Therapeutic Potential of The Low-dose IL-2 Through Targeting the Th17/Treg Axis in Primary Biliary Cholangitis Mice

doi:10.21203/rs.3.rs-2366188/v1

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Therapeutic Potential of The Low-dose IL-2 Through Targeting the Th17/Treg Axis in Primary Biliary Cholangitis Mice

https://doi.org/10.21203/rs.3.rs-2366188/v1

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Journal Publication

published 26 Feb, 2024

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Background/aims

: Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease. The imbalance of Th17/Treg cells has been reported in PBC patients. low-dose IL-2 can alleviate disease severity through modulating CD4 + T cell subsets in patients with autoimmune diseases. Hence, the present study aimed to examine the effects and mechanism of low-dose IL-2 in PBC mouse models.

Methods

PBC models were induced in female C57BL/6 mice by two immunizations with 2OA-BSA at two-week intervals, and poly I: C every three days. PBC mice were divided into the IL-2 treated and untreated groups and low-dose IL-2 was injected at three different time points. Th17 and Tregs were analyzed by flow cytometry, and the related cytokines were analyzed by ELISA. Liver histopathology was examined by H&E and immunohistochemical staining.

Results

Twelve weeks after modeling, the serum AMA was positive and the ALP was significantly increased in PBC mice (P༜0.05). The pathology showed lymphocyte infiltration in the portal area, damage, and reactive proliferation of the small bile duct (P༜0.05). The flow cytometric showed the imbalance of Th17/Treg cells in the liver of PBC mice, with decreased Treg cells, increased Th17 cells and Th17/Treg ratio (P < 0.05). After the low-dose IL-2 intervention, biochemical index and liver pathologies showed improvement at 12 weeks. Besides, the imbalance of Th17 and Treg cells recovered. Public database mining showed that Th17 cell differentiation may contribute to poor response in PBC patients.

Conclusion

Low-dose IL-2 can significantly improve liver biochemistry and pathology by reversing the imbalance of Th17 and Treg cells, suggesting that it may be a potential therapeutic target for PBC.

Primary biliary cholangitis

Interleukin 2

Treg cells

Th17 cells

Mouse model

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease that is characterized by progressive nonsuppurative cholangitis[1]. Without pharmacological interventions, the patients are projected to progress to liver cirrhosis and even hepatocellular carcinoma. At present, the pathogenesis of PBC is not well understood, but it seems to correlate with environmental, genetic, and immunological factors[2]. It is reported in the literature that the prevalence rate of PBC was from 21.7 to 39.2 per 100,000 people from 2004 to 2014[3].

Currently, ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) is the approved first- and second-line drugs for patients with PBC[4]. However, there are certain shortcomings, such as poor clinical response and adverse reactions. The other regimens for PBC are glucocorticoids, immunosuppressants, and monoclonal antibodies, but their efficacy is controversial [5]. Therefore, exploring its early occurrence and developmental mechanism, and finding novel therapeutic targets are key for the treatment of PBC.

Disorders of the immune system exert an important role in the pathogenesis of PBC. The Th17 and Treg cells were a pair of cells with similar developmental pathways and opposite functions, and their imbalance is often associated with autoimmune diseases[6]. As an autoimmune cholestatic liver disease, the high Th17 and low Treg T cell subsets are also observed in PBC patients[7]. Not only that, the same tendency was observed in the PBC mouse model (IL-2 Rα-/- mice)[8]. A recent study also indicated that bile acid metabolites might participate in the differentiation of Th17/Treg cells [9]. Therefore, correcting the Th17/Treg imbalance could be an effective approach for autoimmune diseases, such as PBC.

In recent years, low-dose IL-2 has been employed successfully in patients with autoimmune diseases and has achieved good clinical results. A clinical trial about systemic lupus erythematosus was conducted to evaluate the safety and efficacy of low-dose IL-2 therapy. Meanwhile, it was also confirmed that IL-2 might participate in the regulation of Th17/Treg rather than Th1/Th2 cell balance[10]. With increasing research on the immunomodulatory mechanism of IL-2, it is currently becoming more widely used in a variety of autoimmune disorders. Recent clinical trials have confirmed that IL-2 appears safe and effective in 11 autoimmune diseases, including ankylosing spondylitis (AS), rheumatoid arthritis (RA), and so on[11].

