The Role of TNFα- and IFNγ-induced PANoptosis in Renal Vascular Endothelial Cells: Implications for OMDT

doi:10.21203/rs.3.rs-3844462/v1

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The Role of TNFα- and IFNγ-induced PANoptosis in Renal Vascular Endothelial Cells: Implications for OMDT

https://doi.org/10.21203/rs.3.rs-3844462/v1

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This study mainly investigated the mechanism underlying the joint effects of TNFα and IFNγ on renal injury and preliminarily elucidated the influence of the combination of these two agents on the efficacy of recombinant human tumor necrosis factor-α receptor II fusion protein (rh TNFR:Fc) in the treatment of occupational medicamentose-like dermatitis due to trichloroethylene (TCE) (OMDT) patients. The level of peripheral TNFα and IFNγ of OMDT patients were measured to analyze their joint effects on renal function and vascular endothelial cells (ECs) injury. In vivo and in vitro studies were used to investigate the mechanism of TNFα- and IFNγ-induced ECs PANoptosis. Results showed that the combination of TNFα and IFNγ was significantly associated with kidney function and renal ECs injury. TCE-sensitized positive mice had elevated PANoptosis-related markers in renal ECs, and the injection of a TNFα and IFNγ neutralizing antibody significantly inhibited PANoptosis. In vitro studies revealed that TNFα- and IFNγ-induced ECs PANoptosis could be reversed by silencing interferon regulatory factor 1 (IRF1). In conclusion, peripheral TNFα and IFNγ levels were associated with kidney function. PANoptosis can be induced by the combination of TNFα and IFNγ, IRF1 was the master protein that regulates the assembly of the PANoptosome.

Trichloroethylene

Kidney

Endothelial cell

PANoptosis

Trichloroethylene (TCE) is a popular industrial solvent that has a broad range of applications in industrial and other settings [1]. In recent years, TCE has been widely used as a cleaning agent for electronic components in industry and as a raw material for the production of green refrigerant hydrofluorocarbons (HFCs), which has led to an increase in the use of TCE annually [2]. In China, the amount of output and import of TCE in 2021 had reached to 7,390,000 tons and 6700 tons, respectively [3]. TCE now is becoming one of the most common pollutants in the natural environment [4]. TCE exposure can also result in significant damage to the liver, kidney, respiratory system, cardiovascular system and nervous system [5]. Workers occupationally exposed to TCE may develop a systemic immune disease characterized by extensive skin and mucous membrane damage accompanied by fever, generalized lymphadenopathy, and severe multiorgan damage, such as to the liver and kidney; this disease is called occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) [6]. According to the Chinese National Diagnostic Criteria, OMDT can be classified into four types: erythema multiforme, Stevens-Johnson syndrome, exfoliative dermatitis, and epidermolysis bullosa [7]. Glucocorticoid pulse therapy is still the main treatment for OMDT patients. However, many problems, such as long-term hospitalization and side effects, surround patients and can cause great physical and psychological problems and economic pressure. These problems make OMDT treatment a clinical difficulty in the field of occupational medicine. Exploring the pathogenesis of OMDT and identifying new targets are highly important for ensuring occupational health.

Kidney injury usually occurs at the early stage of OMDT, which is closely related to the disease course and patient prognosis. Our previous studies revealed that TCE-induced immune kidney injury is based on a decrease in glomerular filtration function as well as tubular reabsorption function [8, 9]. However, the mechanism of kidney injury is not fully understood. The kidney is a highly vascularized organ with diverse endothelial cell (EC) populations. Different populations of ECs usually have different architectures and functions. ECs from the microvasculature mainly regulate blood flow, inflammation, and vascular permeability and modulate coagulation. Moreover, peritubular capillary ECs contribute to tubular secretion and reabsorption, whereas glomerular ECs contribute to the glomerular filtration barrier and support podocyte structure. These varied functions of kidney vascular ECs also make them major drivers and targets of inflammatory and thrombotic processes that can be detrimental to the kidney [10]. For example, longer-term exposure to inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ), pathogen-associated molecular patterns (PAMPs) and complement components induces ECs to increase the expression of adhesion molecules while decreasing the production of protective molecules and increasing endothelial permeability [11]. Finally, severely damaged endothelial cells are shed from the basement membrane and form circulating endothelial cells (CECs), resulting in endothelial barrier dysfunction. The number of CECs has been treated as a biomarker of cardiovascular complications and as a reflection of endothelial damage [12, 13]. Li et al. demonstrated increases in peripheral TNFα, IFNγ and IL-6 in OMDT patients [14], and our group revealed activation of the endothelium in TCE-sensitized positive mice [8]. However, whether ECs injury is associated with these inflammatory cytokines and the mechanism of endothelial activation and barrier dysfunction induced by TCE sensitization remain elusive.

