Inter‐alpha‐trypsin inhibitor heavy chain 4: A serologic marker relating to disease risk, activity, and treatment outcomes of rheumatoid arthritis

Abstract Objective Inter‐alpha‐trypsin inhibitor heavy chain 4 (ITIH4) regulates immunity and inflammation, but its clinical role in rheumatoid arthritis (RA) patients remains unclear. Hence, this study was conducted to explore the association of circulating ITIH4 with disease risk, clinical features, inflammatory cytokines, and treatment outcomes of RA. Methods After the enrollment of 93 active RA patients and 50 health controls (HCs), their serum ITIH4 level was analyzed by enzyme‐linked immunosorbent assay (ELISA). For RA patients only, serum ITIH4 level at week (W) 6 and W12 after treatment was also analyzed. Besides, serum tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, and IL‐17A at baseline of RA patients were also detected by ELISA. Results ITIH4 was downregulated in RA patients (151.1 (interquartile range (IQR): 106.2–213.5) ng/mL) than in HCs (306.8 (IQR: 238.9–435.1) ng/mL) (p < 0.001). Furthermore, ITIH4 was negatively related to C‐reactive protein (CRP) (rs = −0.358, p < 0.001) and 28‐joint disease activity score using erythrocyte sedimentation rate (DAS28‐ESR) (rs = −0.253, p = 0.014) in RA patients, but not correlated with other clinical features (all p > 0.05). Besides, ITIH4 was negatively linked with TNF‐α (rs = −0.337, p = 0.001), IL‐6 (rs = −0.221, p = 0.033), and IL‐17A (rs = −0.368, p < 0.001) in RA patients, but not correlated with IL‐1β (rs = −0.195, p = 0.061). Moreover, ITIH4 was gradually elevated in RA patients from baseline to W12 after treatment (p < 0.001). Additionally, the increment of ITIH4 at W6 and W12 was linked with treatment response and remission in RA patients (all p < 0.05). Conclusion Circulating ITIH4 possesses clinical utility in monitoring disease risk, inflammation, disease activity, and treatment outcomes of RA.


| INTRODUC TI ON
Rheumatoid arthritis (RA) is an autoimmune disease characterized by the accumulation of synovial hyperproliferation and inflammation. [1][2][3][4] Moreover, the immune mediate inflammation might further erode articular bone and lead to joint deformity, bone destruction, and disability. [1][2][3][4][5] In addition to severe symptoms described above, excess comorbidities (including fragility fracture, osteoarthritis, etc) are also along with RA patients, which could reduce their quality of life. 6,7 Aiming to control symptoms and progression of RA, many treatments have been applied, which mainly include nonsteroidal anti-inflammatory drug (NSAID), conventional synthetic diseasemodifying antirheumatic drugs (cDMARDs), glucocorticoids (GC), biologic DMARDs, etc. [8][9][10][11] However, many RA patients still suffer from poor treatment response and remission; therefore, exploring biomarkers assisting to predict treatment outcomes can help the clinicians to stratify RA patients, individualize their treatment, and improve the outcomes in RA patients. 12,13 Inter-alpha inhibitor proteins (IAIPs), a family of serine proteases inhibitors, comprise of inter-alpha inhibitor (2 heavy chains and 1 light chain) and pre-alpha inhibitor (1 heavy chain and 1 light chain). 14,15 Several studies disclose the anti-inflammatory properties of IAIPs in some inflammation-implicated diseases, such as sepsis, enterocolitis, stroke, recurrent pregnancy loss, and allergic contact dermatitis [16][17][18][19][20] In terms of inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), a plasma glycoprotein produced by liver, belongs to the family of IAIPs. 21,22 Like other IAIP family members, ITIH4 also has systemic anti-inflammatory properties in some complex diseases, including Alzheimer's disease, acute ischemic stroke, etc. 23,24 With regard to RA, previous studies find that citrullinated form of ITIH4 is differentially expressed in joints of RA patients and it fluctuates with disease activity score, which indicates that citrullinated ITIH4 may participate in the inflammation response of RA. [25][26][27] However, the role of circulating ITIH4 level in clinical management of RA patients has not been examined yet. In our preliminary study with a relatively small sample size, we observed a decrement of ITIH4 in RA patients compared with controls.
Hence, this study detected serum ITIH4 in RA patients (before and after treatment) and health controls (HCs), in order to explore the association of circulating ITIH4 with disease risk, clinical features, inflammatory cytokines, and treatment outcomes of RA.

