Comparison of inflammatory gene networks in dry eye between human and mouse

Abstract

The use of mice in biomedical research to study human diseases and test drugs needs to take into account the differences between species. Hence, we performed a retrospective analysis of 56 inflammatory genes previously identified in human conjunctival cells versus their mouse homologues expressed in the cornea in 3 dry eye (DE) mouse models.

The 3 DE models (I to III) used C57/BL6 mice; I: scopolamine administration associated with a controlled environment exposition for 10 days (n=10); II and III: twice daily instillations for 5 days of hyperosmolar (550 mOsM) NaCl (n=11), and 0.1% benzalkonium chloride (n=10). Control mice (n=12) did not receive any treatment. Total RNAs were extracted from corneas and mRNA copies counted with the mouse inflammation CodeSet (NanoString nCounter). Retrospective statistical analysis comparing the mouse models with the human data was performed using Mann-Whitney non-parametric tests; a p<0.05 was considered significant.

Among the 56 human genes, 45 of their counterpart were detected in the DE mouse corneas, indicating that 80% of gene are common in both species. Nevertheless, Ccl4, Ccl5, Ccl22, Ccl24, Cxcl9, Cxcl10, Il23a, Ccr1, H2-Ea-ps, Nox1 and Hsh2d were not expressed in mouse corneas. Among the 45 inflammatory genes expressed by DE mouse corneas, 24 show a significantly different differential expression (vs control) in the 3 DE models. The genes in common or specific to each group in the different DE models are: 1) Group I, II, III: Mx1, Irf1, Il1rn, Tnfaip3, Keap1 and Mapk8; 2) Group II, III: Tlr3, Nfatc3, Mapkapk2; 3) Group I, II: Mek6; 4) Group I: Tnf, Myd88, Prkca, Maff; 5) Group II: Ltb4r2, H2-Eb1, Ptgfr, Ly96, Traf2, Stat3, Hmgb1, Atf2; 6) Group III: Tradd, Ripk2, Plcb1.
These results suggest that: 1) 54% of the human genes are similarly significantly modulated in the mouse; 2) the different DE models have a common network of genes expressed in the corneas; 3) each DE mouse model expresses a set of genes specific to the ocular stress used to generate DE.

This translational research confirms the usefulness of the mouse as an animal model for studying the underlying mechanisms of DE and for assessing the role of each of the gene identified in humans. This kind of comparative gene expression analysis is useful to help choose the most appropriate animal model to better decipher DE and screen potential new therapeutics.

