Spleen cells were isolated from mice subjected to permanent left coronary artery ligation three days earlier and compared with those of sham-operated mice. Intracellular staining of IL-17-producing cells show that mice suffering from myocardial infarctions (MI) had significantly less IL-17-producing cells in their spleens than non-infarcted mice (p < 0.05; Figure 1A). This difference disappeared upon activating splenocytes with phorbol myristate acetate “PMA” + ionomycin (Figure 1B). On the other hand, there was no statistical differences in the percentage of IL-22 producing splenocytes collected from infarcted and non-infarcted mice, regardless whether spleen cells were non-activated (Figure 1C) or activated with PMA + ionomycin (Figure 1D).

Because IL-17, and possibly IL-22, secreted from inflammatory cells might affect cells of the immune system, we examined the expression of IL-17R and IL-22R on T cells and monocytes, cells presumably activated during myocardial infarction. About 80% of monocytes isolated based on the expression of CD11b also expressed IL-17R, but not IL-22R (Figure 2A). Similarly, T cells, which were isolated based on the expression of CD3, also expressed IL-17R, but not the IL-22R (Figure 2B).

As monocytes expressed the IL-17R, and not the IL-22R, we pursued investigating the effect of IL-17 on these cells, which are known to transmigrate into ischemic myocardial tissue after infarction. First we investigated whether IL-17 affects the expression of chemokine receptors on monocytes. From the chemokine receptors examined (CCR2, CCR5, CCR6, CCR7, CXCR3 and CXCR4), we noticed a tendency towards reduced expression of CCR2 and CXCR4 on the surface of monocytes, although this was not statistically significant (Figure 3A,B).

Based on the above findings, we sought to examine the effect of this cytokine on chemokine-induced monocyte chemotaxis. For this, we used the ligands for CCR2 and CXCR4, namely MCP-1/CCL2 and SDFα-1/CXCL12, respectively. Results in Figure 4A demonstrate that 10 ng/mL of MCP-1/CCL2 significantly induced the chemotaxis of monocytes as compared to the control, where media was used instead of the chemokine (p < 0.05; Figure 4A). This increase in chemotaxis disappeared upon pretreatment with IL-17 (Figure 4B).

Next, we examined the effect of SDF-1α/CXCL12, which binds CXCR4, and observed that 10 and 100 ng/mL concentrations of this chemokine significantly induced the chemotaxis of monocytes (p < 0.04; Figure 5A). Similar to its effect on MCP-1/CCL2-induced chemotaxis, IL-17 pretreatment abolished the chemotaxis induced by SDF-1α/CXCL12 (Figure 5B). Chemotaxis towards SDF-1α/CXCL12 was repeated by measuring the migration index rather than counting the numbers of calcein-AM-labeled cells into the lower wells. Exactly similar results were observed, i.e., 10 and 100 ng/mL of CXCL12 induced the chemotaxis of monocytes into the lower wells of the chemotaxis chamber (Figure 5C), and this effect disappeared upon pretreating these cells with IL-17 (Figure 5D).

Male C57Bl/6 mice 24–28 days old (NOVA-SCB, Nittedal, Norway) were used in this study. All animals were allowed at least five-to-seven days of acclimatization after shipment to the animal stable before the actual experiments. The mice had conventional microbial status and were kept under regulated temperature 22–23 °C and relative humidity 55% ± 5%, with an alternating light: dark cycle (12:12). Animals had free access to water and chow. The experiments were approved by the Norwegian Animal Health Authority and were performed under the principles of laboratory animal care (Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health, NIH Publication no. 85-23, revised 1996).

PE-conjugated monoclonal rat anti-mouse CCR2, PE-conjugated monoclonal rat anti-mouse CCR6, PE-conjugated monoclonal rat anti-mouse CCR7, PE-conjugated monoclonal rat anti-mouse CXCR3, PE-conjugated monoclonal rat anti-mouse CXCR4, PE-conjugated monoclonal rat anti-mouse IL-22Rα, CXCR4 PE-conjugated IgG2A isotype control and PE-conjugated IgG2B isotype control were purchased from R&D Systems (R&D Systems Europe Ltd., Abingdon, UK). PE-conjugated monoclonal rat anti-mouse CCR5 (CD 195) and PE-conjugated IgG2c isotype control were purchased from BD Biosciences. PE-conjugated monoclonal rat anti-mouse IL-17RA (CD 217) was purchased from eBioscience. FITC-conjugated monoclonal rat anti-mouse CD 11b, FITC-conjugated monoclonal rat anti-mouse CD 14 and FITC-conjugated kappa monoclonal mouse IgG2A were purchased from Abcam (Cambridgeshire, UK). FITC-conjugated monoclonal rat anti-mouse CD3 was purchased from Termo Scientific.

