This case–control study evaluated the diagnostic performance of a panel of IPA-associated cytokines in plasma for identifying IPA among patients with HBV-ACLF. Previous studies have reported a prevalence of IPA in patients with HBV-ACLF ranging from 1.85% to 9.88% [5,6,7,8, 10, 25, 26]. In our cohort, the prevalence of IPA was 4.64%, which is consistent with studies conducted both domestically and internationally. Our findings revealed that IL-33 levels were significantly elevated in the IPA group and demonstrated excellent sensitivity and specificity for IPA diagnosis. This highlights the potential of IL-33 not only as a diagnostic biomarker but also as a key player in the pathophysiological mechanisms underlying HBV-ACLF with IPA. Additionally, our data provide compelling evidence that IL-33 can effectively distinguish IPA from bacterial infections. By identifying IL-33 as a reliable biomarker for IPA diagnosis, our study addresses the critical need for improved diagnostic approaches in this high-risk patient population.

IL-33, a member of the IL-1 family identified in 2005, functions both as a secreted cytokine and a nuclear protein involved in gene transcription, with particular abundance in specialized populations of epithelial and endothelial cells [27]. Suppression of tumorigenicity 2 (ST2) is the only well-documented receptor for IL-33 and exists in two primary forms: the transmembrane signaling receptor (ST2L) and the soluble decoy receptor (sST2). The availability of IL-33 is tightly regulated by sST2, the decoy receptor, which prevents productive interaction of IL-33 with ST2 [28, 29]. IL-33 primarily targets immune cells associated with type 2 and regulatory immune responses, including type 2 innate lymphoid cells (ILC2s), Th2 cells, eosinophils, mast cells, basophils, as well as subsets of dendritic cells, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Treg cells) [30]. ILC2s, some Treg cells, and mast cells are the primary tissue-resident cells that constitutively express high levels of ST2, positioning these cells as initial targets of IL-33. IL-33 binds to its receptor ST2 and recruits the accessory protein IL-1 receptor accessory protein (IL-1RacP, an auxiliary receptor shared by other members of the IL-1 family (IL1α, IL1β, IL-36)). Subsequently, the IL-33/ST2/IL1RAcP complex triggers signal transduction through the adaptor protein MyD88, followed by activation of the kinases IRAK1 and IRAK4, and the E3 ubiquitin ligase TRAF6, ultimately leading to the phosphorylation and activation of MAP kinases and NF-κB transcription factors, which drive the expression of type 2 cytokines such as IL-5 and IL-13 [27, 31].

In patients with HBV-ACLF, extensive disruption of the hepatic and intestinal barriers contributes to systemic inflammation and immune dysregulation. Previous studies have demonstrated that serum or plasma IL-33 levels are significantly elevated in patients with ACLF compared to patients with chronic hepatitis B and healthy individuals. Moreover, IL-33 levels correlate with the severity of ACLF and have been shown to predict clinical outcomes [32,33,34]. In our study, after propensity score matching to balance the IPA group with the BP and non-infection groups, IL-33 levels in the IPA group were significantly higher than those in both the BP and non-infection groups (163.07 vs. 12.82 vs. 4.24 pg/mL, P < 0.001). These findings suggest that the marked elevation of IL-33 is not merely attributable to the systemic inflammatory in patients with ACLF but likely reflects a distinct local and systemic immune response triggered by Aspergillus invasion of lung tissue. Recent studies have increasingly focused on the role of IL-33 in fungal infections, revealing that IL-33-mediated immune responses may be detrimental to the clearance of fungi. However, the exact mechanisms by which IL-33 influences Aspergillus infections remain incompletely understood.

Current research suggests that IL-33 may modulate host immune responses and antifungal defenses through several pathways. During pulmonary fungal infections, lung epithelial cells can produce IL-33, serving as a primary source of this cytokine [35]. In Aspergillus fumigatus infections, IL-33 secretion is increased independently of Dectin-1, impairing pulmonary antifungal defense by inhibiting the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway and acting as a negative regulator of IL-17A and IL-22 [36]. Beyond lung epithelial cells, IL-33 activates various immune cells, including eosinophils, Th2 cells, mast cells, and particularly ILC2s. These cells play a critical role in mediating IL-33-induced immune responses, which may exacerbate inflammation and compromise antifungal defenses [27]. In a mouse model of Cryptococcus neoformans infection, the absence of IL-33 or its receptor ST2 significantly reduced lung fungal burden and mortality, suggesting that IL-33-mediated immune responses may impair host defenses against this pathogen [37]. These findings underscore the multifaceted role of IL-33 in fungal infections and its potential as a biomarker for disease diagnosis and progression. As an alarmin, IL-33 is released in response to tissue damage or barrier disruption. Its biological functions are context-dependent and can be considered a double-edged sword. Under physiological or parasitic challenge conditions, IL-33 activates ST2-expressing cells (e.g., ILC2s, Th2 cells) to promote mucus production, epithelial repair, and anti-helminthic immunity [28]. However, in the setting of immune dysregulation characteristic of HBV-ACLF, IL-33-driven type 2 immune polarization may suppress protective Th1/Th17-mediated antifungal responses, thereby impairing the clearance of Aspergillus. Studies in murine models have demonstrated that elevated IL-33 levels impair pulmonary antifungal immunity [36]. Thus, the marked elevation of IL-33 in our patients likely reflects not only the infection but also serves as an integrative marker of severe tissue injury, dysregulated inflammation, and overall disease severity. The precise mechanisms underlying this potential immunomodulatory effect require further investigation in experimental models.

