host-pathogen interactions in mycobacterial infections · host-pathogen interactions in...

Host-Pathogen Interactions in Mycobacterial Infections

Genetic aspects

Olivier Neyrolles

Institute of Pharmacology & Structural Biology

CNRS – University of Toulouse - France

Why (host) genetics matters in infectious disease?

The ability of a microbe to induce a disease is most often not absolute, and

depends on the status of the host, which relies, at least in part, on genetic factors

(e.g. TB & leprosy / BCG infection in children)

Long co-existence of men and microbes had, and still has, a huge impact on

human populations, and on the human genome

The identification of predisposition/protection alleles may help define better

knowledge-based strategies for treatment and prevention at the level of the

community

The efficacy of drugs and vaccines may rely on

genetic factors (e.g. TB meningitis)

Outline

Host genes in microbial pathogenesis

Infectious diseases: transmission & global health issues

Host genes in treatment outcome

The bad date

Host Microbe

Environment

Vector

Susceptible

Infectious diseases rely on the immune status of the host

Host response (immunity)

Insufficient number of

immune effector cells

and/or molecules to prevent

host damage

Overproduction of

inflammatory mediators /

tissue fibrosis / malignant

transformation

Damage

e.g. Genetic immune deficiency

(IFNγ Rmut, IL12Rmut, STAT1mut…)

e.g. Complex genetic susceptibility

(allele combination)

eg P. carinii, L. pneumophila, S. epidermidis…

Dam

age

Host response

Helicobacter pylori

Dam

age

Host responseD

amag

e

Host response

Variable

Infectious diseases rely on the immune status of the host

eg Leishmania sp., M. tuberculosis…

• Severe genetic immune deficiencies

BCG (M. bovis BCG) & mildly virulent mycobacteria (e.g. M. avium)

• Complex genetic susceptibility

Leprosy (M. leprae)

Tuberculosis & TB meningitis (M. tuberculosis)

Host genetics & infectious diseases: Mycobacteria provide a framework

From Casanova & Abel, 2004 Nature Rev Immunol 4, 55-66

Mendelian susceptibility to mycobacterial infections

Nature Genetics 1999

D/+ D/+

+/+D/D

Severe M. avium infection

Mendelian susceptibility to mycobacterial infections

Mycobacterium leprae (Hansen, 1873)

≈220,000 cases in 2009

Wide spectrum of clinical manifestations

Tuberculoid vs lepromatous leprosy

Host genetics is most likely involved

Complex susceptibility to mycobacterial infections: the case of leprosy

TUBERCULOID LEPROSY LEPROMATOUS LEPROSY

Organisms: -/low

Low infectivity

Granulomas & local inflammation

Peripheral nerve damage

Normal serum Ig levels

Normal T-cell response

Specific response to M. leprae antigens

Th1 cytokines (eg IFN): +++

Organisms: +++

High infectivity

Disseminated infection

Bone, cartilage, diffuse nerve damage

Hyper Ig

Local or absent T-cell response

No response to M. leprae antigens

Th2 cytokines (eg IL5): +++



TUBERCULOID (TT) LEPROMATOUS (LL)

BORDERLINE

TUBERCULOID (BT) BORDERLINE (BB)

BORDERLINE

LEPROMATOUS (BL)

PAUCIBACILLARY

LEPROMIN+

TH1

MULTIBACILLARY

LEPROMIN-

TH2


TLR2 Bochud et al. A TLR2 polymorphism that is associated

with lepromatous leprosy is unable to mediate

mycobacterial signaling (2003) J Immunol

+ several others…

TLR1 Johnson et al. A common polymorphism impairs cell

surface trafficking and functional responses of TLR1 but

protects against leprosy. (2007) J Immunol

+ several others…

LTA Alcais et al. Stepwise replication identifies a low

producing lymphotoxin-alpha allele as a major risk factor

for early-onset leprosy. (2007) Nat Genet

+ several others…

PARK2/PACRG Mira et al. Susceptibility to leprosy is associated with

PARK2 and PACRG. (2004) Nature

+ a few others…

+ several others…

TNF

TLR2 (Arg677Trp)

×

TLR2

Microbial signal

TB: The hidden enemy

T

B

Primary TB (~5%)

Latency (95%)

Secondary TB (~5%)

Latency (90%) On a global level, the immune system copes

very well with M. tuberculosis !

However this makes a reservoir of ≈2 billion individuals !

