Pseudorabies Virus Infection Triggers PANoptosis to Enhance Inflammatory Responses Both In Vitro and In Vivo

Abstract

Pseudorabies virus (PRV), an alphaherpesvirus, causes severe neurological and respiratory diseases in multiple mammalian species and poses an emerging threat to public health. Increasing evidence suggests that virus-induced inflammatory cell death plays a pivotal role in shaping host immune responses and disease outcomes. PANoptosis, a newly defined inflammatory programmed cell death pathway integrating pyroptosis, apoptosis, and necroptosis, has been implicated in host defense against diverse pathogens. However, whether PRV infection induces PANoptosis and contributes to inflammatory pathology remains largely unexplored. In this study, we demonstrate that PRV efficiently replicates in Human Acute Monocytic Leukemia Cells (THP-1)-derived macrophages and robustly induces PANoptosis, characterized by the concurrent activation of Gasdermin D, caspase-3, and Mixed Lineage Kinase Domain-Like (MLKL). Pharmacological inhibition of PANoptosis markedly attenuated PRV-induced inflammatory cytokine production in vitro. Furthermore, intranasal inoculation of PRV in Balb/c mice resulted in productive lung infection accompanied by pronounced pulmonary inflammation. Lung tissues from PRV-challenged mice exhibited molecular and histopathological hallmarks of PANoptosis. Importantly, drug-mediated suppression of PANoptosis significantly reduced lung inflammation and inflammatory cytokine expression in vivo. Collectively, our findings identify PANoptosis as a critical mechanism underlying PRV-induced inflammatory responses and suggest that targeting PANoptosis may represent a promising therapeutic strategy for PRV-associated inflammatory diseases.

1. Introduction

Pseudorabies virus (PRV), also known as Suid herpesvirus 1, is a member of the Alphaherpesvirinae subfamily and the causative agent of Aujeszky’s disease, which leads to severe neurological, respiratory, and reproductive disorders in swine [1,2]. Although pigs are the natural hosts, PRV can infect a wide range of mammals, including cattle, dogs, cats, and rodents, often resulting in fatal outcomes. In recent years, sporadic cases of human PRV infection have been reported, raising increasing concerns about its zoonotic potential [3,4,5]. Despite extensive studies on PRV neuroinvasion and immune evasion strategies, the mechanisms underlying PRV-induced inflammatory pathology remain incompletely understood.

Macrophages are key innate immune cells that act as the first line of defense against viral infections. Upon sensing viral components, macrophages initiate antiviral responses and orchestrate inflammation through cytokine and chemokine production [6]. However, excessive or dysregulated inflammatory responses can exacerbate tissue damage and disease severity. Accumulating evidence indicates that virus-induced programmed cell death in macrophages is closely linked to inflammation and disease outcomes [7].

PANoptosis is a recently described inflammatory programmed cell death pathway that integrates components of pyroptosis, apoptosis, and necroptosis into a single coordinated process [8,9,10]. Unlike classical forms of cell death that operate independently, PANoptosis is driven by multiprotein complexes known as PANoptosomes, enabling simultaneous activation of caspase-1–mediated pyroptosis, caspase-8/3–dependent apoptosis, and RIPK3–MLKL–mediated necroptosis [11]. PANoptosis has been implicated in host defense against diverse viral pathogens, including influenza A virus, SARS-CoV-2, and herpesviruses, but also contributes to excessive inflammation and tissue injury [12,13,14].

Whether PRV infection triggers PANoptosis and how this process contributes to inflammatory responses during PRV infection remain unknown. In this study, we investigated the induction and functional significance of PANoptosis during PRV infection using both in vitro and in vivo models. We demonstrate that PRV robustly induces PANoptosis in macrophages and lung tissues, thereby amplifying inflammatory responses. Importantly, pharmacological inhibition of PANoptosis significantly alleviates PRV-induced inflammation, highlighting PANoptosis as a potential therapeutic target.

