Relative contribution of IL-1α, IL-1β and TNF to the

host
response to Mycobacterium tuberculosis and
attenuated M. bovis BCG

Andi Nurul Ilmi

 INTRODUCTION
• 1/3 of the global population considered to be infected

Tuberculosis
• Only 5–10% develop an active disease host immune system efficiently dealing with
the mycobacteria
• Immunodepression of the host  reactivation of latent tuberculosis infection

(TB)
• Neutralisation of TNF for the treatment of severe inflammatory diseases  reactivation
of latent tuberculosis and increased susceptibility to primary tuberculosis infection
• Coordinated innate and adaptive immune responses are required for efficient control of
M. tuberculosis infection

• Early studies  Protective role of the IL-1α/β pathway in M. tuberculosis infection

Studies
• Study by Mayer-Barber  myeloid cell populations co-expressing both IL-1α and IL-1β
that are regulated by Type I and Type 2 interferons
• IL-1β and downstream TNF production  caspase dependent restriction of intracellular
M. tuberculosis growt
 INTRODUCTION
Relative contribution of IL-1α vs. IL-1β in the impairment of host response to acute M.
tuberculosis infection  in the absence of IL-1R1, and compare it to the well established
susceptibility of TNF deficient mice

We confirm that both TNF and IL-1 pathways are required to control M. tuberculosis

Presence of either IL-1α or IL-1β allows some control of acute M. tuberculosis infection

TNF is essential for the early control of infection by virulent or attenuated mycobacteria

IL-1 pathway is dispensable for controlling less virulent infection by M. bovis BCG.
 RESULT: Cross-regulation of IL-1α and IL-1β release by
macrophages in response to mycobacteria

Mutual induction of IL-1α and IL-1β  after stimulation with LPS or turpentine

Bone marrow derived macrophages from mice deficient IL-1α and/IL-1 β, or IL-1R1  stimulated with M.
tuberculosis H37Rv / M. bovis BCG in vitro
• No IL-1α was detected in the IL-1a deficient mice (Fig. 1A) and no IL-1 β was detected in the IL-1β deficient mice (Fig. 1B).
• IL-1α  reduced in IL-1β deficient macrophages stimulated with TLR4 agonist LPS, M. tuberculosis H37Rv or M. bovis BCG
• IL-1β  reduced in the IL-1α deficient macrophages stimulated with LPS, M. tuberculosis H37Rv or M. bovis BCG.
• Absence of functional IL-1R1  little effect on the release of IL-1α or IL-1β (Fig. 1A and B)
• TNF release induced by mycobacteria was independent of the IL-1α/ β /IL-1R1 axis in vitro (Fig. 1C)
RESULT: Lethal M. tuberculosis infection in the absence of both IL-1α
and IL-1β, or TNF pathways
IL-1R1 deficient mice  extremely sensitive to virulent M. tuberculosis H37Rv infection

Mayer-Barber et al a rapid mortality of mice deficient for IL-1α and/or IL-1β, within 30–40 days post M. tuberculosis infection

Deficient for IL-1α or IL-1β  survived the acute phase of infection without bodyweight loss

Deficient for IL-1α and IL-1β  rapidly lost weight and succumbed within 5 weeks

Deficient for IL-1α or IL-1β  survived for the duration of the acute M. tuberculosis infection study (3 months) without clinical symptoms of infection.

Presence of either IL-1α or IL-1β allows some control of acute M. tuberculosis infection.
 RESULT: Exacerbated pulmonary inflammation after M. tuberculosis
infection in the absence of IL-1α, IL-1β or IL-1R1, alike in the absence of
TNF
• IL-1α and IL-1β deficient mice  strongly increased lung weights, 5 weeks post-infection
• IL-1R1 deficient mice and TNF KO mice  increased lung weights at 4 weeks
• Deficient IL- 1α or IL-1β  less pulmonary inflammation on day 35 post-infection
• IL-1α or IL-1β deficient mice  survived to chronic infection and lung inflammation at2 or 3 months post-
Lung infection (Fig. 2F and G).
inflammation • Expression of IL-1α or IL-1β  helped controlling acute lung inflammation induced by M. tuberculosis infection.