PLoS Med 2006

Lancet 2000

Am J Hum Genet 2000

• Nutritional status & hygiene

• HIV co-infection

• Gender & Age

• Infecting strain (virulence)

• Host genetic factors

Complex susceptibility to mycobacterial infections: the case of tuberculosis

Vitamin D Receptor

Lancet 2000

Genotype & status TB contacts (n=42) TB patients (n=71) p Odds ratio (95% CI)

Non-tt & deficient

Yes 10 (24%) 33 (46%) 0.017 2.8 (1.2-6.5)

No 32 (76%) 38 (54%)

Genes may matter in gender-related susceptibility to TB? The case of TLR8

Tlr8

Female Male

(In males)

dbSNP Alleles Cases Contacts p OR (95% CI)

rs3764879 G/C

rs3788935 G/A

rs3761624 G/A

rs3764880 G/A 1069 (79.7%) 997 (76.3%) 0.03 1.2 (1.02-1.48)

(In females)

dbSNP Case:Control p

GG GA AA

rs3764880 78:87 48:56 14:9 0.43

DC-SIGN

From Figdor et al. 2002 Nat ImmunolTailleux et al. 2003 J Exp Med

Tailleux et al. 2005 PLoS Med

Tanne et al. 2009 J Exp Med

Barreiro et al. 2006 PLoS Med

DC-SIGN L-SIGN⁄⁄19p13.2-3

-336A/G -871A/G

DC-SIGN

SNPs & miRNA regulation…

242 VO LU M E 43 | N U M BER 3 | M ARCH 2011 NATU RE G EN ETIC S

L E TTE R S

Susceptibility to Crohn’s disease, a complex inflammatory

disease, is influenced by common variants at many loci.

The common exonic synonymous SNP (c.313C>T) in IRGM,

found in strong linkage disequilibrium with a deletion

polymorphism, has been classified as non-causative because

of the absence of an alteration in the IRGM protein sequence

or splice sites. Here we show that a family of microRNAs

(miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn’s disease and

downregulates the IRGM protective variant (c.313C) but

not the risk-associated allele (c.313T). Subsequent loss of

tight regulation of IRGM expression compromises control of

intracellular replication of Crohn’s disease–associated adherent

invasive Escherichia coli by autophagy. These results suggest

that the association of IRGM with Crohn’s disease arises from

a miRNA-based alteration in IRGM regulation that affects

the efficacy of autophagy, thereby implicating a synonymous

polymorphism as a likely causal variant.

The IRGM region contains multiple polymorphisms that cause tissue-

specific variation in IRGM expression1–3. A synonymous variant

within the IRGM coding region (rs10065172, NM_001145805.1,

c.313C>T) in perfect linkage disequilibrium (r2 = 1.0) with a 20-kb

deletion upstream of IRGM has been strongly associated with

Crohn’s disease in individuals of European descent1,4,5. A recent

study proposed that allelic differences in the promoter region might

be involved in Crohn’s disease pathogenesis1, as the deletion, and

other copy number variants, closely juxtapose several transcription

factor binding sites2. An alternative hypothesis is that the synonymous

exonic (CTG>TTG, leucine) variant might affect protein expres-

sion. In this regard, recent evidence that a polymorphism can alter

miRNA-directed repression of mRNA in a 3 untranslated region6,7 is

of particular interest. Thus, we investigated whether miRNA binding

to IRGM mRNA could be defective in subjects with the T allele and

consequently lead to abnormal regulation of IRGM expression.

For this purpose, we assessed binding of miRNAs to the different

forms of IRGM mRNA in silico using SnipMir, RegRNA and Patrocles

software (see URLs). We observed a predicted loss in binding of two

miRNAs, miR-196A and miR-196B, to the risk haplotype carrying the T allele (Fig. 1a). Indeed, the c.313C>T polymorphism of IRGM is

located within the ‘seed’ region, where mRNA-miRNA forms a complex

within RISC (RNA-induced silencing complex), which is important

for mRNA regulation. Two pre–miR-196A genes (MIR196A1 and

MIR196A2) encode the same mature miR-196A, whereas miR-196B

(MIR196B) is unique within the genome (Supplementary Note).

Both miRNAs share the same ‘seed’ region and target specificity.