The pathogenesis of OMDT is generally believed to be the T-cell-induced type IV hypersensitivity accompanied by the activation of cellular immunity and complement system [15]. After sensitization, the body can produce a variety of proinflammatory factors to participate in the immune response, but an excessive immune response leads to the emergence of cytokine storms and damage. A significantly elevated peripheral TNFα concentration is a common characteristic clinical manifestation in all four dermatitis types of OMDT patients [14]. TNFα has direct cytotoxic effects on target cells and exacerbates the inflammatory response by promoting endothelial cells to express adhesion molecules and mediate macrophage aggregation [16]. TNFα receptor inhibitors were found to partially alleviate liver and kidney injury in TCE-sensitized mice [17, 18]. Recombinant human tumor necrosis factor-α receptor II fusion protein (rh TNFR:Fc) has also been used for the clinical treatment of OMDT [19], but the clinical efficacy of some patients was unsatisfactory. In addition to the significant increase in peripheral TNFα in OMDT patients, especially in patients with severe skin lesions such as exfoliative dermatitis and epidermolysis bullosa, there is also an abnormal increase in IFNγ, which is elevated several fold or even dozens off old compared to that in controls [14, 20, 21]. Like TNFα, IFNγ also has antimicrobial, antiviral and antitumor effects, but its overexpression can exacerbate the inflammatory response and cause tissue damage and necrosis. High levels of TNFα and IFNγ also have joint effects and can influence kidney immune reactions [22]. Therefore, we hypothesized that the synergistic effect of TNFα and IFNγ could be the key factor affecting the efficacy of rh TNFR:Fc therapy in OMDT patients.

How does TNFα synergize with IFNγ to cause renal vascular endothelial cell injury? Karki et al. reported that cotreatment with TNFα and IFNγ activated interferon regulatory factor 1 (IRF1) and induced cell death [23]. IRF1 was the first interferon regulatory factor identified to drive the transcription of the type I interferon gene and is strongly associated with kidney disease [24, 25]; this gene can be regulated by TNF-α and IFN-γ [23, 26, 27]. IRF1 has also been found to play a key role in inducing the formation of PANoptosomes and mediating PANoptosis [23, 28].

PANoptosis, a newly introduced concept, is a unique innate immune inflammatory regulated cell death pathway that is regulated by PANoptosome complexes. The PANoptosome is a platform that contains multiple caspases as well as necrosome components and induces the activation of gasdermin and mixed lineage kinase domain-like protein (MLKL) to form membrane pores and induce cell death [29]. All of these characteristics of PANoptosis cannot be attributed to pyroptosis, apoptosis or necroptosis alone [29, 30]. In terms of the molecular regulatory features of these three types of programmed cell death, apoptosis is executed through caspases [31]; pyroptosis is driven by caspase-1 and ultimately activates the focal death execution protein gasdermin D (GSDMD) to form membrane pores [32]; and necroptosis is executed by MLKL [33]. The assembly of the PANoptosome can initiate a robust immune response in the host to prevent mortality during infection. However, aberrant PANoptosis activation can cause tissue and organ damage [34, 35]. PANoptosis has been found to occur in viral, bacterial and fungal pathogen infections; autoinflammatory diseases; cytokine storms; and cancers [36]. Exploring the mechanism through which PANoptosis is involved in TCE-induced immune renal vascular endothelial cell injury will contribute to the optimization of therapeutic regimens for renal injury in OMDT patients.

In this study, we demonstrated that peripheral blood TNFα and IFNγ levels were significantly elevated in OMDT patients compared to those in the control population and were strongly associated with renal injury. Through animal experiments, we found that PANoptosis occurred in renal vascular endothelial cells of TCE-sensitized mice and that neutralization of peripheral blood TNFα and IFNγ resulted in a decrease in PANoptosis and renal injury. Finally, the mechanism of ECs PANoptosis was further verified by in vitro experiments. The results of this study preliminarily suggest that the reason for the clinical therapeutic effect of rh TNFR:Fc may be related to the joint action of TNFα and IFNγ mediating the occurrence of PANoptosis in vascular endothelial cells.

2.1 Chemicals

TCE and Freund’s complete adjuvant (FCA) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Acetone and olive oil were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Necrostatin-1 (nec-1) (2324) was obtained from Tocris Bioscience (Bristol, UK). Z-IETD-FMK (FMK007), a mouse IFNγ neutralizing antibody (MAB4851) and a mouse TNFα neutralizing antibody (AB-410-NA) used for tail vein injection were purchased from R&D Systems (Minnesota, USA). Anti-human phospho-receptor-interacting protein kinase 1 (pRIPK1) antibody (#65746) and anti-mouse pRIPK1 antibody (#53286) were purchased from Cell Signaling Technology, Inc. (Boston, USA). Anti-human pRIPK3 antibody (ab240383), anti-mouse pRIPK3 antibody (ab205421), anti-MLKL antibody (ab243142), anti-human phospho-MLKL (pMLKL) antibody (ab187091), anti-mouse pMLKL antibody (ab196436), anti-gasdermin E (GSDME) antibody (ab215191), anti-NOD-like receptor family pyrin domain containing 3 (NLRP3) antibody (ab264468), anti-apoptosis associated speck-like protein containing a CARD (ASC) antibody (ab309497), anti-caspase-1 antibody (ab179515), anti-caspase-3 antibody (ab184787), anti-caspase-8 antibody (ab108333), anti-cleaved caspase-3 antibody (ab214430), and anti-GAPDH antibody (ab8245) were purchased from Abcam (Cambridge, UK). The anti-GSDMD antibody (bs-14287R) was purchased from Bioss (Beijing, China). Anti-CD31/PECAM-1 antibody (sc-376764), anti-IRF-1 antibody (sc-514544), and anti-vinculin antibody (sc-25336) were purchased from Santa Cruz Biotechnology (USA). Human TNFα, IFNγ, cystatin c (Cys-c), endothelin-1 (ET-1), vascular cellular adhesion molecule-1 (VCAM-1), and β2-macroglobulin (β2-MG) levels and mouse TNFα, IFNγ, Cys-c, ET-1, VCAM-1, and β2-MG ELISA kits were purchased from Reed Biotech (Wuhan, China). A serum creatinine (Cre) test kit, urea nitrogen (BUN) test kit, MTT test kit, and YO-PRO-1/PI test kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PE-conjugated anti-mouse CD146 antibody (134703) and FITC-conjugated anti-mouse CD146 antibody (134705) were purchased from Biolegend. FITC-conjugated anti-mouse CD45 antibody (30-F11), PE-conjugated anti-mouse CD105 antibody (MJ7/18), and APC-conjugated anti-mouse CD31 antibody (390) were purchased from Biogems (Beijing, China). ECM endothelial cell medium (1001) and Human umbilical vein endothelial cells (HUVECs) (8000) were purchased from ScienCell (California, USA). Human recombinant TNFα protein (300-01A) and human recombinant IFNγ protein (300-02) were purchased from PeproTech (Rocky Hill, NJ, USA).