• Mice deficient for IL-1α + IL-1β could not control the infection, similar to IL-1R1 deficient mice on day 35 post-
infection
• Deficiency in IL-1α or IL-1β  bacterial growth restriction on day 35 post-infection and a limited increase in
bacterial load at 2–3 months post-infection
Bacterial • Deficient in IL-1α+IL-1β  a rapid, exacerbated lung inflammation uncontrolled bacterial infection, as seen in
IL-1R1 or TNF deficient mice
loads
 RESULT: Exacerbated
pulmonary inflammation after M.
tuberculosis infection in the
absence of IL-1α, IL-1β or IL-
1R1, alike in the absence of TNF
 RESULT: Presence of either IL-1β or IL-1α prevents the acute necrotic
pneumonia seen after M. tuberculosis infection in IL-1 pathway defective mice,
but do not compensate for lack of TNF
Granuloma

Deficient for IL- Deficient IL-1α

1α and IL-1β or IL-1β

2–3 months
free alveolar post-
Macroscopic Microscopic limited oedema no necrosis
space infectionlung
pathology

extensive
reduction of increased
pleural large confluent severe massive MN neutrophil confluent abundant reduced free acid-fast bacilli
ventilated inflammatory oedema
adhesions nodules inflammation cell infiltrations necrosis and mycobacteria alveolar space abundance
alveolar spaces cell infiltration
oedema

• Absence of both IL-1α and IL-1β were similar to those seen in

The lung lesions mice deficient for IL-1R1 or TNF

• Recapitulates the acute necrotic pneumonia from defective IL-

Absence of IL-1α and IL-1β 1R1 or TNF pathways

• Limits infiltration of inflammatory cells, oedema and necrosis

Presence of IL-1α or IL-1β after acute M. tuberculosis infection.
Presence of IL-1α or IL-1b prevents acute necrotic Progressive pneumonia after 2–3 months M. tuberculosis infection
pneumonia in response to M. tuberculosis infection. in the absence of IL-1α or IL-1b
RESULT: Regulation of pulmonary cytokine expression by IL-1α/IL-1β
pathway after M. tuberculosis infection

Uncontrolled bacilli growth and pulmonary inflammation  deficient IL-1α and IL-1β

Complete absence of IL-1α + IL-1β  increased IFNg pulmonary levels, but decreased IL-12/23 p40 and IL-23 p19 expression

Absence of IL-1 pathway  on partial reduction of TNF pulmonary levels after in vivo M. tuberculosis infection

No reduction of IL-1α or IL-1β lung content in the absence of TNF.

The levels of IL- IL-1α and IL-1β TNF pulmonary

Lung IFNg levels IL-12/23 p40 levels IL-23 p19 levels
17A levels levels
• Highly elevated in • Reduced in • reduced in TNF- • reduced in • elevated in TNF- • reduced in the
deficient IL-1α and deficient IL-1α and deficient at 4 deficient IL-1α deficient at 4 absence of IL-1α
IL-1β or IL-1R1 at IL-1β weeks of infection and/or IL-1β or IL- weeks of infection and/or IL-1β, or
5 weeks after M. • Lower than wild- 1R1 at 5 weeks • elevated in IL-1R1 IL-1R1
tuberculosis type controls in post-infection deficient
infection deficient IL-1α and • in IL-1α or IL-1β • IL-1α levels were
• TNF deficient at 4 IL-1β deficient at 8 slightly increased
weeks • decreased in IL- weeks post in IL-1β deficient
• Highly elevated in 1R1 deficient or infection mice
IL-1β deficient IL-1α or IL-1β, 5 • IL-1β levels were
IL-1β sufficed to weeks after slightly increased
control IFNg over- infection. in IL-1α deficient
expression.
RESULT: Regulation of pulmonary cytokine expression by IL-1α/IL-1β
pathway after M. tuberculosis infection

Cytokine pulmonary levels in M. tuberculosis infected TNF or

IL-1 deficient mice.
 RESULT: IL-1 pathway is dispensable for controlling attenuated M.
bovis BCG infection, while TNF is essential

Mice deficient for MyD88  highly susceptible to virulent M. tuberculosis infection while they controlled chronic M.
bovis BCG infection and less susceptible to M. tuberculosis

IL-1R1 pathway  controlling acute infection

•Previous reports deficiency IL-1α or IL-1β  early death after infection or survival up to 3–6 months at lower dose infection
•TNF deficient mice  rapidly lost weight and succumbed by 6 weeks of infection
•IL-1R1 and IL-1α+IL-1β deficient  survived systemic M. bovis BCG infection

Histologically
• Liver and lung inflammation were exacerbated in TNF deficient mice
• The pathology was much less severe in IL-1R1 or IL-1α plus IL-1β deficient mice

IL-1 pathway  essential for controlling virulent M. tuberculosis infection is dispensable for the early control
of attenuated M. bovis BCG infection
TNF is crucial, but IL-1α/IL-1β pathway is
dispensable for controlling M. bovis BCG
infection.
 DISCUSSION
• Neutralization of TNF in the treatment of severe inflammatory reactivation of latent tuberculosis and increased
susceptibility to primary tuberculosis infection
• IL-1 has been neutralized using the IL-1R antagonist Anakinra with good safety record  short half-life administered
daily
Neutralization • Efficient and persistent IL-1 neutralization with long-lasting anti-IL-1 antibodies  reduced host resistance and flare-up
of M. tuberculosis infection.