Moreover, tandem affinity purification of miRNA target mRNA

(TAP-Tar)8 showed higher binding of miR-196 to IRGM c.313C mRNA

(hereafter IRGMC) than to IRGM c.313T mRNA (hereafter IRGMT),

confirming in silico predictions (Fig. 1b). In HEK293 cells (C/C for

rs10065172), miR-196 transfection decreased protein expression from

a FLAG-tagged IRGMC construct as well as endogenous IRGM pro-

tein levels, whereas expression from a FLAG-tagged IRGMT construct

remained constant (Fig. 1c). Using a modified miR-196 (miR-196MOD)

with a compensatory c.3G>A mutation (Supplementary Note), we

observed stronger binding to IRGMT than to IRGMC and a concomi-

tant decrease in protein expression from the FLAG-tagged IRGMT

construct (Supplementary Fig. 1). Together, these results indicate that

the Crohn’s disease–associated risk (T allele) and protective (C allele)

haplotypes confer differences in IRGM expression under the control

of miR-196. Notably, the miR-196 family and the miR-196 binding site

within the coding sequence of IRGM family members are conserved in

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn’s disease

Patrick Brest1,2, Pierre Lapaquette3,4, Mouloud Souidi5,6, Kevin Lebrigand2,7, Annabelle Cesaro1,2, Valérie Vouret-Craviari1,2, Bernard Mari2,7, Pascal Barbry2,7, Jean-François Mosnier8, Xavier Hébuterne1,2,9, Annick Harel-Bellan5,6, Baharia Mograbi1,2, Arlette Darfeuille-M ichaud3,4,12 & Paul Hofman1,2,10–12

1INSERM ERI-21, EA4319, Faculty of Medicine, Nice, France. 2University of Nice Sophia Antipolis, Nice, France. 3Clermont Université, Université d’Auvergne,

Jeune Equipe JE 2526, Clermont-Ferrand, France. 4INRA, Institut de Recherche Agronomique, Unité sous contrat USC-2018, Clermont-Ferrand, France. 5Université

Paris-Sud 11, Epigenetics and Cancer, FRE 3239, Villejuif, France. 6Centre National de la Recherche Scientifique (CNRS), Villejuif, France. 7CNRS UMR 6097,

Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France. 8EA4273, University of Nantes, Nantes, France. 9Centre Hospitalier Universitaire de Nice, Hôpital de l’Archet, Service de Gastroentérologie, Nice, France. 10Centre Hospitalier Universitaire de Nice, Hôpital Pasteur, Tumorothèque, Centre de Ressource Biologique

INSERM, Nice, France. 11Centre Hospitalier Universitaire de Nice, Hôpital Pasteur, Laboratoire de Pathologie Clinique et Expérimentale, Nice, France. 12These authors

contributed equally to this work. Correspondence should be addressed to P.H. ([email protected]) or A.D.-M. ([email protected]).

Received 1 October 2010; accepted 10 January 2011; published online 30 January 2011; doi:10.1038/ng.762

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Nat Genet 2011

NATU RE G EN ETIC S VO LU M E 43 | N U M BER 3 | M ARCH 2011 243

L E TTE R S

different species, suggesting that control of IRGM protein expression by

miR-196 is under evolutionary constraint (Supplementary Fig. 2).

To correlate in silico and in vitro data with the pathophysiology of

Crohn’s disease, we analyzed expression of miR-196 in human biopsies

using in situ hybridization and fractional laser capture microdissec-

tion followed by quantitative PCR. Increased expression of miR-196

was restricted to intestinal epithelial cells within inflamed ileum

and colon in individuals with Crohn’s disease compared to healthy

controls, as shown by representative images of global staining for miR-

196 by in situ hybridization (Fig. 2a and Supplementary Fig. 3). To

confirm these data, we determined expression levels of miR-196A and

miR-196B in epithelial and lamina propria fractions isolated by laser

capture microdissection (Supplementary Fig. 4). Notably, expression

of miR-196A and miR-196B in the epithelium (relative to that in the

lamina propria) was lower in healthy tissue from control individuals

or individuals with Crohn’s disease and was progressively increased in

quiescent and inflamed tissues from individuals with Crohn’s disease

(Fig. 2b and Supplementary Fig. 5) independently of IRGM c.313C or

c

2.0 P = 0.05

IRGMC mRNA

IRGMT mRNA

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Biot-m

iR-196

B

Biot-m

iR-2

0IP strep

TA

P-T

ar (r

ela

tive a

ffin

ity)

