Introduction
As the prototype of spondyloarthritis, ankylosing spondylitis (AS) is a chronic inflammatory disease that affects the spine and sacroiliac joints. The pathogenesis of AS can be attributed to both hereditary and environmental factors [
1]. The pathogenesis of AS has still remained unclear, but a strong association with the HLA-B27 [
2] and a perpetual activation of both the innate and the adaptive immune systems [
1], in particular of the IL-23/IL-17 axis and of Th1 effector T cell lineage with the overproduction of tumor necrosis factor-α (TNF-α), are considered to be key steps [
1]. Chronic hyperactivation of T lymphocytes and subsequent skewing of functional subgroups within CD4+ T and CD8+ T cells have been supported by some human studies [
3,
4]. On the other hand, regulatory T cells (Tregs) display decreased prevalence in the blood of AS patients suggesting that their lack may contribute to the pathogenesis of the disease [
5]. However, the data on Tregs have been controversial in AS patients [
6]. Studies on acquired immunity to AS have suggested that T cell-mediated immune regulation may also play an important role. Nevertheless, only limited data are available on the phenotypic and functional status of B cells in AS [
7,
8]. The ability of B cells to negatively regulate cellular immune responses and inflammation has been explored, and the concept of regulatory B cells (Bregs) has emerged. Indeed, defective suppressive functions of Bregs cell subsets have been observed in several chronic inflammatory diseases [
9,
10], but studies into Bregs cell activity in AS patients have been scattered. Some small sample research studies showed regulatory B cells to have a defective function in AS patients but observed no significant change in cell frequency [
11].
Given that immune cells in AS have imbalance, manipulating immune cells may be a new therapeutic strategy for treating AS. Therefore, exploring factors that influence immune cell number and function will play a critical role in understanding the pathogenesis of these diseases and identifying new treatment strategies. TNF-α inhibitor has been shown to have an anti-inflammatory effect in treating AS [
12], but its potential beneficial effects on the bone structure are still challenging to identify and its mechanisms remain unclear. Although targeting of TNF-α is very effective in AS, around one third of treated patients show only a poor response. The symptoms would recur in the vast majority of patients if the drug is stopped; it is important to know the type and degree of the alterations in the immune system developing during the anti-TNF therapy. On the other hand, with respect to adverse reactions and the high costs of anti-TNF agents leading to high economic burden for the health care systems, it is desirable to stratify patients according to treatment predictors prior to biological therapy. In terms of the immune cell subset distribution, we assume that the major mechanism of action of anti-TNF therapy is to obtain inhibition of the inflammatory process, not the restoration of the activated immune system from active disease to a state similar to healthy individuals. We conducted a prospective study of subset distribution of CD4+/CD8+ T cells and negatively regulate immune cells including Tregs and Bregs by identifying the phenotype, and also analyzed the changes in the proportion of lymphocyte subsets in order to indirectly understand the differentiation status, degree of failure, and cell activity of various cell subsets. That will help us to further clarify changes of the immune system caused by AS and to explore resistance that could contribute to relapse after treatment.
Discussion
As we know, the onset of AS suffers from the relationship between the host genetics, the intestinal microbiome, and the immune response [
16]. AS has long been associated with inheritance of the HLA allele B27 [
1], and the pathogenic role of HLAB27 remains unclear despite intensive research. The arthritogenic peptide theory proposes that HLAB27 plays a central pathogenic role in the presentation of joint-specific peptides to CD8+ cytotoxic T cells. Specific self or environmental peptides are proposed to bind to and be presented by HLA-B27, to activate CD8+ cells. Another major theory for the pathogenesis of HLA-B27 in AS revolves around the ability of HLA-B27 to aberrantly fold to form homodimers [
17]. Circulating CD4+ T cells, expressing the killer cell immunoglobulin receptor (KIR3DL2) after activation, recognize HLA-B27 homodimers, and this recognition is associated with the secretion of large amounts of inflammatory cytokines including high levels of IL-17A, and these cells are polarized toward a Th17 phenotype [
18]. Our research is basically consistent with the above immunological concepts in the pathogenesis of AS. We found that the proportion of naïve CD4+/CD8+T cells and memory CD4+/CD8+T cells had increased while terminally differentiated CD4/CD8+T cells had decreased, which just confirmed the role of activation of CD4+/CD8+ T cells in the classic theory of AS pathogenesis.
