Inflammatory bowel disease (IBD) encompasses two primary conditions: ulcerative colitis (UC) and Crohn’s disease (1). UC is a chronic inflammatory disorder characterized by inflammation of the colonic mucosa, typically beginning in the rectum and extending proximally. Its diagnosis hinges on endoscopic and histopathological evidence of colonic inflammation (2). UC follows an unpredictable course, characterized by periods of relapse and remission, which significantly impact patient quality of life and place a substantial burden on healthcare systems (3).

The etiology of UC remains elusive, but current understanding suggests it arises from complex interactions among the gut microbiota, genetics, and various immunological and environmental factors (4). UC affects approximately 1% of the population in some industrialized countries (5) and its prevalence has notably increased in newly industrialized nations in recent years (6), highlighting lifestyle factors as potential risk factors and linking environmental triggers to immune dysregulation. Despite recent advances in UC management and the availability of multiple therapeutic options targeting various inflammatory signaling pathways, the disease remains incurable. A significant proportion of patients experience limited or no response to treatment, resulting in overtreatment, unnecessary exposure to side effects, and delays in receiving optimal therapeutic interventions. Additionally, treatment response is highly variable among patients with similar clinical presentations (7–10), emphasizing the critical need for predictive biomarkers of therapeutic efficacy. Developing tools that link the activation of specific inflammatory pathways with individual patient responses could facilitate personalized treatment strategies, improving therapeutic outcomes and reducing the rate of non-responders.

Central to UC’s pathogenesis is an aberrant immune response, with several inflammatory pathways, including the JAK/STAT signaling pathway, playing crucial roles (11, 12). The Janus kinase (JAK) family—comprising JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2)—mediates cytokine signaling by phosphorylating and activating signal transducer and activator of transcription (STAT) proteins. Once phosphorylated, STAT proteins dimerize and translocate to the nucleus, where they regulate the transcription of genes involved in immune responses and inflammation (12). Overactivation of this pathway can result in a proinflammatory state, driving the chronic inflammation and tissue damage characteristic of UC (13). This dysregulation is reflected in abnormal phosphorylation levels, further perpetuating immune activation (14). This disrupts cytokine signaling, driving the excessive immune activation and chronic inflammation characteristic of UC (13, 15). Various cytokines, such as IFNγ, IL-6, IL-12, IL-15 and IL-23, signal through this pathway to modulate immune responses and contribute to intestinal tissue damage (16), thus playing a central role in UC pathogenesis (12, 17). For example, IL-6 disrupts the intestinal barrier and promotes immune cell infiltration via STAT3 activation (18). These observations underscore the central role of the JAK/STAT pathway in orchestrating inflammatory responses in UC.

Further complicating UC management is the significant heterogeneity observed in disease presentation, complications, and treatment response among patients (19). Increasing evidence indicates that sex-specific factors, including hormonal and genetic differences, modulate inflammatory pathways, contributing to variations between males and females in clinical outcomes such as mortality, surgery rates, and comorbidities (20, 21). Notably, differential immune activation patterns between sexes underscore the importance of incorporating sex as a biological variable in UC research and personalized therapeutic approaches (22, 23). A deeper understanding of these sex-specific influences may improve predictive accuracy for treatment response and facilitate the development of tailored therapeutic strategies.

Thus, our working hypothesis was that profiling the activation of the JAK/STAT inflammatory pathway in UC patients could provide valuable insights into the pathophysiology of UC and help elucidate the variability observed among patients. To address this, our objectives were: i) to analyze the activation of JAK/STAT pathway components in colon samples of a cohort of UC patients; ii) to establish correlations among activation levels of these proteins, revealing interconnections relevant to UC pathophysiology; and iii) to investigate sex-specific differences in pathway activation. These explorations aim to enhance our understanding of UC pathophysiology and lay the groundwork for developing personalized therapeutic strategies in UC.

The primary achievement of this study was the comprehensive profiling of JAK/STAT pathway activation in samples of inflamed and non-inflamed colonic areas from a cohort of sixty-one patients with UC. This approach allowed us to: i) demonstrate a significant increase in the phosphorylation of six of the seven JAK/STAT pathway components analyzed (JAK2, JAK3, TYK2, STAT1, STAT3, and STAT4) in the inflamed colonic areas of UC patients; ii) identify interconnections in the activation of JAK2, JAK3, TYK2 and STAT3 proteins within the JAK/STAT pathway; and iii) uncover a sex-dependent activation of JAK2 and STAT3. These findings align with our hypothesis, providing valuable insights into the pathophysiology of UC, addressing the observed variability among patients, and establishing a foundation for developing novel diagnostic tools and personalized therapeutic strategies.