2.2 Participants

In total, 17 patients (11 males and 6 females) aged 15–49 years were recruited from January 2016 to January 2020 at Guangdong Province Hospital for Occupational Disease Prevention and Treatment and Shenzhen Prevention and Treatment Center for Occupational Disease. All the patients’ plasma and 7 patients’ urine were collected upon admission. Twenty-one controls aged 18 to 49 years were recruited for this study.

The inclusion criteria for patients were as follows: had a clear history of occupational exposure to TCE and were newly diagnosed with OMDT according to the GBZ 185–2006 “Diagnostic Criteria for OMDT”.

The exclusion criteria for patients were as follows: aged older than 60 years, accompanied with kidney or liver disease before exposed to TCE, accompanied with mental diseases, chronic diseases and other serious organic diseases.

2.3 Patients kidney function

Blood BUN and Cre levels were detected by using automatic biochemical analyzer. Plasma Cys-c and urine β2-MG were detected by ELISA to assess glomerular filtration and tubular reabsorption, respectively. The procedure was carried out in accordance with the manufacturer’s instructions.

2.4 Patients peripheral TNFα and IFNγ detection

The peripheral levels of TNFα and IFNγ were detected via ELISA. The procedure was carried out in accordance with the manufacturer’s instructions.

2.5 Patients kidney ECs injury detection

Peripheral and urine ET-1 and urine VCAM-1 were detected by ELISA to determine the kidney EC injury. The procedure was carried out in accordance with the manufacturer’s instructions.

2.6 Establishment of the TCE sensitization mouse model

In total, 125 pathogen-free BALB/c female mice (7–8 weeks old) were purchased from the Experimental Animal Center of Anhui (Anhui, China). Mice were housed in a pathogen-free room with a 12 h/12 h light/dark cycle and were fed normal food. Mice were randomly divided into the blank control group (with 5 replicates), solvent control group (with 5 replicates), PBS control group (with 5 replicates), TCE treatment group (with 20 replicates), TCE + TNFα neutral treatment group (with 15 replicates), TCE + IFNγ neutral treatment group (with 15 replicates), TCE + TNFα neutral + IFNγ neutral treatment group (with 15 replicates), TCE + nec-1 treatment group (with 15 replicates), TCE + Z-IETD-FMK treatment group (with 15 replicates), and TCE + nec-1 + Z-IETD-FMK treatment group (with 15 replicates). The TCE sensitization mouse model was established according to Wang et al.[37]. A 2 × 2 cm² region of dorsal hair was shaved before the first application of TCE. On the first day of this model (after the first application of TCE), all the TCE treatment groups, TCE + TNFα neutral treatment group, TCE + IFNγ neutral treatment group, TCE + TNFα neutral + IFNγ neutral treatment group, TCE + nec-1 treatment group, TCE + Z-IETD-FMK treatment group, and TCE + nec-1 + Z-IETD-FMK treatment group mice received dorsal hypodermic injection of a 100 µl mixture of TCE (FCA: TCE: olive oil: acetone = 10:5:2:3). On the 4th, 7th, and 10th days, all the mice received dorsal skin paintings with 100 µl of 50% TCE (TCE:olive oil:acetone = 5:2:3). On the 17th and 19th days, the mice were painted with 30% TCE (TCE:olive oil:acetone = 3:2:5). Mice in the TCE + nec-1 treatment group also received an intraperitoneal injection of 3.5 mg/kg necrostatin-1 on the 17th and 19th days before painting with TCE. Mice in the TCE + Z-IETD-FMK treatment group also received an intraperitoneal injection of 2.5 mg/kg Z-IETD-FMK on the 17th and 19th days before painting with TCE. Mice in the TCE + nec-1 + Z-IETD-FMK treatment group were intraperitoneally injected with 3.5 mg/kg necrostatin-1 or 2.5 mg/kg Z-IETD-FMK on the 17th and 19th days before they were painted with TCE. Mice in the TCE + TNFα neutral treatment group received a tail vein injection of 2.5 mg/kg TNFα neutralizing antibody on the 15th, 17th and 19th days. Mice in the TCE + IFNγ neutral treatment group received a tail vein injection of 2.5 mg/kg IFNγ neutralizing antibody on the 15th, 17th and 19th days. Mice in the TCE + TNFα neutral + IFNγ neutral treatment group received tail vein injections of 2.5 mg/kg TNFα neutralizing antibody and 2.5 mg/kg IFNγ neutralizing antibody on the 15th, 17th and 19th days. Solvent control group mice and PBS control group mice were treated with identical volume ratios of acetone and olive oil. However, the PBS control group mice received 100 µl of sterile PBS on the 15th, 17th and 19th days. The blank control group mice received no treatment. On the 20th day, according to the cutaneous reactions, all the mice treated with TCE were further divided into TCE-sensitized positive mice (cutaneous reaction score ≥ 1) or TCE-sensitized negative mice (cutaneous reaction score 0). The sensitization rate is shown in Table 1. Then, all the mice were sacrificed, and 24-h urine, peripheral blood, kidney or kidney vascular endothelial cells were collected.

