Recent studies have documented that Th22 cells contribute to the pathology of several autoimmune diseases. However, few studies have examined the roles of Th22 cells in the pathogenesis of SLE, especially the effects of these cells on their target cells. Skin and kidney involvement are common in SLE patients. Our previous study reported that Th22 cells targeted mesangial cells of the kidney and basal keratinocytes of the skin(33). The present study examined the potential roles and the mechanism of Th22 cells in promoting cell proliferation and secretion of proinflammatory cytokines by target cells.
Our results demonstrated increased levels of IL-22 when Th22 cells were co-cultured with target cells (HaCaT or HRMC cells). The proliferation of target cells increased when they were co-cultured with Th22 cells and when rIL-22 was added to the culture, and the proliferation was greater when the cells had direct contact during co-culturing. Co-culturing of target cells with Th22 cells also increased the levels of three pro-inflammatory cytokines (IL-6, TNF-α, and IFN-α). Furthermore, addition of IL-22mAb completely blocked the effect of IL-22 on cell proliferation; addition of IL-22mAb also decreased the levels of pro-inflammatory cytokines, although they were still higher than the controls.
Th22 cells express a variety of skin-homing receptors, including CCR6, CCR4, and CCR10(6, 34). These receptors mediate the infiltration of Th22 cells into skin tissues, suggesting that they function in skin damage. Previous studies reported the role of Th22 cells and IL-22 in psoriasis and other skin diseases, such as allergic dermatitis and scleroderma(35, 36). Our previous study found that SLE patients with skin involvement had higher levels of Th22 cells than those without skin involvement. Additional observations using immunofluorescence staining and confocal microscopy indicated that the expression of IL-22R1 (an IL-22 receptor) on keratinocytes, and that Th22 cells infiltrated the skin around keratinocytes(33). Taken together, these data suggest that Th22 and IL-22 target keratinocytes.
Keratinocytes function as immunomodulators, in that they secrete inhibitory cytokines, stimulate inflammation, and activate Langerhans cells in response to injury(37). Keratinocytes may also be targeted in several chronic inflammatory systemic diseases. When there is an autoimmune attack against the attachment proteins of keratinocytes, they undergo apoptosis and stimulate several subsequent pathological events. For example, in psoriasis, activated T lymphocytes release various cytokines to stimulate the proliferation of keratinocytes(38). Cytokine-activated keratinocytes can also express a broad array of cytokines, chemokines, and membrane molecules that induce the recruitment and activation of T lymphocytes in the skin(39–41). A previous study demonstrated the role of keratinocytes in cutaneous lupus erythematosus (CLE) or SLE with skin involvement(42). A previous study showed that apoptosis of keratinocytes occurred rapidly following exposure to ultraviolet radiation (UVR) and that photosensitivity was associated with increased apoptosis of these cells(43). Apoptotic keratinocytes can also express autoantigens that bind to autoantibodies, leading to complement activation and sustained skin inflammation(42). These many studies thus demonstrate that keratinocytes function as targets and active participants in the inflammatory process.
The presence of lupus nephritis (LN) in patients with SLE is associated with poor prognosis and increased risk of death, possibly because IL-22 targets the kidney. In agreement with this interpretation, a previous study found that IL-22 induced the production of complement proteins, indicating that it may also indirectly affect glomerular epithelial cells through complement and participate in the pathogenesis of LN(44). A recent study by Yang et al. demonstrated increased IL-22 levels in the blood and kidney tissues of LN patients and MRL/lpr mice, and that blocking of IL-22 with anti-IL-22mAb inhibited the progression of LN in the mouse model(11). Our previous study found expression of IL-22R1 in mesangial cells of the renal glomeruli of LN patients, suggesting that IL-22 may target renal mesangial cells(33). Similarly, Lu et al. found that renal mesangial cells and renal tubular epithelial cells induced the infiltration of Th22 cells(45). Renal mesangial cells have an important function in the glomerulus, and they can interact with immune deposits, complement components, and inflammatory mediators(46). The proliferation of mesangial cells and the excessive production of mesangial matrix are typical manifestations of renal biopsies in patients with active LN(47).
