HSF1 knockout mice were donated by Benjamin’s laboratory; wild-type (HSF1^+/+) mice of the same age, strain, and sex were used as control. The protocols for animal breeding and experiments were approved by the Institutional Animal Care and Use Committee of Central South University, approval Number 2018sydw077. All animal experiments were conducted in the Department of Zoology, Central South University.

Sepsis was diagnosed according to the definition of sepsis 3.0, and the Sequential Organ Failure Assessment (SOFA) score was ≥2 points. Each of the six SOFA parameters reflects the function of the organ systems (respiratory, cardiovascular, renal, neurological, hepatic, and hematological) with a score of 0 to 4. The patients had different degrees of lung injury; most of the patients had symptoms such as cough, sputum, and fever. The collection of specimens was approved by the ethical review of the Third Xiangya Hospital of Central South University, Approval Number 2019-S548. The subjects were informed of the collection of this specimen and provided signed informed consent.

The following antibodies were used: rabbit anti-NLRP3 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, dilution 1:1,000); mouse anti-IL-1β monoclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000); mouse anti-Ub monoclonal antibody (Santa Cruz, Dallas, TX, USA, dilution 1:300); rabbit anti-SUGT1 polyclonal antibody (ProteinTech, Chicago, IL, USA, dilution 1:1,000); rabbit anti-HSF1 polyclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000); rabbit anti-pro Caspase-1+p10+p20 monoclonal antibody (abcam, Cambridge, UK, dilution 1:1,000); rabbit anti-IgG (Cell Signaling Technology, USA, dilution 1 μg); mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Cell Signaling Technology, USA, dilution 1:5,000); and mouse anti-ACTIN monoclonal antibody (Cell Signaling Technology, USA, dilution 1:1,000).

Mice were anesthetized with isoflurane inhalation; the skin and muscle layers along the midline of the lower abdomen were cut; the abdominal cavity was exposed to find the cecum; the end of the cecum 1/3 was ligated with line 3; the distal end of the cecum was punctured (through the cecum) with a size 7 needle, squeezing out a little stool gently; then the cecum is gently returned to the abdominal cavity; the peritoneum and the muscular layer are sterilized; and the muscular layer and skin are sewn up and sterilized again. Mice were rehydrated by subcutaneous injection of preheated saline into the back. They were kept warm after the operation, and the general condition of the mice was observed. After the mice were modeled, the state of the mice was observed, including 7 indicators of appearance, consciousness level, activity, stimulus response, eye condition, respiratory rate, and respiratory quality. Each indicator was graded from 0 to 4 points, and the scores of the 7 indicators were added together. The highest score was 28 points, and the dead mice were recorded as having the highest score. H&E staining was used to detect the lung histopathology of mice, and the mice in the cecal ligation and puncture (CLP) group had secondary lung injury, which manifested as a large number of inflammatory cells, debris, and red blood cell (RBC) infiltration in the alveolar cavity and interalveolar septum, destruction of alveolar structure, and obvious edema and thickening of the alveolar septum. The results showed that the CLP-induced sepsis model was successfully replicated and induced lung injury.

Total RNA was purified from cells using TRIzol^® Reagent (Gibco, Grand Island, NY, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using total RNA as a template with PrimeScript^® RTreagent Kit and gDNA Eraser kit (TaKaRa Bio, Siga, Japan). Diluted cDNAs were subjected to qPCR analysis using SYBR^® Green Quantitative PCR kit (TaKaRa Bio, Siga, Japan). Relative mRNA levels were calculated after normalization to actin using the ΔCt method. The PCR primer sequences used in this paper are listed in Table 1 . Primer is designed by Primer5.0 online software, and biosynthesis is performed by TSINGKE (Changsha, China).

Tissue and cell proteins were lysed with radioimmunoprecipitation assay (RIPA) lysates (P0013, Beyotime, Shanghai, China), the supernatant was collected after ultrasound and centrifugation, protein concentrations were measured using a bicinchoninic acid (BCA) protein quantification kit (98L001100, Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China), and the same amount of protein was mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and heated for 10 min at 95°C. The pyrolysis products were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, sealed in TBST containing 5% skim milk powder for 2 h, and incubated overnight with the corresponding primary antibody. After being washed with TBST three times, horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG was used as the secondary antibody to seal for 1.5 h, and the immune response bands were observed by the chemiluminescence imaging system (Aplegen, Pleasanton, CA, USA). Anti-GAPDH or anti-actin antibody was used to normalize equal amounts of proteins.