Current research on the diagnostic value of cytokines for IPA has primarily focused on patients with hematological or respiratory diseases. Several studies have identified associations between IPA and cytokines such as IL-6, IL-8, and IL-10. In patients with hematological malignancies, serum and bronchoalveolar lavage fluid (BALF) levels of IL-6 and IL-8 were significantly elevated in those with probable or proven IPA compared to patients without IPA. Additionally, elevated serum IL-10 levels have been proposed as a predictive marker for IPA [17]. In patients with aplastic anemia (AA), simultaneous increases in serum IL-6 and IL-8 levels have been shown to predict the onset of mixed bacterial and fungal infections [18]. Among stem cell transplant recipients and patients with acute leukemia, IL-6 (AUC = 0.74) and IL-10 (AUC = 0.64) levels demonstrated potential utility in assisting the diagnosis of invasive aspergillosis [19]. Similarly, in patients with chronic obstructive pulmonary disease (COPD), elevated serum and BALF levels of IL-6 (AUC = 0.837 and 0.769) and IL-8 (AUC = 0.876 and 0.825) in IPA cases may serve as auxiliary diagnostic indicators [38].

However, in our study involving an ACLF population, we were unable to fully corroborate previous findings from non-hepatic diseases linking elevated IL-6 and IL-10 levels to IPA. In our analysis, IL-6 (AUC = 0.604) and IL-10 (AUC = 0.615) showed limited diagnostic efficacy for distinguishing IPA from bacterial infections (BP group). In contrast, when comparing the IPA group to the non-infection group, IL-6 (AUC = 0.709) and IL-10 (AUC = 0.680) exhibited moderate diagnostic performance. These discrepancies may reflect the complex immune dysfunction characteristic of patients with ACLF and its influence on cytokine expression patterns.

Previous studies on liver diseases have highlighted the challenges in differentiating fungal from bacterial infections using conventional laboratory markers [39, 40]. Our identification of IL-33 as a biomarker represents the first potential tool for distinguishing Aspergillus infections from bacterial infections. In a study focusing on respiratory diseases, IL-33 levels were significantly higher in patients with chronic pulmonary aspergillosis (CPA) compared to asthmatic groups (59.91 vs. 6.07, P < 0.05), although the diagnostic utility of IL-33 was not further investigated [41]. The elevated IL-33 levels observed in patients with CPA provide a valuable foundation for future research. IL-33 not only demonstrates potential diagnostic value for IPA in patients with ACLF but may also serve as a useful biomarker in other high-risk populations, such as those with chronic respiratory diseases, organ transplant recipients, and individuals with hematologic or autoimmune disorders. By validating the diagnostic efficacy of IL-33 across diverse disease contexts, it is anticipated that IL-33 could be developed into a rapid and reliable diagnostic tool for IPA in a broad spectrum of clinical settings.

Given the single-center prospective design of this study and the low incidence of IPA, a primary limitation is the relatively small sample size of the IPA group. Secondly, although the study examined changes in IL-33 at three time points defined as IPA incubation, IPA development, and IPA recovery, the limited availability of multiple measurements at different time points and the absence of more frequent dynamic monitoring may have affected the comprehensiveness of the data. Thirdly, it is noteworthy that all patients in the IPA cohort were classified as probable rather than proven IPA cases. As a result of coagulation disorders and the specific disease characteristics of patients with ACLF, most did not have access to the tissue samples required for a definitive diagnosis. In addition, the absence of the comparison between IL-33 and GM or other molecular assays also constitutes a study limitation. Future prospective, multicenter studies should validate IL-33 in patient populations undergoing routine fungal diagnostics (e.g., PCR, mNGS), to rigorously assess its diagnostic accuracy and the incremental value of both head-to-head comparisons and integrated models with established biomarkers. We are currently conducting a multicenter prospective study to validate the diagnostic performance of IL-33 in patients with ACLF combined with IPA and to develop a more robust diagnostic model in a larger cohort.