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Submitted on 10 Nov 2020
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IL-7 as a mucosal adjuvant in pulmonary immunization protocols
A Sandouk, A Vieira Antão, S. Figueiredo, B Charmeteau-De-Muylder, F Alby-Laurent, M Benard, E. Véron, Magali Rancez, R Cheynier, A
Couëdel-Courteille
To cite this version:
A Sandouk, A Vieira Antão, S. Figueiredo, B Charmeteau-De-Muylder, F Alby-Laurent, et al.. IL-7 as a mucosal adjuvant in pulmonary immunization protocols. 52rd Annual Meeting of the French society for immunology (SFI 2019), Nov 2019, Nantes, France. �hal-02998897�
A. Sandouk
1, 2
(abdelkader.sandouk@inserm.fr), A. Vieira Antão
1, 2
, S. Figueiredo
1, 2
, B.
Charmeteau-de-Muylder
1, 2
, F. Alby-Laurent
1, 2
, M. Benard
1, 2
, E. Veron
1, 2
, M. Rancez
1, 2
, R. Cheynier
1, 2
, A. Couëdel-Courteille
1, 2
Methods
Intranasal Infection Intra-veinous
Infection
Rapid and transient
IL-7 production
in tissues
Chemokines production in infected organs
Local recruitment of immune cells
Ponte et al.
(Front Immunol. 2017) preliminary results
IL-7
Subcutaneous Injection
Chemokines production in organs
Intestine, lung, skin
Local recruitment of immune cells
Beq et al. Blood 2009
IL-7, is a cytokine produced by stromal cells of lymphoid and non-lymphoid organs, is essential
during thymopoïesis as well as for T lymphocyte homeostasis (survival and proliferation of LT).
IL-7 is also important for B lymphopoiesis.But our data argue for another function.
Could IL-7 act as an adjuvant in mucosal immunization protocols ?
Results
IL-7 (n=6) Sacrifice (n=16) Day 0 D2 Intratracheal administrationIL-7 promotes the production of cytokines involved in both T and B
immune responses rather than tolerogenic cytokines
0 2 4 6 8 *** IL-4 pg/ µg of t ot al prot ei n D0 D2 *** pg/ µg of t ot al prot ei n 0 5 10 15 D0 D2 IL-5 D0 D2 * 0 10 20 30 40 50 IL-6 pg/ µg of t ot al prot ei n * pg/ µg of t ot al prot ei n 0 5 10 15 D0 D2 IL-9 pg/ µg of t ot al prot ei n 0 2 4 6 D0 D2 INF-Ɣ NS pg/ µg of t ot al prot ei n 0 10 D0 D2 IL-2 20 30 40 50 NS pg/ µg of t ot al prot ei n 0 10 D0 D2 IL-10 20 30 40 50 NS pg/ µg of t ot al prot ei n 0 10 D0 D2 IL-13 20 30 40
FIG 2. (A) Mice were intratracheally administered with IL-7 at D0 then sacrificed at D2. Control mice were
not treated with IL-7 (D0). (B) Lung homogenates were assayed for different immune response polarizing cytokines using a liquid phase ELISA. Bars and error bars represent means ± SD.
IL-7 (n=6)Day 0 (n=4)D10 Intratracheal administration D8 D6 D4 D2 (n=4) (n=4) (n=4) (n=4) CD45R CD3 DAPI PP AV BV LA 50 µm 500 µm J0 J2 J4 J6 J8 J10 0 200 400 600 m m 2 d ’A L /c m 2 d e po um on 0 200 400 600 D0 D2 D4 D6 D8 D10 N um be r of L A /c m 2 of l ung J0 J2 J4 J6 J8 J10 0 1000 2000 3000 4000 5000 Su rf ac e m éd ia ne d es A L (µ m 2) 0 1000 2000 3000 4000 5000 M edi an a re a of L A (µ m 2 ) D0 D2 D4 D6 D8 D10 D0 D2 D4 D6 D8 D10 CCL2 (MCP-1) CCL3 (MIP-1α) CCL4 (MIP-1β) CCL5 (RANTES) CCL17 (TARC) CCL20 (MIP-3α) CCL22 (MDC) CXCL1 (KC) CXCL5 (LIX) CXCL9 (MIG) CXCL10 (IP-10) CXCL13 (BLC) CCL11 (EOTAXIN) D0 pg/µg
J0
0
20
40
60
40 60 20 0 Fold increaseJ0 J4 J8
MCP-1 MIP-1b Eotaxin MIP-3a KC MIG BLC1 3 5 7 9
1 3 5 7 9 D0 D2 D4 D6 D8 D10 IL-1α IL-1β IL-11 IL-12p40 IL-12p70 IL-23 IL-27 IL-33 INF- β GM-CSF IL-3 pg/µg 0 90 180 270J0
J4
J8
IL-1a
IL-1b
IL-3
IL-11
IL-12p40
IL-12p70
IL-23
IL-27
IL-33
INF-B
GM-CSF
1 2 3 4 5
J0 J4 J8
MCP-1 MIP-1b Eotaxin MIP-3a KC MIG BLC1 3 5 7 9
J0 0 20 40 60J0
0
90
180
270
J0 J4 J8 IL-1a IL-1b IL-3 IL-11 IL-12p40 IL-12p70 IL-23 IL-27 IL-33 INF-B GM-CSF 1 2 3 4 5 J0 J4 J8 MCP-1 MIP-1b Eotaxin MIP-3a KC MIG BLC1 3 5 7 9
J0 0 20 40 60 J0 0 80 160 240 1 2 3 4 5 Fold increaseLocal administration of IL-7 changes the pulmonary physiology
FIG 1. (A) Mice were intratracheally administered with IL-7 and then sacrificed at different time points (D2, D4,
D6, D8, D10). D0 represents control mice that have not been treated with IL-7. From lung homogenates, both pro-inflammatory chemokines (B) and cytokines (D) were assayed using a liquid-phase multiplex ELISA. (C) Lung sections were labeled for CD3 (LT, red) and CD45R (LB, green). Nuclei were DAPI-stained (blue). Lymphoid aggregates evidenced in the pulmonary mucosa were analyzed with the CaseViewer software allowing to estimate their number (left panel) and surface (right panel). LA: lymphoid aggregate, PP: pulmonary parenchyma, BV: blood vessel, AV: pulmonary alveoli.
