The Inhibition of Bruton Tyrosine Kinase Alleviates Acute Liver Failure via Downregulation of NLRP3 Inflammasome Open Access

Acute liver failure (ALF) is characterized by rapid and severe liver injury, coagulation disorders, and hepatic encephalopathy as a consequence of massive hepatocyte death (1). As an important organ, a dysfunctional liver gradually leads to multiple organ dysfunction syndromes with high mortality. ALF is induced by various causes, such as viral infections (hepatitis B or C or others), alcohol addiction, and drug abuse (2, 3). Dysfunction in the liver immune microenvironment is recognized as the major cause of ALF (4–7). Death of hepatocytes on the one hand activates the innate immune response, which is involved in various liver-resident immunocytes; on the other hand, immune chemokines recruit massive immune cells to the liver, resulting in the imbalance between effector and regulatory immunocytes in the local immune microenvironment (8, 9). The modulation of innate immune function is therefore a potential strategy in the treatment of ALF (7). Currently, there are no practicable therapeutic approaches for ALF other than an artificial liver support system (ALSS) or liver transplant, which are limited by the lack of donor plasma or organs. Therefore, it is necessary to find alternative strategies for the treatment of ALF (10, 11).

Bruton tyrosine kinase (Btk) is an important member of Tec family, along with Tec, Itk, Rlk, and Bmx (12). It was first identified as a key regulator in almost all stages of B cell development, including proliferation, maturation, differentiation, apoptosis, and cell migration (13, 14). Patients with Btk deficiency are prone to bacterial infections (15, 16). Further studies revealed that Btk directly activates multiple signaling pathways, which induced the production of various immune components, such as ILs, IFNs, TNFs, and so forth. Therefore, Btk is also identified as an emerging player in the regulation of the innate immune response (17). However, little research has focused on the possible roles of Btk in the regulation of the liver immune microenvironment, and it is still unknown whether Btk has a role in the pathogenesis of ALF.

The NOD-, LRR- and pyrin domain–containing protein 3 (NLRP3) inflammasome has been demonstrated as one of the most important players in the innate immune response, which is closely related to various liver inflammatory diseases (18–22), including ALF (23–25). Previous studies have confirmed that Btk is also involved in the activation of the NLRP3 inflammasome, an integral functional complex that consists of NLRP3, NLRP3 adaptor, and apoptosis-associated speck-like protein containing a CARD (ASC) (26, 27). On the basis of the theory described above, we speculated that interrupting the signal transduction between Btk and the NLRP3 inflammasome might provide a new strategy in the treatment of ALF.

In this study, we investigated whether Btk-specific inhibitors could protect mice from experimental ALF. We analyzed the effects of Btk inhibition on inflammatory signaling pathways, especially the NLRP3 inflammasome and the production of proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in ALF models. Furthermore, we also observed the dynamic changes of Btk expression levels in the PBMCs of patients with ALF after ALSS treatment, revealing the vital role of Btk in the process of ALF.

All blood samples were obtained from patients who were diagnosed with ALF in The First Affiliated Hospital of School of Medicine, Zhejiang University, and who accepted ALSS treatment for one or more times. The whole blood from patients with ALF was collected before and after ALSS treatment. The study was approved by the clinical research ethics committee of The First Affiliated Hospital, College of Medicine, Zhejiang University.

Human blood from healthy donors or patients with ALF was collected, and PBMCs were isolated by Ficoll density gradient centrifugation according to an instruction manual. Intracellular p-Btk was stained with FITC-ligated CD3 Ab (BioLegend) and Alexa Fluor 647 anti-Btk (pY223)/Itk (pY180) (BD Biosciences) and detected by FCM using the BD LSRFortessa device (BD Biosciences).