Table 1

Sensitization rate of each group of mice
Groups	Blank Control	Solvent control	PBS control	TCE treatment	TCE + TNFα neutral treatment	TCE + IFNγ neutral treatment	TCE + TNFα neutral + IFNγ neutral treatment	TCE + nec-1 treatment	TCE + Z-IETD- FMK treatment	TCE + nec-1 + Z-IETD-FMK treatment
Total	5	5	5	20	15	15	15	15	15	15
Sensitized negative	-	-	-	13	9	10	10	9	10	10
Sensitized positive	-	-	-	7	6	5	5	6	5	5
Sensitization rate	-	-	-	35.0%	40.0%	33.3%	33.3%	40.0%	33.3%	33.3%

2.7 Kidney injury detection

Periodic acid-Schiff (PAS) staining was used to assess pathological changes in the kidney. The 3 µm paraffin sections were dewaxed, rehydrated and stained with hematoxylin and acid-Schiff. Finally, pathological changes were observed under an optical microscope.

Serum BUN, Cre, Cys-c, and urine β2-MG were detected to reflect glomerular filtration and tubular reabsorption, respectively. The procedure was carried out according to the manufacturer’s instructions.

2.8 Mouse serum TNFα and IFNγ detection

The serum levels of TNFα and IFNγ were detected via ELISA. The procedure was carried out according to the manufacturer’s instructions.

2.9 Mouse kidney EC injury detection

Transmission electron microscopy (TEM) was used to observe the microstructure of kidney ECs. In brief, the renal cortex was fixed in glutaraldehyde and then in 1% osmic acid solution. The tissue was subsequently transferred to a uranium acetate aqueous solution and dehydrated with gradient acetic acid. After routine dehydration, embedding, heating, polymerization and staining, glomerular EC and peritubular EC structures were observed.

Urinary ET-1 and VCAM-1 levels were detected to reflect kidney EC injury. The procedure was carried out according to the manufacturer’s instructions.

2.10 Mouse peripheral blood CECs detection

The mouse peripheral blood was collected by using EDTA anticoagulant tubes. The tubes were then divided into test tubes, blank tubes, CD146-PE flag tubes and CD45-FITC flag tubes. All the tubes were filled with 1 ml of blood. Then, test tubes were incubated with 1µl CD146-PE antibody and 1µl CD45-FITC antibody, CD146-PE flag tubes were incubated with 1µl CD146-PE antibody, CD45-FITC flag tube were incubated with 1µl CD45-FITC antibody for 20 min. Then, all the tubes were incubated 3 ml erythrocyte lysate for 8 min, and then 1500r/min centrifuged 7 min. Finally, the supernatant was discarded, and the cells were resuspended in sterile PBS and detected by flow cytometry. CD146⁺CD45⁻ cells were treated as CECs.

2.11 Immunofluorescence

The co-expression of IRF1 and CD31 was detected via immunofluorescence. The 3 µm thick paraffin sections were dewaxed, rehydrated, processed with 0.3% Triton-100 and then incubated with anti-IRF1 and anti-CD31 antibodies overnight at 4°C. Finally, the slices were incubated with secondary antibodies and DAPI, and the immunobinding products were observed with a fluorescence microscope.

2.12 Immunohistochemistry

The 3 µm thick paraffin sections were dewaxed, rehydrated and then placed into citric acid for antigen retrieval. Then, the slices were blocked with goat serum and incubated with antibodies overnight at 4°C. On the second day, the slices were incubated with the secondary antibody horseradish peroxidase. Finally, the antigen-antibody complexes were observed after DAB staining.

2.13 Multiple immunofluorescence

Multiple immunofluorescence assays were used to evaluate the assembly of the PANoptosomes. Briefly, 3 µm paraffin sections were dewaxed, rehydrated, and subjected to microwave antigen retrieval. Then, the slices were blocked with 3% H₂O₂ and BSA. Then, the slices were incubated with CD31 antibodies overnight at 4°C. The next day, the slices were incubated with HRP-conjugated secondary antibodies for 50 min and then incubated with 50 µl of TSA-488 dye solution for 10 min. Then, the slices were subjected to microwave antigen retrieval again and incubated with an anti-ASC antibody, secondary antibody and TSA-555 dye solution. Similarly, cells were incubated with cleaved caspase-3 and pMLKL antibodies. Finally, the slices were incubated with DAPI, and the immunobinding products were observed via laser-scanning confocal microscopy.

2.14 Mouse kidney ECs isolation

Mouse kidney ECs were isolated by the magnetic bead method. First, the magnetic beads were incubated with an anti-CD31 antibody overnight and then washed with sterile PBS. Second, the mice were perfused with 10 ml of sterile PBS through the left ventricle and then perfused with 10 ml of PBS containing 100 µl of magnetic beads. The kidneys were collected and then digested with trypsin and collagenase type I, after which the ECs were allowed to adhere to the tube by using a magnetic frame. Finally, the cells were washed and collected.