b

a5 3 IRGM c.313C

3 5 miR-196A

5 3 IRGM c.313T

3 5 miR-196A

5 3 IRGM c.313C

3 5 miR-196B

5 3 IRGM c.313T

3 5 miR-196B

IRGM and miR-196 binding predictions

P = 0.04

P = 0.038

IRGMC

IRGMT

IRGM

IRGM

+ miR

-196

BNT

IRGM

IRGM

+ miR

-196

BNT

1.4

FLA

G-tagged IR

GM

expre

ssio

nE

ndogenous IR

GM

expre

ssio

n

1.2

1.0

0.8

0.6

0.4

0.2

0

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

+ miR-196B

NT

IRGM-FLAG

IRGM

Actin

IRGMC

IRGMT

IRGMC

IRGMT

Figure 1 Allele-specific regulation of IRGM by miR-196. (a) In silico

prediction of miR-196 and IRGM mRNA interactions showed differences in

binding within the seed region. (b) IRGMC mRNA was significantly enriched

in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1

and transfected with biotinylated miR-196B or control miR-20 as indicated

and then with IRGMC or IRGMT plasmids were submitted to tandem

affinity purification (immunoprecipitation with FLAG antibodies followed

by affinity purification on streptavidin beads). IRGM mRNA variants were

quantified using quantitative RT-PCR; results are presented as the ratio

between miR-196B and miR-20 (non-relevant miRNA) pull-downs and

the mean of three independent experiments standard deviation (s.d.).

IP strep, immunoprecipitation straptavidine. (c) HEK293 cells (IRGMC/C)

were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT

plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM

antibody revealed the specificity of the downregulation effect mediated

by the miRNA IRGM mRNA interaction. Quantification of the immunoblot

signals are presented as IRGM expression relative to actin (mean of at least

three independent experiments s.d.).

a10

8

6

4

P = 0.049

P = 0.013

P = 0.009 miR-196A

miR-196B

P = 0.023

2

–2

Log2 incre

ase, E v

s L

P

0

Healthy Quiescent Inflamed

CD

b

P = 0.047 P = 0.011

Positive c

ell density (cells

/mm

2)

0

50

150

250

350

300

200

100

Healthy

C/C C/CC/T C/T C/TC/C

Quiescent Inflamed

CD

d

c Healthy mucosa Healthy mucosaIRGMC/C

Inflamed mucosa Inflamed mucosa

IRG

M

IRG

M

LPLP

LP

LP

L

LL

L

IRGMC/T

Healthy mucosa Inflamed mucosa

LP

LP

miR

-196

miR

-196

miR

-196

miR

-196

LP

LP

LP

LP

LPL

L L

L

L

L

L

L

IRGMC/C

Healthy mucosa Inflamed mucosaIRGMC/T

Figure 2 miR-196 overexpression in inflamed mucosa correlates with decreased expression of the IRGM c.313C variant ex vivo. (a) Representative in situ

hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn’s disease (CD) (n = 67) without

or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 m; scale bars in the lower panel,

10 m. (b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn’s disease with no

inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After

RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-

individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed

tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the Ct values. (c) Representative in situ staining for IRGM of TMAs

from colon biopsies of healthy individuals or individuals with Crohn’s disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase.

Scale bars, 32 m. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals

with C/C and 8 individuals with C/T) and 67 individuals with Crohn’s disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa.

We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student’s t-test (the one tail P value is indicated on the figure).

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C CCU G G 3’-IRGM c313CACAAC GGA AACUACCU UGUUG CCU UUGAUGGA

GGGU U U 5’-miR-196B

C CCU G U G 3’-IRGM c313TACAAC GGA AACUAC UUGUUG CCU UUGAUG A

GGGU U G U 5’-miR-196B

244 VO LU M E 43 | N U M BER 3 | M ARCH 2011 NATU RE G EN ETIC S

L E TTE R S

T allelic status (Supplementary Note and Supplementary Table 1).

We investigated whether the increase in miRNA expression could be

a consequence of stimulation by bacterial components or cytokines,

as previously reported for various cell lines9–12. However, in vitro

experiments indicated no variation in either miR-196A or miR-196B

under pro-inflammatory cytokine stimulation (IFN- ) or infection

of HEK293 cells with Crohn’s disease–associated adherent invasive

E. coli (AIEC) bacteria (Supplementary Fig. 6).

Next, we analyzed the correlation between miR-196 and IRGM pro-

tein expression in epithelial cells within the human intestinal mucosa

in tissue microarrays (TMAs) from individuals with Crohn’s disease.

Representative immunostaining images from colons of individuals

with Crohn’s disease are shown in Figure 2c. In healthy mucosa, IRGM

basal expression was strongly positive in epithelial cells independent

of genotype and only weakly positive in the lamina propria. Notably,

in active mucosa, a decrease in IRGM expression was restricted to

epithelial cells of individuals with the IRGMC/C genotype. However,

when the T allele was present in individuals with Crohn’s disease,

IRGM expression was maintained under inflammatory conditions

(Fig. 2c,d, Supplementary Note and Supplementary Figs. 3,7 and 8).