The PBMCs from healthy donors were separated via a standard protocol using CD14 MicroBeads (MACS; Miltenyi Biotec), and the selected CD14⁺ monocytes were resuspended with 90% RPMI 1640 and 10% FBS and divided at a density of 10,000 cells per well into four groups. The culture supernatant was collected 6 h after LPS stimulation with or without a pretreatment of Btk inhibitor. An ELISA was used to detect the concentration of inflammatory factors in the culture supernatant following the operating manual.

Mice were injected i.p. with 2 ml Fluid Thioglycollate medium (EMD Millipore). After 3–5 d, peritoneal cells were collected by washing the peritoneal cavity with ice-cold DMEM two times. Then the peritoneal cells were centrifuged and resuspended with 90% DMEM and 10% FBS and seeded into a 6-well plate. Three hours later, the medium and nonadherent cells were removed, and PDMs were obtained, then fresh culture medium (90% DMEM plus 10% FBS) was added for the next experiment.

Male C57BL/6 mice aged 6–8 wk were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. and fed a standard laboratory diet and water and acclimated for 1–3 d in a climate-controlled room with a 12-h/12-h light/dark cycle. LPS, d-galactosamine (D-GalN), and Con A were obtained from Sigma-Aldrich and diluted in sterile normal saline. In the LPS/D-GalN model, mice were challenged with a nonlethal dose of 10 μg/kg LPS and 400 mg/kg D-GalN or a lethal dose of 10 μg/kg LPS and 800 mg/kg D-GalN i.p. In the Con A model, mice were challenged with a nonlethal dose of 20 mg/kg Con A or a lethal dose of 30 mg/kg Con A via tail i.v. injection. Ibrutinib (PCI-32765) and acalabrutinib (ACP-196) (both are Btk inhibitors) were purchased from Selleckchem and dissolved in DMSO. The gavage solvent was a mixture of 2% DMSO, 30% polyethylene glycol 400, 2% Tween 80, and double-distilled water. The dosages of acalabrutinib were 0.1 mg/kg, 1 mg/kg, 5 mg/kg, and 10 mg/kg, and the dosages of ibrutinib were 3 mg/kg and 10 mg/kg. The animal studies were approved by the institutional animal care and use committees of The First Affiliated Hospital of School of Medicine, Zhejiang University.

Serum alanine aminotransferase and aspartate aminotransferase levels were measured using an automatic chemical analyzer 7600-100 (Hitachi). Liver tissues were dissected and fixed in 4% paraformaldehyde, and H&E staining was performed via a standard protocol to assess inflammation and necrosis.

Mouse immune cells in the liver were isolated via 33% Percoll gradient centrifugation. The surface markers (CD3, CD19, NK1.1, and CD11b) and intracellular p-Btk/p-Itk were stained by fluorescein-labeled Abs according to the standard operating manual provided by BD Pharmingen and analyzed using a BD LSRFortessa device (BD Biosciences).

Total RNA in liver tissues and cells was isolated using TRIzol reagent, and the concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was generated using 0.5 μg of total RNA in a final reaction volume of 20 μl using the PrimeScript RT reagent kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed with the 7500 Fast Real-Time PCR System and SYBR Premix Ex Taq II (Takara). All experiments were conducted according to the manufacturers’ protocols. The relative gene quantities were calculated and compared with the expression levels of GAPDH.

Radioimmunoprecipitation assay lysate (Beyotime) and protease inhibitors (Roche) were used to lyse cells and protect proteins from degradation at 4°C. Protein concentration was measured by using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Total cell protein (30 µg) was added to 5× SDS-PAGE sample loading buffer (Beyotime) and heated at 100°C for 10 min. The denatured proteins were loaded and separated on SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked with 5% skim milk or 1% BSA in TBST for 1 h at room temperature and then incubated overnight at 4°C with the primary Ab diluted 1:1000 in 5% skim milk or 5% BSA/TBST. These primary Abs included NLRP3 (D4D8T) rabbit mAb, Btk (D3H5) rabbit mAb, phospho-Btk (Tyr223) (D9T6H) rabbit mAb, and GAPDH (14C10) rabbit mAb (all from Cell Signaling Technology). Then the membranes were incubated with goat anti-rabbit secondary Ab conjugated with HRP (Cell Signaling Technology) diluted 1:5000 in TBST at room temperature for 1 h. Protein bands were detected by Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and recorded by the ChemiScope Chemiluminescence imaging system (Clinx Science Instruments Co., Ltd.).

