Lung-specific CRBN knockout attenuates influenza a virus-induced acute lung injury in mice: a potential therapeutic approach

Abstract

Influenza-related acute lung injury is a life-threatening condition primarily caused by uncontrolled replication of the influenza virus and intense proinflammatory responses. Cereblon (CRBN) is a protein known for its role in the ubiquitin-proteasome system and as a target of the drug thalidomide. However, the function of CRBN in influenza virus infection remains poorly understood. In this study, we investigated the impact of CRBN on A/Puerto Rico/8/34 (PR8) influenza virus-induced lung injury and its potential as a therapeutic target. Knocking down CRBN in vitro significantly reduces PR8-induced cell death. Using Sftpc-Cre; Crbn^flox/flox lung-specific Crbn knockout mice, we demonstrated that Crbn deficiency significantly decreased mortality, weight loss, lung pathology, edema, and viral load in PR8-infected mice. PR8-infected Sftpc-Cre; Crbn^flox/flox mice exhibited a marked reduction in lung inflammatory cell infiltration and suppression of MAPK pathway activation, highlighted by a significant downregulation of the MKK4-JNK-c-JUN signaling cascade. Collectively, these findings indicate that CRBN plays a pivotal role in the pathogenesis of influenza-induced lung injury by modulating MAPK pathway signaling, underscoring its therapeutic potential as a target for intervention.

Introduction

Annual influenza outbreaks result in considerable mortality, particularly among the elderly. The World Health Organization estimates that influenza-related deaths range between 290,000 and 600,000 annually [1]. Influenza in humans is primarily caused by type A and B viruses, with type C playing a minor role. These viruses are negative-sense single-stranded RNA viruses characterized by their high mutation rates and genetic reassortment capabilities. Among them, type A influenza viruses pose the greatest threat, causing the majority of seasonal epidemics.

Currently, treatment options for influenza remain limited. Available antiviral drugs, such as amantadine and oseltamivir, target viral proteins but often encounter challenges like drug resistance driven by the rapid mutation of RNA viruses [2]. This resistance has raised concerns about the potential for new global pandemics. Consequently, there is an urgent need for novel therapeutic strategies.

One promising approach is to target host cell determinants essential for the influenza A virus (IAV) life cycle but temporarily dispensable for the host. For instance, DAS181, a recombinant sialidase, exemplifies a host-directed treatment for influenza and has progressed through various clinical trials [3, 4]. In our preliminary research, a whole-genome RNA interference screen identified the human host protein cereblon (CRBN) as a key factor in IAV-induced cell death.

CRBN is best known as the primary target of the immunomodulatory drug thalidomide. It serves as a substrate recognition receptor, assembling with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and Ring-Box 1 (RBX1) to form the Cullin 4A RING E3 ubiquitin ligase (CRL4A) complex. This complex ubiquitinates various natural substrates, including slowpoke potassium channel 1, myeloid ectopic viral integration site 2, glutamine synthetase, 5’-AMP-activated protein kinase (AMPK) α, and chloride voltage-gated channel, targeting them for proteasomal degradation [5,6,7]. The physiological roles of CRBN are closely linked to these substrates, affecting ion channel regulation related to mental retardation and AMPK-mediated energy metabolism [6]. Tissue-specific Crbn knockout mice have been employed to study non-syndromic intellectual disability [8] and CD4⁺ T-cell activation regulation [9].

To date, no studies have investigated the function of CRBN in influenza virus infection. This study aims to investigate the role of Crbn in mouse ALI induced by a mouse-adapted H1N1 IAV (A/Puerto Rico/8/34 (PR8), a strain known for its high infectivity in mice) using Sftpc-Cre; Crbn^flox/flox lung-specific Crbn knockout mice.

