There is growing evidence that T cell activation and its cytokine expression play a key role in the pathogenesis of CVB3-induced AVMC, such as IFN-γ-producing Th1 cells and IL-17-producing Th17 cells [4–7]. Recently, several works related to Th22 cells have broadened our understanding of T cell-mediated tissue inflammation and immunity [13–16]. Pure Th22 cells represents a distinct CD4+T cell subset characterizing by the secretion of IL-22, but not IL-17 or IFN-γ, thus is identified as IL-22+IL-17-IFN-γ-CD4+T cells [10–12, 16]. IL-22, the crucial cytokine of Th22 cells, mediates its effects via IL-22R and propagates downstream signals including JAK/STAT, ERK, JNK, PI-3K and p38 MAP kinase pathways [19, 23, 24]. A considerable amount of data indicated that the Th22 cell and its crucial cytokine IL-22 could exert either pro-inflammatory or anti-inflammatory activities depending on the context [13–18, 22]. In the present work, we tried to exploit the potential that Th22 cells may participate in the inflammatory and immune response in AVMC. As excepted, higher percentage of the Th22 cell was observed in CVB3-induced mice AVMC, similarly to those of Th17 and Th1 cells. Our data uncovered the evidence of significant elevations in circulating Th22 cells and IL-22 proteins, cardiac mRNA and protein expressions of IL-22 in AVMC. It seems that infective and immunity microenvironment of AVMC is suitable for the differentiation and proliferation of Th22 cells. These novel findings confirm an individual signature for the IL-22-producing Th22 subset in AVMC, and provide a cellular target for therapeutic intervention and may also shed light on thus far unknown pathways in the control of AVMC.
Based on the observation that levels of IL-22 and IL-22 receptor were elevated in AVMC, we examined the efficacy of IL-22 in the AVMC mouse model, with the use of anti-IL-22 antibody (Anti-IL-22Ab) [20, 25]. Functional studies in human or murine model systems have indicated that IL-22 could be either pathologic or protective, depending on the context in which it was expressed [17, 18, 22, 26, 27]. Our results proved that neutralization of IL-22 exacerbated the severity of AVMC, which was verified by the lower survive rate, higher values of HW/BW and pathological scores of heart sections. Meanwhile, down-regulations of circulating Th22 cells and IL-22 were observed in the Anti-IL-22Ab group. The Th22 cell is not the unique source of IL-22. However, Both the pathogenic effect of Anti-IL-22 Ab in our AVMC model and protective role of the IL-22-Ig fusion gene in experimental autoimmune myocarditis (EAM) model , suggest that this cytokine has an important myocardium-protective role regardless of the cell source.
Emerging literature indicate that IL-22 participates in immunity and tissue inflammation through the IL-22–IL-22R pathway [20, 22]. To explore the role of this pathway in AVMC, we firstly investigated the expressions of cardiac IL-22 and IL-22R, the latter which consists of IL-22R1 and IL-10R2. Because signaling through IL-22R1 is restricted to IL-22, we focused on IL-22 and IL-22R1 . Compared with IL-22 and IL-22R1, dramatically higher relative cardiac expression of IL-10R2 was detected in mice, consistence with previous data that IL-10R2 was highly expressed [20, 22]. Compared with those in the control group, significant elevations of IL-22 and IL-22R1 were identified in the AVMC group. Although the major IL-22-producer are immune cells, it has been confirmed that IL-22R1 expression is absent on immune cells but instead restricted to tissues, thus IL-22 acts as a middle-men between the immune system and its environment . These increases of cardiac IL-22 and IL-22R1 may strengthen signaling directionality from the immune system to myocardium, and are thus involved with the pathogenesis of AVMC. After IL-22 was blockade with a neutralizing antibody in AVMC, down-regulations of IL-22 and up-regulations of IL-22R1 were observed. These data are consistent with a previous study that IL-22R1 expression was decreased after treatment with rIL-22 but increased after IL-22 neutralization, while IL-10R2 was not changed significantly in liver ischemia-reperfusion injury model, by RT-PCR analysis . These phenomena may be explained by the following mechanism: IL-22R1 has been recognized as a driver of inflammation, and has a autoregulatory feedback loop that amplifies the level of IL-22 [29, 30]. When IL-22 is decreased by neutralization of anti-IL-22 Ab, IL–22R1 may be increased by autoregulatory feedback loop that promotes the levels of IL-22. And the promotion of IL-22R1 further aggravates the myocarditis in the anti-IL-22 group. On the other hand, IL-10R2 is constitutively expressed on virtually all cells types at high levels and is an component of the receptor complexes for IL-22, IL-10, IL-26, IL-28, and IL-29, thus the effect of anti-IL-22 on the expression of IL-10R2 is not significant. Further investigation with the use of heart-specific IL-22 transgenic mice and mice that lack IL-22R1 will be of particular interest to clarify the dysregulation of the IL-22–IL-22R pathway in AVMC.
Our data suggest that dysregulated IL-22–IL-22R signal can modulate expressions of considerable genes and regulate the productions of inflammatory cytokines, verified by the fact that IL-22 neutralization significantly promoted the circulating and cardiac IL-17, IL-6, TNF-α, which have been identified as important pro-inflammatory cytokines and exacerbating myocarditis by inflammation and immunity [4, 5]. More specifically, dysregulated IL-22–IL-22R pathway in the Anti-IL-22 Ab group contributed to cardiac viral replication, and decreased the level of IFN-γ, which was considered as the vital defenders against viral infection [28, 31]. Our data was consistent with the previous observation that IL-22 contributed to resistance to viral infection , highlighting a role for IL-22-producing Th22 cells in controlling cardiac viral replication in AVMC. It seems whether IL-22 response modulates the expression of IL-1β is context dependent because no significant change of IL-1β production was observed in the Anti-IL-22 Ab group [20, 33, 34].
To our knowledge, this is the first demonstration that Th22 and IL-22–IL-22R pathway may participate in the development of AVMC but with several limitations. First, the neutralization of IL-22 antibody biological activity was not fully quantified, and we have not addressed the mechanism responsible for proliferation of Th22 cells in mice. Whether the decreased Th22 cells in the anti-IL-22Ab group was due to the ab neutralizing or impaired proliferation, required future study. Second, Th22 has been regarded as the most important IL-22-producing CD4+T cells (71.9%) , elucidation of the role of the IL-22–IL-22R pathway would be helpful to understand the whole picture of Th22 cells-involved in AVMC. However, the source of IL-22 was not limited by the Th22 cell. NKT,Th17, Tc17, innate-like γδT, even in Rag2−/− mice which lack of T cells, are IL-22 producers in mice [22, 35]. Thus the myocardium-protective role of IL-22 could not be used to absolutely identify the Th22 function. In future studies on transcription factor AHR, chemokine receptor CCR6, PI-3 kinase pathway based-screening, will be of benefit to understanding of the Th22 cell regulation during inflammatory responses in AVMC [23, 24]. Furthermore, whether RORC reflects the capacity to produce IL-17 and IL-22 in AVMC, is an interesting open issue.