• Both IL-1α and IL-1β are critically required for host resistance
• Absence of both IL-1α+IL-1β recapitulated the overwhelming infection and acute necrotic pneumonia seen in IL-1R1
deficient mice
Mayer-Barber • Presence of IL-1α or IL-1β  allowed to control acute M. tuberculosis infection and pulmonary inflammation,
et al

The presence of IL-1α or IL-1β is sufficient to trigger IL-1R pathway essential

component in the development of innate response to acute M. tuberculosis infection
 DISCUSSION

Inter-dependence of IL-1α and IL-1β expression and/or release

• IL-1β acting as a shuttle for secretion of mature IL-1α

Bone marrow-derived macrophages

• Macrophage deficient for IL-1α  defective in IL-1β production in vitro
• Macrophages deficient IL-1α  reduction IL-1β release in response to mycobacterial antigen stimulation
Indirect IL-1 amplification loop unlikely
• Absence of functional IL-1R1 did not hamper the release of IL-1α or IL-1β.

Coproduction of IL-1α and IL-1β at the single cell level

• In distinct myeloid cell populations in the lung of M. tuberculosis infected mice
 DISCUSSION
• Slightly increased pulmonary IL-1α levels in IL-1β deficient mice during acute infection
• Increased concentrations of IL-1α reported in BAL and lungs of IL-1β deficient mice
• Absence of IL-1α and IL-1β  increased pulmonary levels of IFNg for IL-1R1-deficient mice
• Absence of IL-1α and IL-1β  Increased IFNg levels reported in the BAL of these mice 4 weeks post-infection
Our report • Increased IFNg levels single absence of IL-1α or IL-1β, or of TNF consequence of the failure of the innate immune response

• BAL IL-1α levels unaffected in IL-1β deficient mice, and conversely, 4 weeks after M. tuberculosis infection,
• Ly6GnegCD11bpos myeloid cells producing IL-1α and/or IL-1β were halved in these mice
Recent
study

• Th1 promoting cytokines were upregulated in the absence of IL-1 or TNF pathways
• There was little effect on IL-12/ 23 p40 and rather a decrease in IL-23 p19 expression.
• Increased IL-1β levels in single IL-1α deficient mice  associated with a partial correction of IFNg and IL-23 p19 levels
Our model • IL-17 was strongly reduced in IL-1 deficient mice  contribute to the enhanced susceptibility of these mice
 DISCUSSION
High bacterial burden associated with acute M. tuberculosis infection in mice lacking TNF or functional IL-1
pathway abundant PAMPs to trigger cytokine release directly.

vitaPAMPs Acute, exacerbated M. tuberculosis infection.

High levels of IL-1β and TNF  in the BAL and lung of TRIF/MyD88 double deficient mice classical
TLR agonists, nor TRIF-dependent vitaPAMPs are essential for the increase in cytokine release

DAMPs Released by dying or necrotic cells to entertain the inflammatory context in highly infected
lungs

DAMPsDo not seem essential for in vivo

 DISCUSSION
High levels of IL-1β,
TNF, IFNg and TLR, MyD88-
Mice deficient MyD88 chemokines MCP1 dependent pathways
and MIP1a 5 weeks were not essential
after infection

Mice deficient for NLRP3, ASC or caspase-1,

control acute M.
or for NLRP2, AIM2 or purinergic receptor tuberculosis infection
P2X7

NLRP3 thiol
Immunopathology nitrosylation and
The control by NO of M. tuberculosis and IL-1β production inhibition of NLRP3
inflammasome

Reduced expression of iNOS in IL-1 defective susceptibility to M.

tuberculosis
mice 5 weeks post-infection infection.

Molecular •IL-1β directly activates host resistance to M. tuberculosis

•Similar to Tim3, the ligand of Galectin-9 expressed on M. tuberculosis infected macrophages
•IL-1β inhibited the replication of M. tuberculosis in infected macrophages, Increased TNF release and

mechanisms TNFR1 cell surface expression caspase-3 activation, apoptosis, and restriction of M. tuberculosis
growth
 DISCUSSION
Hypothesised  requirement for IL-1 responses depends on the virulence of the mycobacteria.

