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Par‐C10 culture and treatment

A quantity of 100 μl of growth factor‐reduced Matrigel (GFR‐MG) (8 mg/mL; 2:1 GFR‐MG: DMEM‐Ham's F12 [1:1] medium; Becton Dickinson, Franklin Lakes, NJ) was allowed to solidify in a 37°C incubator for 1 h in eight‐well chambers mounted on No. 1.5 German borosilicate coverglass (Nalge Nunc International, Naperville, IL). Then, Par‐C10 cells (15,000 cells/well; passages 30–60) were plated on the GFR‐MG in DMEM‐Ham's F12 (1:1) medium with supplements and treated with TNF‐α (100 ng/mL; Becton Dickinson), then incubated for 1 h or 6 h at 37°C. Cells were then treated with AT‐RvD1 (100 ng/mL; Cayman Chemical, Ann‐Arbor, MI), and varying doses of DEX (25–100 ng/mL). Three‐dimensional Par‐C10 cell clusters were used for assays after incubation at 37°C with 95% air and 5% CO2 for 72 h.

Treatment with AT‐RvD1 and DEX alone and in combination enhances Par‐C10 cell cluster growth

Par‐C10 cells were plated on GFR‐MG and preincubated with TNF‐α followed by treatment with AT‐RvD1 and DEX alone and in combination as described in . Then, salivary cell clusters were quantified using light microscopy as described in . As shown in Figure , untreated control cells as well as cells subjected to single treatments with AT‐RvD1 (100 ng/mL) or DEX (100 ng/mL) for 72 h showed increased salivary cell cluster growth as compared to cells treated with TNF‐α alone (100 ng/mL). Additionally, cells that were preincubated with TNF‐α (100 ng/mL) for 1 h followed by treatment with AT‐RvD1 (100 ng/mL) and DEX (25–100 ng/mL) alone and in combination recovered the number of salivary cell clusters after 72 h. Surprisingly, cells preincubated with TNF‐α for 1 h and then treated with AT‐RvD1 (100 ng/mL) and ¼ dose of DEX (25 ng/mL) for 72 h displayed a significant increase in the number of cell clusters grown as compared to cells treated with TNF‐α alone. Note that only cells preincubated with TNF‐α without any subsequent treatment showed a statistically significant cell cluster reduction from the control group.

Treatment with AT‐RvD1 and DEX alone and in combination mitigates TNF‐α mediated cellular apoptosis

To assess the effects of AT‐RvD1 and DEX on cell death following preincubation with TNF‐α, we performed a TUNEL assay on the Par‐C10 cell clusters grown on GFR‐MG. As shown in Figure A and B, a basal level of TUNEL‐positive cells (indicating percentage of apoptotic cells) was determined using untreated (control) cells. Both single treatments with DEX (100 ng/mL) and TNF‐α (100 ng/mL) resulted in a significant increase in the degree of apoptosis from the untreated cells, although AT‐RvD1 alone (100 ng/mL) did not increase the percentage of apoptotic cells. After preincubation with TNF‐α for 1 h, AT‐RvD1 alone and in combination with DEX caused a significant reduction in apoptotic cells as compared to cells treated with TNF‐α alone (see asterisks). Interestingly, the treatment consisting of AT‐RvD1 (100 ng/mL) and a reduced dose of DEX (25 ng/mL) was the most effective in reducing apoptosis, with values similar to untreated cells. Figure B shows representative images of percentages of TUNEL‐positive cells in each treatment group.

