AAI Poster Preeyam
1
Early Exposure to Phosphorylcholine-Bearing Microbes Dampens the Development of House Dust Mite Allergy During Adult Life ABSTRACT Preeyam Patel and John F. Kearney Microbiology Theme; Graduate Biomedical Sciences at the University of Alabama at Birmingham INTRODUCTION AND METHODS A suggested mechanism to explain the dramatic rise in the incidence of allergic disease among individuals living in developed countries has been an imbalance in the TH1/TH2 subsets; however, a majority of neonatal pathogens evoke potent B cell responses. These B cell clones, with shared specificity for allergens, can be protective against the development of allergic disease. Streptococcus pneumoniae and house dust mite (HDM) bear similar phosphorylcholine epitopes. Therefore, we hypothesized that B cell clones generated as a result of early pneumococcal exposure would dampen the development of HDM allergy during adult life. To test our hypothesis, we immunized neonatal mice with PC-deficient or PC-bearing pneumococcus and then challenged these mice with HDM as adults. Mice receiving the PC-bearing pneumococcus had a large frequency of PC-specific B cells that resided in their spleen and continually secreted antibodies. Upon pulmonary exposure to PC-bearing HDM, these B cells migrated to the lung and secreted antibody locally into the pulmonary space. As a result, mice immunized with PC-bearing pneumococcus, presented with dampened production of serum IgE and TH2-associated cytokines, development of airway hyperreactivity, and infiltration of allergic mediators into the lung as compared to mice exposed to PC-devoid pneumococcus as neonates. We further demonstrated that neonatal exposure to bacteria was not sufficient enough to disrupt the balance in the TH1/TH2 subsets. RESULTS TH2 TH2 TH2 TH2 T TH2 IgE TH2 TH2 TH2 TH2 B B TH2 TH2 Incidence of Infectious Diseases (%) Incidence of Immune Diseases (%) 1950 1960 1970 1980 1990 2000 0 50 100 Rheumatic Fever Hepatitis A Tuberculosis Mumps Measles Incidence of Immune Diseases (%) 100 200 300 400 Chron’s Disease Multiple Sclerosis Type 1 Diabetes Asthma 1950 1960 1970 1980 1990 2000 QuestDiagnostics Health Trends; Allergy Report 2011 Development of Allergic Disease The Hygiene Hypothesis Graham-Rowe D, Nature 479; Nov 2011; Masoli M., Allergy 59; 2004 Adapted from: Bach J-F, NEJM 347 11; Sept 2002 CONCLUSIONS 100μm 50μm 50μm PBS R36A T15 KI JY2190 Neonatal Immunization with R36A Decreases Mucin Production and Cellular Infiltration in the Bronchioles 0 10 20 30 40 50 1 2 3 4 Vaporized Methacholine (mM) * * C Vaporized Methacholine (mg/mL) Rrs (cmH2O.s/mL) PBS JY2190 R36A T15 KI No HDM IL-2 IL-12p70 IFNg 0 1 2 3 4 5 IL-4 IL-5 IL-6 IL-13 IL-9 0 1 2 3 20 40 60 Cytokines in the BALF (pg/mL) * ** * * * * ** * IL-2 IL-12p70 IFNg IL-4 IL-5 IL-6 IL-13 IL-9 BALF Cytokines (pg/mL) BALF Cytokines (pg/mL) BALF Cytokines (pg/mL) Neonatal Immunization with R36A Decreases the Production of TH2-, but not TH1-, Associated Cytokines in the Lung as well as the Development of Airway Hyperresponsiveness (AHR) PBSJY2190 R36AT15 KI 0 100 200 300 ng/mL Derp1-specific IgE * *** PBSJY2190 R36AT15 KI 0.0 0.5 1.0 1.5 * ** total T cells eos neuts 0.0 0.2 0.4 0.6 2 4 6 Cells in the BALF (x10 6 ) *** ** ** *** ** *** ** ** Baso Mast macs alv mac 0.00 0.01 1 3 6 9 Cells in the BALF (x10 5 ) *** *** *** *** *** ** PBS R36A T15 KI JY2190 Cells (x10 6 ) Cells (x10 5 ) Total IgE (ng/mL) Total T cells