Upregulated Chemokine and Rho-GTPase Genes Define Immune Cell Emigration into Salivary Glands of Sjögren's Syndrome-Susceptible C57BL/6.NOD- Aec1Aec2 Mice

Abstract

The C57BL/6.NOD-Aec1Aec2 mouse is considered a highly appropriate model of Sjögren's Syndrome (SS), a human systemic autoimmune disease characterized primarily as the loss of lacrimal and salivary gland functions. This mouse model, as well as other mouse models of SS, have shown that B lymphocytes are essential for the development and onset of observed clinical manifestations. More recently, studies carried out in the C57BL/6.IL14α transgenic mouse have indicated that the marginal zone B (MZB) cell population is responsible for development of SS disease, reflecting recent observations that MZB cells are present in the salivary glands of SS patients and most likely initiate the subsequent loss of exocrine functions. Although MZB cells are difficult to study in vivo and in vitro, we have carried out an ex vivo investigation that uses temporal global RNA transcriptomic analyses to profile differentially expressed genes known to be associated with cell migration. Results indicate a temporal upregulation of specific chemokine, chemokine receptor, and Rho-GTPase genes in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice that correlate with the early appearance of periductal lymphocyte infiltrations. Using the power of transcriptomic analyses to better define the genetic profile of lymphocytic emigration into the salivary glands of SS mice, new insights into the underlying mechanisms of SS disease development and onset begin to come into focus, thereby establishing a foundation for further in-depth and novel investigations of the covert and early overt phases of SS disease at the cellular level.

Keywords: C57BL/6.NOD-Aec1Aec2 mice; DOCK molecules; GTP-GAP; GTP-GEF; RNA transcriptome microarray; Rho-GTPases; Sjögren’s syndrome; marginal zone B cells.

Figure 1

Histological photomicrographs depicting lymphocytic infiltrations present within the salivary glands of C57BL/6.NOD-Aec1Aec2 mice at 16 (A) and 31 (B) weeks of age. Infiltrations are age-dependent and contain an ever-changing proportion of both T cells (green fluorescence) and B cells (red fluorescence). Lymphocytic infiltration, especially in SS-susceptible C57BL/6.NOD-Aec1Aec2 mice, is strongly periductal. Visualization is at 200X magnification.

Figure 2

Coordinated temporal activation of factors important for MZB cell emigration from splenic marginal zone (MZ) areas to the salivary glands of C57BL/6.NOD-Aec1Aec2 mice at onset of SS-like disease. Transcriptomic data reveal that genes encoding ICAM1 and VCAM1 (Receptors for LFA1), LFA-1 (ItgaL and Itgβ2), Cxcl13, Cxcr5 (the Cxcl13 receptor), Dock2, Ccr6, Ccr7, and Ptk2β are all upregulated in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice at 16 weeks of age (right panel), the approximate time point when the immune response is transitioning to the adaptive immune response phase. In contrast, no similar upregulated expressions of these genes are seen in the salivary glands of C57BL/6J mice (left panel).

Figure 3

Unique upregulation profile of genes encoding for members of the Rho-GTPase family of proteins in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice. Rho-GTPase family proteins are important in regulating the cellular homeostasis of the GTP<>GDP system. The Rho-GTPase family consists of 21 members, of which 16 are divided among 5 subfamilies (Rsf1, Rsf2, Rsf3, Rsf4 and Rsf5), with 5 unassigned (designated Rsfx here). For cells in the non-activated state, Rho-GTP molecules are mostly associated with cellular membranes, but during cell activation the molecules dissociate to the cytoplasm by an as-yet unknown mechanism. In the salivary glands of C57BL/6.NOD-Aec1Aec2 mice, genes encoding for Rhoc, Rhou, Cdc42, Rac3, and Rnd1 are upregulated starting at 8 weeks of age, with Rac2 and Rhobtb1 upregulated starting at 16 weeks of age (lower panel). In contrast, the salivary glands of C57BL/6J mice showed a weak upregulated expression for Rnd1, Rnd2, Rhobtb3, Rhoh, Rhof and Rhov at 8 weeks of age, but this was not prolonged.

Figure 4

Unique upregulation profiles for genes encoding for members of the Rho-GTPase GAP and GEF families of proteins. Transcriptome data showing the temporal expressions of 8 of the 23 Arhgap genes and 7 of the 17 Arhgef genes upregulated in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice (lower panels). In each case, upregulated expressions started around 8 weeks of age. In contrast, expressions of these Arhgap and Arhgef genes remain downregulated.

Figure 5

Upregulated profiles of genes encoding for members of the Rho-GTPase Dock family of proteins. Transcriptome data showing the temporal expressions of the Dock genes upregulated in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice (lower panel) versus C57BL/6J mice (upper panel). In the SS-susceptible C57BL/6.NOD-Aec1Aec2 mice three genes, Dock2, Dock10, and Dock11, reported to be uniquely associated with B and T lymphocyte functions, exhibit a concomitant upregulated short-term expression at 16 weeks of age (arrows), while Dock1, 5, 7 and 8 are each upregulated starting at 4 weeks of age and remain activated out to 16 or 20 weeks of age. In contrast, in the SS-non-susceptible C57BL/6J mice, Dock4, 7, 9, 10 and 11 exhibit an upregulated expression starting at 4 weeks of age, while Dock8 shows a unique expression pattern being upregulated at 16 weeks of age.

Figure 6

Rho-Arhgef pathway activations. Rho-Arhgef molecules are associated with several signal transduction pathways including Rac-GTP, Rho-GTP and CDC42-GP, each leading to downstream activation of a biological process important for cellular migrations. These three pathways interact, respectively, with Wasf, Rock, and Was protein family members. In the salivary glands of C57BL/6.NOD-Aec1Aec2 mouse these appear to be Wasf3, Rock1, Rock2 and Was (right panel). The genes encoding Wasf3, Rock1 and Rock2 show an activation starting at 4 weeks of age, while the gene encoding Was is upregulated uniquely at 16 weeks of age. In the salivary glands of C57BL/6J mice, a strong activation is observed with Wasf3, and possibly a weak response by Rock1.

Figure 7

Temporal and differential gene expressions of various factors involved with signal transduction pathways regulated by members of the Rho-GTPase families of proteins. Transcriptome data of multiple genes (i.e., Cdc42, Vav, and Baiap (brain-specific angiogenesis inhibitor -1 associated protein) family members, plus Scrib1, Setd6, Grb2, Flii, Creb3 and Foxo3a) exhibit upregulated expressions in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice, virtually all starting at 8 weeks of age (lower panel). C57BL/6J mice exhibit a general lack of upregulated expression, except for Vav1 and possibly Cdc42bpg and Vav2 (upper panel).