Materials and methods

Reagents and antibodies

Cell line and virus

The human lung adenocarcinoma A549 cells and Madin-Darby Canine Kidney (MDCK) cells were obtained from the Cell Culture Center of Peking Union Medical College. The A549 and MDCK cells were maintained in Ham’s F12 (Hyclone) or DMEM (Hyclone) medium respectively, with 10% fetal bovine serum (FBS, Gibco) and antibiotics (100 U/ml penicillin and streptomycin) at 37℃ in a 5% CO2 environment. The H1N1 influenza virus strain A/Puerto Rico/8/34 (PR8) was maintained in the State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology. The virus was propagated in specific-pathogen-free embryonated fowl eggs (10 to 11 days old) via the allantoic route. All experiments involving live viruses were conducted in biosafety level 2 facilities.

Cell viability by MTS assay

The A549 cells were seeded in 96-well plates at 1 × 10⁵ cells/ml and subjected to reverse transfection with small interfering RNA (siRNA, Guangzhou RiboBio Co., Ltd.) at the recommended concentration. Twenty-four hours post-transfection, PR8 virus or allantoic fluid was added to the wells at an MOI of 1. Each group had triplicate wells. After a 48-hour incubation, cell viability was measured using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega).

Animal model

B6(Cg)-Crbn^tm1.1Jjh/J(JAX stock #017564) mice were imported for use in a Cre-loxP system. These mice possess loxP sites flanking exons 3 and 4 of the Crbn gene. Homozygous mice for this allele are viable, fertile, normal in size, and do not display any gross physical or behavioral abnormalities. Cre recombinase recognizes loxP sites and excises the target DNA sequence when the orientation of two loxP sites is cis-repeated [9]. To generate Sftpc-Cre; Crbn^flox/flox tissue-specific gene knockout mice, these imported mice are bred with mice that express Cre recombinase driven by the surfactant protein C (Sftpc) promoter, which is exclusively expressed in the type II alveolar epithelial cells. As a result, the offspring have exons 3 and 4 deleted in the type II alveolar epithelial cells.

The Sftpc-Cre; Crbn^flox/flox tissue-specific gene knockout mice were obtained via three steps of breeding(Fig. 1B). First, Crbn^flox/− mice were selfcrossed to obtain homozygous Crbn^flox/flox mice. Next, the Sftpc-Cre transgenic mice were bred with Crbn^flox/flox mice to get Sftpc-Cre; Crbn^flox/− mice. Then these Sftpc-Cre; Crbn^flox/− mice were bred with Crbn^flox/flox mice to get Sftpc-Cre; Crbn^flox/flox mice.

C57BL/6NCrl (WT) mice were purchased from Vital River, Beijing. All animal experiments in this work were approved by the Ethics Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and adhered to the Chinese National Guidelines for the Care and Use of Laboratory Animals and the institutional animal care guidelines.

Lung pathology

Four- to six-week-old Sftpc-Cre; Crbn^flox/flox and WT mice were anesthetized using pentobarbital sodium and then administered 1.33 × 10⁴ TCID50 of PR8 virus or virus diluent intranasally. The mice were sacrificed four days post-infection (DPI). The lungs were fixed in 4% paraformaldehyde/PBS, embedded in paraffin, sectioned into ultrathin slices, and stained with hematoxylin-eosin (HE).

Lung injury score

A blinded investigator independently examined 25 random high-power fields per lung, stained with HE, at 400× magnification to score each condition.

Lung injury score system [10]:

Parameter	Score per field
	0	1	2
A. Neutrophils in the alveolar space	none	1–5	> 5
B. Neutrophils in the interstitial space	none	1–5	> 5
C. Hyaline membranes	none	1	> 1
D. Proteinaceous debris filling the airspaces	none	1	> 1
E. Alveolar septal thickening	2x	2x–4x	> 4x

Score = [(20×A) + (14×B) + (7×C) + (7×D) + (2×E)]/(number of fields ×100).

Data are shown as mean ± SEM (standard error of the mean).

Acute pulmonary edema (wet-to-dry ratio)

To assess acute pulmonary edema, lung wet-to-dry weight ratios were calculated. Four- to six-week-old Sftpc-Cre; Crbn^flox/flox and WT mice were anesthetized with pentobarbital sodium and intranasally inoculated with 1.33 × 10⁴ TCID50 of PR8 virus or allantoic fluid diluent. At 4 DPI, lung wet weights were measured, followed by drying at 60 °C for 48 h to determine dry weights. The wet-to-dry ratios were then calculated.