The serum levels of IL-1β (CSB-E08054m, CUSABIO, Wuhan, China) and IL-18 (CSB-E04609m, CUSABIO, China) in each group were measured using a mouse ELISA kit. A human ELISA kit was used to detect serum levels of IL-1β (CSB-E08053h, CUSABIO, China), IL-18 (CSB-E07450h, CUSABIO, China), and SGT1 (69-32967, MSKBIO, Wuhan, China) in sepsis patients and normal people. All the experimental procedures were carried out according to the kit instructions.

Mice were intraperitoneally injected with 3 ml of 3% thioglycolate broth medium and sacrificed via cervical dislocation after 4 days. After being immersed in 75% alcohol for 10 min, the sacrificed mice were placed in a supine position on an ultra-clean workbench where their abdominal skins were cut open and then flushed twice with 5 ml of pre-chilled Roswell Park Memorial Institute (RPMI) 1640 medium (supplemented with 10 U/ml of penicillin and 100 μg/ml of streptomycin), followed by a collection of their peritoneal lavage fluid (PLF). After centrifugation (1,000 rpm, 5 min), the resulting supernatant of the PLF was discarded, and the pellet was resuspended with 5 ml of 1× RBC Lysis Buffer (555899, BD Biosciences, San Jose, CA, USA) and incubated in an ice bath for 10 min. After a second centrifugation step, the resulting supernatant was discarded again, and the cell pellet was resuspended and plated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) for cultivation.

RAW264.7 cells were treated at 43°C in a 10-cm cell culture dish for 60 min and immediately by the extraction of nuclear proteins (P0027, Beyotime, China). After the protein concentration was determined via the BCA assay, the nuclear protein samples were stored at −80°C in a freezer until use. DNA probes were generated according to the HSE sites as double-stranded ( Tables 2 and 3 ). After being validated with nucleotide blast, the probe sequences were sent to Sangon Biotech (Shanghai, China) Co., Ltd., for probe synthesis. Electrophoretic mobility shift assay (EMSA) was performed in accordance with instructions provided in the kit (20148, Thermo Scientific™, Waltham, MA, USA), and the results were visualized using a chemiluminescence imaging system (Aplegen, USA).

Luciferase reporter plasmids containing SGT1 and TRAF3 were constructed (Sangon Biotech, Shanghai, China). The plasmids were extracted (D6950-01, Omega, Doraville, GA, USA), their concentration was determined, and the DNA was stored at −20°C for subsequent experiments. HEK293 cells were inoculated in a 24-well plate and grown to about 70% to 90% confluence before being co-transfected (L3000015, Invitrogen™, Carlsbad, CA, USA) with pcDNA3.1, HSF1-constitutive plasmids, luciferase reporter plasmids, PGL-basic, and pRL-TK. After 48 h of transfection, the cell lysate was extracted and tested using a dual-luciferase reporter assay kit (E1910, Promega, Madison, WI, USA) and loaded onto a luminometer for the measurement of luminescence (Synergy^HI Microplate Reader, BioTek, Winooski, VT, USA).

Protein A/G Mix Magnetic Beads (50 µl) (LSKMAGAG10, Millipore, Billerica, MA, USA) were pipetted into a 2-ml microcentrifuge tube and washed three times with PBST (phosphate-buffered saline (PBS) containing 0.01% Tween^®20 detergent). After being resuspended with PBS, primary antibodies NLRP3, SGT1, and IgG (rabbit IgG) measuring 1.0–10.0 µl were added and then incubated at room temperature (23°C–26°C) for 1 h. After being washed three times with PBST for 10 min each time, 150 µg of protein sample was added to the antibody-coated magnetic beads, which were then incubated overnight with agitation at 4°C. Magnetic beads were washed three times with PBST for 10 min each, and 1× SDS elution buffer was added, followed by heating at 85°C for 10 min. Subsequently, the supernatant was harvested via aspiration, and 5× SDS loading buffer was added, followed by heating at 95°C for 10 min, before performing SDS-PAGE.