Background
D14 Sacrifice IL-7 (n=8) (n=9) Day 0 D2 IAVi (n=4) PBS (n=8) D29 Intranasal infection (IAV) PBS PBS IL-7 IAVi Intratracheal administration PBS 0 20 10 30 40 50 60 80 70 100 90 2 4 6 8 10 12 14 16 0 P at hol ogy (2x<80%) 0.034 Day post-infection 1.2x10 -6 2 4 6 8 10 12 14 16 Day post-infection 10 30 50 70 90 110 Re cove ry (95%) 0 0.006 IL-7+IAVi PBS+IAVi IL-7+PBS PBS+PBS A nt i-IA V IgA s (O D /[IgA s] PBS+P BS PBS+IA Vi IL-7+IA Vi 4 8 6 2 0 ns ** PBS+ PBS PBS+ IAV IL-7 + IA V 0 2 4 6 8 * ns PBS+ PBS PBS+ IAV IL-7 + IA V 50 60 70 80 90 100 A nt i-IA V IgG s (O D /[IgG s] 60 70 80 90 50 100 PBS+P BS PBS+IA Vi IL-7+IA Vi ** **IAV immunized mice that previously received IL-7 as an adjuvant are
more resistant to influenza pathology
(A) Mice intratracheally pre-treated
with IL-7 or PBS at D0 and immunized against influenza with inactivated IAV (IAVi) or not (PBS) at D2 underwent an intranasal virulent influenza virus (IAV) challenge. Mice were sacrificed at D29.
(B) Monitoring mice weight after
influenza virus infection allowed to estimate the pathology and the recovery. Pathology is defined as two successive measures of body weight <80%. Recovery is defined as a body weight measurement> 95%.
(C) IAV-specific IgAs and IgGs were
quantified by ELISA in bronchoalveolar lavages (BAL). Results are expressed as optical density over IgG or IgA concentration in each sample.
PBS D14 (n=11) Sacrifice (n=10) DT DT IL-7 Day 0 D2 Intratracheal administration *** A nt i-D T IgA s (O D /[IgA ]) ns PBS+DT IL-7+DT 0.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 0.0 1.0 50 100 150 00 50 100 150 0.0003 PBS+DT IL-7+DT A nt i-D T IgG s (O D /[IgG ]) A nt i-D T IgA s (O D /[IgA ]) 0 10 20 30 40 PBS+DT IL-7+DT 10 20 30 0.0 40 0.036
Serum
BAL
IL-7 enhances the specific antibody response
in a mucosal immunization protocol against diphtheria toxoid (DT)
FIG 3. (A) At D0, mice were intratracheally treated with IL-7 or PBS (control) then at D2, immunized against DT, used
as a model antigen. At D14, mice were sacrificed for the analysis. (B) DT-specific IgAs and IgGs were quantified by ELISA in serum and bronchoalveolar lavages (BAL).
Conclusion
LegendPLEX
Technology ImmunoHistoFluorescence (IHF)
Study of the immune cell infiltrate in the pulmonary mucosa Cytokine and chemokine
quantifications Lung C57BL/6 IL-7 DT IAVi Intratracheal administration Dissection Serum Lung Bronchoalveolar lavage (BAL) Samples
Quantification of immunoglobulins (IgG and IgA) by ELISA
BAL Serum
Analysis
1
Immunité Infection Inflammation, Université de Paris, Institut Cochin, Paris, France;
2
INSERM U1016, CNRS, UMR8104, F-75014, Paris, France
Intratracheal administration of IL-7 changes the pulmonary physiology by inducing chemokine production and immune cell infiltration. These modifications seem to prepare the
pulmonary mucosa to better respond to a subsequent administration of antigen by the same route. Indeed, following mucosal immunization against DT, mice pretreated with IL-7
developed stronger specific mucosal immune response than mice not treated with IL-7. In particular, IL-7 pre-treatment promoted a robust production of DT-specific IgAs in the
BAL but not in the serum, as detected by ELISA. Moreover, only mice pretreated with IL-7 before immunization against IAV (inactivated IAV) were protected from the pathology
caused by an intranasal influenza infection. Taken together, these data argue for IL-7 being a good candidate to be further evaluated as a mucosal adjuvant.
A
B
D
C
A
B
A
B
A
B
C
FIG 4. D0IL-7 as a mucosal adjuvant in
pulmonary immunization protocols
SIV
IAV
HistoFluorescence Scanning Analysis
IgA IgG IgA
0,0013 IgA s S p é-D T (D O /mg) IL-7+DT PBS+DT IL-7+DT PBS+DT IgG s S p é-D T (D O /mg) ** 0 20 40 60 80 150 200 * 0 10 20 30 40 PBS+DT IL-7+DT A nt i-D T IgG s (O D /[IgG ]) 20 40 60 0.0 80 150 200 0.0013 IgG
