IL‐22 produced by Th22 cells aggravates atherosclerosis development in ApoE−/− mice by enhancing DC‐induced Th17 cell proliferation

Abstract Th22 cells are a novel subset of CD4+ T cells that primarily mediate biological effects through IL‐22, with both Th22 cells and IL‐22 being closely associated with multiple autoimmune and chronic inflammatory diseases. In this study, we investigated whether and how Th22 cells affect atherosclerosis. ApoE−/− mice and age‐matched C57BL/6J mice were fed a Western diet for 0, 4, 8 or 12 weeks. The results of dynamic analyses showed that Th22 cells, which secrete the majority of IL‐22 among the known CD4+ cells, play a major role in atherosclerosis. ApoE−/− mice fed a Western diet for 12 weeks and administered recombinant mouse IL‐22 (rIL‐22) developed substantially larger plaques in both the aorta and aortic root and higher levels of CD3+ T cells, CD68+ macrophages, collagen, IL‐6, Th17 cells, dendritic cells (DCs) and pSTAT3 but lower smooth muscle cell (SMC) α‐actin expression than the control mice. Treatment with a neutralizing anti–IL‐22 monoclonal antibody (IL‐22 mAb) reversed the above effects. Bone marrow‐derived DCs exhibited increased differentiation into mature DCs following rIL‐22 and ox‐LDL stimulation. IL‐17 and pSTAT3 were up‐regulated after stimulation with IL‐22 and ox‐LDL in cells cocultured with CD4+ T cells and mature DC supernatant, but this up‐regulation was significantly inhibited by IL‐6mAb or the cell‐permeable STAT3 inhibitor S31‐201. Thus, Th22 cell‐derived IL‐22 aggravates atherosclerosis development through a mechanism that is associated with IL‐6/STAT3 activation, DC‐induced Th17 cell proliferation and IL‐22–stimulated SMC dedifferentiation into a synthetic phenotype.


| INTRODUC TI ON
Atherosclerosis (AS) is a highly complex and multifactorial disease, with current data showing that atherosclerosis is the leading cause of death among all human diseases. A growing body of evidence suggests that atherosclerosis is a chronic inflammatory disease that is closely related to innate and acquired immunity. 1,2 During each stage of the disease, progressive atherosclerotic lesions display T-lymphocyte infiltration.
These T cells interact with local macrophages and vascular smooth muscle cells (SMCs) and affect the expression of inflammatory factors.
Inflammatory factors such as MCP-1, tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, which are expressed by macrophages and T lymphocytes, facilitate SMC migration from the tunica media to the intimal or subendothelial space, resulting in the formation of a fibrous cap. [3][4][5] Studies have indicated that the lymphocytes present in a plaque are primarily CD4 + T cells, 6,7 which can be roughly divided into Th1, Th2, Th17 and regulatory T (Treg) cell lineages. The Th1 cell-mediated immune response has been shown to promote the development of AS. 8,9 Th17 cells, which are a powerful inflammatory factor, may play important roles in accelerating AS in specific environments, [10][11][12][13] whereas Treg cells are atheroprotective. 14,15 The overall role of the Th2 immune response during AS remains unclear. [16][17][18] Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and express costimulatory molecules, such as CD80 and CD86. DCs possess a strong capacity to activate and promote T-cell differentiation.
These cells were demonstrated to drive CD4 + T-cell differentiation along both the Th1 and Th17 pathways, 19,20 which partially occurs through STAT3 phosphorylation. 21,22 The Th22 subset is a novel CD4 + T-cell subset that was discovered in humans in 2009 and is distinct from the Th1, Th2 and Th17 subsets. 13 in mice. 25 Basu demonstrated that in the early stages of inflammation, IL-22 is primarily secreted by lymphatic tissue-induced cells. In the middle and late stages of inflammation, IL-22 is pre-dominantly released by Th22 cells. These results indicate that Th22 cells and IL-22 likely play an important role in chronic inflammatory diseases. The biological effects of Th22 cells occur through IL-22, which, though previously considered a Th17 cytokine, belongs to the IL-10 family and has effects that are primarily mediated through IL-22-IL-22R1 interaction. 26,27 IL-22 binding to IL-22R1 pre-dominantly activates the STAT3 pathway. 28 In recent years, the relationship between Th22 cells and clinical disease has attracted increased attention. IL-22 protects against multiple infections of the lungs and intestine as well as liver injury, but it also has pathogenic roles in rheumatoid arthritis and psoriasis. [29][30][31][32][33] However, few studies have investigated the roles of Th22 cells and IL-22 in AS. Our previous study revealed significant increases in the peripheral Th22 cell numbers and IL-22 levels of patients with acute coronary syndrome (ACS) compared to patients with stable angina pectoris (SAP) and control patients, suggesting that the circulating Th22-type response may have a potential role in the onset of ACS symptoms. 23 In this context, we focused on elucidating whether and how Th22 cells and IL-22 act on AS.

