

ed

Days post infection

СВА/N

CBA/N, BCG

CBA/N, FL,BCG

0

20

40

60

80

100

45 90 135 180 225

Ne

utr

op

hil %

time, min

BСВА

СВА/N

G

0

15

30

45

No Ab Isotype

control

anti-

Ly6G

CF

U p

er s

ple

en

, x 1

06

F

0

15

30

45

60

No Ab Isotype

control

anti-Ly6GCF

U p

er lu

ng

, х 10

6

CBA CBA/N

Kondratieva T et al., J. Immunol., 2010

Ly-6G+ neutrophils form necrotic Neutrophil foci are surrounded by the

focus in the lung of M. tuberculosis highly hypoxic tissue (HypoxiprobeR

-infected I/St mouse in vivo staining)

(1 mo post-infection)

Radaeva et al., Tuberculosis, 2008 Kondratieva E. et al., PLoS ONE, 2010

A: CFU counts

100000

1000000

10000000

100000000

Lung Spleen

CF

U p

er

org

an

1A8

Isotype

control

B: Survival curves

0

20

40

60

80

100

0 50 100 150 200

Day post challenge

Pe

r c

en

t s

urv

ive

d

Isotypecontrol

1A8

C: Dynamics of body weight

80

90

100

110

120

1 8 13 22 28 41 54 62 69 77 84 95 105 113 140

Day post challenge

Bo

dy w

eig

ht,

%

Isotype control

1A8

Injections of highly specific anti Ly-6 antibodies are beneficial

for genetically susceptible I/St…

1A8 IC

1A8

Pathology

1000

10000

100000

1000000

10000000

lung spleen

CFU

pe

r o

rgan

D: CFU counts

Isotypecontrol

1A8

0

20

40

60

80

100

120

0 100 200 300

Pe

rce

nt

su

rviv

ed

day post challenge

E: Survival curves

Isotypecontrol

1A8

…but not in resistant B6 mice

What is peculiar in I/St mice? Attempts to dissect by gene

expression analysis

M. Orlova et al., Infection & Immunity, 2006

M. Orlova et al., Infection & Immunity, 2006

0

0,5

1

1,5

control anti-IL-11

CF

U p

er

lob

e (

x 1

06)

0

500

1000

1500

2000

2500

3000

IL-11 IL-6 TNF-a MIP-2 IL-12 IFN-g IL-10

p g

/ m

l

control

anti-IL-11

0

10

20

30

40

Control anti-IL-11P

er c

en

t o

f i

nfil

trated

tis

su

e

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007

0,008

0,009

0,01

IL-11 IL-6 TNF-a Mip-2

Exp

ress

ion

leve

l vs.

GA

PD

H

anti-IL-11 Control

Attenuation of infection with anti-IL-11 antibodies Reduction of inflammatory cytokine levels

0

0,01

0,02

0,03

0,04

0,05

0,06

0 10ng/ml 100ng/ml

rIL-11 concentration

Autocrine regulation

Kapina et al., PLoS ONE, 2011

Administration of anti-IL-11 antibodies selectively decreases

neutrophil influx in the lungs of TB-infected mice

Group Cellular composition

Total

cell

count

CD4+ CD8+ CD19+ F4/80+ Ly-6G+

Per

cent

106/

lobe

Per

cent

106/

lobe

Per

cent

106/

lobe

Per

cent

106/

lobe

Per

cent

106/

lobe

Control

Ig

15.1 ±

1.5

40.6 ±

2.4

6.1

± 0.5

19.6±

1.8

3.0 ±

0.2

14.1 ±

1.2

2.2

± 0.2

8.7

± 0.6

1.3

± 0.2

15.2

± 2.5

2.3

± 0.3

Anti-IL-

11

12.8 ±

1.3

40.7 ±

2.3

5.2

± 0.5

19.4 ±

1.2

2.5

± 0.4

19.9 ±

2.6

2.6

± 0.3

9.1

± 0.6

1.2

± 0.1

8.4

± 1.2

1.1

± 0.2

P 0.278 >0.7 >0.5 0.384 >0.8 0.024

Kapina et al., PLoS ONE, 2011

0 50 100 150 2000

50

100

150

days post infection

% s

urv

ive

d

control

IL-11

mut-IL-11

*

24 days post infection

CFU

pe

r Lu

ng *

32 days post infection

CFU

pe

r Lu

ng

Recombinant IL-11 Recombinant mutated IL-11 with W147A substitution = specific antagonist Aerosol administration at weeks 3 and 5 post aerosol challenge, thrice a week, 2µg/25µl.

P < 0.05

mutIL-11 attenuates lung inflammation

control IL-11

mutIL-11

24 320

2.0106

4.0106

6.0106

8.0106***

***

*

days post infection

CD

4+ T

cell

per

lun

g

24 320

2.0106

4.0106

6.0106

8.0106

***

***

***

days post infection

CD

8+

T c

ell

per

lun

g

24 320

1.0106

2.0106

3.0106

***

***

days post infection

CD

19

+-c

ell

per

lun

g

mutIL-11 diminishes lung infiltration by lymphoid cells

24 320

5.0105

1.0106

1.5106*

**

*

**

**

days post infection

F4/8

0+-c

ell

per

lun

g

24 320

2.0105

4.0105

6.0105

8.0105

1.0106

***

***

*

days post infection

Ly6G

+-c

ell

per

lun

g- control

- mut IL-11

- IL-11

Beneficial effect of neutrophil depletion at the early phase of

infection has an impact on disease progression for a prolonged

period, long after neutrophil levels have returned to normal. This

clearly indicates that the initial stages of mycobacterial infection

play a key role in its further development and immune response

of the host. At least two, not mutually exclusive, sets of our data

may explain a “delayed effect” of neutrophil depletion.

First, according to our observations, neutrophils

suppress mycobacteria-specific T-cell activation, which, in

turn, may affect early interactions between T-cells and

macrophages required for activation of the latter.

Second, neutrophils, as phagocytes migrating to a primary infectious

focus very early, could be readily available for colonization by some

members of a small initial mycobacterial population. Earlier we

demonstrated that mycobacteria engulfed by neutrophils are neither

effectively killed, nor their antigens presented to T-lymphocytes. Thus,

neutrophil-associated mycobacteria may form a privileged population

temporarily hidden from more bactericidal lung macrophages. Nothing

is known yet about shifts in gene expression and biochemistry of

mycobacteria, which interacted with neutrophil, not macrophage,

milieu, and now we try to establish an experimental system to address

this issue.

Tantiana Kondratieva

Elvira Rubakova

Vladimir Yeremeev

Marina Kapina

Irina Linge

Konstantin Majorov

Elena Kondratieva

Michael Averbakh

CBA - CBA/N story

Immune response

Neutrophil reactions

IL-11 antibodies

Microbiology

Pathology

Immune histochemistry

Galya Shepelkova

Vladimir Evstifeev

Gene expression

Immunity

rIL-11

Financial support:

Russian Foundation

for Basic Research

Russian Scientific

Foundation