2. Materials and Methods

2.1. Cells and Virus

THP-1-derived macrophages are widely used as an in vitro model for studying PANoptosis [15]. However, whether pseudorabies virus (PRV) is capable of infecting and replicating in these cells has not been previously reported. Human THP-1 monocytes (ATCC TIB-202) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Shanghai, China), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. THP-1-derived macrophages were generated by treating THP-1 cells with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Saint Louis, MO, USA) for 48 h, followed by a 24 h resting period in PMA-free medium. Pseudorabies virus (PRV) strain (GFP-tagged PRV, laboratory stock) was propagated in Vero cells and titrated using the 50% tissue culture infectious dose (TCID₅₀) method.

2.2. Viral Infection

THP-1-derived macrophages were seeded in 6-well plates at 5 × 10⁵ cells/well and infected with PRV at multiplicities of infection (MOI) of 0.1 or 1.0. Cells were incubated for the indicated time points before downstream assays. For in vivo experiments, six- to eight-week-old female Balb/c mice were anesthetized and intranasally inoculated with 1 × 10⁵ TCID₅₀ of GFP-tagged PRV in 50 μL PBS. Control animals received PBS only (Three mice in each group).

2.3. Viral Titration

Cells were seeded in 96-well plates with Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/0.1 mg/mL streptomycin at 37 °C with 5% CO₂ until grow into a single layer, then incubated with diluted samples containing 2% FBS at 37 °C with 5% CO₂. Perform three replicates for each dilution. After 72 h of incubation, the Reed and Muench method was used to calculate 50% tissue culture infective dose titer (TCID₅₀).

2.4. Mice

Six- to eight-week-old C57BL/6J mice (female, 18–20 g) were purchased from Institute of Comparative Medicine, Yangzhou University [production license number: SCXK (Su) 2022-0009]. Mice were bred in the specific pathogen-free (SPF) grade facility of the Laboratory Animal Center of Yangzhou University [use license number: SYXK (Su) 2022-0044]. Mice were housed in individually ventilated cages and maintained under standard conditions (room temperature: 20–25 °C; relative humidity: 45–65%; 12 h/12 h light/dark cycle; free access to food and water). Mice were acclimatized for at least one week before experiments. Only healthy, non-pregnant female Balb/c mice were included in the study. Mice showing signs of severe illness or distress, such as weight loss exceeding 20%, were excluded.

2.5. Mice Experiments

For establishing a PRV infection model in mice, six C57BL/6J mice (6–8 weeks, female, 18–20 g) were randomly divided into two groups (challenge group and control group), with three mice per group. All mice were acclimated for one week prior to the formal experiment. Each mouse in the challenge group received an intranasal infection of 1 × 10³ TCID₅₀ of PRV, while mice in the control group were inoculated intranasally with an equivalent volume of PBS. Clinical manifestations of the mice were subsequently monitored. Mice exhibiting “ruffled fur and dyspnea with exaggerated abdominal breathing” were subjected to increased monitoring frequency. All mice were euthanized on day 4 post-challenge. Complete lung tissues were then dissected. Half of the tissue samples were fixed in 4% paraformaldehyde (for subsequent paraffin embedding and tissue section preparation), while the remaining half were stored in an ultra-low temperature freezer for subsequent analyses.

For the survival analysis in drug intervention trials in mice, ten C57BL/6J mice (6–8 weeks old, female, 18–20 g) were randomly divided into two groups (5 mice per group). Each group received intraperitoneal injections of either DMSO or a double combination of Z-IETD-FMK + GSK872, with a standardized injection volume of 40 μL per mouse using a 50 μL microliter syringe (Hamilton, Bonaduz, Switzerland). After 12 h of pre-treatment, mice in each group received an intranasal infection of 1 × 10³ TCID₅₀ of PRV. The same drug administration regimen was conducted every 24 h for three days. Starting from the challenge, mice were monitored continuously for 7 days, with close observation of clinical manifestations and rigorous documentation of survival rates across all groups. The survival curves were established upon completion of the experiment.