Thus, our findings show that miR-196 expression in inflammatory

conditions correlates with downregulated IRGM expression in human

epithelial cells. Of note, IRGM staining was maintained at a high level

in Paneth cells (Fig. 2c and Supplementary Fig. 9), indicating a pos-

sible difference in IRGM regulation in these cells. Together with a

previous report showing that the levels of autophagy-related proteins

ATG16L1 and ATG5 were critical in maintaining normal granule

biogenesis13, we hypothesize that alternative mechanisms regulating

autophagy-related events could exist in Paneth cells.

IRGM encodes an autophagic protein that plays an important role in

innate immunity against intracellular pathogens like Mycobacterium

tuberculosis, Salmonella typhimurium and Crohn’s disease–associated

AIEC bacteria1,14,15. Compelling evidence indicates that a critical

threshold of IRGM can regulate the efficiency of the autophagic pro-

cess1,14, but the mechanism of regulation of human IRGM expression

remains unknown3,16. Thus, we investigated whether miR-196 and sub-

sequently modified IRGM expression might influence basal autophagic

flux by monitoring LC3-II conversion. LC3-II levels decrease during

prolonged autophagy due to its degradation after autophagosomal-

lysosomal fusion, so we measured the flux through the autophagic

system by comparing LC3-II levels in the presence or absence of

lysosomal inhibitors that partially (pepstatin + E64D) or completely

(bafilomycin A1) prevent LC3-II degradation. Notably, miR-196, which

reduced IRGM expression (Fig. 1), induced a significant decrease in

LC3-II conversion (Fig. 3a,b; P = 0.02). Moreover, when autophagy

was blocked with lysosomal inhibitors, miR-196 inhibited the accu-

mulation of LC3-II (Fig. 3a,b), which suggests strongly that miR-196

overexpression inhibits the autophagic process at the initiation step.

To determine the effect of the increase in miR-196 expression

observed in individuals with Crohn’s disease, we examined the

autophagic flux in response to Crohn’s disease–associated AIEC infec-

tion. In response to AIEC bacterial infection, we observed increased

formation of LC3-II (Fig. 3c). When we blocked lysosomal LC3-II

degradation, we observed a larger increase in LC3-II in AIEC-infected

P = 0.010

P = 0.028

P = 0.024

P = 0.020

0

2

4

6

8

10

12

14Ctl

4 h0miR-196

Ctl

6 h

Time post infection

0

2.5

1.5

– AIEC

+ AIEC

0.5

1.0

2.0

3.0

% o

f LC3-a

ssocia

ted b

acte

ria

LC3-II / actin (fo

ld incre

ase)

Ctl

Ctl + Inh

miR-196

miR-196

c d

Ctl

g

IRGM

P = 0.005

02468

101214161820

4 h0 6 h

Time post infection

% o

f LC3-a

ssocia

ted b

acte

ria

Ctl

IRGM

12

10

8

6

4 P = 0.02

P = 0.040

P = 0.014

2

0Quantification L

C3-ll / actin

Control

Control + B

af

Control + InhIR

GM

IRGM

+ Baf

IRGM

+ Inh

miR-196

miR-196 +

Baf

miR-196 +

Inh

silRGM

silRGM

+ Baf

silRGM

+ Inh

b

0

2.5

1.5

– AIEC

+ AIEC

0.5

1.0

2.0

3.0

LC3-II / actin (fold

incre

ase)

Ctl

IRGM

Ctl + Inh

IRGM

+ Inh

fP = 0.028

0

1 h

8 h

1.5

0.5

1.0

2.0

Intracellu

lar AIE

C b

acte

ria

(fold

incre

ase)

Ctl

IRGM

hP = 0.00570

60

50

40

30

20

10

0

% A

IEC-c

onta

inin

gacid

ic v

acuole

s

Ctl

IRGM

iP = 0.030

0

4.0

3.51 h

8 h

2.5

1.5

0.5

1.0

2.0

3.0

Intracellu

lar AIE

C b

acte

ria

(fold

incre

ase)

Ctl

miR-196

miR-196

sci

e

Untreated Bafilomycin A1

LC3-II

LC3-II

Actin

Control

Control

IRGM

IRGM

miR1

96

miR1

96

silRGM

silRGM

a

Figure 3 IRGM expression and miR-196 affect autophagic flux and AIEC-bacteria–mediated autophagy. (a) The basal flux of autophagy is affected

by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h

and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments s.d.).

