Anemia in Patients with Takayasu’s Arteritis and its Correlation with Disease Activity


 Background Anemia is a common clinical condition in autoimmune disease, but there are few related studies in Takayasu’s arteritis (TAK). The purpose of this study is to detect clinical characteristics of anemia patients with TAK and to explore the relationship between the hemoglobin level and the disease activity in TAK. Methods This retrospective study included 160 cases of hospitalized patients with TAK and 110 cases of age-and sex-matched healthy people. According to the hemoglobin level, 160 TAK patients were divided into two groups with and without anemia, the immune indexes were compared between the two groups. 28 patients with anemia in TAK were followed up and the changes of immune indexes before and after treatment were compared.Results Hemoglobin in TAK patients is significantly lower than that of healthy control group. Among the 160 cases of TAK, 67 cases of anemia are mild to moderate anemia, and most of which are normocytic anemia. In anemia patients with TAK, women was more common (P=0.014), age at admission was more younger (P=0.002), the disease duration was shorter (P=0.017). The levels of erythrocyte sedimentation rate (ESR, P=0.000), C-reactive protein (CRP, P=0.000), interleukin-6 (IL-6, P=0.036) and disease activity scores (NIH P=0.000, ITASA P=0.001, ITAS2010 P=0.030) were significantly higher in anemia patients with TAK. The risk of anemia in TAK patients with elevated CRP was 2.35 times than that of TAK patients without elevated CRP (OR = 2.350, 95% CI 1.055-5.234, P = 0.037). After followed up for 3-6 months, hemoglobin levels increased significantly (P=0.001), ESR (P=0.000), CRP (P=0.039) and disease activity scores (NIH P=0.001, ITASA P=0.000, ITAS2010 P=0.000) decreased significantly through effective treatment without iron supplementation, and the changes of hemoglobin after treatment were negatively correlated with the changes of ESR (P=0.046) and CRP (P=0.002).Conclusion Anemia patients with TAK were more common in young women, and the disease activity was higher. Anemia can be significantly improved without iron supplementation through effective treatment to reduce disease activity of TAK.


Introduction
TAK is a chronic in ammatory disease that predominantly affects large arteries. The clinical manifestations of TAK include systemic symptoms, local in ammatory symptoms and organ ischemia caused by vascular stenosis or occlusion. Systemic symptoms, including fever, fatigue, and weight loss, are very common in TAK patients, especially in the active phase of disease.
Anemia is a common complication in TAK patients. The etiology and pathogenesis of anemia are complex and diverse, and sometimes is the result of multiple factors. There are many studies on anemia in autoimmune disease such as rheumatoid arthritis (RA). And anemia was signi cantly associated with disease severity and comorbidities of RA [1][2][3]. In TAK patients, the characteristics of anemia patients and the relationship between anemia and disease activity need to be explored. Therefore, we summarized the clinical characteristics of anemia patients with TAK, and analyzed the relationship between anemia and immunological markers.

Patients
The 160 TAK patients recruited from the Department of Rheumatology and Immunology, Beijing Anzhen Hospital during the period from November 2012 to November 2019 were retrospectively analyzed. All TAK patients ful lled the criteria for classi cation of TAK developed by the American College of Rheumatology (ACR) in 1990 [4]. Patients who had current infections, hematologic diseases, tumors, or other autoimmune diseases were excluded.
Disease activity was evaluated by the National Institute of Health (NIH) in 1994 and The Indian Takayasu Clinical Activity Score (ITAS) [5,6].
According to NIH score greater than or equal 2 (≥2) or not, 160 TA patients were divided into active group and inactive group.
The World Health Organization (WHO) de nes anemia as a hemoglobin level lower than 120 g/L in nonpregnant women and 130 g/L in men[7]. According to the hemoglobin level, anemia can be divided into mild (Hb 90g/L), moderate (Hb 60~90 g/L), severe (Hb 60~90 g/L) and very severe (Hb 30 g/L).
According to the cell morphology classi cation, anemia can be divided into macrocytic anemia, normocytic anemia and microcytic anemia.
Among the 160 TAK patients, 28 cases of initial treatment without iron supplement were selected. The guidelines recommend that TAK should be followed up every 1-3 months during the rst year[8]. We followed up for 3-6 months, because disease activity of TAK should be controlled unless treatment was ineffective. The follow-up indexes included ESR, CRP, NIH, ITASA, ITAS2010.
This study was approved by the Ethics Committee of Beijing Anzhen Hospital (2021070X).