PDMs were seeded on glass discs and cultured overnight. After different stimulations, PDMs were fixed with 4% paraformaldehyde for 20 min. After three washes with PBS, PDMs were treated with 0.3% Triton X-100 at room temperature for 20 min and then blocked in 5% BSA for 30 min at room temperature. Subsequently, PDMs were incubated with ASC/TMS1 (D2W8U) rabbit mAb (Cell Signaling Technology) overnight at 4°C. After three washes with PBS, samples were incubated with goat anti-rabbit IgG (H+L chains) and F(ab′)₂ fragment (Alexa Fluor 488 conjugate) (Cell Signaling Technology) at room temperature for 2 h. Cells were stained with DAPI for 15 min. Images were taken by an SP8 DIVE fluorescence microscope (Leica).

To explore the potential role of Btk in the pathogenesis of ALF, we observed the dynamic changes of Btk during the process of ALF. The change of p-Btk expression in liver immunocytes (CD3⁻) was on a rollercoaster ride during the observation time, showing a rapid increase and then decrease in a short period of time (Supplemental Fig. 1). To observe which cell type was changed most obviously in a lethally dosed LPS/D-GalN-induced ALF model, subgroup analysis in CD3⁻ immunocytes was conducted (Fig. 1A). Our data showed that the proportions of total CD3⁻ cells and their subgroup cells, including B cells, myeloid cells, and NK cells, were changed in a dynamic manner, suggesting that the liver immune microenvironment had undergone tremendous changes in a short period of time. In particular, a large number of myeloid cells were recruited into the liver significantly as the stimulation time progressed (Fig. 1B). We also found that the p-Btk expression of total CD3⁻ cells and myeloid cells were enhanced under LPS/D-GalN stimulation and that its percentage reached a significant difference at ∼30 min (Fig. 1C). Similar experiments were also conducted in the Con A–induced ALF mouse model (Supplemental Fig. 2). Our data showed that in experimental ALF, the phosphorylation level of Btk was increased at an early stage, and we speculated that inhibition of Btk phosphorylation might prevent the development of ALF.

To determine the protective effect, we observed the effect of Btk inhibitors on the survival rate of mice with experimental ALF. Our data showed that acalabrutinib (10 mg/kg) significantly decreased the mortality rate in mice treated with a lethal dose of Con A (30 mg/kg) when compared with the untreated group (p < 0.001). Also, the survival time in the medium-dose group (1 mg/kg) was significantly prolonged compared with untreated groups (p < 0.05). Although there was no significant difference in survival rate between 0.1 mg/kg and untreated groups (p > 0.05), the small-dose group also showed an improved survival rate. Meanwhile, we observed a dose–response relationship in median survival time between the four groups; that is, median survival time increased as the dose of acalabrutinib increased (Con A group, 10.25 h; 0.1 mg/kg group, 12 h; 1 mg/kg group, 16.5 h; 10 mg/kg group, 20 h). Those results suggested that a Btk inhibitor can prolong the survival time in a dose-dependent manner (Fig. 2A). In addition, the effect of acalabrutinib on LPS/D-GalN–induced ALF mice was similar to that on Con A–induced ALF (Fig. 2B). Thus, the above results suggested that Btk inhibitor can prolong the survival time and reduce the mortality of experimental ALF mice.