Abstract

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease characterized by diminished secretory function of the exocrine glands. Treatments for hyposalivation are limited to the use of saliva substitutes and medications that provide only temporary relief. In light of the high degree of need and the limitations of current therapies, development of alternative treatments to restore functioning is essential. Resolvins (Rv), which are highly potent lipid mediators, offer a viable alternative for better treating inflammatory diseases such as SS. The goal of this study was to determine whether systemic preventive treatment with Aspirin-triggered RvD1 (AT-RvD1) reduces inflammation and preserves secretory functioning in NOD/ShiLtJ SS-like mice. Our results indicate that systemic treatment with AT-RvD1 diminishes the progression of the disease in salivary epithelium from female mice as follows: (a) improves secretory function, (b) reduces pro-inflammatory molecule gene expression, (c) increases anti-inflammatory molecule gene expression and (d) induces M2 macrophage polarization. Finally, AT-RvD1 decreases lymphocytic infiltration into the salivary glands when used with small doses of the steroid, dexamethasone, and promotes the tissue healing process.

Experimental animals

Female and male NOD/ShiLtJ mice were treated twice a wk starting at 4 wk for 16 wk via tail vein injections with a vehicle control (EtOH, 8.9%) and AT-RvD1 (Cayman Chemical, Ann Arbor, MI) at 0.1 mg/kg. We used DEX (Sigma Aldrich, St. Louis, MO) at 8.25 mg/kg as a positive control and for a combination of AT-RvD1 and DEX (4.25 mg/kg). The doses of AT-RvD1 and EDX used in this study were chosen based on previous studies indicating that 0.1 mg/kg produces a significant downregulation of systemic inflammatory genes as compared to 0.05 mg/kg (data not shown). At 12 wk and 20 wk of age, the mice were anesthetized with 80–100 mg/kg Ketamine +5 mg/kg Xylazine and subsequently euthanized by abdominal exsanguination. 20 wk old untreated C57BL/6, NOD/ShiLtJ and ALX/FPR2−/− mice were used for [Ca2+]i measurements. The animal protocol (14-006007) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at University of Utah, according to the animal welfare act of the United States (7 U.S.C. 2131 et. seq.). All mouse experiments were carried out at the animal care facility of the University of Utah, Colorow 383 building, in accordance with approved guidelines.

Treatment with AT-RvD1 before disease onset maintains healthy saliva flow rates in female NOD/ShiLtJ mice

In order to verify whether AT-RvD1 was able to stimulate its receptor, ALX/FPR2, in SMG from NOD/ShiLtJ mice, we measured [Ca+2]i mobilization in response to this agonist in mouse SMG cells. As shown in , SMG cells from 20 wk NOD/ShiLtJ mice were able to mobilize [Ca2+]i in response to AT-RvD1. In contrast, SMG cells from 20 wk ALX/FPR2−/− mice (used as a negative control) did not show such increase. However, SMG cells from both NOD/ShiltJ and ALX/FPR2−/− mice responded to carbachol (Cch; acetylcholine analog; used as a positive control). Finally, SMG cells from C57BL/6 control mice showed responses to both AT-RvD1 and Cch ( and ). In order to determine whether AT-RvD1 was able to preserve salivary secretory function in NOD/ShiLtJ mice, we treated them with AT-RvD1 before disease onset as described in the Materials and Methods, then measured saliva flow rate at 20 wk. As shown in , female NOD/ShiLtJ mice treated with AT-RvD1 displayed saliva flow rates comparable to healthy controls (i.e., 4 wk old mice) and significantly higher flow rates when compared to vehicle controls. In contrast, male NOD/ShiLtJ mice subjected to the same treatment displayed saliva flow rates lower than healthy controls and comparable to vehicle controls ().

Treatment with AT-RvD1 prior to disease onset reduces pro-inflammatory and increases anti-inflammatory molecule gene expression in SMG

Upregulation of pro-inflammatory cytokines has previously been observed in the NOD mouse. To determine whether AT-RvD1 altered the inflammatory cytokine profiles, we performed a quantitative PCR analysis in SMG from female NOD/ShiLtJ mice. As shown in , SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed significant downregulation of expression of the pro-inflammatory cytokines, TNFα, IL-1β and IFNγ, as compared to vehicle-treated controls. In addition, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant downregulation of the pro-inflammatory cytokines, TNFα, IL-1β, IFNγ and IL-17 as compared to vehicle-treated controls (). In contrast, SMG from 20 wk male NOD/ShiLtJ mice treated with AT-RvD1 did not display these changes ().