2.15 Western blot

RIPA buffer was used to extract total protein from kidney ECs or human umbilical vein endothelial cells (HUVECs). The protein concentration was measured by a BCA kit. Equal amounts of protein (20 µg) were loaded and subsequently separated via SDS-PAGE. The proteins were subsequently transferred to a PVDF membrane. The membranes were blocked and then incubated with primary and secondary antibodies. Finally, the signals were detected by using enhanced chemiluminescence. The band intensity was calculated by ImageJ.

2.16 Real-time quantitative PCR

Total RNA was isolated by using TRIzol reagent. cDNA was generated by using a RevertAid First Strand cDNA Synthesis Kit. Real-time quantitative PCR was performed using a Light Cycler 480 SYBR Green I Master Kit. The primers utilized in this study were as follows: mouse Irf1 (forward, 5’- AACAGGGGACCATCCTCCTT-3’; reverse, 5’- GATCGACGCATGTCAATGCT-3’). Gene expression was analyzed by the 2^−ΔΔCt method.

2.17 Cell culture

HUVECs were cultured in ECM containing 10% FBS, 1% endothelial cell growth supplement (ECGS) and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. The cells were divided into the PBS control group, empty vehicle (EV) control group, TNFα group, IFNγ group, TNFα + IFNγ group, and IRF1-siRNA group. IRF1-siRNA cells were transfected with siIRF1 and then exposed to TNFα and IFNγ. The sequence of siIRF1 was as follows:

(Forward, 5’-GACAUCAACAAGGAUGCCUTT-3’; Reverse, 5’-AGGCAUCCUUGUUGAUGUCTT-3’).

2.18 Determination of the treatment ratio, time and concentration of TNFα and IFNγ

The ratio of TNFα to IFNγ required to incubate cells is based on the ratio of the two inflammatory factors in the serum of OMDT patients. In the present study, the ratio of TNFα to IFNγ was 1:2.

The MTT test was used to determine the tumor concentration and duration of TNFα and IFNγ treatment in HUVECs. 100µl (5000) cells were seeded into 96-well plate, then were treated with TNFα recombinant protein according to following concentration gradient: 0 ng/ml, 8 ng/ml, 16 ng/ml, 32 ng/ml, 64 ng/ml, 128 ng/ml, 256 ng/ml, 512 ng/ml, or treated cells with IFNγ recombinant protein according to following concentration gradient: 0 ng/ml, 8 ng/ml, 16 ng/ml, 32 ng/ml, 64 ng/ml, 128 ng/ml, 256 ng/ml, 512 ng/ml, or treated cells with TNFα + IFNγ recombinant protein according to following concentration gradient: 0 ng/ml + 0 ng/ml, 8 ng/ml + 16 ng/ml, 16 ng/ml + 32 ng/ml, 32 ng/ml + 64 ng/ml, 64 ng/ml + 128 ng/ml, 128 ng/ml + 256 ng/ml, for 0 h, 6 h, 12 h, 24 h, 48 h, respectively. After the incubation, 10 µl of MTT working solution was added, 150 µl of DMSO was added, and the absorbance was detected at 570 nm. The treatment concentration and time were chosen according to the cell viability.

2.19 Propidium iodide (PI) staining

PI stains necrotic cells or apoptotic cells that lack cell membrane integrity, as indicated by red fluorescence. After treated with TNFα and IFNγ, discarded the cell culture medium and treated cells were PI 300 µl and Hoechst 200 µl for 30 min. Then, observed under fluorescence microscope, and calculate the PI positive rate.

2.20 Statistical analysis

SPSS V.23.0 and R V.4.2.2 were used for the statistical analyses. The data are shown as the mean ± standard deviation (M ± SD). One-way analysis of variance (ANOVA) with the least significant difference (LSD) test was used for differences between groups. P < 0.05 was considered to indicate statistical significance in this study. The relationships between TNFα or IFNγ and ET-1, Cys-c, VCAM-1, β2-MG, BUN, and Cre were tested by linear regression analysis. The relationships between TNFα and IFN-γ and ET-1, Cys-c, VCAM-1, β2-MG, BUN, and Cre were tested by multiple linear regression analysis.