Subsequently, we analyzed the liver function and pathogenic changes in those acalabrutinib-pretreated mice with ALF. Btk inhibitor protected mice from LPS/D-GalN–induced ALF, showing that the serum levels of both alanine aminotransferase and aspartate aminotransferase in ALF mice were downregulated in the acalabrutinib-pretreated group (Fig. 2C, 2D). Moreover, H&E staining also showed that acalabrutinib significantly alleviated liver injury (Fig. 2E). The results in LPS/D-GalN–induced ALF were repeatable in Con A–induced ALF (Supplemental Fig. 3). Thus, treatment with Btk inhibitor can significantly alleviate liver injury to decrease the mortality of ALF.

On the basis of the above results that Btk inhibitors had protective effects on experimental ALF mice, we checked whether the inflammatory cytokines could be regulated by Btk inhibitors. The results showed that acalabrutinib significantly decreased the protein levels of TNF-α, IL-1β, IL-6, and IL-10 in the culture supernatant of human primary CD14⁺ monocytes stimulated by LPS compared with the untreated group (Fig. 3A).

Our quantitative PCR assay also showed that the mRNA levels of cytokines TNF-α, IL-1β, IL-6, and IL-10 in the liver tissue were significantly increased at 6 h after LPS/D-GalN treatment compared with the normal control group. Acalabrutinib significantly decreased the mRNA levels of TNF-α, IL-1β, and IL-6 (Fig. 3B), which indicated that the Btk inhibitor downregulated the expression of proinflammatory cytokines and improved the immune microenvironment of the ALF model induced by LPS/D-GalN. These results were similar in the Con A–induced ALF model (Fig. 3C). The above results indicated that inhibition of Btk phosphorylation can reduce the production of inflammatory cytokines in liver tissue to maintain the immune balance in the liver microenvironment.

Our previous work confirmed that the NLRP3 inflammasome was involved in the development of ALF (25), and it was clarified that Btk regulated the formation of the NLRP3 inflammasome (26, 27). Hence, we supposed that a Btk/NLRP3 inflammasome signaling pathway participated in the ALF process. To verify this hypothesis, the expression of the NLRP3 inflammasome in the liver of the ALF mouse model was detected at protein and mRNA levels separately. As shown in (Fig. 4A and 4B, the expression of NLRP3 was upregulated significantly at 6 h after LPS/D-GalN injection, and acalabrutinib pretreatment partially inhibited its expression. The mRNA levels of NLRP3 in acalabrutinib pretreatment group were also decreased obviously compared with those in the LPS/D-GalN group (Fig. 4C). However, the expression level of Btk did not show the expected significant difference (Fig. 4D).

Because dual-signal stimulation was required for the full activation of the NLRP3 inflammasome, priming signal LPS and activation signal ATP were applied for NLRP3 inflammasome activation in vitro (28). Our study on mouse PDMs showed that the mRNA expression of NLRP3 inflammasome components (NLRP3, caspase-1, and IL-1β, except ASC) was upregulated by LPS combined with ATP, and this upregulation could be inhibited by acalabrutinib treatment (Fig. 4E). We also analyzed the protein expression of the main NLRP3 inflammasome components in mouse PDMs. In the presence of both LPS and ATP, the expression of NLRP3, cleaved caspase-1, pro–IL-1β, and cleaved IL-1β was increased at the protein level, and acalabrutinib significantly inhibited their expression but had no effect on pro–caspase-1 and ASC (Fig. 4F). Then, we investigated whether Btk inhibition hampered ASC oligomerization during NLRP3 inflammasome activation, a critical step for subsequent caspase-1 activation (29). Our immunofluorescence staining on ASC specks showed that both LPS and ATP were required to induce inflammasome assembly, and the Btk inhibitor hampered ASC oligomerization significantly to affect the assembly of the NLRP3 inflammasome (Fig. 4G). The above results suggested that the Btk inhibitor protected mice from ALF partially via decreasing the activation of the NLRP3 inflammasome (Fig. 5).

