The results of this study demonstrated that Th17 cells are positively correlated with COPD symptom score, but negatively correlated with pulmonary function index. We also characterized the immunophenotypes of patients with AECOPD, noting abnormal activation of Th1 cells, Th17 cells and Treg cells. To the best of our best knowledge, this study is the first to describe the immunophenotype of patients with AECOPD.
There are two main methods to detect CD4 + T cell subtypes in human peripheral blood by flow cytometry. One method is the traditional method of isolating peripheral blood mononuclear cells, stimulating the cells in vitro, and detecting cell surface markers and intracellular factor staining. Although this method allows for a detailed analysis of the functional characteristics of cells, it requires more blood samples, is time-consuming, and involves a large amount of cell loss. Another method is to use cell surface staining exclusively to label cell surface markers such as CX-chemokine receptor 3 or CD183, CCR6 or CD196 to detect Th1, Th2 and Th17 cells; this method is a simple and rapid alternative, but these surface markers are not specific, so surface markers alone may increase the proportion of cell subsets. Acosta-Rodriguez et al  found that the CD183 + CD196 + cells can produce IFN-γ and IL-17, which are also produced by Th1 and Th17 cells, respectively; therefore, we labeled Th1 cells as CD3 + CD4 + CD183 + and Th17 cells as CD3 + CD4 + CD196+.
Th1 cells participate in the cellular immune response by secreting pro-inflammatory cytokines such as IL-2 and IFN- γ. Previous studies on Th1 cells mainly focused on COPD airway inflammation, demonstrating that the expression of Th1 cell-related factors in the airway and lung tissues was increased, while there have been relatively few studies in peripheral blood. Our study found that the proportion of Th1 cells and their related cytokines in patients with AECOPD were higher than in HCs. This is consistent with the study by Sun et al. which included 30 patients with AECOPD, 15 patients with SCOPD and 15 normal controls, and found higher levels of IFN-γ in AECOPD and SCOPD groups compared to the HC group, but lower IFN-γ levels in patients with AECOPD than patients with SCOPD. This inconsistency may be due to differences in patient populations between the latter study and our own. The above results also suggest that there is a difference in serum levels of cytokines produced by Th1 cells in patients with COPD, which may be related to different stages and severity of the disease. Some scholars have shown that IFN-γ secreted by Th1 cells can induce the production and release of matrix metalloproteinase (MMP)-12 and MMP-9 in vivo; MMP can degrade the extracellular matrix components in the lung parenchyma and inhibit alpha trypsin, causing tissue cell infiltration and tissue destruction, thereby causing lung inflammation and emphysema. Therefore, Th1 cells and their related cytokines contribute to the onset and development of COPD and show different characteristics in different stages of the disease, but further research is required to elucidate the specific regulatory mechanisms involved.
Th2 cells can secrete pro-inflammatory cytokines such as IL-5, IL-6, and TNF- α, as well as anti-inflammatory cytokines such as IL-4, IL-10, and IL-13, which participate in humoral immunity and allergic responses[25, 26]. Therefore, Th2 cells are not only immune effector cells, but also have immune regulation functions. In their study of 245 patients with AECOPD, 193 patients with SCOPD and 50 HCs, Wei et al showed that the levels of IL-4 in the peripheral blood of patients with AECOPD were higher than those of patients with SCOPD and HCs. Our study found that the percentage of Th2 cells in the peripheral blood of patients with AECOPD was significantly lower than that of patients with SCOPD and HCs, while there was no significant difference in the levels of IL-4 and IL-5. A possible reason for this inconsistency may be related to the individuals selected by the specimens and the experimental methods. Therefore, the levels of Th2 cells and their related cytokines in COPD may also be related to different stages of disease development.
The ratio of Th1 to Th2 cells is relatively balanced in the normal body but can be altered in the presence of pathological changes. Previous studies have shown that the imbalance of Th1/Th2 cells is involved in the occurrence and development of COPD . In both animal models of COPD and in the lung tissue of COPD patients, levels of Th1 cells and Th1-secreted IFN-γ are increased, while those of Th2 cells and Th2-secreted IL-4 are decreased. A previous study also demonstrated that patients with COPD had weaker Th2-mediated responses, including a weaker inhibitory effect on Th1 cells, and stronger Th1 cell responses, suggesting that there was an imbalance of Th1/Th2 cells in COPD patients. Our study also showed that the ratio of Th1/Th2 cells in peripheral blood increased significantly in patients with AECOPD. Taken together, these results suggest that the immune mechanism of COPD involves an imbalance in the number, ratio, and function of Th1 and Th2 cells and their corresponding cytokines, and there is a Th1 cell-type immune inflammatory response in which the balance of Th1/Th2 cells is biased to Th1 cells.
Th17 cells can secrete cytokines such as IL-17A, IL-17F, IL-21, IL-22 and IL-23 which subsequently play a role in recruiting neutrophils and maintaining inflammation. Previous studies have shown that Th17 cells[14, 15] and IL-17 levels in the peripheral blood of patients with AECOPD are increased. These results are consistent with our own observations in patients with AECOPD and SCOPD. Th17 cells were positively correlated with COPD symptom scores (CAT and mMRC score), but negatively correlated with pulmonary function index (FVC). The latter finding is similar to that of Vargas et al.  who showed that the level of Th17 cells is negatively correlated with FEV1% predicted and FEV1/FVC. Therefore, there is a correlation between the level of Th17 cells and the severity of COPD; this finding has important clinical implications for COPD patients, and may represent a novel index for the evaluation of COPD. In addition, our study also found that the proportion of Th17 cells was positively correlated with the proportion of Th1 cells, but negatively correlated with the proportion of Th2 cells. Some scholars found that the ratio of Th1 cells to Th17 cells increased significantly in the lung parenchyma of patients with COPD, while our study found that the increase of Th17 cells was mainly found in patients with AECOPD. These results suggest that there is co-activation of Th1 cells and Th17 cells in AECOPD, and these can interact to promote the onset and progression of COPD and emphysema.
Among the CD4 + T cell subtypes, there are also anti-inflammatory cell subtypes, namely Treg cells, which play an important role in immune tolerance and maintenance of immune balance. Treg cells exert their effect mainly by producing and secreting IL-10. Previous reports on the distribution of Treg cells in COPD are not consistent. Most studies have shown that compared with HCs, the proportion of Treg cells in AECOPD[5, 6] and SCOPD[7–10] is lower. However, other studies have reported the opposite result[11, 12]. The reason for this inconsistency may be related to the number of subjects and experimental methods. There are two methods for flow detection of Treg cells: one is to stain CD4 + CD25 + FoxP3 + by breaking the cell membrane, and the other is to label CD4 + CD25highCD127-/low by cell surface staining (this study). Our study increased the sample size, and the results showed that the proportion of Treg cells in AECOPD and patients with SCOPD was significantly higher than that of HCs. Previous researchers have proposed that the imbalance of Th17/Treg cells plays an important role in the occurrence and development of COPD. Our study showed that the expression of Th17 cells and Treg cells increased in AECOPD, but the increase in Th17 cells was more significant. Therefore, the above results suggest that there is co-activation of Th17 cells and Treg cells in AECOPD, and the increase of Th17 cells is dominant. The imbalance of Th17/Treg cells plays an important role in the occurrence and development of AECOPD, but further basic research is required to elucidate the underlying mechanism.