Role of interferon regulatory factors in predicting the prognosis of Crimean-Congo hemorrhagic fever

Introduction

Crimean-Congo hemorrhagic fever (CCHF) is induced by a negative-sense, single-stranded RNA virus. Despite ongoing research, the intricate mechanisms underlying its pathogenesis remain incompletely understood. The immune system, however, emerges as a pivotal player in the progression of CCHF. Saksida’s findings in 2010 underscored that a delayed and downregulated immune response could potentially lead to heightened viral replication and increased spread, resulting in severe outcomes [1]. This notion gains further support in a study that elucidated how CCHF virus (CCHFV) strategically delays the activation of the innate immune response, thereby facilitating rapid viral dissemination [2]. Highlighting the critical importance of understanding the pathogenesis of CCHF, it has been emphasized the role of the interferon response in controlling CCHFV replication. A notable observation was made in a STAT-1 knockout mouse model, where the absence of this key signaling molecule led to a cytokine storm and delayed activation of immune cells [3]. This emphasizes the significance of comprehending the underlying mechanisms of CCHF pathogenesis, as it not only sheds light on the dynamics of the disease but also provides valuable insights for the development of targeted therapeutic interventions.

Interferons (IFNs) constitute a protein family crucial for antiviral defense, cell growth regulation, and immune activation. Their transcription is facilitated by interferon regulatory factors (IRFs), which form a family of nine members, each serving diverse functions in response to external stimuli [4], 5]. Both IRFs and IFNs play a central role in antiviral defense and immunoregulation [6]. IFNs, being essential proteins in the immune response against viral infections, are released by cells in reaction to viral invasion, thereby inhibiting viral replication and spread. IRFs, as transcription factors, regulate the expression of IFNs and other genes involved in the antiviral response. Activation of IRFs has been observed during human coronavirus infections, including human coronavirus 229E, OC43, and severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) [7]. However, no previous studies have examined the IFNs and IRFs in patients with CCHF.

This study endeavors to augment the current understanding of the pathogenesis of CCHF. Focusing on the intricate interplay between CCHF, IFNs, and IRFs, our investigation aims to shed light on the intricate mechanisms that drive the progression of the disease. Through this exploration, we aspire to provide valuable insights that can inform the development of more effective therapeutic strategies.

Materials and methods

Results

A significant difference was observed in the levels of serum IRF-1, IRF-2, IRF-3, IRF-4, IRF-7, IFN-alpha, and IFN-beta between patient and healthy control groups. As shown in Table 2 and Figure 1, the patient group exhibited higher levels of all parameters except IRF-1. Furthermore, significant differences were detected in various markers between patients who survived and those who died, including BUN, creatinine, AST, LDH, CK, PT, aPTT, INR, fibrinogen, D-dimer, white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), platelet (Plt), ferritin, and IL-6. However, no statistically significant differences were found in IRF-1, IRF-2, IRF-3, IRF-4, IRF-7, IFN-alpha, and IFN-beta levels between patients who survived and those who died. These results are shown in detail in Table 3. As shown in Table 4, notably, a negative correlation was identified between IRF-1 and the SGS. In patients with a severe clinical course, IRF-7 levels were higher compared to mild and moderate groups, while IRF-1 levels were lower in the severe group than in the mild group. Other clinical parameters did not show significant differences among mild, moderate, and severe groups. The strongest and positive correlations between interferons and IRFs were observed between IFN-beta and IRF-4 (Spearman’s rho=0.738) and IFN-beta and IRF-7 (Spearman’s rho=0.699) (Figure 2). Positive correlations were observed between IFN-alpha and lymphocyte count, IRF-1 and platelet count, as well as fibrinogen. Negative correlations were found between IRF-1 and BUN, ALT, AST, LDH, CK, aPTT, and ferritin. Additionally, IRF-2 showed a positive correlation with aPTT, and IRF-3 had a positive correlation with lymphocyte count. These results are expressed in Figure 3.

Figure 1:

Violin plots of interferons and IRFs in CCHF patients and controls. CCHF, Crimean-Congo hemorrhagic fever; IFN, interferon; IRF, interferon regulatory factor.

Figure 2:

Scatter plots showing the relationships between interferons and IRFs in CCHF patients. Spearman correlation analysis was performed. *p<0.001. CCHF, Crimean-Congo hemorrhagic fever; IFN, interferon; IRF, interferon regulatory factor.

Figure 3:

Heatmap graphs of Spearman correlation coefficients among interferons, IRFs, and laboratory parameters in CCHF patients. ALT, alanine aminotransferase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CCHF, Crimean-Congo hemorrhagic fever; CK, creatine kinase; Hgb, hemoglobin; IFN, interferon; IL, interleukin; INR, international normalized ratio; IRF, interferon regulatory factor; LDH, lactate dehydrogenase; Lym, lymphocyte; Neu, neutrophil; Plt, platelet; PT, prothrombin time; WBC, white blood cell.