ALSS treatment can quickly remove the harmful factors in plasma, correct the immune imbalance within a very short period, and facilitate the recovery of liver function in patients with ALF. Thirty-one patients with ALF with ALSS treatment were collected and tested for p-Btk/Btk protein levels via Western blotting. Among them, 1 patient received ALSS treatment five times, 2 received ALSS treatment four times, 6 received ALSS treatment three times, 7 received ALSS treatment two times, and the remaining 15 received ALSS treatment one time. Western blotting showed that ALSS treatment could reduce the expression levels of p-Btk/Btk in 21 patients (Fig. 6A). However, there were 10 patients in whom a decrease in the expression of p-Btk/Btk could not be measured (Fig. 6B). Statistically, among the 31 patients with ALF with ALSS treatment, 18 patients showed clinical improvement after ALSS treatment; among them, 16 patients (16 of 18; 88.9%) showed a decrease in p-Btk/Btk expression. Among the other 13 patients who showed no improvement or even deteriorated, only 5 patients (5 of 13; 38.5%) showed a decrease of p-Btk/Btk (improved versus unimproved; p < 0.01) (Fig. 6C). These results suggested that p-Btk/Btk expression level might be a predictor of the effect of ALSS treatment on ALF.

Furthermore, to validate the above results, we performed the FCM analysis again to check the p-Btk expression in PBMCs of another 10 patients with ALF who gained benefits from ALSS. FCM analysis revealed that the expression of p-Btk in CD3⁻ PBMCs of these patients decreased in various degrees (Fig. 6D, 6E). Our results indicated that Btk might be a new potential biomarker for not only evaluating the curative effect of ALSS treatment on patients with ALF but also predicting disease outcomes.

Previous studies have demonstrated that Btk inhibition prevents liver damage from partial warm ischemia and in situ reperfusion in a mouse model via modulating neutrophil recruitment and activation (30), and they have shown that the neutrophil Btk signalosome is involved in integrin activation of liver sterile inflammation (31). In this study, we showed that the expression of Btk was upregulated in animal models of ALF and that a Btk inhibitor significantly improved the survival rate and attenuated liver damage in experimental ALF models. The protective effect of Btk inhibition was associated with the reduction of NLRP3 inflammasome signaling (Fig. 5). Moreover, we found that Btk might be a feasible biomarker to indicate whether patients with ALF could benefit from ALSS treatment.

Although it is well known that immune factors play important and complex roles in the pathogenesis of ALF, the mechanism of ALF is not completely clear. To explore the potential role of Btk in the pathogenesis of ALF, we observed the dynamic changes of Btk during the process of ALF. We confirmed that the expression level of p-Btk changed quickly in a dynamic process under the stress of acute liver injury, and our data were consistent with those from Weber’s group (27). Some researchers have pointed out that Btk protein has its own internal mechanism in its autoinhibition and activation (32), which might partially explain this dynamic change of Btk in the inflammatory state. After analyzing the dynamic changes of Btk signaling during mouse ALF, we hypothesized that the inhibition of Btk phosphorylation might protect mice from ALF. We found that by inhibiting the phosphorylation of Btk, liver damage in mice with ALF induced by either LPS/D-GalN or Con A was alleviated significantly, and the survival rate was improved, along with a decreased expression of proinflammatory cytokines in liver tissues, such as IL-1β, TNF-α, and IL-6. To elucidate whether the data obtained from the animal model are applicable to patients with ALF, we used normal PBMCs from healthy donors and isolated CD14⁺ monocytes. We found that the expression of those inflammatory cytokines was significantly increased in the supernatant of cultured human CD14⁺ PBMCs treated by LPS, and the Btk inhibitor significantly reduced these cytokines. Thus, Btk inhibition effectively protected liver injury from ALF in vivo and weakened the LPS-mediated mononuclear macrophage immune response in vitro. Unfortunately, we have not observed a meaningful reduction in liver damage or in mortality using an LPS/D-GalN– or Con A —induced ALF model in Btk-null mice, which was reported by the research using a mouse polyinosinic:polycytidylic acid–induced acute hepatitis model (33), and we have also discussed this discrepancy, which might be caused by a variety of complex factors, in a published review (34). For example, Btk has many sites in every domain to bind with different molecules and regulate their physiological activities, which means that Btk is involved in multiple signaling transduction pathways and has complex and pleiotropic functions (35). Furthermore, other Tec family members, such as Tec and Itk, were confirmed to have an overlap with Btk in some functions, especially that Tec has similar functions of Btk in some degree (36, 37). In addition, this discrepancy is not unique to Btk; other molecules, such as receptor-interacting protein kinase 1 (38), are also multifunctional regulators of cell death.

