Immunity to Chlamydiae

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Immunity to Chlamydiae

• Xi Yang
• Canada Research Chair in Infection and Immunity
• Professor
• Departments of Medical Microbiology and
  Immunology
• University of Manitoba
• For Medical Immunology, March 2013

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Objectives

• Chlamydial species which cause human diseases
• Major diseases caused by or associated with
  chlamydial infections
• Immune protective mechanisms
• Immune pathological mechanisms
• Interaction between innate and adaptive immune
  responses in chlamydial infection

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Chlamydia

• Obligate intracellular bacteria
• Gram negative
• Characteristic development cycle
• Elementary body (infectious form)
• Reticulate body (metabolic form)

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The developmental cycle of Chlamydia trachomatis

• Genus Chlamydia
• Chlamydia, single
• Chlamydiae, plural
• Not life cycle

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Major C. trachomatis diseases of humans
C. trachomatis
Disease
Significance
Worlds leading cause of infectious preventable
blindness (86 M 3.1 M are blind or have
severely impaired vision, women gt risk men)c
Serovars A, B, C
Trachomaa
Serovars D-K, L1-L3
92 million new cases per year globally
(cervicitis, urethritis) women PID gt HIV risk
factor)c
Sexually transmitted diseasesb
a One of the Worlds top seven most neglected
tropical diseases b Most common bacterial caused
sexually transmitted disease c WHO reports on the
2nd global scientific meeting on trachoma (2003)
and global STD (2007).
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Paradox of Chlamydial Host and Tissue Tropisms

• MoPn vs D gt99 coding ORFs conserved
• D vs A gt99.6 nucleotide identity

Conserved and unique genes In C. trachomatis
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Trachoma
Mabey D. et al., Lancet, 362 2003

• Disease of increasing severity with age.
• Re-infection/persistent infection drives damaging
  immunopathology.
• Thought to be mediated by combination of
  ineffective protective and damaging pathologic
  immune responses.

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The Range of Trachoma
Countries with cases in 2004
Source World Health Organization
The New York Times
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NORMAL CONJUNCTIVA
7-30 mm thick with 3 layers - Oil, aqueous,
mucin defensins, PLA, Antibodies,
Chemokines cytokines
Tear film
Squamous epithelial cells (producing membrane
tethered MUC1, 4 16)
Goblet cells secreting mucin (MUC5AC)
Stratified 3 7 layers thick containing goblet
cells
DC and IEL can traverse Basement membrane via
pores
Basal lamina - secreted by Epithelial cells
Basement membrane
Reticular lamina - secreted by fibroblasts
fibrillar collagen
Loose arrangement of lymphoid spots which
contain HEV, intraepithelialLymphocytes,
Langerhans cells, CD8 T cells
Lymphocyte layer
Lamina propria
Extra cellular Matrix Random arrangement of loose
packed Type I, III and IV collagen fibrils.
Matrix contains Pro IL-1b, Pro TGF-b.
Connective tissue
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INFECTION ACTIVE DISEASE
Infection with Chlamydial Elementary bodies (0.3
mm)
Uptake by Receptor mediated endocytosis
Tear film
Avoidance of phagolysosome fusion and formation
of specialised inclusion (upto 1.5 mm in
size) containing chlamydial reticulate bodies and
Elementary bodies
Thinning of stratified squamous epithelial cell
layer loss of goblet cells
DC and IEL infiltrate epithelial cell layer
Organisation of lymphoid spots into follicles or
germinal centres which contain HEV, migratory DC,
T and B Lymphocytes, plasmacytes
Basement membrane
HEV
HEV
Lymphoid Follicle
Migratory DC
Lymphoid follicle formation Visible to naked eye
3 5 mm In diameter Angiogenesis
B cell areas
Breakdown of Extra cellular Matrix By secretion
of inflammatory cytokines and MMPs. Cleavage of
Pro IL-1b Pro TGF-b and influx of
fibroblasts with collagen deposition
T cell areas
Fibroblasts
Between follicles a diffuse mixed Infiltrate of
T B cells, Macrophage, plasma cells and
neutrophils
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TRICHIASIS
Disrupted tear film
Tear film
Infection with C. trachomatis rarely detected
Squamous epithelial cell layer may be one cell
thick in places
Loss of normal function increased risk of
Opportunitistic secondary infection
Basement membrane
DC and IEL may infiltrate epithelial cell layer
Poor organisation of lymphoid spots
Lymphoid follicle Resolution tissue repair and
remodelling
Reformation of Extra cellular Matrix Influx of
fibroblasts. Non random deposition of tight
bundles of types I, III IV collagen with
additional deposition of type V collagen. Fibrils
are deposited on longitudinal axis anchored on
posterior tarsal plate leading to loss of
elasticity and tightening of tarsal plate.
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NORMAL CONJUNCTIVA
Goblet cells
Stratified squamous epithelial cells
Basement membrane
Lymphocyte layer Langerhans cells CD8 T
cells IEL
HEV