• MyD88 deficient mice highly susceptible to virulent M. tuberculosis infection control chronic M. bovis BCG infection and less susceptible to M.
tuberculosis
•TNF deficient mice highly susceptible to virulent M. tuberculosis and attenuated M. bovis BCG

Different regulation of caspase-1 dependent IL-1β release by type I IFNs

•Absence of IL-1R1, or IL-1α plus IL-1β did not compromise the control of a M. bovis BCG infection

The IL-1 pathway is dispensable for the immediate, innate response to control M. bovis BCG infection

•IL-1β requires TNF pathway to inhibit M. tuberculosis replication

•T-cell derived TNF is important at later stages of the infection

TNF is essential for the early control of mycobacterial infection

• TNF antimicrobial effects are independent of IL-1β

•TNF of macrophage/neutrophil origin being crucial for the immediate response

TNF essential for the IL-1 pathway  not Long-lasting

Presence of IL-1a or IL-
early control of infection central for controlling neutralizing monoclonal
1b  allows some
by either virulent or less virulent antibodies preferable
control of acute M.
attenuated mycobacteria such as to target specifically IL-
tuberculosis infection
mycobacteria M. bovis BCG. 1b, or IL-1a
 MATERIALS AND METHODS
Mice
• Mice deficient for IL-1α or
IL-1β , or both IL-1α and
IL-1β , IL1-R1, or TNF
• Backcrossed at least 7–
10 times on C57BL/6
genetic background.
• For experiments, adult
(8–12 weeks old) animals
were kept in isolators
• The infected mice 
monitored regularly for
clinical status and
weighed twice weekly.
Bacteria and infection Pulmonary infection with
clumping was disrupted M. tuberculosis H37Rv
• M.
M. tuberculosis
tuberculosis H37Rv
H37Rv diluted in sterile saline
(Pasteur) aliquots kept by 30 repeated
containing 0.05% Tween • Performed
Performed by
by delivering
delivering
frozen at 80 C were thawed
20
aspirations through a 26
gauge needle
1600±300 CFU/lung
Bacterial load in tissues
•• Evaluated at different time
Alternatively, M. bovis
inoculum size was
injected intravenously at BCG or fluorescent
points after infection with M.
tuberculosis
tuberculosis H37Rv
H37Rv 1066 CFU/mouse. GFP-expressing M.
bovis BCG
verified 24 h after
infection
Organs were weighed
and defined aliquots
Tenfold serial dilutions
Plated in duplicates onto
Middlebrook 7H11
Colonies were
enumerated at 3 weeks
were homogenised in
PBS in a Dispomix
of organ homogenates
in 0.05% Tween 20
(Difco) agar plates
containing 10% OADC
and results are
expressed as log10
containing 0.9% NaCl
homogeniser and incubated at 37C. CFU per organ
Determination of IL-1a, The supernatants
Pulmonary cytokine
determination
Immediately frozen on
IL-1β , IL-12/IL-23p40, sterilised by
dry ice and stored at -80 •• Lung
Lung homogenates
homogenates were
were
IL-23p19, TNF and IFNg centrifugation through centrifuged
C centrifuged
levels by ELISA 0.22 mm filter

Histopathological analysis
• Left lobe of lungs from M.
tuberculosis infected mice
Primary macrophage
cultures
• Murine bone marrow cells
Cell supernatants 
harvested after 24 h of
stimulation for IL-1a, IL-1b
and TNF quantification by
ELISA
MATERIALS AND METHODS
Fixed in 4% phosphate
buffered formalin and
paraffin-embedded.
Isolated from femurs and
differentiated into
macrophages after culturing
at 1066 cells/ml for 7 days in
DMEM
Statistical analysis
• Determined with Graph Pad
Prism
Liver and lung from M. bovis
Free alveolar space, lung
Two to 3-mm sections were BCG infected mice  H&E
cellular infiltration, oedema
stained with Hematoxylin staining and the slides
and necrosis quantified
and Eosin and a modified scored for inflammatory cell
using a semi-quantitative
Ziehl-Neelsen method. infiltration and granuloma
score
lesions.
Three days after washing
Macrophages were plated in Stimulated with LPS, heat
and re-culturing in fresh
96 well microculture plates killed M. tuberculosis
medium  contained a
(at 105 cells/well in H37Rv, live or heat-killed M.
homogenous population of
supplemented DMEM) bovis BCG (HKBCG).
macrophages.
Differences between
multiple in vivo groups  Non-parametric Mann–
Values of P 0.05 
analysed by means of one- Whitney t-test analyzing in
significant Two-tailed
way non-parametric ANOVA vitro results
test
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