The effect of IL-2 on Th17/Treg has not been studied in PBC, but it has been reported in other autoimmune liver diseases. One study showed that IL-2 reduces the ALT levels and increases Treg cell induction in a late autoimmune hepatitis mouse model (emAIH)[12]. Another study using the primary sclerosing cholangitis mouse model (MDR2-/-) revealed that IL-2 attenuates related chronic biliary injury and prevents fibrosis progression by increasing the number of Treg cells[13]. These suggest that IL-2 is a potentially powerful means of treatment of autoimmune liver disease. Therefore, the present study is to investigate the potential role of Th17/Treg cells in the pathogenesis of PBC and the regulatory role of IL-2 in the PBC mouse model.

2.1 Animal experiments

4–6 weeks old female C57BL/6 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology (Beijing, China), and raised in a specific pathogen-free (SPF) facility at the Animal Center of Peking University People’s Hospital. The animal house was well-ventilated with 12-hour light/dark cycles in 25–30°C ambient temperatures. The study was approved by the Ethics Committee of Peking University People’s Hospital (2020PHE081) and was performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. The study followed ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines).

PBC was induced by two immunizations with 2-nonynoic acid (2OA-BSA) at two-week intervals. The first injection was prepared with the same volume of complete Freund’s adjuvant, and the second was administered with incomplete Freunds adjuvant, and the final concentration of the reagent was 100 µg/100µL. Polyinosinic polycytidylic acid (poly I: C) was injected i.p. every three days (5mg/kg) (Supplementary Fig.S1). [14]. The control group was injected with PBS instead of 2OA-BSA and poly I: C. PBC mice were divided into the treated and untreated groups, and low-dose IL-2 (Supplementary Fig.S2) was injected s.c. every three days at three-time points, three days before modeling, four and eight weeks after modeling in the treated group, and the untreated group was replaced with saline (Supplementary Fig.S3).

2.2 Flow cytometry

Flow cytometric analysis of Th17 and Treg populations was performed from mononuclear cells (MNCs) in the liver. Livers were removed and single-cell suspensions were prepared and purified by Percoll density gradient centrifugation. Cell suspensions with 1×10⁶ cells were stained in dark. After surface staining of CD3, CD4, and CD25 and permeabilization/fixation, cells were stained for intracytoplasmic IL-17A and intranuclear Foxp3. In addition, the levels of IL-17A were detected after in vitro activation with a cell activation cocktail and blocking with brefeldin. Th17 cells were defined as CD3⁺CD4⁺IL-17A⁺ and Treg cells were defined as CD3⁺CD4⁺ Foxp3⁺ (Supplementary Fig.S4, Table.S1).

2.3 H&E and immunohistochemistry

For Hematoxylin and eosin (H&E) staining, the liver sections were paraffin-embedded, dewaxed, rehydrated, and stained. H&E staining was conducted according to routine protocols, and the images were captured by an inverted microscope. Liver pathological scores were evaluated according to the scoring system adapted from the previous literature by two pathologists[15]. The pathological scoring ranges from zero to eleven, including biliary duct involvement (0–3), biliary epithelial proliferation (0–4), and mononuclear leucocytic infiltration (0–4).

For immunohistochemistry, the liver sections were incubated overnight at 4°C with the primary antibodies (CD4, CD8, CK-19, Rorγt), and with secondary antibodies for 30min at 37°C.

2.4 Enzyme-Linked Immunosorbent Assay

The levels of IL-17A, IL-10, TGF-β, and PDC-E2 antibodies in the mouse serum were measured by ELISA. The operations were conducted strictly according to the kit instructions. In brief, the diluted detection antibody was added to diluted sera and incubated at room temperature for 1.5h. After plates were washed 5 times, horseradish peroxidase (HRP)-conjugated streptavidin was added to each well followed by incubation at room temperature. After incubation, the substrate solution was added and measured at 450nm.

2.5 Data sources and differentially expressed gene (DEGs) analysis

RNA-sequencing dataset GSE79850, including UDCA responders (n = 7), UDCA non-responders (n = 9) and non-liver disease controls (n = 9) was retrieved from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/).

In the GSE79850 dataset, we analyzed DECs by using the “llimma” package in the software R. DEGs were defined as the absolute value of log2FC ≥ 1 as well as the false discovery rate (FDR) < 0.05[16]. Gene ontology (GO) and pathway enrichment analysis (Kyoto Encyclopedia of Genes and Genomes (KEGG)) were implemented using the “ClusterProfiler” package, and the threshold for significance was a p-value less than 0.05.

2.6 Statistical analysis

Data were expressed as mean ± standard deviation and were analyzed using student t-tests or one-way ANOVA by GraphPad Prism 7 software (GraphPad Software Inc., San Diego, CA, USA). A P-value of less than 0.05 was considered to be statistically significant.

