In developing countries, Rheumatic heart disease (RHD) is the leading cause of heart valve disease. In 2015, India (13.17 million cases), China (7.07 million), Pakistan (2.25 million), and Indonesia (1.18 million) were the countries with the most significant estimated number of RHD cases.¹ In RHD patients, the thrombus is a common manifestation. Atrial thrombus was found in 38% of patients with severe mitral stenosis (MS) and atrial fibrillation (AF).² Intracardiac thrombus formation is associated with endothelial injury, abnormal blood flow, and hypercoagulability (Trias Vircow).³ Those conditions are affected by pro-inflammatory cytokines produced by subendothelial inflammatory cells, such as TNF-α and IFN-ɣ.⁴ To inhibit thrombus formation, it is necessary to have anti-inflammatory cytokines that oppose the pro-inflammatory action, such as IL-4. IL-4 is an anti-inflammatory cytokine that plays a role in reversing the inflammatory state and regulating autoimmunity.⁵

Currently, RHD therapy aims to prevent disease progression and optimize heart function. Secondary prophylactic antibiotics are the only therapy confirmed to reduce disease severity by preventing recurrent infections.⁶ New research into the pathogenesis of the disease is being conducted to find alternative therapies. However, the role of cytokines at the site of inflammation still requires further investigation, as most studies have been conducted using peripheral blood.⁷ This study aims to determine the role of IL-4 in the thrombus pathogenesis of RHD patients directly at the site of injury, serving as a basis for developing alternative therapies.

The result showed that 16 patients (64%) were female and were dominated by patients older than 30 years (88%) (p = 0.001) with echocardiographic findings listed (Table 1 and 2). The normality test revealed that the data for LVEF was normally distributed, and the data for LAD was not normally distributed. The distribution of LVEF data in the patient with a thrombus was abnormal, while without a thrombus, it was normal. The distribution of LAD data in patients with thrombus was normal, while those without thrombus were abnormal. There were seven patients (28%) with intracardiac thrombus, and there was no significant difference based on age (p = 1.000), sex (p = 0.673), LVEV (p = 0.317), and LAD (p = 0.739) in thrombus and non-thrombus patients. All patients had mitral valve calcification, and patients with thrombus significantly correlated with AF (p = 0.001) (Table 3).

Patients with moderate and high IL-4 expression did not have less thrombus than patients with low IL-4 expression (Table 5/ central figure). IL-4 expressions are shown in Figure 1 (Central Illustration), and intracardiac thrombus events are displayed by echocardiography in Figure 2.

RHD is an autoimmune disease that can affect both males and females, with a higher prevalence in females.^9,10 This study showed similar results, with female patients comprising the majority of subjects (65%). Sex-linked hormones are known to regulate adaptive and acquired immune responses, explaining the higher incidence of autoimmune diseases in females.⁹

The research subjects were 27-68 years old, and the majority (88%) were over 30, averaging 43.4 years. Patients in this study were younger than those in the previous survey by Jiao et al. in 2022, which showed mitral valve surgery caused by rheumatic disease occurring at 40-70 years old.¹¹ This finding may be related to limited access to secondary prophylaxis antibiotics in Indonesia, which means that heart valve damage requiring surgery occurs earlier.¹² A study from Huntley in 2019 also stated that RHD manifested at younger ages in developing countries. This condition is due to limited access to health services and the use of prophylactic antibiotics, poverty, and overcrowding environments.¹³

The results showed that 28% of patients had intracardiac thrombus in LA and LAA without significant differences in sex and age. Previous studies have shown different prevalence rates, ranging from 24.9-38%.^2,13 Our study is consistent with a previous study from Ozal et al. in 2016, which showed that 24.9% of RHD patients had thrombus in the left atrial without significant differences in sex and age compared to those without thrombus.¹⁴