The demographic and clinical features of our cohort are representative of real-world UC patient populations, aligning well with data from previous studies (26), and providing a robust framework for investigating the activation of the JAK/STAT pathway in UC. Importantly, the majority of patients included had not received advanced therapies such as biologics, and none were treated with tofacitinib or other small-molecule JAK inhibitors at the time of sampling, ensuring that observed patterns of JAK/STAT activation were not influenced by direct pharmacological inhibition. Moreover, the inclusion of six treatment-naïve patients (9.8%) further enhances the representativeness and generalizability of our findings.

Although considerable variability in the phosphorylation levels of JAK/STAT pathway components was observed among the patients included in the analysis, significant increases were detected in the phosphorylation of JAK2, JAK3, TYK2, STAT1, STAT3, and STAT4 in inflamed colonic samples of UC patients compared to non-inflamed samples. In contrast, no significant changes were observed in JAK1 phosphorylation.

The observed increase in the phosphorylation of JAK2, JAK3, and TYK2 in our cohort aligns closely with findings in the existing literature, which highlight their critical roles in the pathophysiology of UC. JAK2 is a key mediator in the signaling pathways of several cytokines involved in UC-related inflammation, including IL-6, IL-12 and IL-23 (27, 28). Similarly, JAK3 is involved in the signaling pathways of cytokines relevant to UC, such as IL-15 (16, 17), further supporting its role in intestinal inflammation as documented in prior studies (29). TYK2, which plays a pivotal role in the signaling of IL-12 and IL-23 (27, 30), is also recognized as a key driver of chronic inflammation in UC (31). Thus, our data corroborate these findings, further reinforcing the importance of JAK2, JAK3, and TYK2 as central mediators in UC-associated inflammation as described previously in the literature.

The increased phosphorylation of STAT1, STAT3, and STAT4 observed in our study is consistent with their roles as downstream effectors of JAK activation, reflecting the enhanced inflammatory signaling characteristic of UC (32–34). STAT3, in particular, plays a key role in regulating immune responses and epithelial repair, but its persistent activation has also been associated with chronic inflammation and disease progression in UC (35).

The lack of a significant increase in JAK1 phosphorylation in biopsies from inflamed areas of the colon in our cohort is intriguing, particularly in light of the development of selective JAK1 inhibitors for UC treatment (36). This observation may reflect a high degree of variability in JAK1 activation among UC patients, underscoring the heterogeneous nature of the disease. Such variability highlights the potential importance of personalized approaches to UC management, where understanding individual signaling profiles could inform the selection of the most appropriate therapeutic strategies. These findings emphasize the need for further research to elucidate the factors influencing JAK1 activation and to assess their implications for tailored treatment in UC.

The observed phosphorylation levels within the JAK/STAT pathway provide a foundation for exploring the functional correlations among its components. These correlations offer further insights into the molecular dynamics underpinning UC-related inflammation.

The significant correlation between the phosphorylation of JAK2 and STAT3 (r = 0.534, p < 0.001) underscores the essential role of JAK2 in STAT3 activation (37). STAT3, a downstream effector of the JAK/STAT pathway, is a central mediator of IL-6 signaling and a key driver of tissue inflammation and repair dysregulation in UC (38). This correlation aligns with previous findings that STAT3 activation contributes to the severity of inflammatory processes underlying UC pathophysiology and is involved in the progression to colitis-associated cancer (35, 39). However, it is worth noting that STAT3 also exhibits a protective role by enhancing the production of mucus in the gut, adding a layer of complexity to its function in UC pathophysiology (40). Taken together, these results reinforce the notion that JAK2 serves as a central node in coordinating inflammatory pathways in UC.

Furthermore, there was a strong correlation between the phosphorylation of JAK2 and TYK2 (r = 0.545, p < 0.001) highlighting their synergistic roles in amplifying inflammatory signals, consistent with their established involvement in IL-12 and IL-23-mediated pathways (41, 42). TYK2, a kinase critical for the signaling of these cytokines, has been shown to work in concert with JAK2 to drive pro-inflammatory responses in various immune-mediated conditions (43). This synergy may reflect their roles in promoting the differentiation and activation of Th17 cells, which are pivotal in sustaining chronic inflammation in UC (44, 45).

Another layer of interconnection comes from the significant correlation between the phosphorylation of TYK2 and JAK3 (r = 0.539, p < 0.001), suggesting a functional interplay between these kinases in UC. As discussed before, JAK3 is known to participate in signaling pathways activated by cytokines such as IL-15, which are central to UC pathogenesis (16, 17). The correlation between the phosphorylation of TYK2 and JAK3 may indicate an interdependency in their activation, a relationship that, to our knowledge, has not been extensively explored. These findings suggest that further investigation into the interplay between these JAK kinases could provide novel insights into their roles in UC pathophysiology.