Viral titration

Virus titers were determined from the supernatants of lung homogenates collected from Sftpc-Cre; Crbn^flox/flox mice and WT mice on day 4 post-infection. Briefly, MDCK cells were seeded into 96-well plates at a density of 3 × 10⁴ cells per well and cultured for 24 h. Supernatant samples were added to the first column of the 96-well plate and subjected to 10-fold serial dilutions across subsequent columns. The infected cells were cultured for 96 h, and virus titers were calculated using the Reed-Muench method, expressed as TCID50 per milliliter of supernatant.

Survival rate and body weight

Four- to six-week-old Sftpc-Cre; Crbn^flox/flox and WT mice were anesthetized with pentobarbital sodium and intranasally inoculated with 1.33 × 10⁴ TCID50 of PR8 virus or allantoic fluid diluent. Survival rates of each group (25 male mice per group) were recorded daily for 14 days. Body weights of the mice were measured and documented daily.

Western blot

Four- to six-week-old Sftpc-Cre; Crbn^flox/flox and WT mice were anesthetized with pentobarbital sodium and intranasally inoculated with 1.33 × 10⁴ TCID50 of PR8 virus or allantoic fluid diluent. After 4 days, the mice were sacrificed, and their lungs were homogenized in ice-cold RIPA buffer containing a protease inhibitor cocktail. The lung lysates were separated using SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then probed with primary antibodies followed by HRP-conjugated secondary antibodies. Target proteins were visualized using a chemiluminescent substrate (Thermo Fisher Scientific) and a digital imaging camera.

Reagents and antibodies

Primary antibodies for p-MKK4, MKK4, p-JNK, JNK, p-c-Jun, and c-Jun were obtained from Cell Signaling Technology. HRP-conjugated secondary antibodies were sourced from Multisciences. PVDF membranes and western blot luminal reagents were acquired from Thermo Fisher Scientific. siRNAs targeting human CRBN were purchased from RiboBio.

Results

CRBN deficiency reduces PR8-induced cell death and mouse mortality in vitro and vivo

The PR8 influenza virus strain is an attenuated virus that has lost its ability to replicate in humans due to being passaged over 100 times in mice. During this process, the strain acquired mutations that enhanced its replication efficiency and allowed it to evade the immune response, resulting in high virulence in mice [11].

In our preliminary research, we used siRNA to knock down the CRBN gene in A549 cells and observed a significant improvement in the viability of PR8-infected A549 cells compared to controls (Fig. 1A).

To investigate the role of CRBN deficiency during IAV infection in vivo, we generated and confirmed the conditional Crbn knockout mice using the Cre-loxP system (Fig. 1B, supplementary data). Four- to six-week-old Sftpc-Cre; Crbn^flox/flox mice and WT mice were infected with the PR8 virus via intranasal instillation. Survival rates and body weights were monitored daily for 14 days.

The survival rate of Sftpc-Cre; Crbn^flox/flox mice was significantly higher than that of WT mice, demonstrating a protective effect of Crbn deficiency against PR8-induced mortality (Fig. 1C). Notably, by the second day post-infection, Crbn knockout mice showed a significant increase in body weight and maintained a statistically significant improvement in average body weights, in contrast to the continuous weight loss observed in WT controls (Fig. 1D).

Fig. 1

CRBN plays a critical role in the infection of PR8 virus. (A) CRBN knockdown increases A549 cell viability infected with A/Puerto Rico/8/34 (PR8) virus. (B) Generation of Sftpc-Cre; Crbn^flox/flox tissue-specific gene knockout mice. (C) Survival rates of WT mice and Sftpc-Cre; Crbn^flox/flox mice(n = 25). P < 0.05. (D) Changes in body weights of WT mice and Sftpc-Cre; Crbn^flox/flox mice. The values are means ± SEM from 25 mice. n = 3 replicates for A, n = 25 WT, Sftpc-Cre; Crbn^flox/flox mice for C-D, Student’s t test for A and plotted as means ± SD, Kaplan-Meier survival analysis for C. *P < 0.05, **P < 0.01