3.1 General conditions and liver pathologies of the PBC mouse model

During modeling, there were no significant differences in the general conditions of mice, including eating, drinking, body weight, activity, and coat color (Fig. 1A). No signs of jaundice were observed in the PBC mouse model. ALP levels (119.10 ± 6.20 vs 64.67 ± 27.72 U/L, P༜0.05) were significantly higher in the PBC mouse model. No significant difference was observed in ALT (72.67 ± 13.77 vs 72.6 ± 22.64 U/L, P༞0.05) and AST (118.3 ± 9.16 vs 116.7 ± 10.67 U/L, P༞0.05) between the model and control groups (Fig. 1C). Besides, the serum anti-PDC-E2 could be detected in the PBC mouse model (Fig. 1B).

After injection of 2OA-BSA and poly I: C, there was visible evident inflammatory infiltration in the portal tracts, massive bile duct destruction, and marked bile duct proliferation in the PBC mouse model (Fig. 1D). The relevant pathological change could be observed 4 weeks after modeling, and with the extension of modeling time, the liver pathologies gradually aggravated. A significant difference in liver pathology scores was calculated (0 vs 6.3 ± 1.23, P༜0.05, Fig. 1E).

3.2 The disturbance of Th17/Treg balance in the liver in the PBC mouse model

Th17 and Treg cells infiltrating the liver were analyzed by flow cytometry for model and control group mice. Compared with control mice, Th17 cell populations were significantly increased in PBC mice (0.38 ± 0.05 vs 0.84 ± 0.09%, P༜0.05), while the number of Treg cells was decreased in PBC mice (5.70 ± 0.53 vs 4.32 ± 0.29%, P༜0.05) (Fig. 2A). The imbalance of Th17/Treg cells was progressively aggravated with the extension of modeling time. We also tested the localization of Th17 cells in the liver by immunohistochemical staining and observed that Th17 cells infiltrated the section of portal tracts, mainly in the periphery of bile duct epithelial cells (Fig. 2B). With the aggravation of hepatic inflammation, the number of Th17 cells increased suggesting that Th17 cells may initiate and aggravate the progression of bile duct destruction.

Besides, we examined the Th17- and Treg-related cytokines in mice sera with ELISA. The results suggested that compared with control mice, the serum levels of IL-17A increased significantly in the PBC mice (0 vs 4.55 ± 0.60 ng/ml, P༜0.01). While the Treg-related cytokines, TGF-β (51.78 ± 0.89 vs 36.48 ± 13.00 ng/ml, P = 0.19) and IL-10 (0.89 ± 0.02 vs 0.97 ± 0.02 ng/ml, P = 0.49), there was a decreasing tendency in PBC mice, although the difference did not reach statistical significance (Fig. 2C). The above results suggested that the level of Th17- and Treg-related cytokines have the same changing trend as Th17 and Treg cells.

3.3 Biochemical and pathological changes after Low-dose IL-2-treated in PBC mouse model

After 4 weeks of modeling, the PBC mice were treated with low-dose IL-2 until the end of 12 weeks. It was observed that ALP (119.10 ± 6.20 vs 82.36 ± 12.50 U/L, P = 0.04) was significantly decreased in the treated mice compared with PBC mice, whereas the ALT and AST did not (Fig. 3C). Treatment with low-dose IL-2 significantly reduced inflammatory infiltration in the portal area and improved the destruction and proliferation of small bile ducts. The Liver pathological scores of the low-dose IL-2 treatment mice decreased significantly (6.00 ± 1.00 vs 2.50 ± 0.50, P < 0.05) (Fig. 3E).

In addition, flow cytometry showed that the number of liver infiltrating Treg cells increased while the number of Th17 cells decreased, and the imbalance of Th17 and Treg cells recovered (Fig. 3A). ELISA showed that the level of serum IL-17A decreased (4.55 ± 0.60 vs 19.28 ± 0.39 ng/ml, P = 0.02), TGF- β levels increased (36.48 ± 13.00 vs 51.48 ± 1.87 ng/ml, P = 0.32) (Fig. 3D). The infiltration and distribution of Th17 cells in the liver were observed by immunohistochemistry and showed that the number of Th17 cells in the portal area decreased (Fig. 3B).