Full text

Comparison of inflammatory gene networks in dry eye between human and mouse
KESSAL K1,2, DAULL P3, FERAILLE L4, CIMBOLINI N4, LIANG H1,2, DOCQUIER M5, BARABINO S6, MELIK PARSADANIANTZ S1,
GARRIGUE JS3 , BAUDOUIN C1,2,7, BRIGNOLE-BAUDOUIN F1,2,8
1Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France; 2CHNO des Quinze-Vingts, INSERM-DGOS CIC 1423, Paris, France; 3Santen SAS, Evry, France; 4Iris Pharma, Nice, France;
5iGE3 Genomics Platform University of Geneva, Switzerland; 6Clinica Oculistica, Università degli Studi di Genova, Italy; 7Université Versailles-Saint-Quentin-en-Yvelines, UVSQ,
Hôpital Ambroise Paré, APHP, Boulogne-Billancourt, France; 8Université de Paris, Faculté de Pharmacie de Paris, USPC, France,
1Animal models of dry eye: a critical assessment of opportunities and limitations. Barabino S, Dana MR; IOVS, 2004
2What We Have Learned from Animal Models of Dry Eye. Stern ME; Int Ophthalmol Clin, 2017
3Conjunctival Inflammatory Gene Expression Profiling in Dry Eye Disease: Correlations With HLA-DRA and HLA-DRB1.
Kessal K ; Front Immunol, 2018
4Correlation of clinical symptoms and signs with conjunctival gene expression in primary Sjögren syndrome dry eye patients.
Liang H ,Kessal K; Ocul Surf, 2019
5The controlled-environment chamber: a new mouse model of dry eye. Barabino S; IOVS, 2005
6Modulation of Inflammation-Related Genes in the Cornea of a Mouse Model of Dry Eye upon Treatment with Cyclosporine Eye
Drops A. Daull P; Curr Eye Res, 2019
7A mouse dry eye model induced by topical administration of benzalkonium chloride. Lin Z; Mol Vis, 2011
8A Comparison of the Effects of Benzalkonium Chloride on Ocular Surfaces between C57BL/6 and BALB/c Mice.
Yang Q; Int J Mol Sci, 2017
9Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface. Luo L; Eye Contact Lens, 2005
10Direct multiplexed measurement of gene expression with color-coded probe pairs. Geiss GK; Nat Biotechnol, 2008
This translational research confirms the usefulness of
the mouse as an animal model for studying the
underlying mechanisms of DE and for assessing the
role of each of the gene identified in humans.
This kind of comparative gene expression analysis is a
useful strategy to evaluate consistency between
species, and should be done to correctly assess the
most appropriate mouse models to better decipher DE
and screen potential new therapeutics.
INTRODUCTION
MATERIALS and METHODS
CONCLUSIONSREFERENCES
RESULTS
Contact : karima.kessal@inserm.fr
Session Dry Eye (113)
274 - B0413
1-Samples: Total RNAs were provided
from cornea extracts and mRNA copies were
counted with the mouse inflammation
CodeSet (NanoString® nCounter)[10].
2- Retrospective statistical analysis:
Mice (mm) and human (hs)gene expressions
was compared by calculation of fold change
of each selected mouse gene (56) against its
corresponding control. Significant differential
expressed genes were analyzed using Mann-
Whitney non-parametric tests; a p<0.05 was
considered significant.
Key points
- Similarities with 80 % genes expressed between mouse and human
- Among 56 human genes, 24 mouse genes are differentially expressed
-Specific gene inflammatory network is envolved in each DE mouse model
- Induced mice [NaCl, 550mOsM] (n=11)
The use of mice in biomedical research to
study human diseases[1,2] and test drugs needs
to take into account the differences between
species.
Hence, we performed a retrospective analysis
of 56 inflammatory genes previously identified
in human conjunctival cells[3,4] versus their
mouse homologues expressed in the cornea in
3 dry eye (DE) selected mouse models:
DE-I[5,6]: Tear deficiency
DE-II and III[7,8,9]: Subchronic toxic stress
Figure 1. Fifty six modulated genes involved in inflammatory process in human tissues [3,4]
Homo Sapien (hs)
Conjunctival Imprint
52-hs-MX1
53-hs-OAS2
54-hs-IRF1
55-hs-IFI44
56-hs-HSH2D
IFN & ISGs
TLR Receptors
&
Adaptators
47-hs-TLR2
48-hs-TLR3
49-hs-MYD88
50-hs-LY96
51-hs-NOD2
43-hs-PLCB1
Phospholipid
enzymes
29-hs-HMGB1
30-hs-HMGB2
31-hs-ATF2
32-hs-NFATC3
33-hs-MAFF
Transcription
factor
35-hs-MAPK8
36-hs-MK2
37-hs-MEK6
MAPKs family
41-hs-RIPK2
42-hs-PRKCA
Ser/Thr
kinases
Enzymes &
Adaptators
44-hs-NOX1
45-hs-NOS2
46-hs-KEAP1
1-hs-HLA-DRA
2-hs-HLA-DRB1
MHC Class II
3-hs-TNF
4-hs-CD40
5-hs-TRAF2
6-hs-TRADD
7-hs-TNFAIP3
TNF superfamily
17-hs-IL6
18-hs-IL15
19-hs-IL23A
20-hs-IL1RN
Interleukins
& Receptors
38-hs-STAT1
39-hs-STAT2
40-hs-STAT3
STAT family
25-hs-C2
26-hs-CFB
27-hs-C1QB
28-hs-ITGB2
Complement
& CRP
GPCR
& SubUnits
21-hs-LTB4R1
22-hs-LTB4R2
23-hs-PTGFR
24-hs-GNGT1
HSP family
34-hs-HSPB2
Chemokines & Receptors
8-hs-CCL4
9-hs-CCL5
10-hs-CCR1
11-hs-CCL22
12-hs-CCL24
13-hs-CXCL2
14-hs-CXCL9
15-hs-CXCL10
16-hs-CXCR4
Figure 2. Distribution of expressed hs-Genes in induced mouse cornea
DE-I
Tear deficiency [5,6]
Scopolamine Injection
In controlled environmental chamber (CEC)
Transdermal patch
administration
Scopoderm® TTS
(0.5 mg/72h)
- Temperature: 20–22 °C;
- Relative humidity: <25%
- Airflow: 15 L/min
Days
D1 D3 D5 D7 D9
- Induced mice (n=10)
- Control mice (n=10)
Duration 10 Days
D10
End
Cornea
harvesting
mus musculus (mm)
Figure 3. A Differentially Expressed mm-genes in mouse cornea according to DE model
(III) HyperOsmotic
17 targets
(I) Scopolamine-CEC
11 targets
(II) BAK
12 targets
MX1*
IRF1***
MAFF***
TNFAIP3***
KEAP1***
ATF2**
MYD88*
MAPK8 ***
TNF ***
PRKCA**
HMGB1*** NFATC3**
4
6
3
7
0
1
3
STAT3**
IL1RN *
MEK6***
TRADD***
RIPK2*
PLCB1*
MK2**
TLR3**
LTB4R2 **
TRAF2*LY96*H2-EB1*
mm-genes
hs-genes
C. Baudouin
DE-II and DE-III
Toxic stress on ocular surface
Benzalkonium chloride, BAK[7,8]
Hyperosmolar saline [9]
- Induced mice [BAK, 0.1% ] (n=10)
- Topical Administration (TA)
- One Eye
-Twice daily
Days
TA1
(a.m)
TA 2
(p.m)
[4h] //
D1
- Control mice (n=12)
Duration 5 Days
D2 D3 D4 D5
D5
End
Cornea
harvesting
x 5 days
24 Differentially
Expressed genes
43%
11 Non Expressed genes
20%
45 Expressed genes
80%
21 Non Differentially
Expressed genes
37%
H2-EA-PS
HSH2D
NOX1
LY96
MYD88
TLR3
Non Differentially
Expressed genes
37 %
Differentially
Expressed genes
43%
Commercial relationships: Santen, Iris Pharma