Reduced lung pathology and edema in CRBN conditional knockout mice

Lung pathology and wet-to-dry weight ratio measurements indicated that Sftpc-Cre; Crbn^flox/flox mice experienced milder acute lung injury compared to WT mice. In WT mice, histological analysis revealed that PR8 infection caused severe lung pathology, including extensive alveolar damage, septal thickening, inflammatory cell infiltration in both alveolar and interstitial spaces, proteinaceous debris filling the airspaces, and hemorrhage.(Fig. 2A).

In contrast, PR8-infected Sftpc-Cre; Crbn^flox/flox conditional knockout mice exhibited significantly reduced lung damage, characterized by decreased inflammation, preserved alveolar structure, and lower lung injury scores (Fig. 2A and B). Quantification of infiltrating neutrophils and macrophages further demonstrated a significant reduction in immune cell infiltration in the lungs of Crbn conditional knockout mice compared to WT controls (Fig. 2C).

Additionally, pulmonary edema, as assessed by the wet-to-dry lung weight ratio, was markedly reduced in Sftpc-Cre; Crbn^flox/flox mice, highlighting a significant amelioration of lung injury (Fig. 2D).

Lower viral titers in CRBN knockout mice

A high viral load is a critical factor contributing to the progression of acute lung injury [12]. To assess whether PR8 virus replication is affected in Sftpc-Cre; Crbn^flox/flox mice, we measured viral titers in lung homogenates from WT and Sftpc-Cre; Crbn^flox/flox mice on the fourth day post-infection.The viral titer in the lungs of PR8-infected Sftpc-Cre; Crbn^flox/flox mice was significantly lower compared to that of WT mice (Fig. 2E). These results suggest that Crbn deficiency inhibited the replication of PR8 in vivo.

Fig. 2

Lung-specific Crbn knockout ameliorates PR8 induced mice lung injury. (A) H&E staining of lung tissue of WT mice and Sftpc-Cre; Crbn^flox/flox mice at 4 days post infection (DPI). Scale bar, 50 μm. (B) The lung injury score and (C) the infiltrating cell count in 25 random high-power ( Magnification, 400×) fields of lung tissue stained with H&E of WT and Sftpc-Cre; Crbn^flox/flox mice at 4 days post infection with PR8 or saline. (D) Wet-to-dry ratios of lung of WT mice and Sftpc-Cre; Crbn^flox/flox at 4 DPI. (E) Viral titer of lung of WT mice and Sftpc-Cre; Crbn^flox/flox at 4 DPI. n = 3–5 WT, Sftpc-Cre; Crbn^flox/flox mice for B-E, Student’s t test and plotted as means ± SD. *P < 0.05, **P < 0.01

In summary, our results demonstrate that the Crbn gene plays a pivotal role in mouse mortality and disease severity associated with PR8 infection.

Crbn knockout mice show suppressed PR8-mediated activation of MAPK pathways

The activation of host cell signaling cascades, such as the mitogen activated protein kinase (MAPK) pathway, has been implicated in IAV entry, replication, and the production of excessive inflammatory mediators [13,14,15]. MKK4, a key upstream kinase, is known to activate MAPK, i.e. JNK [16]. In this study, we found that PR8 virus infection led to dramatically increased phosphorylation levels of key MAPK pathway signaling molecules (MKK4, JNK, c-Jun) in the lungs of WT mice, whereas the total protein levels of MKK4, JNK and c-Jun remained relatively unchanged. In contrast, phosphorylation of MKK4, JNK and c-Jun was significantly suppressed in PR8-infected Sftpc-Cre; Crbn^flox/flox mice.(Fig. 3A, B).

These findings suggest that the MKK4-JNK-c-Jun signaling pathway plays a critical role in PR8-induced ALI in WT mice. Furthermore, Crbn knockout in mouse type II alveolar epithelial cells effectively inhibits the activation of the MKK4-JNK-c-Jun pathway in response to PR8 infection.