Mice were anesthetized with isofluran and intubated with the maintenance of isofluran 1.5% mixed with pure oxygen. Mechanical ventilation (Model 874 092, B. Braun) was used to keep a respiratory rate of 133 per min. For induction of myocardial infarction, in vivo permanent occlusion of the left anterior descending coronary artery was performed after a left-sided thoracotomy, as previously described in detail [28]. The body temperature was kept at 37 °C during surgery with a heating plate. After ligation, the thoracic cavity was closed and skin and muscle was reattached. For the sham operation, all these steps were performed with the exception of coronary artery ligation. Sodium saline (0.5 mL) and Temgesic (0.1 mg/kg) were administered subcutaneously for rehydration and analgesia after the operation. Mice were extubated when spontaneous respiration occurred and kept in a “mini intensive care unit” with 100% humidity and 30 °C during the first postoperative day. Temgesic was administered postoperatively by the veterinarian any time pain-associated behavior of the mice was observed.

In the first series of experiments, wild-type C57BL/6 mice were subjected to either in vivo coronary artery occlusion (n = 5) or sham operation (n = 5). Spleens were harvested for cell isolation three days later from isofluran anesthetized mice and compared with spleens from sham operated mice. In subsequent experiments, spleens were harvested from C57Bl/6 after cervical dislocation for the end-points of flow cytometry analysis or chemotaxis and migration, as described below.

Spleen cells harvested from mice with coronary artery ligation or sham-operation were subjected to flow cytometry analysis after stimulation with phorbol myristate acetate (PMA, 20 ng/mL) combined with ionomycin (2 µM) for 22–24 h, and compared with non-treated cells collected the same day as the spleens were sampled. Spleens were extracted, placed in culture medium and homogenized under sterile conditions. The culture medium consisted of RPMI-1640 supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 1% nonessential amino acids (Gibco BRL Life Technologies, the Netherlands) and 50 µM β-mercaptoethanol (Sigma-Aldrich, Oslo, Norway). After histopaque (Sigma-Aldrich, Oslo, Norway) separation, the monocyte population was purified using either adherence or a Mouse Monocyte Enrichment negative selection kit (StemCell Technologies SARL, Grenoble, France). T-cells were positively selected using a Monocyte Enrichment negative selection kit (StemCell Technologies SARL, Grenoble, France). After isolation, cells (1 × 10⁶/mL) were divided in two groups, incubated with culture medium and with or without recombinant murine IL-17A (250 ng/mL, PeproTech) overnight at 37 °C in a 5% CO₂ incubator.

Cells incubated with or without IL-17 were resuspended in PBS containing 0.1% sodium azide and labeled with 1 µg/mL PE-conjugated monoclonal rat anti-mouse CCR2, CCR5, CCR6, CCR7, CXCR3, CXCR4 (R&D Systems Europe Ltd., Abingdon, UK), IL-17RA (eBioscience, San Diego, CA, USA), IL-22Rα (R&D Systems) or FITC-conjugated monoclonal rat anti-mouse CD3, CD11b and CD14 for 45 min at 4 °C. As a control, cells were incubated with isotype control antibodies. The cells were washed twice and examined in flow cytometer (FACSCalibur, Becton Dickinson Biosciences, San Jose, CA, USA). Gating was performed according to the isotype control. To investigate if the spleen cells express IL-17 or IL-22 receptors (IL-17R and IL-22R), spleen cells were incubated with antibodies against CD3 (T-cell marker), CD-11b (monocyte marker) together with antibodies against IL-17R and IL-22R and sorted by flow cytometric analysis. As spleen cells contained the receptor for IL-17, but not IL-22, spleen cells were isolated and incubated with recombinant murine 250 ng/mL IL-17A (PeproTech EC Ltd., England) over night, compared with unstimulated cells, and sorted by FACS to evaluate the expression of CCR2, CCR4, CCR5,CCR6, CCR7, CXCR3 and CXCR4.

To evaluate a possible role of IL-17 on chemotaxis, monocytes were incubated in the presence or absence of 250 ng/mL recombinant murine IL-17 overnight. Chemotaxis assay was done using Nuclepore blind well chemotaxis chambers with a lower well volume of 200 µL used. A maximum volume of 200 µL medium containing RPMI plus 0.1% BSA was placed in the lower wells in the presence or absence of 0.1–100 ng/mL of MCP-1/CCL2 or SDF-1α/CXCL12 chemokines (PeproTech, England). Cells (5 × 10⁵) were placed in the upper compartments and incubated for three hours at 37 °C in a 5% CO₂ incubator. The filters were removed, dehydrated, cells were fixed with methanol, stained with 15% modified Giemsa for 10 min and then mounted on glass slides. Cells in 10 high-power fields were counted and averaged for each sample. Migration index (MI) was calculated as the number of cells migrating toward the concentration gradients of chemokines divided by the number of cells migrating toward medium only. In the second protocol, cells (1 × 10⁶/mL) were incubated with 10 µg Calcein-AM and incubated for 45 min at 37 °C in 24 well plates. The cells were spun down at 500 rpm for 5 min, washed and collected. Calcein-AM labeled cells (5 × 10⁵) were incubated in the chemotaxis chambers, as described above for 3 h. After this time, cell suspensions were collected from the lower wells and fluorescence intensity of the calcein AM-loaded cells was measured in a BioTek FLX TBI plate reader using 485/528 nm fluorescence filters.

A t-test was used when data were evenly distributed, while a Mann-Whitney test was used for data with a non-gaussian distribution. p < 0.05 was considered significant.