Total cellular protein measuring 150 μg was added to a 1.5-ml microcentrifuge tube, and primary antibodies NLRP3 (#15101, Cell Signaling Technology, USA) and IgG (#2729, Cell Signaling Technology, USA) measuring were added and incubated for 1 h at 4°C. The resuspended volume of Protein A/G plus-agarose (sc-2003, Santa Cruz, USA) measuring 20 μl was added and incubated at 4°C on a rotating device for 1 h overnight. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C, and the pellet was washed four times with PBS, each time repeating the above centrifugation steps. After the final wash, the supernatant was aspirated and discarded, and the pellet was resuspended in a 1× electrophoresis sample buffer. Samples were boiled for 2–3 min and analyzed by SDS-PAGE. Proteins were transferred onto methanol-activated 0.2-μm PVDF membranes. Membranes were washed three times in TBST and blocked in 5% non-fat dried milk in TBST. Membranes were then incubated overnight with an anti-Ub antibody.

Results are expressed as means ± SEM. All data were analyzed using PASW Statistics 21.0. Student’s t-test was applied for comparison between two groups and one-way ANOVA for multiple comparisons. Survival data were analyzed using the log-rank test. Differences were considered statistically significant when p < 0.05.

In order to demonstrate the protective effects of HSF1 against sepsis-induced lung injury, CLP was used to prepare the sepsis model, and the survival curve was plotted. As shown in Figure 1A , the survival rate of HSF1 knockout (HSF1^−/−) mice was significantly lower than that of HSF1^+/+ mice. Subsequently, the histological examination of lung tissues after CLP was detected. H&E staining showed hyperemia and edema in the alveolar cavity, the alveolar septa were significantly widened, accompanied by a large number of inflammatory cells infiltration, and the HSF1^−/− group was significantly more serious than the HSF1^+/+ group ( Figure 1B ). In addition, ELISA showed that the expression of inflammatory cytokines IL-1β and IL-18 in lung tissue homogenate and alveolar lavage fluid of mice after CLP was significantly increased, and the HSF1^−/− group was significantly higher than the HSF1^+/+ group ( Figures 1C–F ). The above results indicated that HSF1 can significantly alleviate CLP-induced ALI and improve the survival rate of septic mice by inhibiting the inflammatory response.

To clarify the NLRP3 inflammasome activation in sepsis, we performed CLP on mice to observe the NLRP3 inflammasome expression in lung tissue. It is found that the mRNA and protein levels of NLRP3 and IL-1β were both increased in the HSF1^+/+ and HSF1^−/− groups after CLP, but the enhanced expression was significantly higher in the HSF1^−/− group ( Figures 2A–D ). The inflammasome-related molecule expression was also detected in lung tissue. CLP intervention significantly increased the production of IL-1β and IL-18 in serum in both the HSF1^−/− and HSF1^+/+ groups, with a significantly higher cytokine increase in the HSF1^−/− group ( Figures 2E, F ).

Peritoneal primary macrophages were isolated from HSF1^−/− and HSF1^+/+ mice. The peritoneal primary macrophages were stimulated with ATP, an activator of the NLRP3 inflammasome (17). A total of 1 day later, the cells were stimulated with LPS for 3 h, followed by ATP (2.5 mM) for 30 min. As shown in Figure 3 , the mRNA expressions of NLRP3, caspase-1, IL-18, and IL-1β in macrophages were sharply increased in the HSF1^−/− group than in the HSF1^+/+ group after LPS and ATP stimulations. The protein expressions of NLRP3, activated caspase-1, and mature IL-1β also increased much higher in the HSF1^−/− group. The secretion of mature IL-1β and IL-18 in supernatants also increased much higher in HSF1^−/− mice. These results collectively indicate that HSF1 can inhibit the activation of NLRP3 inflammasome in the lung tissue of septic mice and primary peritoneal macrophages.

NF-κB activation is required for NLRP3 and pro-IL-1β production (18); therefore, we investigated whether HSF1 impaired LPS-induced NF-κB activation. TRAF3 is an important class of intracellular signal transduction factors involved in the signal transduction of a variety of receptor families, including the TNF receptor (TNFR) family, Toll-like receptor (TLR) family, interleukin-1 receptor (IL-1R) family, and RIG-I-like receptor (RLR) family, and plays an important role in innate and acquired immunity. When the receptor is activated, TRAF3 directly or indirectly recruits the intracellular domain of the downstream receptor, participates in signal transduction, and ultimately inhibits the NF-κB signaling pathway, which is involved in the occurrence and development of inflammatory immune diseases (19, 20). To further investigate the role of HSF1 in the NF-κB signaling pathway in septic ALI, we detected the expression of NF-κB related proteins TRAF3, p-IκBα, and p-p65 in the lung tissues of septic mice. As shown in Figure 4 , compared with the control group, TRAF3 and IκBα in the CLP group were decreased, while the expressions of p-IκBα and p-p65 were increased. Compared with the HSF1^+/+ group, the expressions of TRAF3 and IκBα in the HSF1^−/− group were decreased more obviously, and the expressions of p-IκBα and p-p65 were increased more obviously.