Fig. 3

PR8-induced activation of MAPK pathways is suppressed in Sftpc-Cre; Crbn^flox/floxmice. (A) Western blotting analysis of p-MKK4, p-JNK, and p-c-Jun in lung tissues of AF or PR8 infected WT or Sftpc-Cre; Crbn^flox/flox mice. The blots were analyzed with anti-p-MKK4, anti-MKK4, anti-p-JNK, anti-JNK, anti-p-c-Jun, and anti-c-Jun antibodies. Student’s t test and plotted as means ± SD. **P < 0.01 and ***P < 0.001

Discussion

Sftpc-Cre; Crbn ^flox/floxmice—a Conditional Crbn Knockout Model in Lung AEC2s

Alveoli, the primary sites of gas exchange in the lungs, are lined by type 2 and type 1 alveolar epithelial cells (AEC2s and AEC1s). AEC2s play crucial roles in producing surfactant proteins normally and facilitating epithelial repair after injury [17]. They are also highly susceptible to influenza virus infection, serving as a primary site of viral replication and a key determinant of disease severity.

CRBN itself is a multifunctional protein involved in ubiquitination and degradation pathways [18]. Although global knockout of Crbn is not lethal in mice, these animals frequently display physiological abnormalities, including altered fat metabolism and neurobehavioral functions, dysregulated inflammatory and immune responses [19,20,21]. These systemic changes can obscure or complicate the investigation of CRBN’s role in lung.

We deleted the Crbn gene from murine AEC2s cells to investigate the cell-specific role of Crbn during influenza infection, particularly in regulating antiviral responses, and signal pathway activation post-infection. We utilized a conditional knockout approach by employing mice with a Cre recombinase driven by the Sftpc (surfactant protein C, specifically expressed in AEC2s) promoter and mice possessing loxP sites flanking exons 3 and 4 of the Crbn gene. This strategy enables the targeted knockout of Crbn in AEC2s, avoiding the confounding effects associated with whole-body knockout. It provides a more precise and focused framework for investigating the role of CRBN in influenza pathogenesis.

Crbn deficiency protects against PR8-induced ALI

Our findings demonstrate that Crbn plays a significant role in the pathogenesis of PR8 influenza virus-induced lung injury. In vivo, lung-specific Crbn knockout significantly reduced mortality, weight loss, lung pathology, and viral load. The observed decrease in viral replication in the lungs, though modest, suggests that Crbn may directly affect viral replication or modify host cell susceptibility to infection. Furthermore, the reduced inflammatory response, as evidenced by lung pathology findings, indicates that Crbn modulates immune signaling pathways that exacerbate lung injury during infection.

Crbn deficiency-mediated protection from PR8-induced ALI involves suppressed MAPK signaling

The MAPK pathway is a crucial cellular signaling mechanism that converts various stimuli—including cytokines, hormones, growth factors, pathogens, stress, and heat—into cellular responses such as proliferation, differentiation, transformation, and apoptosis. This pathway consists of a three-tiered module with evolutionarily conserved proteins: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK. In mammals, at least four distinct MAPK groups are expressed, including ERK1/2, p38, JNK1/2/3, and ERK5. Among these, JNK and p38 MAPKs are particularly responsive to cellular stress, influencing processes such as cell cycle regulation, inflammation, antiviral responses, and apoptosis [22].

During viral infections, the activation of cell signaling pathways is a key part of the cellular defense mechanism, aiming to establish an antiviral state. However, viruses can also exploit these enhanced signaling activities to support their replication cycles. For example, activation of JNK signaling by IAV proteins plays a crucial role in facilitating efficient IAV replication [23, 24]. Additionally, JNK is pivotal in initiating inflammatory responses and is implicated in the virus-induced expression of inflammatory cytokines and chemokines, contributing to cytokine storms and immunopathology [25, 26].