(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected

with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments s.d.). (d) Confocal microscopic examination of LC3

revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control

cells (mean s.d.). (e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase s.e.m.

of intracellular bacteria. (f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC

infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h

(mean of three independent experiments s.d.). (g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC

bacteria in IRGM cells compared to control cells (mean s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria.

(i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments s.d.).

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Cell 2010

ITA4H

T/C

TB (Vietnam) OR=0.71, padj=0.011

MB Leprosy (Nepal) OR=0.62, padj=0.001 T/G

TB (Vietnam) OR=0.64, padj=0.0003

MB Leprosy (Nepal) OR=0.70, padj=0.021

ITA4H

• 20% long-exposed individuals never become TST+ (although not anergic)

• Suggests T cell-independent resistance to TB

• Strong heritability

Innate resistance to TB?

J Exp Med 2009

Take-home

Complex susceptibility to infectious agents is mostly due to combinations of both

common and rare variants, each with relatively minor effects, in important, although

not « key » , immunity-related genes (e.g. NRAMP1, DC-SIGN, TLR2, VDR etc.)

Fatal cases of infection with poorly virulent microbes are mostly due to rare

mendalian mutations in key immunity-related genes (e.g. IFNγ R, IL-12R etc.)

Outline

Host genes in microbial pathogenesis

Infectious diseases: transmission & global health issues

Host genes in treatment outcome

1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400

Days after enrollment

EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0

Surv

iva

l

P = 0.02


100 200 300 400

D

P = 0.2All Genotypes

C/T (73)

T/T (25)

C/C (84)All Patients

1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400


EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0

Surv

iva

l

P = 0.02


100 200 300 400

D


C/T (73)

T/T (25)


How your genes may save (or kill !) you upon treatment: ITA4H in TB meningitis

Cell 2012

T ⁄ TC ⁄ TC ⁄ C

ITA4H

LTB4

LXA4

TNF

Poor inflammation

Bacterial proliferation

Strong inflammation

Tissue injury

rs1978331

MtbMtb

TNF

DEX

TNF

DEX

TNFMtb


1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400


EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0

Surv

iva

l

P = 0.02


100 200 300 400

D


C/T (73)

T/T (25)


1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400


EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0

Surv

iva

l

P = 0.02


100 200 300 400

D


C/T (73)

T/T (25)



Tobin et al. 2012 Cell

1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400


EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0S

urv

iva

l

P = 0.02


100 200 300 400

D


C/T (73)

T/T (25)


TREATED

1

10

100

1000

WB

C/µ

L C

SF

C/C C/T T/T

A B C

0.4

0.6

0.8

1.0

Su

rviv

al

No DEX (68)

DEX (114)

0 100 200 300 400


EGenotype

P = 0.006

C/T (27)

C/C (27)

T/T (12)P = 0.042

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


P = 0.005

C/T (46)

C/C (55)

T/T (13)

0.4

0.6

0.8

1.0

Su

rviv

al

0 100 200 300 400


No DEX DEX

00.6

0.7

0.8

0.9

1.0

Surv

iva

l

P = 0.02


100 200 300 400

D


C/T (73)

T/T (25)


UNTREATED

Genes matter in TB treatment: the case of VDR

Lancet 2011

Days of treatment

Spu

tum

cultu

re n

egat

ive

(%)

Nr patients Median time 95% CI p

to sputum

conversion

+Vitamin D 62 36 31.8-40.2

Placebo 64 43.5 36.5-50.5 0.41

VDR genotype Hazard ratio 95% CI p

TaqI tt 8.09 1.36-48.01 0.02

TaqI Tt 0.85 0.45-1.63 0.63

TaqI TT 1.13 0.60-2.10 0.71

Some references…

Rappuoli. 2004. From Pasteur to genomics: progress and challenges in infectious

diseases. Nature Medicine 10:1174

Morens et al. 2004. The challenge of emerging and re-emerging infectious diseases.

Nature 430:242

Wolfe et al. 2007. Origins of major human infectious diseases. Nature 447:279

Quintana-Murci et al. 2007. Immunology in natura: clinical, epidemiological and

evolutionary genetics of infectious diseases. Nat Immunol 8:1165

Okeke & Wain. 2008. Post-genomic challenges for collaborative research in infectious

diseases. Nat Rev Microbiol 6:858

http://www.who.int

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