Recombinant human IL-1β, recombinant human TNF-α and recombinant human IFN-γ were purchased from PEPROTECH (Rocky Hill, NJ). Oxytocin was purchased from Sigma-Aldrich (Saint Louis, MO). Cell media Dulbecco’s Modified Eagle (DMEM) and RPMI-1640 medium (Roswell Park Memorial Institute), collagenase type II, penicillin-streptomycin, L-glutamine, and fetal bovine serum (FBS) were from Gibco (Waltham, MA). TrypLE Express was from Invitrogen (Carlsbad, CA). Rabbit polyclonal antibody against αSMA (ab5694 at 1:400 dilution) and rabbit monoclonal antibody against calponin (ab46794 at 1:2000 dilution) were from Abcam (Waltham, MA). All secondary antibodies were from LI-COR (Lincoln, NE).

All experiments described herein were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research and were approved by the Tufts Medical Center Institutional Animal Care and Use Committee. Mice were maintained in constant temperature rooms with fixed light/dark intervals of 12 h length and were fed ad libitum. To obtain MEC, SMA‐GFP mice (C57BL6)/SMA^CreErt2 strain was used for this study that was described by Yokota (19) and were a kind gift of Dr. Ivo Kalajzic (UConn Health, Farmington, CT). In these mice, the lacrimal gland MEC, which express SMA, are therefore labeled with GFP.

Four- to 6-week-old SMA-GFP mice were euthanized and the exorbital lacrimal glands were removed and minced into lobules for collagenase digestion using our previously described protocol (5, 8). Briefly, lacrimal glands were washed in cold DMEM, gently minced with a scalpel and forceps to prepare 2-3 mm lobules and placed in digestion media (1.5 mL/gland of DMEM and 1.65 mg/mL of collagenase type II). Samples were then incubated in a shaking water bath (37°C and 100 rpm) for 20-30 minutes. At regular 5 min intervals, lobules were gently pipetted, 10 times, through tips of decreasing diameter. Digested media was filtered through a sterile cell strainer (100 µm nylon mesh; Thermo Fisher Scientific, Waltham, MA), remaining tissue pushed through the mesh using the pipette tip, and collected cells washed with 1-2 mL DMEM. Cells were then centrifuged at 100 x g for 5 minutes, resuspended in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100 µg/mL penicillin-streptomycin and centrifuged again at 100 x g for 5 minutes. Pelleted cells were resuspended in 10 mL complete RPMI media, plated in 100 mm culture dishes (VWR, Radnor, PA) and placed in a 37°C incubator (5% CO₂).

Lacrimal gland MEC were seeded in 6-well plates in 5% FBS RPMI Media. Confluent to sub-confluent MEC cultures were treated with 10 ng/ml of IL-1β alone or in combination with 10 ng/ml each of TNF-α and IFN-γ, for a total of 7 days. Every other day media was changed, and 4-5 pictures were randomly taken from each well to perform further analysis of GFP intensity and cell area measurements. In other experiments, cells were trypsinized to perform the contraction assay, as described below. After the last day of treatment (day 7), cells were lysed, and protein samples were prepared for western blotting studies, as described below.

Images of MEC cultures were taken on day 0 (before addition of cytokines) then 2, 4 and 7 days of cytokines treatment using a digital camera (SPOT Insight CMOS; SPOT Imaging, Sterling Heights, MI) mounted on an inverted light microscope (Eclipse TE2000-S; Nikon Instruments Inc., Melville, NY). Total GFP intensity was analyzed, in each of these time frames from control and treated MEC, using ImageJ/Fiji software (ImageJ 1.53, National Institutes of Health, USA). Analyzing GFP intensity is an indirect indicator of SMA protein levels since GFP expression in MEC is under the control of the SMA promoter. In addition, a minimum of 5 to 6 cells per photograph were selected to measure the cell area (µm²) using SPOT Advanced Imaging software (Version 5.6).

At the end of the cytokine treatment, lacrimal gland MEC from treated and control groups were lysed in 0.2 mL ice-cold radio-immunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS supplemented with protease inhibitors). Cell lysates were centrifuged at 20,000 x g for 30 minutes and the supernatant collected. Proteins were separated by SDS-PAGE on NuPage 4–12% Bis-Tris gels in MOPS-SDS buffer (Invitrogen, Carlsbad, CA). Protein in the gels were transferred to nitrocellulose membranes using NuPage transfer buffer (Invitrogen) and processed for immunoblotting. After transfer, nitrocellulose membranes were stained with REVERT Total Protein Stain (LI-COR) following the manufacturer’s instructions and prior to being blocked using Odyssey blocking buffer (LI-COR) for 1 hour at room temperature. Membranes were then incubated overnight at 4°C with the appropriate primary antibody for SMA and calponin diluted in blocking buffer + 0.1% tween-20. Following washing with Tris-buffered saline + tween-20 (TBS; 50 mM Tris-HCl, 150 mM NaCl, 0.1% tween-20, pH 7.6) membranes were then incubated for 1 hour at room temperature with their appropriate secondary antibodies followed by detection on a LI-COR Odyssey Infrared Imager. Staining in each lane for REVERT total protein stain (see Supplementary Material), and band intensity for immunoblotting was quantified using the LI-COR Image Studio software (v.4.0). Western blot band quantifications were then normalized to the total amount of protein in each lane.