3.1 Relationships between TNFα and IFNγ levels and kidney injury indices in OMDT patients

In OMDT patients, the peripheral TNFα (86.83 ± 9.78 pg/ml) and IFNγ (163.93 ± 48.57 pg/ml) levels were significantly greater than those in controls (22.10 ± 4.05 pg/ml for TNFα and 9.94 ± 1.39 pg/ml for IFNγ) (Figs. 1a and 1b). The levels of blood Cre (74.13 ± 5.28 µmol/l), BUN (6.72 ± 0.36 mmol/l), peripheral Cys-c (2068.07 ± 278.00 ng/ml) and urine β2 MG (360.43 ± 99.73 ng/ml) were also greater than those in the controls (57.58 ± 2.74 µmol/l for Cre, 4.14 ± 0.23 mmol/l for BUN, 595.10 ± 99.76 ng/ml for Cys-c, and 38.61 ± 2.84 ng/ml for β2 MG) (Fig. 1c-1f). Linear regression models were subsequently used to analyze the relationships between TNFα and Cre, BUN, Cys-c, and β2 MG and between IFNγ and Cre, BUN, Cys-c, and β2 MG. TNFα was positively correlated with Cre (R² = 0.3476, P < 0.01) and nearly correlated with BUN (R² = 0.231, P = 0.051) and Cys-c (R² = 0.223, P = 0.056) (Fig. 1g-1i) but was not correlated with β2 MG (R² = 0.081, P = 0.536) (Fig. 1j). IFNγ was positively correlated with Cre (R² = 0.455, P < 0.01) (Fig. 1k) and Cys-c (R² = 0.310, P < 0.05) (Fig. 1m) and was nearly correlated with β2 MG (R² = 0.450, P = 0.099) (Fig. 1n) but was not correlated with BUN (R² = 0.023, P = 0.560) (Fig. 1l). Furthermore, we constructed a multiple linear regression model to analyze the synergistic relationship between TNFα and IFN-γ and Cre, BUN, Cys-c, and β2-MG. The results showed that the synergistic effect of TNFα with IFN-γ was positively correlated with Cre (R² = 0.612, P < 0.01) (Fig. 1o) and Cys-c (R² = 0.356, P < 0.05) (Fig. 1q) but not with BUN (R² = 0.244, P = 0.142) (Fig. 1p) or β2 MG (R² = 0.451, P = 0.302) (Fig. 1r).

3.2 Relationships between TNFα and IFNγ levels and the ECs injury indices in OMDT patients

Peripheral ET-1 and urine ET-1 and VCAM-1 were detected to reflect kidney EC injury. The results showed that the peripheral ET-1 (38.44 ± 6.45 pg/ml), urine ET-1 (3.63 ± 0.47 pg/ml) and VCAM-1 (8.08 ± 1.18 pg/ml) levels were significantly greater in the patients than in the controls (9.25 ± 1.53 pg/ml for peripheral ET-1, 1.67 ± 0.06 pg/ml for urine ET-1 and 2.59 ± 0.23 pg/ml for urine VCAM-1) (Fig. 2a-2c). The linear regression model showed that TNFα was positively correlated with peripheral ET-1 (R² = 0.405, P < 0.01) but not with urine ET-1 or VCAM-1 (Fig. 2d-2f). IFNγ was positively correlated with peripheral ET-1 (R² = 0.562, P < 0.01), urine ET-1 (R² = 0.642, P < 0.05) and urine VCAM-1 (R² = 0.747, P < 0.05) (Fig. 2g-2i). A multiple linear regression model was used to analyze the synergistic relationships of TNFα with IFNγ and peripheral ET-1 with urine ET-1 and VCAM-1. The results showed that the synergistic effect of TNFα with IFN-γ was positively correlated with peripheral ET-1 (R² = 0.646, P < 0.01) and urine VCAM-1 (R² = 0.778, P < 0.05) (Figs. 2j and 2k) and nearly positively correlated with urine ET-1 (R² = 0.705, P = 0.087) (Fig. 2l).

3.3 TCE sensitization leads to kidney injury, and PANoptosis-related proteins are highly expressed

The results showed that TCE sensitization led to reddish skin and edema (Fig. 3a), decreased kidney function (Figs. 3b and 3c), vacuolated kidney epithelial cells, and hyperplastic glomerular mesangial cells (Fig. 3d). Additionally, IRF1 was abundantly expressed and colocalized with endothelial cells in TCE-sensitized positive mouse kidneys (Fig. 3e-3g). Western blot analysis revealed that the expression of the PANoptosis-related proteins NLRP3, cleaved caspase-1 (p20), N-terminal GSDMD (p30), N-terminal GSDME (p34), cleaved caspase-3 (p17), cleaved caspase-8 (p18), phospho-RIPK 1, phospho-RIPK 3 and phospho-MLKL was significantly elevated in TCE-sensitized positive mice compared to those in the solvent control group (Fig. 3h-3j).

3.4 PANoptosis was involved in TCE-induced immune kidney injury

The RPK1 inhibitor necrostatin-1 and the caspase8 inhibitor Z-IETD-FMK were applied to determine whether PANoptosis was involved in TCE-induced immune kidney injury. Treatment with necrostatin-1 or Z-IETD-FMK alone reduced necroptosis (phospo-RIPK 1, phospo-RIPK 3 and phospo-MLKL) and apoptosis (cleaved caspase 3 (p17) and cleaved caspase 8 (p18)-related proteins only. However, the simultaneous application of these two inhibitors significantly reduced necroptosis, apoptosis and pyroptosis (NLRP3, cleaved caspase-1, N-terminal GSDMD (p30), and N-terminal GSDME (p34))-related proteins (Fig. 4a-4c). Furthermore, compared with those in the TCE + nec-1 + Z-IETD-FMK-sensitized group, renal function (cre and BUN) (Figs. 4d and 4e) and pathological damage (Fig. 4f) in the TCE + nec-1 sensitized positive group mice, and in the TCE + Z-IETD-FMK-sensitized positive group mice, renal function were significantly restored.

