CD4 naive 0 2 4 6 0 10 20 30 40 DPBS GH W eeks of treatment % o f c el ls
Severe deficiency of the somatotrope GHRH/GH/IGF-1 axis induces a
dramatic susceptibility to Stre
p
tococcus pneumoniae infection
Khalil Farhat
1, Gwennaelle Bodart
1, Chantal Renard
1, Christophe Desmet
1, Michel Moutschen
2, Fréderic Baron
3, Yves Beguin
3,
Roberto Salvatori
4, Vincent Geenen
1& Henri Martens
11GIGA Research Institute, GIGA-I3 Center of Immunoendoc rinology, University of Liege, Belgium 2Clinical Immunology and Infectiology, University Hospital of Liege, Belgium
3GIGA Research Hematology, University of Liege, Belgium
4Division of Endocrinology & Metabolism, Johns Hopkins University, Baltimore, USA
The question of a physiological role of the somatotrope axis in immunity was reassessed in GHRH-deficient (Ghrh
-/-) mice,
a new dwarf mouse model with a severe deficiency of the GHRH/GH/IGF-1 axis.
Prev 13 Poly 23
A- Polysaccharide vaccine (Poly 23):
• Primarily elicit Ab responses in the absence of T-cell help
• B-1 cells are envisioned as key players in this immune response along with marginal zone (MZ) B cells.
B- Conjugated pneumococcal vaccine (Prev-13)
• Conjugated to highly immunogenic cross-reactive material 197.
• Stimule FO B cells that engage in T-cell–B-cell cooperation, class-switching, yielding IgG antibodies of high affinity..
S.pneumoniae Bacteremia Age : 6-8 weeks Vaccine administration: S.C ELISA against CWPS1 GH supplementation : I.P : Blood sample : Sacrifice : GH supplementation : Day D
IgM without GH supplementation
n=6 Pneumovax 23 D 0 0 W T D 0 0 K O D 0 7 W T D 0 7 K O D 1 4 W T D 1 4 K O D 2 1 W T D 2 1 K O D 2 8 W T D 2 8 K O D 3 5 W T D 3 5 K O 0 50 100 150 200 250 ** * p = 0.0574 * * P e r c e n t o f c o n t r o l Prevnar 13 D 0 0 W T D 0 0 K O D 0 7 W T D 0 7 K O D 1 4 W T D 1 4 K O D 2 1 W T D 2 1 K O D 2 8 W T D 2 8 K O D 3 5 W T D 3 5 K O 0 50 100 150 200 250 ** ** WT KO
IgM with GH supplementation
n=6-7
Test Mann-Withney one tailed D 0 0 KO D 00 GH D 07 KO D 07 GH D 14 KO D 14 GH D 21 KO D 21 GH D 28 KO D 28 GH D 35 KO D 35 GH D 42 KO D 42 GH 0 50 100 150 200 250 * p = 0.0507 * ** P e r c e n t o f c o n t r o l D 0 0 K O D 0 0 G H D 0 7 K O D 0 7 G H D 1 4 K O D 1 4 G H D 2 1 K O D 2 1 G H D 2 8 K O D 2 8 G H D 3 5 K O D 3 5 G H 0 50 100 150 200 250 * Experimental protocol : Sacrifice : Hour h
Mice age: 6-12 weeks
Cytometry of lung immune cells
bacterial load in lung and spleen (septicemia)
S.Pneumoniae serotype 1, clinical isolate E1586
Unpaired t test with welch's correction one-tailed
WT Ghrh-/-
6h 0/7 0/7 24h 0/13 8/12 48h 0/13 12/13 72h 0/3 3/3
Ghrh
-/-mice:
severe deficiency the somatotrope GHRH/GH/IGF-1 axis
Thymic subpopulations Lymphoid subpopulations in periphery
Thymus (g) (g) Spleen Thymopoeisis Thymus weight(mg) Spleen weight(mg) IGF-1 expression LB 0 2 4 6 0 20 40 60 80 DPBS GH W eeks of treatment % o f c el ls
Basal immunological phenotype of Ghrh
-/-mice without and
with GH supplementation
.