Eos Neuts Baso Mast Macs Alv Macs PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI A.M. A.M. neut neut eo eo A.M. neut eo 20μm A.M. neut Derp1-IgE (ng/mL) PBS JY2190 R36A T15 KI Neonatal Immunization with R36A Decreased the Number of Cells infiltrating the Bronchoalveolar Space and IgE Production mm PBS JY2190 R36A T15 KI CD44 PBS R36A T15 KI JY2190 % of Max Neonatal Immunization with R36A Decreases Priming of Antigen-Experienced T Cells in the Mediastinal Lymph Node CD44 0.0 0.5 1.0 1.5 ) *** * CD4 + T Cells CD44 MFI (x10 4 ) DCs imm DCs macs alv macs 0.0 0.5 1.0 1.5 2.0 * *** * *** * ** total T cells Eos Neuts 0.00 0.02 0.2 2 4 Cells in the Lung (x10 7 ) * * * *** ** *** * *** macs DCs Mast Baso 0.0 0.5 1.0 2 4 10 20 Cells in the Lung (x10 5 ) *** *** ** *** * * ** * Total T cells Eos Neuts Macs DCs Mast Baso Cells (x10 7 ) Cells (x10 5 ) DCs imm DCs macs alv macs CD86 MFI (x10 3 ) PBS JY2190 R36A T15 KI No HDM Neonatal Immunization with R36A Decreased Cellular Infiltration into the Pulmonary Parenchyma 0 10 20 30 ng/mL anti-PC IgM ** * NS PBS JY2190 R36A T15 KI ng/mL anti-PC IgM PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI Neonatal Immunization with R36A Increased the Number of PC-Specific B Cells in the Lung IgM PC-BSA Laminin PC-Specific IgM-Expressing 50μg anti-PC IgM ab i.t. 1 hour prior to challenge with 5ug HDM ISO BH8 mm CD44 ISO BH8 % of Max Passive Antibody Administration CD44 0 2 4 6 8 MLN CD4+ T Cells: M uMT uMT uMT JY2190 R36A mm CD4+ T Cells CD44 MFI DC imm DC macs 0 5 10 15 20 CD86 MFI: Lung CD86 MFI DC imm DC Macs Mice Deficient in Mature B Cells HDM (ISO) S107 (IgA) BH8 (IgM) R36A (ISO) S107 (IgA) BH8 (IgM) JY2190 (ISO) S107 (IgA) BH8 (IgM) Streptococcus pneumoniae D. pteronyssinus 0.5mm ISO BH8 D. pteronyssinus 3-4 day PFA-fixed PC-bearing pneumococcus R36A Neonatal Immunization TEPC15 KI mouse Inc population of T15 id PC-specific B cells PFA-fixed PC-devoid pneumococcus JY2190 HDM Allergy Model 5ug 5 x 5ug 0 7 8 91011 14 OR FSC SSC B220 FSC CD19 PC-BSA PC-BSA PC-BSA PC-BSA TC68 TC68 TC68 B220 + CD19 + IgM PC-BSA B220 + CD19 + TC68 PC-BSA B220 + CD19 + AB1-2 TC68 CD19 B220 IgM PC-BSA PC-BSA AB1-2 IgM TC68 TC68 0 10 2 10 3 10 4 10 5 0 2 4 5 85.9 2.59 13.5 94.4 SiglecF Ly6G Alveolar macrophages Eosinophils lymphocytes, monos eosinophils neutrophils HDM onlyISO BH8 Media 0.0 0.5 1.0 1.5 *** CD86 CD86 MFI (x10 3 ) Alv Mac Pulm APCsMac cell line 0 5 10 20 40 60 80 HDM-ISO HDM-10 BH8 HDM-20 BH8 * ** * ** * ** HDM only HDM Positive Cells (%) Alv Mac Pulm APCs Mac cell line HDM ISO BH8 Media 4 3 2 1 1 HDM 2 ISO 3 BH8 4 Media um H8 um H8 0 2 4 6 8 10 * NS * AL H8 AL H8 0 5 10 15 20 * NS * NS nly SO H8 0 2 4 6 HDM Positive Cells (%) NS *** A B C Serum + + + + BH8 - + - + HI - - + + BALF + + + + BH8 - + - + HI - - + + HDM Positive Cells (%) HDM Positive Cells (%) Media + + + BH8 - - + ISO - + - HDM Positive Cells (%) Flow Cytometry Identifying Antigen-Specific B Cells by Flow Cytometry IgM Antibodies Can Decrease Phagocytosis Dendritic cells migrate from the lung to the mediastinal lymph node, where they prime a TH2-directed response. Br Br Br Br V V V PBS JY2190 R36A T15 KI IgM Neonatal Immunization with R36A Results in Increased IgM Secretion in the Lung and Decreased Eosinophil Infiltration Into the Lung Br V V V V Br Br Br Siglec-F CD11c • Neonatal immunization with PC-bearing pneumococcus (R36A) generates a large population of PC-specific B cells, which migrate to the lung