| Subjects
Between July 2018 and April 2021, 93 active RA patients were consecutively recruited in this study. Eligible patients were ≥18 years with a diagnosis of RA and fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism Rheumatoid criteria. 28 All patients had active RA, defined as 28-joint disease activity score using erythrocyte sedimentation rate (DAS28-ESR) > 3.2 at screening. The exclusion criteria included serious infections within 6 months before enrollment, hematological or autoimmune diseases, severe liver and kidney diseases, malignancies, or a history of cancer. The HCs group was formed by a total of 50 healthy individuals who were gender (male vs. female, 1:4) and age (40-70 years) matched with RA patients. The exclusion criteria for HCs were consistent with those for RA patients; besides, subjects with immune-related diseases were also excluded. The Ethics Committee approved the study, and written informed consent was obtained from each subject.

| Data recording
Demographic data, medication histories, laboratory tests, and physical examinations were recorded at enrollment. Laboratory tests in-

| Sample collection
Blood samples were taken from RA patients at baseline (before treatment), week 6 (W6), and week 12 (W12) after treatment. Meanwhile, blood samples of HCs were also taken after enrollment. All samples were centrifuged (1500 g, 10 min, 25℃) to separate the serums for further detection. The serums of RA patients and HCs were used to detect the level of ITIH4. Moreover, the serum levels of tumor necrosis factor-alpha (TNFα), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and interleukin-17A (IL-17A) of RA patients at baseline were also measured.

| Quantification of Cytokines and ITIH4 in Serum
The ITIH4 in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) using Human ITIH4 DuoSet ELISA (DY8157-05; R&D, Minneapolis, Minnesota, USA). The TNFα, IL-1β, IL-6, and IL-17A in serum were detected using commercial ELISA kits (R&D, Minneapolis, Minnesota, USA) containing antibodies raised against target cytokines. The tests were performed strictly according to the manufacturer's instructions. The brief steps were as follows: 100 μL of sample or standards were added in diluent per well and incubated for 2 h at 24℃. Each well was aspirated and washed for 3 times. Then, 100 μL of the diluted detection antibody was added to every well and incubated for 2 h at 24℃, followed by repeated washing for 3 times. Following that, 100 µL of substrate solution was added to every well and incubated for 20 min at 24℃ avoiding direct light. After that, 50 μL of stop solution was added to each well. Finally, determination of the optical density of each well was immediately performed by a microplate reader set to 450 nm.

| Treatment
Considering disease situation, doctor's advice, and patients' wishes, some patients chose biologics-based regimen including tumor necrosis factor inhibitor (eg, etanercept 25 mg twice a week, subcutaneous injection) or interleukin-6 inhibitor (eg, tocilizumab 8 mg/kg, once every 4 weeks intravenously), while others received monotherapy or combination therapy of conventional disease-modifying antirheumatic drugs (cDMARDs) including methotrexate (7.5~20 mg once a week orally), sulfasalazine (2~3 g three times a day orally), and leflunomide (20 mg once a day orally).

| Follow-up and evaluation
Patients were followed up at W6 and W12 after treatment. Two  SPSS

| Level of ITIH4 in RA patients and HCs
ITIH4 level was declined in RA patients compared with HCs (p < 0.001, Figure 1A); meanwhile, median ITIH4 of RA patients and

| Correlation of ITIH4 with clinical features and inflammatory cytokines in RA patients
In RA patients, ITIH4 was negatively related to CRP (r s = −0.358,  In terms of the association of ITIH4 with treatments, there was no correlation of ITIH4 with history of treatment or current treatment regimens in RA patients (all p > 0.050) ( Table 3). Abbreviations: RA, rheumatoid arthritis; NSAID, nonsteroidal antiinflammatory drug; GC, glucocorticoid; cDMARD, conventional diseasemodifying antirheumatic drug; TNFi, tumor necrosis factor inhibitor; IL-6i, interleukin-6 inhibitor.