3.5 Multiple immunofluorescence results of PANoptosis

Multiple immunofluorescence techniques were used to visualize the assembly of the PANoptosome intuitively (Fig. 5). The results showed that there was abundant coexpression of ASE, pMLKL, cleaved caspase 3 and PECAM-1 in the TCE-sensitized positive group of mice. The immuno-binding products are expressed mainly on endothelial cells. The injection of necrostatin-1 or Z-IETD-FMK inhibited the expression of pMLKL or cleaved caspase 3, respectively. However, the combined injection of necrostatin-1 and Z-IETD-FMK significantly inhibited the coexpression of ASE, pMLKL, cleaved caspase 3 and PECAM-1. Furthermore, the coexpression of ASE, pMLKL, cleaved caspase 3 and PECAM-1 can be inhibited slightly by the application of a TNFα neutralizing antibody or IFNγ neutralizing antibody independently. The immunobinding products can be inhibited significantly by administering a TNFα neutralizing antibody and IFNγ neutralizing antibody jointly.

3.6 Neutralization of peripheral TNFα and IFNγ inhibits renal vascular EC IRF1 expression and PANoptosis

The flow cytometry results showed that injection of both the TNFα neutralizing antibody and IFNγ neutralizing antibody reduced the number of ECEs compared to that in the TCE-sensitized group of mice. Administration of a TNFα neutralizing antibody combined with an IFNγ neutralizing antibody reduced the number of ECEs to normal levels (Figs. 6a and 6b). The gene and protein levels of IRF1 and the necroptosis, apoptotic and pyroptotic protein levels also decreased significantly after the administration of the TNFα neutralizing antibody combined with the IFNγ neutralizing antibody (Fig. 6c-6f).

3.7 Neutralization of peripheral TNFα and IFNγ alleviates kidney injury induced by TCE sensitization

Compared to those in TCE-sensitized mice, the injection of TNFα neutralizing antibody alone alleviated kidney pathological damage (Fig. 7a) and reduced the serum Cre, BUN, Cys-c, and urine β2 MG levels, as well as the reduction in peripheral TNFα and IFNγ levels (Fig. 7b-7g). Furthermore, the kidney EC injury-related proteins urine ET-1 and VCAM-1 were also reduced after administration of the TNFα neutralizing antibody (Figs. 7h and 7i). However, the ability of IFNγ alone to alleviate kidney damage and endothelial cell injury was not significant. Injection of a TNFα neutralizing antibody combined with an IFNγ neutralizing antibody can ameliorate pathological kidney damage; reduce serum Cys-c, Cre, BUN and urine β2 MG levels; and reduce EC injury nearly to normal levels. TEM also revealed glomerular ECs and tubular EC membranes ruptured and exhibited cytoplasmic efflux in TCE-sensitized mice, while structural integrity of the cell membrane was observed in mice in the TCE + TNFα neutralizing + IFNγ neutralizing positive group (Fig. 7j).

3.8 IRF1 is the key protein that regulates HUVEC PANoptosis induced by TNFα in combination with IFNγ

Treatment with TNFα or IFNγ recombinant protein in HUVECs dose- and time-dependently decreased cell viability (Figs. 8a and 8b). TNFα in combination with IFNγ at doses of 64 ng/ml and 128 ng/ml significantly inhibited cell viability (Fig. 8c) and induced the expression of IRF1 (Fig. 8d), which promoted cell PANoptosis; these effects included the overexpression of phospho-RIPK 1, phospho-RIPK 3, phospho-MLKL, cleaved caspase 3 (p17), cleaved caspase 8 (p18), cleaved caspase 1 (p20), N-terminal GSDMD (p30), and N-terminal GSDME (p34). However, this cell death was rescued by silencing IRF1 (Fig. 8e-8h).

Growing concerns over the side effects of glucocorticoid pulse therapy combined with OMDT has led to increased attempts to find new drug therapies. The neutralizing antibody rh TNFR:Fc has been used for the treatment of OMDT patients, but the clinical treatment efficacy has been unsatisfactory for some patients. Our previous studies also treated TCE-sensitized mice with the tumor necrosis factor receptor 1 (TNFR1) inhibitor R7050, and the results showed that R7050 significantly alleviated liver and kidney dysfunction in TCE-sensitized mice, but hepatocytes and tubules still showed obvious pathological damage [17, 38]. These results suggested that targeting TNFα alone is not enough.

The activation of CD4⁺ T cells is a key cellular event in the development of OMDT, and the expression of IFNγ and TNFα is obviously increased [6]. We reviewed the clinical case information and found that in addition to the increase in peripheral TNFα, especially in patients with severe skin lesions, there was also an abnormally elevated level of IFNγ [14, 20, 21]. The present study also showed increased levels of TNFα and IFNγ in both OMDT patients and TCE-sensitized positive mice. TNFα in combination with IFNγ can induce a variety of diseases, such as TNFα/IFNγ-induced HaCaT keratinocyte dysfunction [39], TNFα/IFNγ-mediated intestinal epithelial barrier dysfunction [40], and TNFα/IFNγ coexposure-induced myotube atrophy [41].

Vascular ECs, the first and most exposed cells to peripheral inflammatory factors, are vulnerable to IFNγ and TNFα, which are secreted by CD4⁺ T cells. The kidney is one of the targets of TCE sensitization, and a decrease in kidney function may be related to kidney vascular EC injury. The results from the population study showed that synergism between IFNγ and TNFα not only contributed to more severe kidney damage but was also associated with higher levels of urine ET-1, VCAM-1 and peripheral ET-1. We subsequently performed animal experiments by administering an IFNγ neutralizing antibody and a TNFα neutralizing antibody. The results from animal experiments showed that coinjection of IFNγ neutralizing antibody and TNFα neutralizing antibody strongly alleviated kidney damage and inhibited the activation of kidney vascular ECs. However, the mechanism by which IFN-γ and TNF-α activate kidney vascular ECs is unclear.