3.4 The effect of low-dose IL-2 at different points differed in each group

The serum biochemical indexes, the degree of liver inflammatory infiltration, and the destruction and proliferation of bile duct were compared in the mouse models treated with low-dose IL-2 at three-time points, including three days before modeling, four and eight weeks after modeling. The results showed that compared with the other two groups, the improvement of biochemical indexes ALP in the three days before modeling was limited (94.50 ± 14.64 vs 68.40 ± 13.79 vs 62.40 ± 16.22 U/L, P < 0.05) (Fig. 4A), and the improvement of inflammation in the portal area, and the degree of bile duct destruction and proliferation was not obvious compared with other two groups (5.22 ± 0.52 vs 3.67 ± 0.33 vs 3.33 ± 0.44, P < 0.05) (Fig. 4B, C). The above results suggest that compared with the post-treated group, the improvement of mice in the pre-treated group is limited.

3.5 Increased Th17 cell populations were associated with UDCA suboptimal response in PBC patients

Analysis was performed on GSE79850 in the GEO database, including 8 non-liver disease patients, 7 UDCA responders, and 9 UDCA non-responders. The expression of the key transcription factor RORC of Th17 cells was observed and found that the expression of RORC in PBC patients was significantly higher than in non-liver disease patients (6.36 ± 0.42 vs 8.49 ± 0.10, P༜0.01), suggesting that the liver microenvironment of PBC patients was disordered and Th17 cells were significantly increased (Fig. 5A).

Besides, GO and KEGG analyses were also performed on differentially expressed genes between UDCA responders and non-responders. GO analysis showed that among the 10 signal pathways enriched with more than 2 times of DEGs, adaptive immune response, immune receptor recombination constructed by immunoglobulin superfamily domain, humoral immunity, lymphocyte-mediated immunity, and other pathways were related to immunity, suggesting that immune factors may be the cause of poor UDCA response (Fig. 5B). KEGG analysis of the enrichment pathway showed that Th17 cell differentiation was involved (Fig. 5B).

The liver is a multifaceted organ with a unique immune constitution, which has the functions of phagocytosis, immune defense, and immune regulation. PBC is characterized by the non-suppurative destruction of small bile ducts, and multiple mechanisms are involved in the destruction of bile duct epithelial cells, in which immune response plays an extremely important role[17]. Different immune cell subsets are directly or indirectly involved in the destruction of bile ducts. Through GO and KEGG analysis of differentially expressed genes in the liver of PBC patients with good or poor UDCA response in GSE79850, it is found that enriched genes are associated with the immune pathways, suggesting that regulating immunity may be the key factor to improve the therapeutic efficacy in PBC patients.

The normal immune function of the body depends on the balance between proinflammatory factors and immunosuppressive factors. Th17 and Treg cells are a pair of immunomodulatory cells with different functions but closely related differentiation. In the presence of IL-6, with a low concentration of transforming growth factor β (TGF-β), initial CD4 + T cells differentiate into Th17 cells and inhibit Foxp3 expression[18], while a high concentration of TGF-β can inhibit the expression of IL-23R and promote the differentiation of Treg. Th17/Treg cells play a bipolar role in inflammation, promoting inflammatory response and anti-inflammatory actions[19]. When the immune proinflammatory effect is too strong, the balance of the body is broken, thus inducing the occurrence of some diseases[20]. Th17 cells and Th17-related cytokines play a vital role in many diseases, such as autoimmune liver disease, alcoholic liver disease, viral liver disease, and other diseases[21]. The imbalance of Th17/Treg cells has also been confirmed in PBC patients[22], suggesting that Th17 and Treg cells are involved in the occurrence and development of PBC. In addition, KEGG analysis of differentially expressed genes in patients with good and poor UDCA responses was also found to be related to the Th17 differentiation pathway, suggesting that Th17 cells may be potential therapeutic targets for PBC.

In previous studies, regulating the imbalance of Th17/Treg cells was identified as a potential therapeutic target for diseases. Arsenic trioxide[23], hypoxia-inducible factor (HIF-1α) [24], IL-21[25], zinc [26], and other drugs have been proved to regulate Th17 /Treg cells in different diseases, playing a beneficial role in improving disease models.

Currently, low-dose IL-2 has been proved to play a role in the treatment of autoimmune diseases by regulating Th17 and Treg cells[10]. There is a high-affinity IL-2 receptor on the surface of Treg cells, it activates the downstream JAK1 and JAK3 after binding with IL-2. And then promoting the activation of STAT5 after phosphorylation and maintaining the stability and function of Treg. IL-2 inhibits Th17 cells by promoting the phosphorylation of STAT5 and reducing the phosphorylation of STAT3. Activated STAT5 competes with STAT3 to bind the IL17A site, thereby inhibiting the transcription of IL17A and playing a negative regulatory role[27].