For the ELISA analysis in drug intervention trials in mice, ten C57BL/6J mice (6–8 weeks old, female, 18–20 g) were randomly divided into two groups (5 mice per group). Each group received intraperitoneal injections of either DMSO or a double combination of Z-IETD-FMK + GSK872, with a standardized injection volume of 40 μL per mouse using a 50 μL microliter syringe (Hamilton, Bonaduz, Switzerland). After 12 h of pre-treatment, mice in each group received an intranasal infection of 1 × 10³ TCID₅₀ of PRV. The same drug administration protocol was conducted every 24 h for three days. All mice were euthanized on day 4 post-challenge (24 h after the final drug administration), and complete lung tissue samples were dissected and stored in an ultra-low temperature freezer for subsequent analyses.

Sample sizes were determined based on prior experience and similar studies in the literature to ensure sufficient power for detecting differences between groups. Mice were randomly assigned to control or treatment groups using a computer-generated randomisation sequence. Mice were housed in separate cages according to treatment group to avoid cross-contamination, and treatments were applied in a randomised order to reduce potential bias from environmental factors. The experimenter performing outcome assessments (histopathology, cytokine analysis) was blinded to group allocation. The investigators conducting the data analysis were also blinded to the groupings.

2.6. Pharmacological Inhibition of PANoptosis

Caspase-8 activity was inhibited using Z-IETD-FMK (Selleckchem, Houston, TX, USA), and RIPK3 kinase activity was inhibited using GSK872 (Selleckchem, Houston, TX, USA). THP-1-derived macrophages were pretreated with Z-IETD-FMK (50 μM) and/or GSK872 (5 μM) for 2 h prior to PRV infection. For in vivo studies, mice were administered Z-IETD-FMK (5 mg/kg) and GSK872 (1 mg/kg) via intraperitoneal injection 2 h before viral challenge. Control groups included uninfected THP-1-derived macrophages or cells treated with vehicle (DMSO) in parallel to the treatment groups. For the in vivo experiments, mice receiving only the DMSO vehicle served as control. The experimental unit was a single THP-1-derived macrophage culture well or a single Balb/c mouse.

2.7. Western Blot Analysis

Cells or lung tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay (Thermo Fisher, Shanghai, China). Equal amounts of protein (30–50 μg) were resolved on SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk and incubated overnight at 4 °C with primary antibodies against PRV glycoprotein B (gB) (a key viral structural protein indicative of productive infection). Caspase-3, Gasdermin D (GSDMD), phosphorylated MLKL (p-MLKL), and β-actin (Cell Signaling Technology, Danvers, MA, USA). Horseradish Peroxidase (HRP)-conjugated secondary antibodies were applied for 1 h at room temperature, and protein bands were visualized using an enhanced chemiluminescence (ECL) system.

2.8. Immunofluorescence Assay

Immunofluorescence staining cells were infected with PRV for designated time periods, then fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, blocked with 3% bovine serum albumin in PBS for 1 h at room temperature or 30 min at 37 °C, and incubated with primary antibodies at 4 °C overnight. After washing with PBS, cells were incubated with secondary body at 37 °C for 1 h, followed by staining with DAPI for 7 min at room temperature. Finally, the slides were observed under an inverted fluorescence microscope (U-HGLGPS; Olympus, Tokyo, Japan).

2.9. Immunohistochemistry Staining

After the mice were euthanized, the lung tissues were immediately removed, trimmed into 5 × 5 × 3 mm pieces, and placed in 10% neutral-buffered formalin (NBF) for 24 h fixation to preserve tissue morphology. For IHC, sections were incubated overnight at 4 °C in a humid chamber with rabbit anti-PRV gB polyclonal antibody—normal rabbit immunoglobulin G (IgG) at the same concentration was used as the negative control instead of the primary antibody. After rinsing, slides were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (diluted 1:500, Jackson ImmunoResearch, 111-035-003, Lancaster County, PA, USA) at room temperature for 1 h, followed by 3,3′-diaminobenzidine (DAB) substrate (Sigma-Aldrich, D4293) development for 5–10 min (monitored under an optical microscope).