1. Vascular
2. Lymphocytes
3. Plasma cells
4. HEV
5. Follicles
6. Fibroblasts

Lamina propria
Connective tissue
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ACTIVE TRACHOMA WITH LYMPHOID FOLLICLES
Epithelial cell layer
Deep and superficial lymphoid follicles
Basement membrane
HEV
T cell areas
B cell areas
Mixed cell infiltrate
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Immune responses in trachoma

• Lymphoid follicle formation
• Human studies in Africa
• Pathological inflammation and fibrosis associated
  with higher IL-4 production
• Association with FoxP3 Treg
• Vaccine trial in 1960s killed whole organism
  induce partial protection (reduction of
  infection) but more pathology.

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Challenges to trachoma vaccine development

• Poor natural immunity.
• Vaccine has to be superior.
• Non human primates are the only animal model that
  mimics human infection and disease.
• Protective and pathologic immune mechanisms are
  not well understood.
• Complex host-pathogen interactions.
• Infection is restricted to conjunctival
  epithelial cells.
• Target mucosal immunity.
• Extracellular infectious (EB) and intracellular
  replicative (RB) forms.
• Represent multiple structural and secreted
  targets of protective immunity that could require
  native antigen presentation.
• Recombinant subunit or acellular trachoma
  vaccines have been poorly efficacious in
  non-human primates.

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Infection of the female genital tract with
Chlamydia trachomatis

• Genital tract infection
• Pelvic inflammatory diseases (PID)
• Infertility and ectopical pregnancy
• Sensitive to antibiotics
• Azithromycin and doxycycline.
• Tetracycline and erythromycin groups

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Animal models

• Non-human primates
• Mouse
• Genital tract
• Lung infection
• -complex of immune responses location, species
• -MoPn is used most often

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Immunity to genital tract infection

• No vaccine
• T cell immunity is most important
• CD4 Th1 cells (IFN?) are most protective
• Th17?. Protective in lung model
• The role of CD8 is not clear, CTL ?
• Antibody is generally not protective but local
  IgA appears associated with protection
• Antibody may play a role in prevent reinfection
• Natural immunity is weak, short lasting and
  strain specific

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protective
pathologic
Protective
pathologic
pathologic
From T. Wynn
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McClarty, Grant/Development of a Live-attenuated
Chlamydial Vaccine


Immunity To murine C. muridarum Primary Infection
and Reinfection. Genital infection with C.
murdiarum produces robust long-lived adaptive
immunity. The immune responses that are elicited
during infection resolve primary infection in
approximately 4 to 5 weeks, and upon rechallenge,
those adaptive immune responses result in an
infection of much shorter duration (3 to 10
days), and far fewer infectious bacteria (gt104
fewer) are shed. Generally speaking, adaptive
immune responses elicited during infection
consist of CD4 T cells, CD8 T cells, and
antibody. CD4 T cells are absolutely essential
to bring about the resolution of primary genital
infection, whereas CD8 T cells and antibody are
dispensable. In the absence of CD4 T cells,
primary infection persists. Interestingly,
immunity to reinfection is governed by a more
complex set of responses. First, as with primary
infection, CD4 T cells protect against
reinfection and resolve infection in the absence
of CD8 T cells and/or antibody responses.
However, in the context of reinfection/rechallenge
, antibody is now protective and resolves
secondary infection in the absence of CD4 and/or
CD8 T cells. An indispensable element of the
antibody-mediated protective immunity is the
priming of the genital tract tissues by CD4 T
cells (infection-primed genital tract). Once the
genital tract has been primed, CD4 T cells are
dispensable. Therefore, while the protective
efficacy of antibody is dependent on CD4 T-cell
priming of the genital tract, antibody functions
independently of CD4 T cells. Adapted from
Farris C M , Morrison R P Infect. Immun.
201179986-996
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Chlamydial antigens related to immunity and
pathology

• Major outer membrane proteins (MOMP) is the
  protective antigen which has been studied the
  most. Surface exposed, 60cell wall protein.
• Heat shock protein 60 appears associated with
  pathology
• Levels of antibody to HSP60 are correlated the
  degree of PID.
• Higher IL-10 production was detected in patients
  with persistent infection and PID.

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Recent results of vaccination trials using
Chlamydia muridarum major outer-membrane protein
as antigen
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Chlamydia trachomatis proteins that are
recognized by human or mouse T cells
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Inhibition of chlamydial growth by interferon-?