Clinical and laboratory data
Clinical data including sex, age at admission, age at symptom onset, Body Mass Index (BMI) and disease duration were extracted from the medical records. Laboratory parameters were also recorded, including white blood cell (WBC), platelets (PLT), hemoglobin, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), ESR, CRP, IgG, IgA, IgM, C3, C4.
Statistical analysis SPSS program V.25.0 (SPSS, Chicago, Illinois, USA) was used for statistical analysis. According to the normality, variables were described as means ± SE or as medians. We performed univariate analysis by the Student's T-test or Wilcoxon-Mann-Whitney test for numerical data according to normality. Also, the One-Way Anova test and Jonckheere-Terpstra test were used to determine the statistical difference between the means of continuous variables, and the chi-square test was used to compare the categorical variables. The Spearman correlation analysis was employed to validate the relationship between age, duration and biomarkers. Logistic regression was used to analyze the related factors of anemia in TAK patients. P<0.05 was considered statistically signi cant.

Description of 67 cases in 160 TAK patients
There were 67 cases of anemia in 160 TAK patients. According to cell morphology, 38 cases of normocytic anemia, 29 cases of microcytic anemia, no macrocytic anemia. According to the hemoglobin level, 60 cases of mild anemia, 7 cases of moderate anemia, no severe anemia(  (Table 3).

Relationship between hemoglobin and biomarkers with disease activity
Among the 160 TAK patients, hemoglobin decreased with the increase of NIH score signi cantly ( Figure   1a, 1b). Taking anemia as dependent variable, age, disease duration, BMI, elevated CRP and IL-6 as covariates, binary logistic stepwise regression analysis was performed. Finally, the variables entering the regression equation were elevated CRP and age. The risk of anemia in TAK patients with elevated CRP was 2.35 times than that of TA patients without elevated CRP(OR = 2.350, 95% CI 1.055-5.234, P = 0.037). Age is a protective factor of anemia in TAK patients. The older the age, the lower the risk of anemia in TAK patients (Table 4).