2.10. Cell Viability and Cytotoxicity Assays

Cell viability was quantitatively assessed using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China), which is based on the reduction in the water-soluble tetrazolium salt WST-8 to an orange-colored, water-soluble formazan dye by intracellular dehydrogenases in metabolically active cells. Briefly, cells in the logarithmic growth phase were seeded into 96-well plates at an optimal density and treated accordingly. After the treatment period, a specific volume of the CCK-8 reagent was directly added to each well, and the plates were incubated at 37 °C for 1 to 4 h to allow the colorimetric reaction to occur. The absorbance of the formed formazan was then measured at a wavelength of 450 nm using a microplate reader (BioTek, Winooski, VT, USA). The percentage of cell viability was calculated by normalizing the absorbance of the treated groups to that of the untreated control groups. This method is recognized for its sensitivity, convenience, and suitability for high-throughput screening.

2.11. Cytokine Quantification

Supernatants from infected cells or homogenized lung tissues were collected for quantification of High Mobility Group Box 1 Protein (HMGB1), Interleukin-1 Beta (IL-1β), and Monocyte Chemoattractant Protein-1 (MCP-1) using commercially available ELISA kits (R&D System, Minneapolis, MN, USA) following the manufacturer’s protocols. Collect the cell culture supernatants from the infected group and the control group, as well as the supernatant of lung tissue homogenates from the infected group and the control group of mice, and measure the concentrations of pro-inflammatory factors, respectively. Use a dual-wavelength microplate reader (BioTek Epoch, Windsor, VT, USA) to detect the absorbance (OD) values at the primary wavelength of 450 nm and the reference wavelength of 630 nm (to eliminate non-specific background interference).

2.12. Outcome Measures

Outcome measures in the study included viral replication, cell viability (via CCK-8 assay), PANoptosis marker activation (Western blot analysis), and inflammatory cytokine production (ELISA). The primary outcome measure for in vitro experiments was cell viability, while for in vivo experiments, the primary outcome was pulmonary inflammation and cytokine levels.

2.13. Ethics Statement

Animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University (Approval Number: 202206009). All experimental procedures were executed in strict compliance with the ARRIVE guidelines 2.0.100 Animal care followed the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011). Euthanasia procedures adhered to the American Veterinary Medical Association (AVMA) Guidelines (2020 Edition).

2.14. Statistical Analysis

Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 9.0. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test or Student’s t-test where appropriate. Kaplan–Meier survival curves were analyzed using the log-rank test. p-values < 0.05 were considered statistically significant. Effect size (Cohen’s d) and 95% confidence intervals were calculated for comparisons between groups. No animals were excluded from the analysis in this study.

3. Results

3.1. Pseudorabies Virus Can Efficiently Replicate in THP-1-Derived Macrophages

To explore the infectivity and replication ability of Pseudorabies virus (PRV) in THP-1-derived macrophages, Western blot analysis was employed to detect the expression of PRV glycoprotein B (gB). These macrophages were infected with PRV at multiplicity of infection (MOI) values of 0.1 and 1.0, respectively. Subsequently, viral replication was assessed at multiple time-points post-infection. gB protein expression was detectable as early as 12 h post infection (hpi) in THP-1-derived macrophages infected with either MOI. The gB expression level markedly increased and reached its peak at 24 hpi, followed by a gradual decline at later time points (36 and 48 hpi) (Figure 1A,B). To further confirm PRV infection at the cellular level, immunofluorescence assay (IFA) was conducted using a monoclonal antibody against PRV gB. Consistent with the Western blot results, gB-specific fluorescence signals were clearly observed at 12 hpi, became most prominent at 24 hpi, and were reduced at 48 hpi in cells infected with PRV at both MOIs (Figure 1C). In addition, viral replication kinetics were evaluated by measuring viral titers in the culture supernatants collected at 12, 24, 36 and 48 hpi. TCID₅₀ assays demonstrated a significant increase in viral titers between 12 and 36 hpi for both infection doses (p < 0.05), indicating active viral replication in THP-1-derived macrophages (Figure 1D). Collectively, these results demonstrate that PRV can efficiently infect and replicate in THP-1-derived macrophages, establishing this cell model as a suitable system for investigating PRV-induced PANoptosis and associated inflammatory responses.