following exposure to HDM. • Early exposure to R36A decreases: -infiltration of allergy-associated cells into the BALF and lungs -cellular composition of draining lymph nodes -activation of APCs in the lung and T cells in the mediastinal lymph node -formation of mucin-producing cells in the bronchioles -migration of cells around the bronchioles -development of airway hyperresponvieness -production of TH2-associated cytokines -secretion of IgE We suggest that a PC-bearing pneumococcal vaccine be used during the regimen of neonatal vaccine exposure among at-risk children to prevent the development of allergies and asthma • Early exposure to PC-deficient pneumococcus was not sufficient to dampen the development of HDM-induced allergic disease during adult life • Early microbial exposure did not result in a significant imbalance in the TH1/TH2 T cell subsets • Adult C57BL/6 mice passively administered anti-PC IgM antibodies were significantly protected against the development of HDM-induced allergic disease • Mice deficient in mature B cells, that were immunized with PC- bearing pneumococcus (R36A) as neonates were not significantly protected against the development of HDM-induced allergic disease 6-8 weeks of age PBSJY2190R36AT15 KI 0.0 0.2 0.4 0.6 0.8 1.0 ** * NS PBS JY2190 R36A T15 KI 0 10 20 30 40 *** ** NS PBSJY2190R36AT15 KI 0 2 4 6 8 ug/mL anti-PC IgA *** PBS JY2190 R36A T15 KI TC68 Anti-PC IgM (μg/mL) Anti-PC IgA (μg/mL) PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI 1.07 1.11 1.72 4.71 Anti-PC IgG3 (OD 405) Sustained Antibody Production and Cells in the Spleen PC-BSA
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Transcript of AAI Poster Preeyam
Early Exposure to Phosphorylcholine-Bearing Microbes Dampens the Development of House Dust Mite Allergy During Adult Life
ABSTRACT
Preeyam Patel and John F. Kearney Microbiology Theme; Graduate Biomedical Sciences at the University of Alabama at Birmingham
INTRODUCTION AND METHODS
A suggested mechanism to explain the dramatic rise in the incidence of allergic disease among individuals living in developed countries has been an imbalance in the TH1/TH2 subsets; however, a majority of neonatal pathogens evoke potent B cell responses. These B cell clones, with shared specificity for allergens, can be protective against the development of allergic disease. Streptococcus pneumoniae and house dust mite (HDM) bear similar phosphorylcholine epitopes. Therefore, we hypothesized that B cell clones generated as a result of early pneumococcal exposure would dampen the development of HDM allergy during adult life. To test our hypothesis, we immunized neonatal mice with PC-deficient or PC-bearing pneumococcus and then challenged these mice with HDM as adults. Mice receiving the PC-bearing pneumococcus had a large frequency of PC-specific B cells that resided in their spleen and continually secreted antibodies. Upon pulmonary exposure to PC-bearing HDM, these B cells migrated to the lung and secreted antibody locally into the pulmonary space. As a result, mice immunized with PC-bearing pneumococcus, presented with dampened production of serum IgE and TH2-associated cytokines, development of airway hyperreactivity, and infiltration of allergic mediators into the lung as compared to mice exposed to PC-devoid pneumococcus as neonates. We further demonstrated that neonatal exposure to bacteria was not sufficient enough to disrupt the balance in the TH1/TH2 subsets.
