AT-RvD1 combined with DEX is highly effective in treating TNF-α-mediated disruption of the salivary gland epithelium

Abstract

Sjögren's syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands leading to dry mouth and dry eyes, respectively. Currently, the etiology of SS is unknown and the current therapies have no permanent benefit; therefore, new approaches are necessary to effectively treat this condition. Resolvins are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Previous studies indicate that the resolvin family member, RvD1, binds to the ALX/FPR2 receptor to block inflammatory signals caused by tumor necrosis factor-alpha (TNF-α) in the salivary epithelium. More recently, the corticosteroid, dexamethasone (DEX), was shown to be effective in reducing salivary gland inflammation. However, DEX, as with other corticosteroids, elicits adverse secondary effects that could be ameliorated when used in smaller doses. Therefore, we investigated whether the more stable aspirin-triggered (AT) epimer, AT-RvD1, combined with reduced doses of DEX is effective in treating TNF-α-mediated disruption of polarized rat parotid gland (Par-C10) epithelial cell clusters. Our results indicate that AT-RvD1 and DEX individually reduced TNF-α-mediated alteration in the salivary epithelium (i.e, maintained cell cluster formation, increased lumen size, reduced apoptosis, and preserved cell survival signaling responses) as compared to untreated cells. Furthermore, AT-RvD1 combined with a reduced dose of DEX produced stronger responses (i.e., robust salivary cell cluster formation, larger lumen sizes, further reduced apoptosis, and sustained survival signaling responses) as compared to those observed with individual treatments. These studies demonstrate that AT-RvD1 combined with DEX is highly effective in treating TNF-α-mediated disruption of salivary gland epithelium.

Keywords: ALX/FPR2; AT‐RvD1; RvD1; Sjögren's syndrome; salivary glands.

Figure 1

Treatment with aspirin‐triggered resolvin D1 (AT‐RvD1) and dexamethasone (DEX) alone and in combination enhances Par‐C10 cell cluster growth on growth factor‐reduced Matrigel (GFR‐MG). Par‐C10 cells were grown on GFR‐MG in eight‐well chambers as described in Materials and Methods and preincubated in the absence or presence of TNF‐α (100 ng/mL for 1 h), then treated with AT‐RvD1 (100 ng/mL) and DEX (25–100 ng/mL) alone and in combination. Cell clusters were then quantified using light microscopy. Cell cluster growth was quantified and expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicate significant differences from cells incubated with TNF‐α alone, and ^† P < 0.05 indicate significant differences from control (untreated) cells.

Figure 2

Treatment with aspirin‐triggered resolvin D1 (AT‐RvD1) and dexamethasone (DEX) alone and in combination enhances cell cluster and lumen formation in Par‐C10 cells grown on growth factor‐reduced Matrigel (GFR‐MG). To establish baseline data, Par‐C10 cells were subjected to single treatments (A–D). Next, cells were preincubated with TNF‐α (100 ng/mL) for 1 h (E–I) or 6 h (J–N) and treated for 72 h with AT‐RvD1(100 ng/mL) and DEX (25–100 ng/mL) alone and in combination. Then, cell clusters were fixed with 4% of paraformaldehyde and stained with rabbit anti‐ZO‐1 and Alexa Fluor^® 488 (green), followed by the F‐actin stain Alexa Fluor^® phalloidin 568 (red) and the nuclear stain TO‐PRO ^®‐3 iodide (blue). White arrows point to apical localities of ZO‐1 (2A‐C, E‐N), while the red arrow shows an improper collapsed lumen in cells treated with TNF‐α (2D). Images were obtained and analyzed using a Carl Zeiss 710 confocal microscope and ZEN software. All scale bars represent 20 μm. Lumen sizes were measured following either a 1 h (O) or 6 h (P) preincubation with TNF‐α, followed by 72 h treatments with AT‐RvD1 (100 ng/mL) and DEX (25–100 ng/mL) alone and in combination. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicate significant differences from cells incubated with TNF‐α alone and ^† P < 0.05 indicate significant differences from control (untreated) cells.

Figure 3

Treatment with aspirin‐triggered RvD1 (AT‐RvD1) and a reduced dose of dexamethasone (DEX) mitigates TNF‐α mediated apoptosis. Par‐C10 cells were grown on GFR‐MG in eight‐well chambered coverglass slides as described in Materials and Methods. Then, cells were preincubated in the absence or presence of TNF‐α (100 ng/mL for 1 h) and treated with AT‐RvD1 (100 ng/mL) and DEX (25–100 ng/mL) alone and in combination for 72 h. (A) Percentages of apoptotic cells in Par‐C10 cell clusters were determined by a terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) assay. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicate significant differences from cells incubated with TNF‐α alone, ^† P < 0.05 indicate significant differences from control (untreated) cells, and ^# P < 0.05 indicate significant differences from the combination treatment group consisting of AT‐RvD1 (100 ng/mL) and DEX (25 ng/mL) after 1 h preincubation with TNF‐α. (B) Representative images depicting varying degrees of apoptosis per treatment group. TOPRO‐3^® Iodide was used to visualize all nuclei (blue) and TUNEL Alexa Fluor^® 488 was used to visualize apoptosing cells (green). Scale bars represent 50 μmol/L.

Figure 4

Aspirin‐triggered RvD1 (AT‐RvD1) and dexamethasone (DEX) alone and in combination cause transient phosphorylation of Akt in the salivary epithelium. Par‐C10 cells were incubated with (A) AT‐RvD1 (100 ng/mL), (B) DEX (100 ng/mL), (C) AT‐RvD1 (100 ng/mL) + DEX (25 ng/mL), and (D) TNF‐α (100 ng/mL) for 0–60 min. Cells were then lysed with Laemmli buffer, and the expression of phosphorylated Akt (p‐Akt) was detected by western blot analysis. Phosphorylated Akt values were normalized to total protein using a Pan Akt antibody. Data are expressed as means ± SE of results from 3 or more experiments. Significant differences in protein expression as compared to the basal value (0 min) are indicated by asterisks. Results from a representative experiment are shown below graphs.