One of the most specific pathologic features of AS is inflammation at the enthesis [
19]. Recent genetic and immunological research has highlighted a key role for IL-17A/IL-23 cytokine dysregulation of the Th17 immune pathway in AS [
1]. IL-23 signaling through the IL-23 receptor (IL-23R) on CD4+ Th cells is required for the differentiation and expansion of Th17 cells. Mechanical stress and/or infectious stress resulting in overexpression of the IL-17A/IL23 axis with activation of resident entheseal cells that can respond to IL-23 including CD4+T cells, CD8+ T cells, γδT cells, and other innate immune cells. This leads to production of IL-17A, IL-22, TNF-α, and other cytokines that mediate spinal and peripheral inflammation directly or through tissue-resident effector cells [
20]. A perpetual activation of both the innate and the adaptive immune systems, in particular of the IL-23/IL-17 axis and of Th1 effector T cell lineage with the overproduction of TNF-α, is considered to be key steps in the pathogenesis of AS. We also found that the proportion of Th17 cells increased while the proportion of Th1 cells decreased, confirming the key role of IL-17A/IL-23 signaling pathway in the pathogenesis of AS.
The inflammatory effects seen throughout AS are associated with immune imbalance. Previous studies have found changes in the immune cells of patients with AS [
21‐
23]. Our study, which included an extensively validated large sample size, showed that active AS causes abnormalities in the proportion of lymphocyte subsets and that help us to indirectly understand the differentiation status, degree of failure, and cell activity of various cell subsets. Our results, which showed changes in the frequency of Th cells, Tfh cells, Tc cells, and Treg cells in patients with AS were consistent with the findings of past studies [
3,
8,
22]. We also made a discovery regarding abnormal changes in CD4+T cells and CD8+T cells at different stages of differentiation of AS, which further suggests that AS patients have an irregular proportion of T cells at different stages of differentiation. Some previous studies based on small sample sizes found only abnormal changes in CD4+T cell subsets at different stages of differentiation but not in CD8+T cell subsets [
24].
TNF-α induces the production of multiple inflammatory factors and has an impact on the differentiation and activation of various T cell subtypes, and therefore, it has a significant role in shaping the adaptive immune system [
25]. Our results showed that Anbainuo injections could address partial immune imbalance in AS patients by affecting differentiation and activation of immune cell subtypes, including naïve CD4+ T cells, Tregs, and B10 cells. Among these, the decreased proportion of naïve CD4+ T cells after Anbainuo treatment was consistent with the findings of past research [
26]. Our observation that naïve CD4+ T cells decreased but effector memory CD8+ T cells increased after Anbainuo treatment is in line with our expectations, because there are many reasonable explanations for this result. One possible explanation is that migration of T cell precursors from the thymus or release of naïve T cells from the secondary lymphoid organs may have been impaired, but differentiation of memory T cells from naïve cells may have accelerated. This could be one of the mechanisms by which Anbainuo regulates immunity and plays an anti-inflammatory role.
However, we think it makes more sense that Anbainuo treatment for AS can increase the proportion of negative regulatory cells such as Tregs and Bregs in inflammation that are significantly lower than normal before treatment. We have described the decreased frequency of Tregs, immature Bregs, and B10 cells, in AS patients who had previously received no biological treatment agents (we found that B10 cells did not display a significant change between AS and HCs). This may suggest that the lack of negative regulatory cells in inflammation may contribute to the pathogenesis of AS. The parallel compensatory increase seen in Tregs and B10 cells after Anbainuo treatment for 12 weeks suggests that TNF-α inhibitor may assist with the recovery of patients’ immune tolerance.
Tregs are a small subset of CD4+ T cells that play a pivotal role in the maintenance of immunological tolerance and prevention of autoimmunity [
27]. It has been reported that decreases in Treg number and function lead to abnormal immune responses toward self-antigens, thus resulting in rheumatic diseases including AS. Recent report showed that the mean fluorescence intensity (MFI) of FOXP3 in circulating Treg cells was significantly decreased in active AS patients, and Treg cells could not effectively inhibit the proliferation of naïve T cell [
6]. In addition, active AS patients harbor Treg cells that are defective in using IL-2, have relatively little STAT5 phosphorylation, and have higher CpG methylation levels in CNS2 region of the foxp3 gene [
6]. It suggested that the functional defects of Treg cells are present and play important roles in AS. Although higher values of CRP, together with the presence of HLA-B27, have been reported to be useful baseline predictors for a successful anti-TNF-α therapy response in AS, the robustness, sensitivity, and specificity fail if applied to individual patients. Given that Treg cells are powerful immunosuppressive agents, manipulating Treg cells is a new therapeutic strategy for treating autoimmune diseases. An increase in Tregs during long-term anti-TNF therapy for AS has also been noted [
26]. Our research further reveals a positive correlation between the reduction in CRP value and a higher frequency of Tregs. We propose a hypothesis that low Tregs frequency at the initiation of anti-TNF therapy may be a good predictor of treatment response to anti-TNF treatment in AS. Although our findings are promising, a further validation of Tregs as a potential biomarker for TNF responsiveness is necessary in an independent cohort of AS and other rheumatic and gastrointestinal diseases where anti-TNF blockers are successfully administered.