On days 2, 4 or 7, cells were trypsinized from the 6-well plate and seeded overnight in a 24-well-plate, at a density of around 5,000 cells/well. Cells treated with cytokines and untreated cells (control) were stimulated with OXT (10^-6 M) for 20 minutes. Video recording of the contraction process was performed using a digital camera (SPOT Insight CMOS; SPOT Imaging, Sterling Heights, MI) mounted on an inverted fluorescence microscope (Eclipse TE2000-S; Nikon Instruments Inc., Melville, NY). Also, still images were taken before and 20 min after OXT stimulation for image analyses. At least 10 random cells from each well and each condition were used for image analyses, using ImageJ/Fiji software (ImageJ 1.53, National Institutes of Health, USA). The perimeter of the same cell was calculated before and after OXT stimulation, and the difference between these two values that represents the decrease in cell size after OXT stimulation, was expressed in percentage.

Statistical analyses were performed using GraphPad Prism Software (version 9.0; San Diego, CA). Where appropriate, data are presented as means ± standard deviation (SD). Data consisting of 2 groups were analyzed using a 2-tailed unpaired Student’s t-test or the Mann-Whitney U test for non-normally distributed data with significant results being considered at p-value < 0.05.

Since GFP expression in the transgenic mouse used in our studies is under the control of the SMA promoter, a decrease in GFP intensity would imply a decrease in SMA expression. Lacrimal gland MEC were left untreated or incubated with IL-1β for up to 7 days. Images were taken at days 0, 2, 4, and 7 and used for image analyses to quantify GFP intensity or MEC size, as described in the Methods section. As shown in Figure 1, at day 0, control and treated cells showed no difference in GFP intensity. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing at day 4 (25%; p=0.007) and day 7 (30%; p=0.0001) (Figure 1).

The data in Figure 2 summarizes the effect of IL-1β treatment on MEC size. At day 0 there was no significant difference in cell size between the treated and control group. However, mean cell area was reduced at day 2 (34%; p=0.0005), day 4 (51%; p<0.0001) and day 7 (30%; p=0.0015) in treated MEC treated compared with the control group (Figure 2).

These data suggest that chronic treatment of lacrimal gland MEC with IL-1β lead to degradation of SMA protein, which resulted in smaller sized cells. Quantification of SMA and calponin protein expression levels, as discussed below, lend support to this hypothesis.

The data from the GFP intensity analyses suggested that chronic treatment of lacrimal gland MEC with IL-1β lead to lower expression of SMA protein. To test this hypothesis, we prepared cell lysates from control and IL-1β treated MEC and performed western blotting analyses to quantify the level of expression of SMA as well as calponin. As shown in Figure 3, there was a 33% decrease in the amount of SMA and calponin protein in treated MEC compared with the control group (p=0.0016 and p=0.0206; respectively). Please note that, as expected, SMA expression decreased to a similar level as did GFP intensity (Figure 1).

These data suggest that chronic treatment of MEC with IL-1β lead to lower expression of SMA and calponin proteins.

So far, our data suggest that chronic IL-1β treatment led to degradation of contractile proteins which could result in impaired lacrimal gland MEC contraction. Cells incubated with IL-1β for 2, 4 or 7 days were trypsinized, seeded overnight on 24-well plates to perform contraction assay as described in the Methods section. As shown in Figure 4, compared to controls, day 2 treated MEC showed a 70% (p<0.0001) decrease in OXT-induced contraction. Contraction was further decreased by 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 (Figure 4).

These data suggest that IL-1β induced lower expression of SMA and calponin proteins led to inhibition of OXT-induced MEC contraction.

The proinflammatory cytokines TNF-α and IFN-γ, are known to be elevated in chronically inflamed lacrimal glands as occurs in Sjogren’s syndrome. Therefore, we tested their effect, when added with IL-1β, on MEC functions. At day 0, GFP intensity and cell size of treated and untreated MEC was not significantly different (data non shown). In contrast, both GFP intensity and MEC size were significantly decreased following treatment with the cytokine cocktail for 7 days (Figures 5A, B). When compared to IL-1β alone, the cytokine cocktail decreased GFP intensity and cell size to a larger extent: 57% vs. 30% and 41% vs. 30%; respectively (Figures 1, 2, and 5). Similarly, the amounts of the contractile proteins, SMA and calponin, were also decreased, although not statistically significantly, following cytokine cocktail treatment (Figure 5C).

These data suggest that TNF-α and IFN-γ can synergize with IL-1β to further impact MEC functions in chronically inflamed lacrimal glands.