Discussion

IFN-alpha and beta are produced by infected cells in viral infections and trigger a cascade of signals leading to the activation of numerous interferon-stimulated genes (ISGs). These ISGs encode proteins with various antiviral functions, including hindering viral replication and bolstering the immune response [9], 10]. The presence of certain gene polymorphisms in IFN-alpha has been linked to susceptibility to viral infections, including CCHF [10]. Additionally, the activation of interferon-stimulated genes during CCHF infection suggests the involvement of both type I and II interferon-mediated antiviral mechanisms [11]. Research has shown that type I interferon effectively inhibits CCHF virus in human cells, and studies in mice lacking the interferon α/β receptor have highlighted the importance of interferon signaling in controlling viral loads and inflammation [12]. Despite these insights, clinical trials specifically investigating the effect of IFN-alpha and beta on CCHF are lacking, representing a significant gap in knowledge. In this study, which fills this gap, we observed increased levels of IFN-alpha and IFN-beta in patients with CCHF compared to healthy individuals. On the other hand, our analysis did not reveal statistically significant differences between surviving and non-surviving patients. Additionally, no significant correlations were found among IFN-alpha, beta levels, coagulopathy indicators, and hepatic injury biomarkers. The upregulation of IFN-alpha and beta is believed to have a significant impact on the immune response, bolstering the body’s antiviral defenses in cases of CCHF. Understanding the complex interplay between interferons, viral pathogenesis, and clinical outcomes holds promise for the development of targeted therapies. Our study contributes to this understanding by demonstrating elevated levels of IFN-alpha and beta in CCHF patients. Despite this observation, we did not find a significant correlation between these biomarkers and coagulopathy or hepatic biomarkers. This suggests that while IFN-alpha and beta likely contribute to the pathophysiology of CCHF by bolstering the immune response, they may not directly influence the development of coagulopathy or hepatic dysfunction in affected individuals.

In this study, elevated levels of IRF-2, 3, 4, and 7 were detected in patients compared to healthy controls. Moreover, we found a weak positive correlation between IRF-2 and aPTT levels. Previous research has explored the involvement of IRFs in various viral infectious diseases [13], [14], [15], [16]. Ousman et al. observed increased expression of the IRF-7 gene in a mouse model of central nervous system viral infection [13]. Ainsua-Enrich demonstrated the regulatory role of IRF4 in T-cell differentiation during influenza infection [14]. Li et al. identified a protective function of IRF-2 in viral neuroinfection [15]. Furthermore, IRF-3 has been shown to induce apoptosis in virus-infected cells, thereby limiting virus propagation within the host [16], 17]. IRFs are a family of transcription factors that play a central role in regulating the expression of interferons and other genes involved in the immune response. Various pattern recognition receptors (PRRs) detect viral nucleic acids and activate signaling pathways that lead to the production of IFN-alpha and IFN-beta [13], 18], 19]. This activation involves the recruitment and activation of IRF proteins. These IRFs translocate to the nucleus and bind to specific DNA sequences in the promoters of the IFN-alpha and IFN-beta genes, leading to their transcription. IRFs are critical regulators of the IFN response to viral infections. They control the induction, amplification, and regulation of IFN-alpha and IFN-beta production, as well as the triggering signaling pathways that mediate the antiviral effects of these cytokines. Therefore, we think that IRF-2, 3, 4, and 7 have a role in the expression of IFN alpha and beta expressions and these IRFs are crucial for antiviral response in CCHF.

It was notable that patients showed decreased levels of IRF-1 compared to the healthy control group. Negative associations were observed between IRF-1 and various biomarkers, including BUN, ALT, AST, LDH, CK, aPTT, and ferritin. Additionally, IRF-1 exhibited a negative correlation with SGS. Moreover, IRF-1 levels were lower in the severe group compared to the mild group. IRF-1 plays a crucial role in the innate immune response against viral infections [20]. It’s known to activate antiviral defenses and induce IFN-β and other protective genes during viral infections [20]. Moreover, IRF-1 has been found to protect against diseases caused by various viruses, such as Chikungunya and vesicular stomatitis virus, by inhibiting viral replication [21], 22]. In accordance with our study, previous studies have also highlighted diminished IRF-1 expression in viral infections. For instance, hepatitis C virus core proteins, human immunodeficiency virus-1 transactivator protein, and rotavirus NSP1 proteins have been shown to suppress IRF-1 expression or degrade it, affecting its antiviral activity [23], [24], [25]. The decreased IRF-1 levels observed in patients with CCHF could stem from various factors. It’s possible that the CCHF virus actively suppresses IRF-1 to evade the immune response or the infection disrupts the interferon pathway, where IRF-1 plays a vital role. Additionally, individual factors such as underlying health conditions or genetic variations might influence IRF-1 production during viral infections. Further research is necessary to understand the impact of CCHF infection on IRF-1 and the interferon response pathway. Overall, these findings suggest that the diminished levels of IRF-1 in CCHF patients may play a role in the pathogenesis and prognosis of the disease, potentially contributing to immune suppression or dysregulation during infection. Targeted interventions, particularly those aimed at modulating IRF-1, could be beneficial in reducing the severity of disease symptoms and lessening the overall impact of the illness.

In conclusion, our findings shed light on the complex interplay between the immune response and CCHFV. While type I interferons, IFN-alpha and beta, and IRF-2, 3, 4, and 7 contribute to the immune response against CCHF, their direct impact on patient prognosis might be limited. Our study’s intriguing finding of diminished IRF-1 levels in CCHF patients warrants further investigation. This downregulation of IRF-1 could potentially play a significant role in the disease’s pathogenesis. Insufficient IRF-1 might lead to impaired IFN-alpha and beta production, ultimately resulting in compromised antiviral immunity and dysregulation of the immune response during CCHF infection. These findings open new avenues for exploring targeted interventions. By modulating IRF-1 function, we might be able to develop therapeutic strategies to mitigate disease severity and improve patient outcomes in CCHF.