Discussion
Our study found that anemia was more common in TAK patients, especially in young women. The hemoglobin level in TA patients was negatively correlated with ESR, CRP, IL-6 and disease activity. Degree of anemia is positively associated with the disease activity of TAK. Moreover, for the rst time, we found that anemia can be signi cantly improved without iron supplementation through effective treatment to reduce disease activity of TAK.
Autoimmune disease is one of important causes of anemia in chronic disease (ACD). Previous studies have reported that incidence rate of anemia in RA was 30.4% in men and 32% in women. More than 50% of Systemic lupus erythematosus (SLE) patients have anemia during the course of disease [9]. We found that anemia is also a common problem in TAK patients. the incidence of anemia in TA patients was higher(41.88%). Zhang ying etc. reported the incidence of anemia in TAk patients was 36.7% [10], which was similar to our study.
Anemia patients in RA is usually mild to moderate anemia, most normocytic anemia, few microcytic anemia, which was a typical example of ACD, may also combine with iron de ciency anemia (IDA) [1].
Similarly, in this study, all anemia patients in TA were mild to moderate anemia, without severe anemia. The type of anemia in TA was mostly normocytic anemia (56.72%, 38/67), 43.28% (29/67) of anemia patients with TA were microcytic anemia, which may be ACD, or ACD combined with IDA.
The prevalence of anemia in women is higher than that in men [11], TAK is more common in women, the male/female ratio ranges from 1:5 to 1:9 [12], and anemia patients with TAK are also more common in women. We found anemia patients with TAK were younger. Previous studies have shown that the incidence of anemia in TAK patients decreases with age, but the mechanism has not been elucidated [10,13,14]. In our study, we found the same results that the hemoglobin level was positively correlated with age at admission. Furthermore, we found the age at admission was negatively correlated with CRP and IL-6, which was associated with disease activity of TAK. Therefore, we speculated that the younger the patient, the higher the disease activity(Supplementary Table 1). The shorter disease duration in anemia patients with TAK may be related to the fact that the disease was not effectively controlled in the early stage of onset.
The etiology of TA is still unclear, which may be related to infection, genetic, and immune dysfunction [15].
Cell-mediated autoimmunity has been strongly con rmed to play an important role in the pathogenesis of TA. More in ammatory cells in ltration was shown in the aortic tissue of TA patients by immunohistochemistry, including macrophages, CD4 + T cells, CD8 + T cells, γδ T cells, natural killer (NK) cells and neutrophils [16]. Activated in ammatory cells can secrete a large number of cytokines, such as IL-6, which are involved in the pathogenesis of aortic tissue and peripheral blood of TA patients [17]. At There are many studies on anemia of RA. The mechanism of anemia caused by RA includes shortening of RBC lifespan, insu cient RBC production in bone marrow and abnormal iron metabolism [1]. Moreover, hepcidin plays an important role in the occurrence and development of rheumatoid anemia. Many cytokines such as IL-1 and IL-6 induce hepcidin production, which is involved in the occurrence and development of rheumatoid anemia [19]. The pathological mechanism of anemia caused by TA may be similar to RA.
As an autoimmune disease, the autoantibodies produced by B lymphocytes may be one of the key points in the pathogenesis of TAK. Some studies found that the levels of IgA and IgG in patients with active TAK were signi cantly increased, but there was no difference in the levels of IgM between active and inactive TAK [20]. We found the level of serum IgM in anemia patients with TAK was signi cantly increased, which may be related to the combined effect of in ammation and anemia on humoral immunity[21], but the speci c mechanism is still unclear. ESR and CRP are commonly used to evaluate the disease activity of TAK. It had also been reported that ESR and CRP can re ect the systemic in ammatory state of TAK patients [22]. A series of studies also showed that IL-6, IL-8, TNF-α These cytokines are not only involved in the pathogenesis of TA, but also related to disease activity [17]. In the baseline data of TAK patients, we found that ESR, CRP and IL-6 were signi cantly increased in anemia patients with TAK. But these serum indexes including ESR, CRP and IL-6 are lack of speci city, especially ESR, which is often affected by hemoglobin. For lack of speci c biomarkers, NIH score and ITAS score seem to be helpful in assessing disease activity and vascular damage in TAK [23]. We also found that NIH score and ITAS score were higher in anemia patients with TAK. Meanwhile, we analyzed the risk factors of anemia in TAK, and found that the risk of anemia in TAK patients with elevated CRP was higher, while the risk of anemia in older TAK patients was lower. As mentioned above, age may be negatively correlated with disease activity. The older the age, the lower the disease activity. We inferred that anemia was closely related to disease activity of TAK.
Glucocorticoids and immunosuppressants are the basis of drug therapy in TAK patients. For the treatment of anemia in TAK patients, previous studies have shown that iron supplementation can improve the condition [10]. So we wanted to explore whether anemia could be improved by just controlling the disease activity of TAK. Therefore, we followed up 28 anemia patients without iron supplementation in TAK, we found that anemia could be improved without iron supplementation.

Limitation
The main limitation of this paper is that it is a retrospective study; In addition, there was lack of indicators related to iron metabolism.

Conclusion
In this study, we found that hemoglobin levels may be associated with disease activity. By controlling the in ammation of TAK itself, anemia can be effectively controlled without iron supplement.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee of Beijing Anzhen Hospital A liated to Capital Medical University 2021070X . Written informed consent was waived because of the retrospective chart review design of this study.

Not applicable
Availability of data and materials The datasets use and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The present study was supported by grants from National Natural Science Foundation of China (81900448).
Authors' contributions YG conceived the study, performed the statistical analysis, and drafted the manuscript. JD,TL, KZ and NG collected data and revised the manuscript. LP guided the design of this study and modi ed the paper. There are no con icts of interest to declare.    Figure 1 The relationship between hemoglobin and TAK disease activity. a. The distribution of HGB in different levels of NIH score. b. The mean value of HGB in different subgroups based on the cut-off value (NIH score ESR CRP).