The results showed that patients with moderate (++) and high (+++) IL-4 expression did not have less intracardiac thrombus than those with low IL-4 expression. This result is discordant with previous studies, which showed that IL-4 can potentially prevent thrombus formation by inhibiting TNF-α formation¹⁵ and polarizing M2 macrophage.¹⁶ It was stated that TNF-α is directly associated with endothelial dysfunction by mediating NF-kB translocation, increasing Reactive Oxygen Species production, and influencing endothelial nitric oxide synthase expression. TNF-α also induces tissue factor expression and suppresses thrombomodulin and endothelial cell protein C receptors.⁴ By inhibiting this TNF-α, thrombus formation can be prevented. The study by Celik et al. in 2020 showed that IL-4 has a role in increasing the number of macrophages and polarizing them toward M2 macrophages.¹⁷ The role of macrophages in thrombus pathogenesis depends on the phenotype. M1 proinflammatory macrophages promote thrombus formation, and M2 anti-inflammatory macrophages have the opposite effect. M2 macrophages inhibit thrombus formation by producing IL-10, TGF-β, and IL-1Ra15. The shifting of M2 macrophages will cause the amount of M1 macrophages to decrease so that the production of pro-inflammatory cytokines also decreases. While performing, IL-4 is influenced by the amount, time, and location of the expression and other signals received by cells together with IL-4. These factors affect whether these cytokines have a protective or harmful effect.¹⁵

This difference in results in our study may be due to a condition that thrombus formation in RHD is not only associated with inflammation but also AF and MS. Our study results confirm that AF is strongly associated with thrombus events. AF triggers thrombus formation, especially in LA and LAA. The pathogenesis of thrombus in AF conditions involves three aspects: abnormalities in the heart wall (endothelial dysfunction), stasis (disturbances in blood flow), and abnormalities in blood components.¹⁸ There are two opinions regarding the relationship between AF and endothelial dysfunction. The first opinion states that AF causes endothelial dysfunction, and the second opinion states that endothelial dysfunction increases atrial arrhythmic substrates, thereby increasing the incidence of AF.¹⁹ In AF condition, there are also abnormal changes in platelets and blood clotting components such as von Willebrand factor (vWF), tissue factors, β-thrombomodulin, and VEGF. The increase of platelets and VEGF play a role in hypercoagulable status, and the vWF elevates the stasis condition. The accumulation of tissue factors in significant amounts causes thrombin formation and platelet activation. The increase of β-thrombomodulin also activates platelets.¹⁸

Our study also indicates that thrombus formation in RHD patients is associated with MS. All of the patients with intracardiac thrombus had mitral valve calcification and stenosis. Valve calcification causes stiffness resulting in MS. MS can cause left atrial enlargement, blood stasis in the cardiac chambers, and increased risk of thrombus formation in the LA and LAA.³ However, this study also showed that not all patients with mitral calcification and stenosis had intracardiac thrombus because several factors, including AF, influence thrombus formation in patients with MS, left atrial size, duration of symptoms, advanced age, the severity of MS, inflammation, oxidative stress, and platelet levels.^20,21 This study exhibited that all patients with thrombus had AF, left atrial dilatation, and severe MS.

In addition, the results of the study that did not show a protective function of IL-4 against thrombus incidence may also be related to the fact that while performing IL-4 is influenced not only by the amount but also by time, location of the expression and other signals received by cells together with IL-4. These factors affect whether these cytokines have a protective or harmful effect.¹⁵

This study's limitation arises from the necessity to exclude specific samples due to technical difficulties encountered during the IHC staining process. These technical challenges resulted in the depletion of the block material, rendering these samples unsuitable for analysis and thereby necessitating their exclusion from the study. As a consequence, the sample size is small. Another area for improvement is that this study was single center.

In conclusion, intracardiac thrombus formation in RHD patients is not associated with IL-4 expression but is strongly associated with AF. Further research that identifies other cytokines co-existing with IL-4 at the injury site may reveal better insight into the event.