3.2. Pseudorabies Virus Infection Triggers PANoptosis in THP-1-Derived Macrophages

To visualize the efficiency and cellular impact of Pseudorabies virus (PRV) infection, THP-1-derived macrophages were inoculated with GFP-expressing PRV at a multiplicity of infection (MOI) of 1.0. Fluorescence microscopy images revealed a high infection efficiency, as indicated by widespread GFP-positive cells at 24 hpi (Figure 2A). However, we identified distinct cytopathic features in a substantial proportion of infected macrophages: membrane blebbing, cell shrinkage, and the formation of apoptotic bodies, which are hallmarks of apoptosis, alongside cell swelling and ballooning, characteristics typically associated with necroptosis and pyroptosis. Collectively, the concurrence of these morphological features defines a complex cytopathic effect that could be considered consistent with the integrated morphological spectrum of PANoptosis (Figure 2C). To quantitatively assess PRV-induced cytotoxicity, cell viability was examined using a CCK-8 assay. A significant reduction in cell viability was observed beginning at 12 hpi, which progressively declined thereafter (Figure 2B), suggesting an active induction of cell death following PRV infection. Given that PANoptosis represents an integrated form of programmed cell death that encompasses apoptosis, pyroptosis, and necroptosis, we further investigated whether PRV triggers this pathway in THP-1-derived macrophages. Western blot analysis revealed the cleavage and activation of hallmark molecules associated with each death modality: Caspase-3 (apoptosis), Gasdermin D (GSDMD; pyroptosis) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL; necroptosis). The concurrent activation of these pathways strongly indicates that PRV infection induces PANoptosis in THP-1-derived macrophages (Figure 2D). Collectively, these results demonstrate that PRV infection not only efficiently invades THP-1-derived macrophages but also elicits extensive PANoptotic cell death, characterized by simultaneous activation of apoptotic, pyroptotic, and necroptotic signaling cascades.

3.3. Pharmacological Inhibition of PANoptosis Effectively Reduces the Inflammatory Responses During Pseudorabies Virus Infection In Vitro

To further determine whether PRV-induced cell death in THP-1-derived macrophages is mediated by PANoptosis, we employed pharmacological inhibitors targeting key components of the PANoptotic pathway [16]. Z-IETD-FMK, a selective Caspase-8 inhibitor, and GSK872, an inhibitor of RIPK3 kinase activity, were tested for cytotoxicity to establish safe working concentrations. THP-1-derived macrophages were treated with gradient concentrations of each compound (100, 75, 50, 25, 10, and 5 μM) for 36 h, followed by cell viability assessment using the CCK-8 assay. As shown in Figure 3A, GSK872 exhibited no detectable cytotoxicity at concentrations below 5 μM, whereas Z-IETD-FMK was well tolerated even at concentrations up to 100 μM. Based on these findings, 50 μM Z-IETD-FMK and 5 μM GSK872 were selected for subsequent experiments. To evaluate the impact of PANoptosis inhibition on PRV-induced cytotoxicity, THP-1-derived macrophages were pretreated with Z-IETD-FMK, GSK872, or a combination of both for 2 h prior to PRV infection (MOI = 1.0) at 36 hpi, CCK-8 assays revealed that combined pretreatment with Z-IETD-FMK and GSK872 significantly rescued cell viability compared to PRV-infected controls (p < 0.05; Figure 3B). In contrast, either inhibitor alone exerted a moderate but non-significant protective effect, suggesting that the simultaneous inhibition of Caspase-8 and RIPK3 is required to efficiently block PRV-induced PANoptosis. We next examined whether the inhibition of PANoptosis modulates the inflammatory milieu associated with PRV infection. Since the combined pretreatment with Z-IETD-FMK and GSK872 significantly rescued cell viability, this treatment was used in the following experiments. Supernatants from the treated and infected macrophages were collected for ELISA quantification of key inflammatory mediators, including HMGB1, IL-1β, and MCP-1. Consistent with the suppression of cell death, combined treatment with Z-IETD-FMK and GSK872 markedly reduced the secretion of these cytokines compared to the PRV-infected group (p < 0.001; Figure 3C). Collectively, these results demonstrate that pharmacological blockade of PANoptosis via dual inhibition of Caspase-8 and RIPK3 effectively mitigates PRV-induced cytotoxicity and dampens the associated inflammatory responses in THP-1-derived macrophages.