| Animal models and interventions
Eight-week-old homozygous ApoE −/− male mice (in a C57BL/6J background) and age-matched C57BL/6J mice with bodyweights ranging from 20 to 25 g were obtained from The Jackson Laboratory housed under specific pathogen-free conditions and fed a Western-type diet (Clinton/Cybulsky Rodent Diet D12108 with 1.  Figure S1).

| Tissue preparation and atherosclerosis lesion evaluation
At 0, 4, 8 and 12 weeks, the mice were killed, and blood was collected retro-orbitally to assess the IL-22, IL-6, MCP-1 and TNF-α concentrations by enzyme-linked immunosorbent assays (ELISAs). The spleen, aorta and heart of each mouse were rapidly removed after perfusion with PBS. The spleens were mechanically dissociated to isolate lymphocytes for analysis of Th1, Th17 and Th22 cell frequencies by flow cytometry. The hearts and aortas were snap-frozen at −80°C for further analysis. Atherosclerotic lesions were measured by lipid staining. Hearts with an attached aortic root were snap-frozen in OCT compound, and the aortic sinus cross-sections (10 μm per section) were excised, fixed in 4% formalin and stained with oil red O. For further analysis, five sections from each mouse were obtained from five different locations (each separated by 100 μm), and the lipid deposits in the aorta were fixed and stained as described above. Before euthanasia, all mice were anesthetized with 3% chloral hydrate at 10 μL/g bodyweight. All images were captured by a single observer blinded to the experimental protocol and analysed with computer image analysis software (Image-Pro Plus 6.0).

| Cell preparation
Mice were killed at 0, 4, 8 and 12 weeks, and lymphocytes from the spleens were isolated. Immediately after being removed from the mice, the fresh spleens were gently crushed with a clear glass pestle in cold PBS, and the splenocytes were passed through a 100 mesh stainless steel screen to prepare single-cell suspensions. The cells were extracted with an equal volume of Ficoll-Hypaque and washed twice in PBS to isolate the splenic lymphocytes.

| Analysis of IL-22-related T cells
Cells were resuspended in PBS at a density of 10 5 -10 6 cells/mL and incubated with a fluorescein isothiocyanate (FITC)-conjugated antimouse CD4 antibody at 4°C for 30 minutes for cell surface staining. After surface staining, the cells were washed twice and resuspended in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated foetal calf serum (FCS; Gibco, BRL), also referred to as complete medium, and then transferred to 24well plates. The lymphocytes were stimulated with 2 μL of Cell Stimulation Cocktail (plus protein transport inhibitors, eBioscience). The cells were incubated at 37°C under a 5% CO 2 atmosphere. After 4 hours of culturing, the contents of the wells were
All assays were performed in triplicate.

| Immunohistochemistry and Masson trichrome staining
Hearts with an attached aortic root were snap-frozen in OCT compound and then cut into 4-μm-thickaortic sinus cross-sections.

| Real-time quantitative reverse transcriptionpolymerase chain reaction (RT-PCR)
Total RNA was extracted from the aortas and cells with TRIzol  Table 1.

| Western blot analysis
Total protein was extracted from the aortas and cells and then detected with a BCA Protein Assay kit (Thermo Fisher Scientific).

| Statistical analysis
All data are presented as the means ± SD. Multiple-group comparisons were performed by ANOVA. Differences between two groups were compared by Student's t test, and P < .05 was considered significant.

| Basic physiological parameters
The bodyweight gain of the ApoE −/− mice and age-matched

| Th22 cells are the major source of IL-22 during atherosclerosis
To elucidate the role of Th22 cells in the development of AS, we

| IL-22 and IL-22R1 are expressed in mouse atherosclerotic plaques, and their expression levels are increased in ApoE −/− mice
As noted previously, IL-22R is expressed in various non-immune tissues, such as the skin, lungs and kidneys, and in cardiomyocytes.
Through immunohistochemical analyses, we showed that IL-22R1 is also expressed in mouse atherosclerotic plaques, and the expres-

| IL-22 promotes the phenotypic dedifferentiation of SMCs
Given the observed trend of the variation in SMC α-actin, we sub-

| IL-22 increases DC and Th17 cell numbers by activating IL-6/STAT3
To investigate the inflammatory mechanisms by which circulating

ACK N OWLED G EM ENTS
The authors thank the teachers of Laboratory of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology for their offers of equipment and helpful suggestions.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.