Next, we detected the key proteins involved in pyroptosis, apoptosis and necroptosis in mouse kidney vascular ECs and found that all the proteins, including pRIPK 1, pRIPK 3, phospo-MLKL, cleaved-caspase3 (p17), cleaved-caspase8 (p18), NLRP3, cleaved-caspase1 (p20), N-terminal GSDMD (p30) and N-terminal GSDME (p34), were significantly greater in the TCE-sensitized positive control group than in the solvent control group. Recent studies have indicated extensive crosstalk between pyroptosis, apoptosis and necroptosis. These three different kinds of regulated cell death can simultaneously occur in one disease and can be regulated at the same time [42]. This newly defined cell death mode was PANoptosis.

PANoptosis has been proven to play a large role in the regulation of cancer [43], infectious diseases [44], and nervous system diseases [45]. All of these findings suggested that identifying PANoptosis was vital for informing the development of targeted regulators of inflammatory cell death for therapeutic modulation of the immune response. The master regulators of PANoptosis include Z-DNA binding protein 1 (ZBP1) [46], TGF-beta-activated kinase 1 (TAK1) [42], RIPK1 [47], absent in melanoma 2 (AIM2) [48], NLRP12 [49], and IRF1 [50]. IRF1 is an important molecule that regulates downstream inflammation and cell death and can be induced by innate immune signaling. IRF1 has also been proven to be an integral component of the PANoptosome that drives PANoptosis in inflammatory and infectious diseases [50]. Furthermore, IRF1 can regulate and be regulated by TNFα and IFNγ [23, 51–53]. In the present study, IRF1 was highly expressed in kidney vascular ECs from TCE-sensitized mice, and neutralization of TNFα and IFNγ downregulated the expression of IRF1 in ECs. To determine the key role of IRF1 in PANoptosis, we designed in vitro experiments. The results showed that TNFα in combination with IFNγ at doses of 64 ng/ml and 128 ng/ml significantly inhibited cell viability and promoted cell apoptosis, while silencing IRF1 rescued cell death.

In conclusion, this study preliminarily revealed the occurrence of PANoptosis in TCE-induced immune kidney injury. The PANoptosis of ECs can be induced by the combination of TNFα and IFNγ. Neutralizing peripheral TNFα and IFNγ can protect the integrity of the ECs barrier by inhibiting ECs apoptosis. Furthermore, IRF1 was indicated to be the master protein that regulates the assembly of PANoptosomes. Neutralizing peripheral TNFα and IFNγ may be a promising option for OMDT therapy because OMDT provides good vascular endothelial cell protection and fewer side effects.

Author Contributions HX, BL, ZL, HL and ZQ contributed to data curation. HX and BL contributed to investigation. HX contributed to formal analysis. HX contributed to original drafting. YW, JZ and QZ contributed to conceptualization. HX, JZ and QZ contributed to funding acquisition. YW and QZ contributed to review and editing. All authors read and approved the final manuscript.

Ethics approval All animal experiments were complied with the ARRIVE guidelines and carried out in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and, as applicable, the Animal Welfare Act. The animal experiments were approved by Anhui Medical University (Ethical approval number: LLSC 20230343). The ethical approval was obtained on 1 March 2023.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the First Affiliated Hospital of Anhui Medical University ((Date: 1 March 2023/No. 2023010). Informed consent was obtained from all individual participants included in the study.

Statements and Declarations

Competing interests The authors declare no competing interests.

Funding This work was supported by the National Natural Science Foundation of China (Grant numbers 82304106, 82173494, and 82273602); and the Key Laboratory of Dermatology, Anhui Medical University, Ministry of Education, China (Grant number AYPYS-2023-5).

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The Role of TNFα- and IFNγ-induced PANoptosis in Renal Vascular Endothelial Cells: Implications for OMDT

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Chemicals

2.2 Participants

2.3 Patients kidney function

2.4 Patients peripheral TNFα and IFNγ detection

2.5 Patients kidney ECs injury detection

2.6 Establishment of the TCE sensitization mouse model

2.7 Kidney injury detection

2.8 Mouse serum TNFα and IFNγ detection

2.9 Mouse kidney EC injury detection

2.10 Mouse peripheral blood CECs detection

2.11 Immunofluorescence

2.12 Immunohistochemistry

2.13 Multiple immunofluorescence

2.14 Mouse kidney ECs isolation

2.15 Western blot

2.16 Real-time quantitative PCR

2.17 Cell culture

2.18 Determination of the treatment ratio, time and concentration of TNFα and IFNγ

2.19 Propidium iodide (PI) staining

2.20 Statistical analysis

3 Results

3.1 Relationships between TNFα and IFNγ levels and kidney injury indices in OMDT patients

3.2 Relationships between TNFα and IFNγ levels and the ECs injury indices in OMDT patients

3.3 TCE sensitization leads to kidney injury, and PANoptosis-related proteins are highly expressed

3.4 PANoptosis was involved in TCE-induced immune kidney injury

3.5 Multiple immunofluorescence results of PANoptosis

3.6 Neutralization of peripheral TNFα and IFNγ inhibits renal vascular EC IRF1 expression and PANoptosis

3.7 Neutralization of peripheral TNFα and IFNγ alleviates kidney injury induced by TCE sensitization

4 Discussion

Declarations

References

Additional Declarations

Supplementary Files

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