Anti-pneumococcal vaccination
2 ways of vaccination against S.pneumoniae
Characteristics of Ghrh
-/-immune system in basal conditions:
↗ TREC, ↗ naive T cells,
↙↙
B cells
.
Therefore, we investigated the
B-dependent
vaccine and immune
responses of Ghrh
-/-mice to 2 anti-pneumococcal vaccines
and to a
sublethal infection by S.pneumoniae.
Test Mann-Withney one tailed
KO KO GH
Prevnar 13 Pneumovax 23
Ghrh-/- are unable to elicit vaccinal response to S.pneumoniae vaccines.
GH treatment restores vaccinal response of Ghrh-/- mice but only to Pneumovax 23.
Sublethal infection by S.pneumoniae
Experimental protocol
Bacteremia
Ghrh-/- mice develop S.pneumoniae fatal septicemia afer intranasal instillation while
WT/HZ mice totally clear infection after 24 hours.
There is also a marked decrease of B and T lymphocytes with high level of IFN-γ,
IL-10, CD40 and CXCL-9 expression in the lungs of infected Ghrh-/- mice.
Adapted from Vale et al, 2015
Ghrh
-/-mice: Phenotype
Alba, M. and R. Salvatori (2004) Endocrinology 145(9): 4134-4143.
• GH and IGF-1 deficiency
• Dwarf phenotype
• Normal bodily function
• Supplementation at different level of somatotropic
axis
Ghrh
-/-mice: Immune system
Shohreh, R., C. A. Pardo, F. Guaraldi, A. V. Schally and R. Salvatori (2011).
Endocrinology 152(10): 3803-3810.
• Less prone to develop experimental autoimmune
encephalomyelitis (EAE).
• GH administration but not GHRH restores the
original susceptibility of EAE.
Pivotal role of GH on the regulation of different components of the immune system independent from GHRH
Bacterial load
Determine bacterial concentration
Phenotype cells via flow cytomety
Assay in situ mRNA expression qPCR
Histological section
Results
4.2x 10x WT
KO
Histological section 48 h post-infection
20x Leucocyte 0 10 20 30 48 72 24 p=ns p=ns p=ns % CD 4 5 + Monocyte 0 10 20 30 48 72 24 ** * p=ns p=ns % C D 45 + C D 11 b + Ly 6g -Neutrophil 0 10 20 30 48 72 24 * * * p=ns % C D 45 + C D 11 b - L y6 g + Macro Infilt 0 10 20 30 40 50 48 72 24 * * p=ns p=ns % C D 45 + C D 11 b + F 4/ 80 + F S C + B lymphocytes 0 10 20 30 40 50 48 72 24 * * * ** p=ns % C D 45 + C D 11 b - F 4/ 80 - C D 19 + T lymphocytes 0 10 20 30 40 50 48 72 24 * p=ns p=ns % C D 45 + C D 11 b - F 4/ 80 - C D 3 +
Phenotype cells flow cytomety In situ transcripts quantification
n=3 n=3 n=3 n=2 1000 10000 100000 1000000 24 heures 48 heures * *** ** * IFN- 2^ CT IF N -HPRT *1000 000
n=3
n=3
n=3
n=2
100 1000 10000 100000 24heures 48heures ** *** p=0.0885 p=0.5463 IL-102^
CT IL10-HPRT *1000000
n=3 n=3 n=3 n=2 10000 100000 1000000 24heures 48heures ** *** p=0.0734 p=0.0784 CD402^
CT CD40-HPRT *1000000
n=3 n=3 n=3 n=2 103 104 105 106 107 24 heures 48 heures 2^ CT C X CL 9-H P RT * 10 00 00 0 ** * * * * p=0.4833 ** CXCL-9 n=3 n=3 n=3 n=2 100000 1000000 24 heures 48 heures ** p=0.42 * * TGG- 2^ C T T G F -H P R T *1 0 0 0 0 0 0 n=3 n=3 n=3 n=2 1000 10000 24 heures 48 heures * p=0.5176 p=0.7425 p=0.1448 IgG-j 2^ CT IG G J-HP RT * 10 00 00 0Mann Whitney test Gaussian Approximation