3.4. Pseudorabies Virus Efficiently Infects the Lungs of Balb/c Mice Through Intranasal Inoculation

Having established that PRV infection triggers PANoptosis in THP-1-derived macrophages in vitro, we next sought to determine whether similar processes occur in vivo. To this end, a murine model of PRV infection was established by intranasal inoculation of six- to eight-week-old Balb/c mice with our GFP-tagged PRV strain. The infected mice exhibited marked respiratory distress characterized by pronounced abdominal breathing and labored respiratory movements, suggesting a preferential pulmonary tropism of the virus. Histopathological analysis was performed to assess PRV distribution within the lung tissues. Immunohistochemical (IHC) staining using PRV-specific antibodies revealed robust viral antigen expression in bronchial epithelial cells and alveolar regions (Figure 4), confirming that PRV efficiently infects the lungs of Balb/c mice following intranasal exposure, which provided a physiologically relevant system for subsequent analysis of PANoptosis and inflammatory responses in vivo.

3.5. Lung Cells of Balb/c Mice Undergo PANoptosis upon Pseudorabies Virus Infection

To determine whether PRV infection induces PANoptosis in vivo, we examined the activation of molecular markers associated with apoptotic, pyroptotic, and necroptotic pathways in the lungs of infected Balb/c mice. Western blot analysis revealed pronounced cleavage and activation of Caspase-3, Gasdermin D (GSDMD), and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) in lung tissue lysates collected from PRV-infected mice, whereas these proteins remained inactive in mock-infected controls (Figure 5). The concurrent activation of these three hallmark molecules provides compelling evidence that PRV infection triggers PANoptosis in the pulmonary tissues of Balb/c mice. These findings, together with our in vitro results, demonstrate that PRV-mediated PANoptotic cell death represents a conserved pathogenic mechanism that occurs across both cellular and organismal levels.

3.6. Drug-Mediated Suppression of PANoptosis Significantly Reduces the Level of Pulmonary Inflammation in PRV-Challenged Mice

To investigate the impact of PANoptosis on PRV-induced pulmonary pathology, Balb/c mice were pretreated via intraperitoneal injection with a combination of Z-IETD-FMK and GSK872 prior to intranasal challenge with PRV. Survival analysis revealed that mice receiving the dual-drug pretreatment exhibited a significant delay in mortality compared with the untreated controls (p < 0.05), indicating a protective effect of PANoptosis inhibition on disease progression (Figure 6A). To further assess the inflammatory response, the supernatants of homogenized lung tissues were analyzed for cytokine levels using ELISA. Dual-drug pretreatment markedly suppressed the production of key pro-inflammatory mediators in the lungs of PRV-infected mice (p < 0.001), demonstrating that targeted inhibition of PANoptotic signaling effectively mitigates virus-induced pulmonary inflammation (Figure 6B–D). Collectively, these findings establish that PANoptosis contributes substantially to the inflammatory pathology in PRV infection, and its pharmacological suppression provides significant protective effects against PRV-induced lung injury.