We further studied the effect of Th22 cells on keratinocytes and renal mesangial cells by performing in vitro experiments with HRMC and HaCaT cells. In particular, we used the Transwell system to co-culture these target cells with Th22 cells or treat them with rIL-22. Our results showed that IL-22 promoted the proliferation of these target cells and their production of pro-inflammatory cytokines. Consistent with our present results, previous studies suggested that IL-22 promoted the proliferation of multiple epithelial and mesenchymal cells, such as keratinocytes, renal mesangial cells, synovial fibroblasts, and intestinal epithelial cells(48). To maintain a homeostatic balance, these pathologically proliferated cells underwent apoptosis. Due to the reduced function of apoptotic clearance in SLE, necrosis and local inflammation worsens, and this may activate the immune system to produce autoantibodies and immune complexes, causing systemic damage(49). Other research showed that IL-22 promoted the production of IL-1, IL-6, IL-18, and other proinflammatory cytokines by skin cells exposed to UVR(43). Another study demonstrated that Th22 cells promoted the production of IL-6 and TNF-α by HRMC cells when co-cultured together with contact(45). We also found that Th22 cells induced the proliferation and secretion of pro-inflammatory cytokines in HaCaT and HRMC cells when they were co-cultured. Co-culturing of these cells also increased the level of IL-22, thus indicating that Th22 cells act upon their target cells via the production of IL-22. When Th22 cells were co-cultured in contact with the target cells (rather co-cultured without contact), this further increased the production of IL-22 and cell proliferation.
Our results suggest that Th22 cells promote the proliferation of HaCaT cells by secreting of IL-22 and by physical contact during co-culture. In contrast, Th22 cells had similar effects on HRMC cells during co-culture with and without physical contact. This difference is intriguing and suggests the need for further research on additional cell lines. We also found that IL-22mAb completely blocked the effect of IL-22, in that it prevented cell proliferation, suggesting that the promotion of target cell proliferation depended on IL-22. IL-22mAb significantly decreased the levels of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-α), but their levels remained higher than the controls. This suggests that although Th22 cells enhance the secretion of pro-inflammatory cytokines by target cells, other factors are also important. Considering all these results together, we speculate that during the pathogenesis of SLE, Th22 cells infiltrate their target cells and have full physical contact with them via cytokine receptor-ligand interactions, and that this leads to enhanced secretion of IL-22, thereby promoting disease progression.
The binding of IL-22 to IL22R leads to activation of JAK1 (mobile kinase IL22R1) and Tyk2 (mobile kinase IL10R2), which trigger multiple intracellular pathways, such as MAPK, AKT, P38, JNK, and ERK1/2(23). The PI3K/AKT pathway has links with many intracellular signaling pathways, and functions in the regulation of various cellular events, including cell proliferation, angiogenesis, invasion, and metastasis. Our results indicated that Th22 cells markedly activated PI3K/AKT signaling in vitro. Other studies reported similar molecular effects in different diseases, such as UVB-induced skin inflammation and experimental autoimmune uveitis(22, 50–52). To confirm the role of PI3K/AKT signaling, we assessed the effects of separate additions of LY294002 and IL-22mAb. The results indicated that LY294002 downregulated the protein levels of AKT, p-AKT(Thr308), p-AKT(Ser473), and also downregulated the secretion of IL-6, TNF-α, and IFN-α. LY294001 also blocked the proliferation of co-cultured HaCaT and HRMC cells; IL-22mAb had a similar but weaker suppressive effect. These results suggest that PI3K/AKT signaling functions in target cell-mediated proliferation and secretion of pro-inflammatory cytokines. Thus, inhibition of this pathway using LY294001, IL-22mAb, or another agent may have potential for the therapy of SLE.
In summary, our findings provide a preliminarily description of the potential mechanism by which Th22 cells interact with their target cells — keratinocytes and renal mesangial cells. We also identified a possible role of PI3K/AKT signaling in SLE, and IL-22 as a possible therapeutic target for the treatment of SLE. Our future studies will examine the effects of different interventions involving Th22 cells and IL-22 in animal models. We will also investigate other T cell subsets, such as TFH and Treg cells, to examine organ involvement in SLE by using a large sample size and performing assessments at multiple times during disease progression.