Differential gene expressions in the lacrimal gland during development and onset of keratoconjunctivitis sicca in Sjögren's syndrome (SJS)-like disease of the C57BL/6.NOD-Aec1Aec2 mouse

Abstract

Recently, we reported development of the C57BL/6.NOD-Aec1Aec2 mouse carrying two genetic intervals derived from the NOD mouse. These two genetic regions confer Sjögren's syndrome (SjS)-like disease in SjS-non-susceptible C57BL/6 mice. In an attempt to define the molecular bases underlying onset of dacryoadenitis and subsequently keratoconjunctivitis sicca (or xerophthalmia) in the C57BL/6.NOD-Aec1Aec2 mouse model, we have carried out a study utilizing microarray technology. Using oligonucleotide microarrays, gene expression profiles of lacrimal glands at 4, 8, 12, 16 and 20weeks of age were generated for C57BL/6.NOD-Aec1Aec2 male mice. Analyses using Linear Models for Microarray Analysis package and B-statistics, 552 genes were identified as being differentially expressed (adjusted p-value <0.01 and B <1.5) during the development of SjS-like disease. These 552 genes could be arranged into four clusters, with each cluster defining a unique pattern of temporal expression, while the individual genes within each cluster could be grouped according to related function. Using a pair-wise analysis, temporal changes in gene expressions provided profiles indicating that individual genes were differentially expressed at specific time points during development of SjS. In addition, multiple genes that have been reported to show, either in humans or mouse models, an association with autoimmunity and/or SjS, e.g., ApoE, Baff, Clu, Ctla4, Fas/Fasl, Irf5, Lyzs, Nfkb, Socs3, Stat4, Tap2, Tgfbeta1, Tnfa, and Vcam1 were also found to exhibit differential expressions, both quantitatively and temporally. Selecting a few families of genes, e.g., cystatins, cathepsins, metalloproteinases, lipocalins, complement, kallikreins, carbonic anhydrases and tumor necrosis factors, it was noted that only a limited number of family members showed differential expressions, suggesting a restricted glandular expression. Utilizing these genes, pathways of inter-reactive genes have been constructed for apoptosis and fatty acid homeostasis, leading to modeling of possible underlying events inducing disease. Thus, these different approaches to analyze microarray data permit identification of multiple sets of genes of interest whose expressions and expression profiles may correlate with molecular mechanisms, signaling pathways and/or immunological processes involved in the development and onset of SjS in this mouse model, thereby providing new insight into the underlying cause or regulation of this disease.

Figure 1

Heat-map of differentially expressed genes (n = 552) in the lacrimal glands of individual C57BL/6.NOD-Aec1Aec2 male mice at 4, 8, 12, 16 and 20 weeks of age (n = 4-6 mice per age group), grouped into 4 clusters based on temporal expression profiles. Up-regulated gene expressions are shown in red, down-regulated gene expressions are shown in green.

Figure 2

Temporal changes in gene expressions in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice for genes that are associated with SjS, SLE and/or RA in humans (A) and genes considered important for disease development in the salivary glands of NOD and NOD-derived mice (B). Expression profiles are presented for these genes at 8, 12, 16 and 20 weeks of age relative to 4 weeks of age.

Figure 3

Temporal gene expression of cystatins (A), cathepsins (B), matrix metalloproteinases (C) and lipocalins (D) in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, 16 and 20 weeks of age relative to 4 weeks of age.

Figure 4

Temporal gene expression of the C1q-associated (A) and other components (B) of complement in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, 16 and 20 weeks of age relative to 4 weeks of age.

Figure 5

Temporal gene expressions of chemokine-associated Ccl (A), Cxcl (B) and Ccr (C) genes in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, 16 and 20 weeks of age relative to 4 weeks of age.

Figure 6

Temporal gene expression of various members of the kallikrein (A), carbonic anhydrase (B) and tumor-necrosis factor (C) gene families in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, 16 and 20 weeks of age relative to 4 weeks of age.

Figure 7

The inter-relationships of genes associated with apoptosis differentially expressed in the lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice. The inter-relationships were created by Pathway Studio Version 5.0 software using the ResNet mammalian database. Interactions identified were manually validated and collated for final pathway visualization.

Figure 8

Schematic showing inter-relationships of clustered genes associated with fatty acid, lipid and lipoprotein homeostasis based on differentially-expressed genes identified in lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice. Associations of the differentially-expressed genes to fatty acid, lipid and lipoprotein homeostasis were annotated using Pathway Studio and PANTHER softwares.