RESULTS
TH2$TH2$
TH2$TH2$T$
TH2$
IgE$TH2$TH2$TH2$TH2$
B$B$
TH2$TH2$
Inci
denc
e of
Infe
ctio
us D
isea
ses
(%)
Inci
denc
e of
Imm
une
Dis
ease
s (%
)
1950 1960 1970 1980 1990 2000 0
50
100
100
200
300
400 Rheumatic Fever Hepatitis A Tuberculosis Mumps Measles
Chron’s Disease Multiple Sclerosis Type 1 Diabetes Asthma
1950 1960 1970 1980 1990 2000
Inci
denc
e of
Infe
ctio
us D
isea
ses
(%)
Inci
denc
e of
Imm
une
Dis
ease
s (%
)
1950 1960 1970 1980 1990 2000 0
50
100
100
200
300
400 Rheumatic Fever Hepatitis A Tuberculosis Mumps Measles
Chron’s Disease Multiple Sclerosis Type 1 Diabetes Asthma
1950 1960 1970 1980 1990 2000
Quest Diagnostics Health Trends; Allergy Report 2011
Development of Allergic Disease The Hygiene Hypothesis
Graham-Rowe D, Nature 479; Nov 2011; Masoli M., Allergy 59; 2004
Adapted from: Bach J-F, NEJM 347 11; Sept 2002
CONCLUSIONS
0 10 20 30 40 50
1
2
3
4
Vaporized Methacholine (mM)
Rrs
(cm
H2O
.s/m
L)
**
GATA3 TBET0
1
2468
10
Cells
in th
e Lu
ng (x
103 )
*
GATA3 TBET0.0
0.1
0.20.51.01.5
Cells
in th
e BA
LF (x
104 )
****
GATA3 TBET0.00.20.4
123
Cells
in th
e M
LN (x
104 )
*****
IL-2 IL-12p70 IFNg0
1
2
3
4
5
Cyto
kines
in th
e BA
LF (p
g/m
L)
IL-4 IL-5 IL-6 IL-13 IL-90123
204060
Cyto
kines
in th
e BA
LF (p
g/m
L)
* ***** *
**
*
CXCL1CXCL2RANTESCCL4024
10
20
30
Cyto
kines
in th
e BA
LF (p
g/m
L)
*
*****
*
***
*
100μm"
50μm"
50μm"
PBS! R36A! T15 KI!JY2190!
GATA 3 Tbet" GATA 3 Tbet" Vaporized Methacholine (mg/mL)"Cells
in th
e Lu
ng (x
103 )"
Cells
in th
e BA
LF (x
104 )"
Rrs
(cm
H2O
.s/m
L)"
IL-2 IL-12p70 IFNg" IL-4 IL-5 IL-6 IL-13 IL-9" CXCL1 CXCL2 RANTES CCL4"BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)" PBS"
JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"No HDM"
Neonatal Immunization with R36A Decreases Mucin Production and Cellular Infiltration in the Bronchioles
0 10 20 30 40 50
1
2
3
4
Vaporized Methacholine (mM)
Rrs
(cm
H2O
.s/m
L)
**
GATA3 TBET0
1
2468
10
Cells
in th
e Lu
ng (x
103 )
*
GATA3 TBET0.0
0.1
0.20.51.01.5
Cells
in th
e BA
LF (x
104 )
****
GATA3 TBET0.00.20.4
123
Cells
in th
e M
LN (x
104 )
*****
IL-2 IL-12p70 IFNg0
1
2
3
4
5
Cyto
kines
in th
e BA
LF (p
g/m
L)
IL-4 IL-5 IL-6 IL-13 IL-90123
204060
Cyto
kines
in th
e BA
LF (p
g/m
L)
* ***** *
**
*
CXCL1CXCL2RANTESCCL4024
10
20
30
Cyto
kines
in th
e BA
LF (p
g/m
L)
*
*****
*
***
*
100μm"
50μm"
50μm"
PBS! R36A! T15 KI!JY2190!