Another noteworthy observation is the significant correlation between the phosphorylation of effector proteins STAT1 and STAT3 (r = 0.429, p = 0.010), suggesting cooperative roles for these transcription factors in regulating inflammation. STAT1 and STAT3 are both downstream effectors of the JAK/STAT pathway and are activated by overlapping yet distinct cytokines. This interplay aligns with their shared involvement in mediating responses to pro-inflammatory cytokines such as IL-6, which is central to the pathogenesis of UC (12). IL-6 is known to lead to the activation of STAT3 (46), promoting inflammatory responses and impairing epithelial barrier function in UC (47). STAT1, typically activated by IFN-γ and other proinflammatory cytokines (33), may synergize with STAT3 under certain inflammatory conditions to amplify or modulate downstream transcriptional responses, contributing to the chronic inflammation characteristic of UC.

Taken together, all these correlations emphasize the complex and interdependent nature of JAK/STAT signaling in UC. The interactions among JAK2, JAK3, TYK2 and STAT3 likely reflect a coordinated effort to propagate and sustain inflammatory responses across multiple immune cell types.

Building on these findings, an analysis of phosphorylated JAK/STAT pathway components by sex revealed additional nuances in pathway activation. Figure 4 highlights significant sex-dependent differences in JAK2 (p = 0.049) and STAT3 (p = 0.033) phosphorylation, while the remaining proteins showed no statistically significant variation. These results introduce a potential dependence on sex-specific factors in shaping the activation dynamics of the JAK/STAT pathway.

The higher median intensity of JAK2 phosphorylation in male patients compared to females suggests that JAK2 may play a more prominent role in propagating inflammatory responses in males. This finding aligns with evidence indicating that JAK2-mediated signaling is crucial for amplifying pro-inflammatory cytokines such as IL-6, which, as discussed before, plays a central role in UC pathogenesis (12). Similarly, the significant sex-dependent difference in STAT3 phosphorylation underscores its importance as a downstream effector, potentially reflecting sex-specific modulation of the IL-6/JAK2/STAT3 axis. This observation may correspond with prior reports of elevated IL-6 expression and activity in males across various pathological contexts, such as severe trauma and COVID-19, which are associated with exacerbated inflammatory responses (48, 49). Moreover, given the established association between STAT3 activation and an increased risk of developing colorectal cancer due to colitis, the observed increase in STAT3 phosphorylation in males may be linked to the higher incidence of colorectal cancer reported in male UC patients (50).

Although statistically significant sex-related differences were only observed for JAK2 and STAT3 phosphorylation, these findings are noteworthy given the moderate effect sizes and their achievement of statistical significance despite the relatively limited sample size. Post hoc power analyses indicated that the study may have been underpowered to detect small differences in the remaining pathway components (power range: 0.065–0.573; see Table 4 ). Consequently, the absence of statistical significance for other proteins should be interpreted with caution, and the observed trends in effect sizes may justify further investigation in larger, adequately powered cohorts. Overall, these results support the potential contribution of sex-related variability in JAK2 and STAT3 activation to differential immune responses in UC.

Taken together, these findings underscore the potential importance of considering sex as a biological variable in both research and treatment in inflammatory diseases, such as UC. Understanding these differences could pave the way for more personalized therapeutic strategies that account for sex-specific variations in pathway activation.

While the present findings provide valuable insights into JAK/STAT pathway activation in UC, certain aspects warrant cautious interpretation. Due to sample constraints, only the phosphorylated forms of each protein were analyzed, and total-protein levels were not assessed. Although phosphorylation serves as a widely accepted surrogate for activation, future studies incorporating both total and phospho-protein quantification could offer more comprehensive insights. Additionally, although we explored potential associations between phosphorylation levels and endoscopic disease activity, no significant correlations were identified in this cohort. This may be attributed to the limited sample size or variability in disease presentation, and future investigations in stratified or larger populations will be necessary to clarify these relationships.

In summary, this study offers a comprehensive analysis of JAK/STAT pathway activation in UC patients, emphasizing phosphorylation patterns and their functional correlations. Utilizing data from a real-world clinical cohort, we identified significant increases in the phosphorylation of six pathway components—JAK2, JAK3, TYK2, STAT1, STAT3, and STAT4—despite marked inter-patient variability in their activation. Notably, significant correlations between the activation of specific JAK kinases (JAK2, JAK3, TYK2) and STAT3 suggest a coordinated effort to amplify cytokine-driven inflammatory responses. Furthermore, the observed sex-specific differences, with enhanced activation of the JAK2/STAT3 axis in male UC patients, shed light on their elevated risk for colorectal cancer development. These findings reinforce the need to consider sex as a biological variable in therapeutic strategies to establish a foundation for developing personalized therapeutic approaches in inflammatory diseases.