In humans, plasmablasts, immature B cells, B10 cells, B regulatory 1 (Br1) cells, and Granzyme B (GrB)+ B cells comprise the identified Breg subsets [
28]. Bregs cells contain a high proportion of IL-10-producing Bregs cells, named B10 cells [
29], which, via the production of IL-10, suppress TNF-α production by monocytes. Human regulatory plasmablasts are suggested to drive from immature B cells and act through IL-10 production [
30,
31]. This may explain our observation that immature Bregs decreased but B10 cells increased after Anbainuo treatment. However, another small sample size study about Bregs led to the opposite conclusion [
7]. IL-10 secreted by B10 cells plays a role in the development of intestinal immunity [
32]. Environmental factors, which may include the microbiome and infections, probably also play a significant role in AS pathogenesis [
1]. Bowel inflammation and AS are frequently clinically associated [
33]. Our finding that B10 cells increased after Anbainuo treatment may provide further support to the hypothesis that AS is caused by interactions between the host gut immune system and the gut microbiome. IL-10 secreted by B10 cells could be used as potential indicators for the therapeutic efficacy of TNF-α inhibitors in AS with bowel inflammation.
However, Anbainuo is not able to regulate all of the immune imbalances attributed to AS. Our results show that the proportion of effector memory CD8+ T cells and Tfh17 cells, which was significantly higher in AS patients (110 AS patients vs. 55 HCs), and the proportion of Tc1 cells, which was significantly lower in AS patients (110 AS patients vs. 55 HCs), have not been rescued after treatment. Initially, we found the frequency of Tc1 cells to be significantly decreased and to be negatively associated with CRP levels. Although we have to admit that the actual significance of the correlation coefficient between CRP and Tc1 is indeed limited, we still expected that after treatment with TNF inhibitors, the frequency of Tc1 cells will increase because the inflammatory process has been put to quiescence clinically as reflected by routine laboratory inflammatory marker (CRP) and disease activity index (ASDAS and BASDAI). However, after Anbainuo treatment, although the disease activity of AS patients improved, the frequency of Tc1 cells was still lower than the baseline, and at the same time, the results of correlation analysis showed that neither BASDAI nor ASDAS had anything to do with the changes of immune cell phenotype. The present measurements confirm our previous hypothesis that the major mechanism of action of anti-TNF therapy is to obtain inhibition of the inflammatory process, not the restoration of the activated immune system from active disease to a state similar to healthy individuals, in terms of the immune cell subset distribution. It seems that the generation of differentiated CD8+ T cells (such as effector memory CD8+T cell and Tc1 cell in the result) remains an ongoing process despite TNF blockers, and the T cell repertoire necessary for an effective antitumor or antimicrobial defense is not compromised. Now that some immunological abnormality cannot be rescued despite the effects of Anbainuo, further study is required to clarify whether the abnormalities of the CD8+ T cells in the frequency or functions are related to resistance that could contribute to relapse after treatment. The value of these immune cells as new potential targets for AS therapy is also worthy of attention in future research.
In the past, there have been a small number of reports on the abnormal distribution of B cells in the peripheral blood of AS patients [
23]. However, the distribution of different subtypes of B cells or different stages of differentiation has not yet been fully revealed. Our study found that AS patients experienced a significant increase in the total of B cells and class-switched B cells but a significant reduction in the proportion of antibody-secreting subtype B cells during the B cell effector phase. At the same time, no significant difference was observed in terms of autoimmune-related changes in the AS patients compared with the HCs. This shows that AS is better defined as an autoinflammatory disease than an autoimmune disease. In the future, potential immune regulation against B cells is likely to benefit patients with AS.
This study has a few weak points. It was found that the average onset age of the first stage (primary screening phase) is lower than general AS population [
34]. That may be related to the limited sample size of the first stage without artificial screening for demographic characteristics. That may cause some bias especially the result of immune imbalance associated with the course of the disease. However, the onset age of the second stage (expanded validation phase) (22.7 ± 7.85) is consistent with the general AS population. Here we truthfully report the true statistics and plan to further expand the verification study in the future.
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