| Association of ITIH4 with treatment outcomes in RA patients
ITIH4 was gradually elevated in RA patients from baseline to W12 after treatment (p < 0.001, Figure 4A). Moreover, ITIH4 at W0 was not correlated with treatment response (p = 0.335), while higher ITIH4 at W6 (p = 0.035) and W12 (p = 0.007) was related to treatment response in RA patients ( Figure 4B). Besides, ITIH4 at W0 was not linked with treatment remission (p = 0.061), while higher ITIH4 at W6 (p = 0.007) and W12 (p = 0.005) was associated with treatment remission in RA patients ( Figure 4C).

| DISCUSS ION
ITIH4 is known as a negative acute-phase inflammatory response protein, which belongs to IAIPs family and protects against the damaging effects of several proteases. 16,21,32 Previous studies have determined the abnormal level of ITIH4 in different diseases, including chronic obstructive pulmonary disease (COPD), recurrent pregnancy loss, acute ischemic stroke, etc. 19,33 For example, one study finds that ITIH4 is downregulated in COPD patients than in HCs. 33 However, the circulating level of ITIH4 in RA patients has not been examined yet. In this study, serum ITIH4 was decreased  ITIH4 promoted the formation of HA·HC complexes, which inhibited TNFα activity via regulating tumor necrosis factor-stimulated gene-6 (TSG-6) production. 36,44 Hence, ITIH4 was negatively correlated with TNFα in RA patients. (3) As described above, ITIH4 was negatively linked with IL-6, IL-17A, and TNFα, while the decline in those pro-inflammatory cytokines was related to alleviated disease activity of RA patients. 45 Hence, ITIH4 was negatively associated with some disease activity scores (including CRP and DAS28-ESR) in RA patients.

TA B L E 3 Correlation of ITIH4 expression with treatments in RA patients
Apart from the findings mentioned above, this study also investigated that ITIH4 was gradually increased in RA patients during the treatment; meanwhile, increased level of ITIH4 was related to better treatment outcomes in RA patients. The probable reasons could be that (1) as mentioned, ITIH4 was negatively correlated with inflammation in RA patients, whose inflammation level was declined after treatment. 46 48 Therefore, elevated ITIH4 was related to treatment response and remission in RA patients.
Some limitations occurred in this study. First, the number of patients in the current study was relatively small; therefore, studies with a larger sample size to valid the findings were necessary. Second, this study enrolled HCs to evaluate the diagnostic value of ITIH4 for RA patients, while we did not recruit disease controls, which was required in the future studies. Third, the 12-week follow-up duration was relatively short; hence, a further study with longer follow-up duration needed to be conducted. Fourth, the upstream pathway of ITIH4 was still unclear, which needed to be explored in the future study. Fifth, as mentioned above, citrullinated form of ITIH4 might be involved in the pathogenesis of RA, while we did not collect relevant data in the current study. Sixth, ITIH4 had been reported to regulate the expression of mannan-binding lectin-associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which were key proteases for intravascular host defense, while the correlations of ITIH4 with MASP-1, MASP-2, and plasma kallikrein were unanswered and needed further study. 47 Seventh, the underlying mechanism of how ITIH4 participated in the inflammation response of RA was not completely explored; hence, in vivo and in vitro studies were necessary.
In conclusion, we suggest that circulating ITIH4 correlates with disease risk, disease activity, and treatment outcomes of RA; consequently, it can be used as a potential biomarker which helps clinicians to stratify RA patients, individualize their treatment, and improve the outcomes in RA patients.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.