4. Discussion

In this study, we provide the first comprehensive demonstration that pseudorabies virus (PRV) infection robustly induces PANoptosis in both macrophages and pulmonary tissues, thereby amplifying inflammatory responses. Our findings significantly advance the understanding of PRV pathogenesis by linking viral infection to a coordinated inflammatory cell death pathway, highlighting PANoptosis as a pivotal contributor to PRV-induced tissue damage.

Our in vitro data indicate that PRV efficiently infects THP-1-derived macrophages and triggers simultaneous activation of caspase-3, GSDMD, and p-MLKL, hallmarks of apoptosis, pyroptosis and necroptosis, respectively. This multi-modal activation strongly supports the engagement of PANoptosis rather than isolated forms of cell death [8]. The pharmacological inhibition of key PANoptotic components, Caspase-8 and RIPK3, effectively attenuated cell death and reduced the secretion of inflammatory mediators such as HMGB1, IL-1β, and MCP-1. These results suggest that PANoptosis is not merely a byproduct of viral infection but actively contributes to the pro-inflammatory milieu, consistent with reports in other viral models, including influenza and SARS-CoV-2 [13,14,15].

In vivo, our findings demonstrate that PRV exhibits a pronounced pulmonary tropism in Balb/c mice following intranasal inoculation. Histopathological and molecular analyses revealed that PRV-infected lung cells underwent PANoptosis, recapitulating the in vitro phenotype. Importantly, dual-drug inhibition of PANoptosis in mice significantly mitigated pulmonary inflammation and delayed mortality, establishing a causal relationship between PANoptotic cell death and tissue pathology. This observation underscores the potential of targeting PANoptosis as a therapeutic strategy to control virus-induced inflammation without directly affecting viral replication.

Mechanistically, PANoptosis represents a unique intersection of canonical cell death pathways, enabling the host to mount a potent anti-viral response while simultaneously triggering inflammation [9]. While this can facilitate pathogen clearance, excessive or uncontrolled PANoptosis may exacerbate tissue injury, as observed in our PRV infection model. The identification of PANoptosis in PRV-infected macrophages and lung tissue provides a mechanistic explanation for the severe inflammatory manifestations observed in both natural and experimental infections, including cytokine release and pulmonary damage.

Our study also has broadened implications for understanding herpesvirus pathogenesis. Alphaherpesviruses, such as herpes simplex virus and PRV, are known to manipulate host cell death pathways to promote replication and evade immune detection [1]. The induction of PANoptosis may represent a conserved strategy by which these viruses modulate the balance between host defense and tissue injury. Future studies should aim to dissect the upstream signaling events and molecular triggers of PRV-induced PANoptosis, including the involvement of pattern recognition receptors, inflammasome activation, and viral proteins.

There are two limitations in this study, one is the model system of this study. It should be noted that the reliance on human-derived THP-1 macrophages and a mice infection model may limit the physiological relevance of our findings. Since pigs are the natural host for pseudorabies virus (PRV), the absence of porcine-specific primary cells or in vivo porcine models restricts the direct translation of our results to the natural host–pathogen interaction. Another limitation is the sample size of the in vivo mice experiments; it (e.g., n = 3–5 per group) might be relatively small, potentially limiting the statistical power to detect more subtle effects.

In summary, we demonstrate that PRV infection triggers PANoptosis, which amplifies inflammatory responses both in vitro and in vivo. Pharmacological inhibition of this pathway effectively reduces inflammation and lung injury, highlighting PANoptosis as a potential therapeutic target. These findings not only provide novel insights into PRV pathogenesis but also establish a framework for exploring PANoptosis in other viral infections and inflammatory diseases.