GATA 3 Tbet" GATA 3 Tbet" Vaporized Methacholine (mg/mL)"Cells
in th
e Lu
ng (x
103 )"
Cells
in th
e BA
LF (x
104 )"
Rrs
(cm
H2O
.s/m
L)"
IL-2 IL-12p70 IFNg" IL-4 IL-5 IL-6 IL-13 IL-9" CXCL1 CXCL2 RANTES CCL4"BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)" PBS"
JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"No HDM"
0 10 20 30 40 50
1
2
3
4
Vaporized Methacholine (mM)
Rrs
(cm
H2O
.s/m
L)
**
GATA3 TBET0
1
2468
10
Cells
in th
e Lu
ng (x
103 )
*
GATA3 TBET0.0
0.1
0.20.51.01.5
Cells
in th
e BA
LF (x
104 )
****
GATA3 TBET0.00.20.4
123
Cells
in th
e M
LN (x
104 )
*****
IL-2 IL-12p70 IFNg0
1
2
3
4
5
Cyto
kines
in th
e BA
LF (p
g/m
L)
IL-4 IL-5 IL-6 IL-13 IL-90123
204060
Cyto
kines
in th
e BA
LF (p
g/m
L)
* ***** *
**
*
CXCL1CXCL2RANTESCCL4024
10
20
30
Cyto
kines
in th
e BA
LF (p
g/m
L)
*
*****
*
***
*
100μm"
50μm"
50μm"
PBS! R36A! T15 KI!JY2190!
GATA 3 Tbet" GATA 3 Tbet" Vaporized Methacholine (mg/mL)"Cells
in th
e Lu
ng (x
103 )"
Cells
in th
e BA
LF (x
104 )"
Rrs
(cm
H2O
.s/m
L)"
IL-2 IL-12p70 IFNg" IL-4 IL-5 IL-6 IL-13 IL-9" CXCL1 CXCL2 RANTES CCL4"BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)"
BALF
Cyt
okin
es (p
g/m
L)" PBS"
JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"
PBS"JY2190"R36A"T15 KI"No HDM"
Neonatal Immunization with R36A Decreases the Production of TH2-, but not TH1-, Associated Cytokines in the Lung as well as the Development of Airway Hyperresponsiveness (AHR)
PBSJY2190R36AT15 KI0
100
200
300
ng/m
L De
rp1-
spec
ific Ig
E
*
***
PBSJY2190R36AT15 KI0.0
0.5
1.0
1.5
ng/m
L To
tal I
gE
***
total T cells eos neuts0.00.20.40.6
246
Cells
in th
e BA
LF (x
106 )
*****
***** *********
Baso Mast macs alv mac0.00
0.01
1369
Cells
in th
e BA
LF (x
105 )
************ *****
PBS! R36A! T15 KI!JY2190!
Cel
ls (x
106 )!
Cel
ls (x
105 )!
Tota
l IgE
(ng/
mL)!
Total T cells Eos Neuts! Baso Mast Macs Alv Macs! PBS JY2190 R36A T15 KI! PBS JY2190 R36A T15 KI!
A.M.!
A.M.!
neut!
neut!
eo!
eo!A.M.! neut!
eo!20μm!
A.M.!
neut!
Der
p1-Ig
E (n
g/m
L)!
PBS!JY2190!R36A!T15 KI!
Neonatal Immunization with R36A Decreased the Number of Cells infiltrating the Bronchoalveolar Space and IgE Production
mm
PBS JY2190
R36A T15 KI
CD44!
PBS! R36A! T15 KI!JY2190!
% o
f Max!
Neonatal Immunization with R36A Decreases Priming of Antigen-Experienced T Cells in the Mediastinal Lymph Node
CD440.0
0.5
1.0
1.5
CD4
T Ce
ll MFI
(x10
4 )
****
CD4+ T Cells!CD
44 M
FI (x
104 )!
DCs imm DCs macs alv macs0.0
0.5
1.0
1.5
2.0
CD
86 M
FI (x
103 )
*****
***
***
total T cells Eos Neuts0.000.02
0.2
24
Cel
ls in
the
Lung
(x10
7 )
**
********* ****
macs DCs Mast Baso0.00.51.0
24
10
20
Cel
ls in
the
Lung
(x10
5 )
******
***** ** ***
Total T cells Eos Neuts ! Macs DCs Mast Baso!
Cel
ls (x
107 )!
Cel
ls (x
105 )!