• IFN? upregulate indoleamine 2,3 deoxygenase (IDO)
  expression, leading to decyclization and cellular
  depletion of tryptophan, an amino acid essential
  for chlamydial growth
• Induce iNOS, nitric oxide-based killing

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C. pneumoniae

• High prevalence of infection (upto 60 of the
  population is antibody positive)
• Respiratory diseases, bronchitis and pneumonia
• Most are mild and asymtomatic.
• Association with coronary artery and neurological
  diseases
• CD8 cells are more important than CD4 T cells in
  protection

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Immune cells studied in chlamydial infections

• Neutrophils
• NK
• NKT
• DC
• T cell
• CD4 T cell, Th1, 2, 9, 17
• CD8 T cell
• ?d T cell
• B cell

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Interaction between innate and adaptive immune
cells

• ?s T cells produce IL-17 promote Th17 responses
• NKT and NK cell modulate DC function for
  directing T cell responses
• NKT promote NK cells

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Experimental Strategy

• Comparison between NKT-KO (V?14J?18 KO) and wt
  mice
• Effect of a-GalCer administration on wt mice in
  following aspects
• -Susceptibility to infection
• -CD4 and CD8 T cell responses
• -DC phenotype and function
• -DC subset function

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More severe disease in NKT mice following Cpn
infection
D9 D15 D15 D9 D15
D9
D15
WT
KO
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Effect of a-Galcer on chlamydial infection and
immune responses
WT- ? GC KO- ? GC WT-Veh KO-Veh
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Kinetics of NKT responses following Cpn infection
(lung)
uninfected
infected
NKT cytokine profile following Cpn infection (day
3)(CD1 tetramer CD3)
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Effect of NKT responses on T cell cytokine
profile(NKT enhances IFN? production)
CD8
CD4
Cpn infection only
A-GalCer Cpn infection
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Effect of NKT responses on T cell cytokine
profile(NKT decreases IL-4 production)
Cpn infection only
A-GalCer Cpn infection
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Restoration of NKT enhance type 1 T cell
responses and Cpn clearance
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Conclusion (1)

• NKT cells are protective in C. pneumoniae
  infection
• NKT produces higher IFNg than IL-4, thus more
  NKT1-like, following Cpn infection
• NKT promotes type 1 T cell response (CD4 and CD8)
  following C. pneumoniae infection

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NKT ?
NKT ?
Lambrecht BN, 2001. Clin Exp Allergy
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approaches
Compare NKT-KO (or aGalcer treated) vs wild-type
mice in -Dendritic cell surface markers and
cytokine pattern -DC function in vitro (DCT
coculture) -DC function in vivo (adoptively
transfer of DC) -effect of NKT restoration in KO
mice (adoptive transfer of NKT) on DC function

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Altered surface phenotype and cytokine production
pattern of DC in NKT-KO mice following
C.pneumoniae infection.
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Direct effect of NKT on DC NKT Enhanced DC
IL-12p70 production through CD40L/CD40
interaction and IFN? (dependent on cell-cell
contact)
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Adoptive transfer to test DC function in vivo
WT-DC
KO-DC
PBS
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Adoptive transfer to test DC function in vivo
(cytokine responses)
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What about DC subset?
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Adoptive transfer of NKT to NKT-KO mice increases
IL-12 producing DC (CD8)
Restoration of NKT leads to increase of CD8a DC
especially IL-12 producing CD8a DC
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NKT selectively enhance IL-12p70 production by
CD8? DC subset and the effect is dependent on
CD40-CD40L interaction and IFN? (in vitro
co-sulture)
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NKT enhance the ability of CD8a DCs for
polarizing type 1 CD8 and CD4 T cell response (in
vitro)

• AB, co-culture DC subsets from infected wt and
  NKT mice with T cell from Cpn immunized mice
• CD, co-culture CD8DC from infected wt mice with
  T cell from Cpn immunized mice

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Adoptive transfer of CD8? DCs from WT mice, but
not those from NKT-KO mice, generated protective
type-1 immunity in vivo.
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Conclusion (II)

• NKT can influence co-stimulating molecule
  expression on DCs
• NKT can influence cytokine production patterns in
  DCs
• NKT can modulate the function of DC in directing
  CD4 and DC8 T cell responses and host defense
  against infection
• NKT can preferentially modulate CD8a DC subset

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NKT plays important regulatory role in linking
innate and adaptive immune response
NKT
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Further reading

• Brunham RC, Rey-Ladino J, Nat Rev Immunol. 2005
  Feb5(2)149-61 Immunology of Chlamydia
  infection implications for a Chlamydia
  trachomatis
• Farris CM, Morrison RP. Infect Immun. 2011
  Mar79(3)986-96. Vaccination against Chlamydia
  genital infection utilizing the murine C.
  muridarum model.