Treatment with AT-RvD1 prior to disease onset reduces pro-inflammatory and increases anti-inflammatory molecule gene expression in SMG

Anti-inflammatory molecules were also investigated in this study. As shown in , SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant upregulation of Del-1 (endogenous leukocyte-endothelial adhesion inhibitor), TGFβ and IL-10 as compared to vehicle-treated controls. Moreover, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant upregulation of Annexin A1 (ANXA1), Del-1, TGFβ and IL-10 expression (). However, SMG from 20 wk male NOD/ShiLtJ mice treated with AT-RvD1 did not show significant changes as compared to vehicle-treated controls ().

Treatment with AT-RvD1 prior to disease onset triggers macrophage (Mϕ) M2 polarization in SMG

Previous studies have shown that treatment with AT-RvD1 polarizes pro-inflammatory M1-type macrophages (Mϕ) toward a pro-resolution M2-type in mouse and human cells. Therefore, we aimed to characterize the Mϕ phenotype in SMG from different treatment groups. Specifically, gene signatures of either M1 (IL-23 and iNOS) or M2 (IL-1ra and Arg-1) type Mϕ were analyzed in this study. Our results showed that SMG from 12 wk female NOD/ShiLtJ mice treated with AT-RvD1 displayed a significant downregulation of M1-type Mϕ genes (IL-23 and iNOS, ) and upregulation of pro-resolution M2-type Mϕ genes (IL-1ra and Arg-1, ) as compared to vehicle-treated controls (). Furthermore, SMG from 20 wk female NOD/ShiLtJ mice treated with AT-RvD1 demonstrated similar effects (i.e. M1-type downregulation/M2-type upregulation) as compared to vehicle-treated controls ().

Methods

VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.

Results

Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.

Conclusions

Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Cell culture and reagents

Primary VSMCs were isolated from human great saphenous veins discarded at the time of bypass operation under a University of California San Francisco Institutional Review Board-approved protocol (UCSF Committee on Human Research- Number: 10–03395; the committee waived the need for informed consent) as described previously []. VSMCs were cultured at 37°C and 5% CO2 in low glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, Logan, UT, USA) containing 10% FBS (Life Technologies, Grand Island, NY). VSMCs between passages 2 and 5 were utilized for all experiments. AT-RvD1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid), 17S-RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) were obtained from Cayman Chemical (Ann Arbor, MI, USA). The anti-GPR32 antibody was purchased from GeneTex (Irvine, CA, USA) and the anti-ALX antibody from Abcam (Cambridge, United Kingdom). PDGF-BB was purchased from Sigma-Aldrich (St. Louis, MO, USA) and Rp-8-Br-cAMP from Enzo Life Sciences (Farmingdale, NY, USA). Phorbol 12,13-dibutyrate (PDBu) was purchased from Biolog (Hayward, CA, USA) and chelerythrine chloride from Sigma-Aldrich (St. Louis, MO, USA).

cAMP measurement

Confluent VSMCs grown in 12-well plates were serum-starved overnight in DMEM containing 0.1% FBS. Cells were treated with AT-RvD1 (10nM) and harvested at different times using a 0.1N HCl, 1% Triton X-100 lysis buffer. After spinning the lysates at 12,000g for 10 minutes and collecting the supernatants, cAMP and total protein contents were measured via ELISA (Enzo Life Sciences, Farmingdale, NY, USA) and BCA assay (Thermo Fisher Scientific, Waltham, MA, USA), respectively, following the manufacturers’ protocols. cAMP concentrations in the lysates were calculated from the absorbance readouts using a four-parameter logistic fit and then normalized by total protein content.