DCs imm DCs macs alv macs!C
D86
MFI
(x10
3 )!PBS!JY2190!R36A!T15 KI!
No HDM!
Neonatal Immunization with R36A Decreased Cellular Infiltration into the Pulmonary Parenchyma
PBSJY2190R36AT15 KI0
10
20
30
ng/m
L an
ti-PC
IgM **
*NS
PBS JY2190 R36A T15 KI!
ng/m
L an
ti-PC
IgM! PBS! JY2190! R36A! T15 KI!
PBS! JY2190!
R36A! T15 KI!
Neonatal Immunization with R36A Increased the Number of PC-Specific B Cells in the Lung
IgM!
PC-BSA!
Laminin!
PC
-Spe
cific
IgM
-Exp
ress
ing
50μg anti-PC IgM ab i.t. 1 hour prior to
challenge with 5ug HDM!
ISO BH8!
mm!CD44!
ISO!BH8!
% o
f Max!
Passive Antibody Administration
CD440
2
4
6
8
MLN
CD
4+ T
Cel
ls: M
FI
uMT uMT uMT ! JY2190 R36A!
mm!CD4+ T Cells!
CD
44 M
FI!
DC imm DC macs0
5
10
15
20
CD
86 M
FI: L
ung
CD
86 M
FI!
DC imm DC Macs!
Mice Deficient in Mature B Cells
HDM (ISO) S107 (IgA) BH8 (IgM)
R36A (ISO) S107 (IgA) BH8 (IgM)
JY2190 (ISO) S107 (IgA) BH8 (IgM)
Streptococcus pneumoniae D. pteronyssinus
0.5mm!
ISO! BH8!D. pteronyssinus
3-4 day
PFA-fixed PC-bearing
pneumococcus R36A
Neonatal Immunization TEPC15 KI mouse
Inc population of T15 id PC-specific B cells
PFA-fixed PC-devoid
pneumococcus JY2190
HDM Allergy Model
5ug$ 5$x$5ug$
0$ 7$ 8$ 9$10$11$ 14$
OR
FSC! CD19! PC-BSA! PC-BSA! AB1-2!
PC-BSA! PC-BSA! PC-BSA! GATA3! GATA3!
SSC!
B220!
IgM!
TC68!
TC68!
TC68!
TC68!
TC68!
CD
4!
CD
4!
B220+CD19+!
IgM PC-BSA!B220+CD19+!
TC68 PC-BSA!B220+CD19+!
AB1-2 TC68!0.81 3.8
FSC! CD19! PC-BSA! PC-BSA! AB1-2!
PC-BSA! PC-BSA! PC-BSA! GATA3! GATA3!
SSC!
B220!
IgM!
TC68!
TC68!
TC68!
TC68!
TC68!
CD
4!
CD
4!
B220+CD19+!
IgM PC-BSA!B220+CD19+!
TC68 PC-BSA!B220+CD19+!
AB1-2 TC68!0.81 3.8
FSC! CD19! PC-BSA! PC-BSA! AB1-2!
PC-BSA! PC-BSA! PC-BSA! GATA3! GATA3!
SSC!
B220!
IgM!
TC68!
TC68!
TC68!
TC68!
TC68!
CD
4!
CD
4!
B220+CD19+!
IgM PC-BSA!B220+CD19+!
TC68 PC-BSA!B220+CD19+!
AB1-2 TC68!0.81 3.8
FSC! CD19! PC-BSA! PC-BSA! AB1-2!
PC-BSA! PC-BSA! PC-BSA! GATA3! GATA3!
SSC!
B220!
IgM!
TC68!
TC68!
TC68!
TC68!
TC68!
CD
4!
CD
4!
B220+CD19+!
IgM PC-BSA!B220+CD19+!
TC68 PC-BSA!B220+CD19+!
AB1-2 TC68!0.81 3.8
0 102 103 104 105
<PE-A>: CD11b/SiglecF
0
102
103
104
105
<APC
-A>:
Ly6
G
85.9
2.59
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A
13.5
94.4
SiglecF
Ly6G
Alveolar macrophages
Eosinophils
lymphocytes, monos
eosinophils
neutrophils
HDM onlyISO BH8 Media0.0
0.5
1.0
1.5
CD
86 M
FI (x
103 ) ***
CD86!