Overactivation of the NLRP3 inflammasome is a central step in the development of various deleterious inflammations, such as ALF. Regulation of NLRP3 function is recognized as an effective strategy in the treatment of inflammatory diseases (39). Previous studies had clarified that Btk regulated the formation of the NLRP3 inflammasome (26, 27), and our team’s work also revealed that the NLRP3 inflammasome was involved in the development of ALF (25). Hence, we supposed that the Btk/NLRP3 inflammasome signaling pathway participated in ALF progress. To verify this hypothesis, the expression of the NLRP3 inflammasome in the liver of an ALF mouse model was detected at protein and mRNA levels. We found that the NLRP3 inflammasome was significantly increased in these models.

According to our previous work, miR-17 from the mesenchymal stem cell–derived exosome could alleviate LPS/D-GalN–induced mouse ALF via reducing the activation of the NLRP3 inflammasome (25). Another study also demonstrated that Btk was essential for NLRP3 inflammasome activation via linking NLRP3 with ASC to eventually form bioactive IL-1β and IL-18 (26). In this study, Btk inhibitors interrupted the signaling transduction of the NLRP3 inflammasome at both RNA and protein levels in vivo and in vitro, indicating that the protective effect of the Btk inhibitor on ALF was related to the inhibition of the NLRP3 inflammasome.

Systemic inflammatory responses are closely related to the development of ALF (40). Therefore, it is an important and feasible strategy to substitute fresh healthy plasma for ALF patient plasma, which contains various toxic substances and inflammatory mediators, such as endotoxin, bilirubin, TNF-α, ILs, and so forth. On the basis of this theory, ALSS treatment is currently proved to be an effective therapeutic strategy that can reduce the mortality of patients with ALF in addition to liver transplant (41). ALSS treatment can relieve persistent injuries and repair the liver tissue, which provides valuable time for liver transplant or other treatments. However, not all patients with ALF benefit from ALSS treatment, and some of them even have no response to ALSS. Currently, there is still no mature and popularized systematic tool to evaluate the therapeutic effect of ALSS (42, 43). In clinical practice, the improvement of ALF patient symptoms and the recovery of liver function are mainly considered positive outcomes, and the observation of the recovery of immune function is rarely included. In our study, we analyzed the expression of p-Btk in PBMCs before and after ALSS treatment. We found that the phosphorylation level of Btk was decreased in patients with ALF who gained benefits from ALSS treatment, and those with no response to ALSS treatment also showed that no significant decrease in the expression of Btk. As a central immune regulator, the expression levels of Btk before and after ALSS treatment can reflect the changes in the patient’s systemic inflammatory and immune responses, and we thought the change of p-Btk might have a relationship with the therapeutic effect of ALSS on the liver immune system. We found that Btk was not only a potential therapeutic target for ALF treatment but also a marker for the outcomes of patients with ALF treated with ALSS. It should be noted that the number of patients included in this study was limited, so large-scale investigations are needed in further research.

In conclusion, our work demonstrated that Btk participated in ALF progress via NLRP3 inflammasome signaling and that Btk inhibition played a protective role in ALF. Moreover, Btk was an important molecule that indicated the therapeutic effect of ALSS on ALF. Therefore, Btk was a therapeutic target for the treatment of ALF, which might have great value in clinical practice.

We thank Hongqun Liu for reviewing the manuscript.

The authors have no financial conflicts of interest.