IL-2 has not been reported in PBC, but in other autoimmune liver diseases have been studied, such as autoimmune hepatitis mouse model (emAIH) [12]and primary sclerosing cholangitis (PSC) preclinical mouse model[13]. The results showed that IL-2 can improve biochemical indicators and reduce bile duct injury, suggesting that IL-2 may be a potential treatment. Besides, it should be noted that different doses of IL-2 may play different physiological roles. Low-dose IL-2 plays a role in regulating Treg and Th17, while high-dose IL-2 may activate Teff cells and promote immune activation[28]. Therefore, it is particularly important to select the appropriate dose of IL-2.

In our previous experiments, we treated C57BL/6 mice with 10000 U, 30000 U, and 100000 U IL-2, and observed the changes of Th17/Treg and liver pathological changes. It was found that 10000 U dose of IL-2 had no significant effect on Treg and Th17 cells, and 30000 U and 100000 U doses of IL-2 had similar regulatory levels on immune cells, but 100000 U dose of IL-2 had inflammatory cell infiltration in the liver of mouse models. To rule out the impact of IL-2 on liver pathology, Therefore, a 30000 U dose of IL-2 was finally selected to treat the PBC mouse model. After low-dose IL-2 treatment, in the PBC mouse model, the unbalanced Th17/Treg cells have recovered, and the expression of related cytokines tends to be consistent with that of cells. In addition, the biochemical indexes and pathological changes of the model were improved.

Mice treated with low-dose IL-2 at different time points have different effects on the biochemical indexes and pathological changes. Compared with the post-treated group, the improvement of mice in the pre-treated group is limited. The different effects of low-dose IL-2 at different time points may be that the preventive use of IL-2 played a physiological function of stimulating T cell proliferation and activation in advance, resulting in the activation of the mouse immune system. Poly I: C is an inducer of type-1 interferon (IFN). In clinical practice, it has been found that type-1 interferon induces autoimmune characteristics in patients with viral hepatitis [29], while 2OA-BSA, as a high-affinity reactant of AMA [30], can also lead to immune system disorders. The "double hit" of IL-2 and modeling reagents on the immune system may lead to poor improvement of the PBC mouse model by preventive administration. While the use of low-dose IL-2 in the middle and late stages of modeling had a therapeutic effect by improving the imbalance of Th17 and Treg cells.

In conclusion, the disorder of Th17 and Treg cells is involved in the process of PBC disease. Low dose IL-2 could suppress the occurrence and progression of PBC disease by improving the imbalance of Th17 and Treg cells, and significantly improve liver biochemistry and pathology, suggesting that it may be a potential therapeutic target for PBC.

PBC: primary biliary cholangitis; UDCA: ursodeoxycholic acid; OCA: obeticholic acid; 2-OA: 2-nonynoic acid; PSC: primary sclerosing cholangitis.

Ethical Statement

The study was approved by the Ethics Committee of Peking University People’s Hospital (2020PHE081) and was performed in accordance with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals. The study followed ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines).

Consent for publication

No applicable.

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author, who received permission from Ethics Committee of Peking University People’s Hospital.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work has been supported by the National Natural Science Foundation of China (grant number 82000557)

Author contributions

FB and XY supervised and conceived the project. WZ, LZ, ZJ and HL designed and carried out most of the experiments. JR, WX and CD conducted experiment instruction.

Acknowledgements

No applicable.

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Therapeutic Potential of The Low-dose IL-2 Through Targeting the Th17/Treg Axis in Primary Biliary Cholangitis Mice

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Abstract

Background/aims

Methods

Results

Conclusion

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1 Introduction

2 Materials And Methods

2.1 Animal experiments

2.2 Flow cytometry

2.3 H&E and immunohistochemistry

2.4 Enzyme-Linked Immunosorbent Assay

2.5 Data sources and differentially expressed gene (DEGs) analysis

2.6 Statistical analysis

3 Results

3.1 General conditions and liver pathologies of the PBC mouse model

3.2 The disturbance of Th17/Treg balance in the liver in the PBC mouse model

3.3 Biochemical and pathological changes after Low-dose IL-2-treated in PBC mouse model

3.4 The effect of low-dose IL-2 at different points differed in each group

3.5 Increased Th17 cell populations were associated with UDCA suboptimal response in PBC patients

4 Discussion

5 Conclusion

Abbreviations

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References

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