CD
86 M
FI (x
103 )!
Alv Mac Pulm APCsMac cell line 0
5
1020406080
HDM
Pos
itive
Cells
(%)
HDM-ISOHDM-10 BH8HDM-20 BH8
***
***
***HDM only
HD
M P
ositi
ve C
ells
(%)!
Alv Mac Pulm APCs Mac cell line!
HDM ISO BH8 Media!
4!3!2!1!
1 HDM!2 ISO!3 BH8!4 Media!
Serum
Serum +
100u
g BH8
HI Seru
m
HI Seru
m + 10
0ug B
H802468
10
HDM
Pos
itive
Cells
(%)
*
NS
*
BAL
BAL + 10
0ug B
H8
HI BAL
HI BAL +
100u
g BH8
0
5
10
15
20
HDM
Pos
itive
Cells
(%)
* NS *
NS
HDM only
HDM + 10
0ug I
SO
HDM + 10
0ug B
H80
2
4
6
HDM
Pos
itive
Cells
(%)
NS ***A! B! C!
Serum! +! +! +! +!
BH8! -! +! -! +!
HI! -! -! +! +!
BALF! +! +! +! +!
BH8! -! +! -! +!
HI! -! -! +! +!
HDM
Pos
itive
Cells
(%)!
HDM
Pos
itive
Cells
(%)!
Media! +! +! +!
BH8! -! -! +!
ISO! -! +! -!
HDM
Pos
itive
Cells
(%)!
Flow Cytometry
Identifying Antigen-Specific B Cells by Flow Cytometry IgM Antibodies Can Decrease Phagocytosis
Dendritic cells migrate from the lung to the mediastinal lymph node, where they prime a TH2-directed response.
Br!
Br! Br!Br!
V!
V!
V!
PBS! JY2190! R36A! T15 KI!IgM!
Neonatal Immunization with R36A Results in Increased IgM Secretion in the Lung and Decreased Eosinophil Infiltration Into the Lung
Br!V!
V!
V!
V! Br!Br!
Br!
Siglec-F!CD11c!
• Neonatal immunization with PC-bearing pneumococcus (R36A) generates a large population of PC-specific B cells, which migrate to the lung following exposure to HDM.
• Early exposure to R36A decreases: -infiltration of allergy-associated cells into the BALF and lungs -cellular composition of draining lymph nodes -activation of APCs in the lung and T cells in the mediastinal lymph node -formation of mucin-producing cells in the bronchioles -migration of cells around the bronchioles -development of airway hyperresponvieness -production of TH2-associated cytokines -secretion of IgE
We suggest that a PC-bearing pneumococcal vaccine be used during the regimen of neonatal vaccine exposure among at-risk children to prevent the development of allergies and asthma
• Early exposure to PC-deficient pneumococcus was not sufficient to dampen the development of HDM-induced allergic disease during adult life
• Early microbial exposure did not result in a significant imbalance in the TH1/TH2 T cell subsets
• Adult C57BL/6 mice passively administered anti-PC IgM antibodies were significantly protected against the development of HDM-induced allergic disease
• Mice deficient in mature B cells, that were immunized with PC-bearing pneumococcus (R36A) as neonates were not significantly protected against the development of HDM-induced allergic disease
6-8 weeks of age
PBSJY2190R36AT15 KI0.00.20.40.60.81.0
O.D
. 405
***
NS
PBS JY2190 R36A T15 KI0
10
20
30
40
ug/m
L an
ti-PC
IgM ***
**NS
PBSJY2190R36AT15 KI0
2
4
6
8
ug/m
L an
ti-PC
IgA ***
PBS! JY2190! R36A! T15 KI!
TC68!
Anti-
PC Ig
M (μ
g/m
L) !
Anti-
PC Ig
A (μ
g/m
L) !
PBS JY2190 R36A T15 KI! PBS JY2190 R36A T15 KI! PBS JY2190 R36A T15 KI!
1.07! 1.11! 1.72! 4.71!
Anti-
PC Ig
G3
(OD
405
) !Sustained Antibody Production and Cells in the Spleen
PC-BSA
