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. 2018 Jul;188(7):1555-1562.
doi: 10.1016/j.ajpath.2018.04.003. Epub 2018 Apr 22.

The G-Protein-Coupled Receptor ALX/Fpr2 Regulates Adaptive Immune Responses in Mouse Submandibular Glands

Ching-Shuen Wang  1 , Olga J Baker  2
Affiliations

The G-Protein-Coupled Receptor ALX/Fpr2 Regulates Adaptive Immune Responses in Mouse Submandibular Glands

Ching-Shuen Wang et al. Am J Pathol. 2018 Jul.

Abstract

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A4, and the Ca2+-dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences.

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Figures

Figure 1
Figure 1
Saliva flow rates are altered in female but not in male knockout (KO) mice. A: Saliva flow rate was measured in both female and male wild-type (WT; closed circles) and KO (open circles) mice at 12 and 32 weeks of age. Specifically, mice were anesthetized, stimulated with pilocarpine HCl/phosphate-buffered saline at 10 mg/kg i.p. Then, saliva was collected for 15 minutes. B: Body weight of mice in each group were measured at both 12 and 32 weeks of age. Data are expressed as means ± SEM. n = ≥6 mice. ∗P < 0.05.
Figure 2
Figure 2
Serum from female knockout (KO) mice contains serum reactive antibodies against healthy submandibular gland (SMG) tissue. Five-micron paraffin-embedded SMG sections from both sexes of healthy wild-type (WT) mice were obtained and processed for immunofluorescence as described in Materials and Methods. Serum from each group (WT male, KO male, WT female, and KO female) was applied to healthy SMG sections, and positive reactivity of antibodies was detected by confocal microscopy. Note the presence of serum reactive antibodies (green fluorescent signal) in female KO mouse SMG section. TO-PRO-3 Iodide (InvitroGen, Carlsbad, CA) was used to distinguish DNA (blue). Scale bars = 50 μm.
Figure 3
Figure 3
Immunoglobulin levels are altered in female but not in male knockout (KO) mice. Saliva and serum were collected from both female and male wild-type (WT) (closed circles) and KO (open circles) mice at 12 weeks of age. Expression levels of serum IgG (A), IgA (B), and IgM (C), and salivary IgG (D), IgA (E), and IgM (F) were measured by enzyme-linked immunosorbent assay. Data are expressed as means ± SEM. n = 12 mice per group. ∗P < 0.05, ∗∗P < 0.01.
Figure 4
Figure 4
Up-regulation of Sjögren syndrome–related genes and proteins occurs in female but not in male knockout (KO) mice. A and B: Submandibular glands (SMGs) were collected from male (A) and female (B) wild-type (WT) and KO mice at 12 weeks of age and total RNA were obtained and reverse-transcribed into cDNA. Then quantitative PCR reactions were performed on 96-well plates according to the manufacturer's instructions. Relative gene expression of Baff, Cxcl13, and Cxcr5 were normalized using β-actin. C–E: Protein lysates were prepared from male (C) and female (D) WT and KO mouse SMG. B lymphocyte activating factor (BAFF) and chemokine (C-X-C motif) ligand (CXCL)-13 and receptor (CXCR)-5 expression were detected by Western blot analysis, as described in Materials and Methods, using β-actin as normalization control (representative image shown); data are shown in E. F and G: Male (F) and female (G) SMG sections were subjected to immunofluorescence using rabbit anti-BAFF and mouse anti–E-cadherin (E-cad) antibodies followed by Alexa Fluor 488–conjugated goat anti-rabbit IgG antibody (green), Alexa Fluor 568–conjugated goat anti-mouse IgG antibody (red), and TO-PRO-3 Iodide (InvitroGen, Carlsbad, CA) nuclear stain (blue). xy Images were obtained and analyzed using a confocal microscope (model 510; Carl Zeiss, Oberkochen, Germany). Images were taken at the widest point of the xy plane. Data are expressed as means ± SEM. n = ≥6 mice (A), n = ≥3 experiments (E). ∗P < 0.05. Scale bars = 50 μm.
Figure 5
Figure 5
Overexpression of CD20 and increased CD20-positive B cells in female knockout (KO) mice. A and B: Protein lysates were prepared from male and female wild-type (WT) and KO mouse submandibular gland. CD20 expression was detected by Western analysis as described in Materials and Methods using β-actin as normalization control (representative image shown); data are shown in B. C: Five-micron representative sections from each group were stained with CD20 antibody and counterstained with TO-PRO-3 Iodide (InvitroGen, Carlsbad, CA). Arrows indicate CD20-positive cells within SMG. D: ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov.ij) quantification of CD20-positive cells from 12 representative fields at ×20 magnification per animal. Data are expressed as means ± SEM. n ≥ 3 experiments (B); n = 3 mice per group (D). ∗P < 0.05. Scale bars = 50 μm.
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