Therefore, in IAV infections, targeting the JNK signaling pathway offers dual benefits: restricting viral production within target cells and mitigating immunopathology caused by excessive immune response activation. For instance, JNK inhibitors, such as SP600125 and AS601245, have been shown to suppress IAV replication and alleviate virus-induced cytokine storms [15, 25]. Our findings suggest that Crbn deficiency leads to reduced phosphorylation of JNK during PR8 infection. This reduction may underlie the mechanisms that inhibit viral replication, decreases immunopathology, and promote cell survival during PR8 infection.

c-Jun and activating-transcription-factor-2 (ATF-2) together form activator protein-1 (AP-1), a transcription factor activated by JNK. Research has indicated that AP-1 contributed to IAV-mediated NLRP3 inflammasome activation and inflammatory response [27]. In our study, we observed downstream activation of c-Jun during PR8 infection, as evidenced by increased phosphorylation. However, in Crbn-deficient mice, c-Jun phosphorylation was significantly reduced, suggesting that Crbn deficiency attenuates the inflammatory response by disrupting AP-1 activation.

CRL4/CRBN-AMPK-MAPK axis potentially underlines PR8-induced ALI

Cullin-RING ligases (CRLs) represent the largest family of E3 ubiquitin ligases, responsible for ubiquitinating roughly 20% of cellular proteins targeted for degradation via the ubiquitin-proteasome system. Typically, CRLs are composed of four key components: scaffold cullins (eight members, including CUL-1, CUL-2, CUL-3, CUL-4 A, CUL-4B, CUL-5, CUL-7, and CUL-9), RING proteins (RBX, essential for ligase activity), adaptor proteins (e.g., DDB1 in the context of CUL-4 A/B), and substrate recognition receptors (e.g., CRBN). These components work in concert to transfer ubiquitin from an E2 enzyme to a specific substrate. Based on the scaffold cullin, CRLs are classified into eight subfamilies, one of which is CRL4A.

In the CRL4A/CRBN complex, CRBN serves as the substrate recognition receptor, interacting with CUL4A, RBX1, and DDB1 to mediate substrate ubiquitination [18, 28] (Fig. 4). The physiological roles of CRBN are partly mediated by its native substrates, which undergo ubiquitination and subsequent degradation. Identified substrates of the CRL4A/CRBN complex include slowpoke potassium channel 1, myeloid ectopic viral integration site 2, glutamine synthetase, chloride voltage-gated channel, and AMPKα.

AMPK is a highly conserved intracellular serine/threonine kinase and a critical energy sensor activated under metabolic stress, such as ATP depletion [29]. AMPK dysfunction has been linked to metabolic diseases, including obesity, diabetes, and hypertension. Beyond its metabolic functions, AMPKα is increasingly recognized for its role in antiviral host immunity [30]. Activation of AMPKα has been demonstrated to attenuate inflammation-related diseases by suppressing the MAPK signaling pathways and reducing excessive inflammatory responses [31, 32].

Since the CRL4A/CRBN complex can polyubiquitinate and degrade AMPKα via the proteasome, Crbn deficiency has been associated with increased AMPK levels and its constitutive activation in mice [33, 34] (Fig. 4). We hypothesize that in the lung tissue of lung-specific Crbn knockout mice (Sftpc-Cre; Crbn^flox/flox), elevated levels of AMPK and its constitutive activation may inhibit downstream MAPK pathways and mitigate excessive inflammatory responses during IAV infection (Fig. 4).

Fig. 4

A proposed model suggesting that CRBN deficiency or thalidomide may inhibit viral replication and immune response during influenza virus infection by suppressing the MAPK pathway

Conclusion

Our findings demonstrate that disrupting CRL4A/CRBN activity through lung-specific Crbn knockout inhibited MAPK signal transduction during PR8 infection. This disruption resulted in reduced mortality, milder lung pathology, lower viral titers, and a diminished inflammatory response in Sftpc-Cre; Crbnflox/flox mice infected with PR8. This study identifies CRBN as a critical factor in the pathogenesis of H1N1 influenza virus-induced lung injury and highlights its potential as a therapeutic target for influenza.

Notably, our results suggest that small-molecule inhibitors, such as thalidomide or its derivatives, which suppress CRBN activity, may offer promising avenues for the development of influenza treatments.