Similarly, peritoneal primary macrophages were isolated from HSF1^−/− and HSF1^+/+ mice. The cells were stimulated with LPS and ATP as described previously. We detected the expression of TRAF3, p-IκBα, and p-p65, and the results were consistent with those in the lung tissue of septic mice. Immunofluorescence detected the localization of phosphorylated P65 in primary peritoneal macrophages, compared with the control group, the fluorescence intensity of P65 in the LPS+ATP treatment group was increased while increased in the HSF1^−/− group compared with the HSF1^+/+ group. The results suggested that the activation of the NF-κB signaling pathway was increased in the HSF1^−/− group. These results indicated that HSF1 can effectively inhibit the activation of the NF-κB signaling pathway in the lung tissues of septic mice and primary peritoneal macrophages induced by LPS.

TRAF3 plays an important role in the activation of the NF-κB signaling pathway, and TRAF3 expression is decreased in both the lung tissues of septic mice and the primary peritoneal macrophages of LPS-stimulated. The decrease was more obvious in the HSF1^−/− group, suggesting a certain correlation between TRAF3 and HSF1. In order to investigate whether HSF1 has a transcriptional regulation effect on TRAF3, we used the Jaspar database (http://jaspar.genereg.net/) to predict the presence of HSF1 transcriptional binding elements in the TRAF3 promoter region (21). As shown in Figure 5 , the dual-luciferase reporter genes showed that the transcriptional activity of TRAF3 was significantly increased in the cells transfected with HSF1 composed of activated plasmid, while the transcriptional activity of the mutant TRAF3 was significantly decreased. Furthermore, the EMSA showed that the activation of HSF1 significantly increased the binding between HSF1 and TRAF3 DNA promoter region. At the same time, we co-transfected pcDNA3.1-HSF1 and siTRAF3 to detect the expression of NF-κB signaling pathway-related proteins and found that TRAF3 interference can reverse the inhibition of HSF1 on the NF-κB signaling pathway. These results suggest that HSF1 inhibited the NF-κB signaling pathway by upregulating TRAF3 expression, thereby inhibiting the production of NLRP3 at the transcriptional level and ultimately inhibiting the activation of the NLRP3 inflammasome.

Diverse PTMs determine the active state of NLRP3, among which polyubiquitination is critical for subsequent NLRP3 activation. FBXL2 is an SCF (SKP1-cullin-F-box) E3 ligase complex subunit that can recognize Trp73 in NLRP3 and target it for ubiquitination and proteasomal degradation (22). This finding established that the E3 ligase is required for NLRP3 ubiquitination-dependent activation, indicating that the SCF complex is most likely a key E3 ligase responsible for NLRP3 ubiquitination-dependent degradation in sepsis development. SGT1 (a suppressor of the G2 allele of SKP1) is a ubiquitin ligase-associated protein that associates with SKP1 and CUL1, subunits of the SCF ubiquitin ligase complex (23). Jaspar database (http://jaspar.genereg.net/) prediction identified the presence of HSF1 transcriptional binding elements in the SGT1 promoter region. Then we hypothesized that SGT1 may be involved in HSF1-regulated ubiquitination of NLRP3. We found that compared with the control group, the mRNA, and protein expression of SGT1 after CLP were decreased. Compared with the HSF1^+/+ group, the expression of SGT1 in the HSF1^−/− group decreased more obviously. Similarly, we extracted primary peritoneal macrophages to detect the expression of SGT1, and the results were consistent with those in the lung tissue of septic mice. These results indicated that SGT1 expression was decreased in both septic mice and primary peritoneal macrophages, and the knockout of HSF1 could inhibit the expression of SGT1 ( Figure 5 ).

The above findings suggest that HSF1 may be involved in the activation of the NLRP3 inflammasome by affecting the expression of SGT1. To prove whether HSF1 has a transcriptional regulation effect on SGT1, as shown in Figure 5 , the dual-luciferase reporter genes showed that the transcriptional activity of SGT1 was significantly increased in the cells transfected with HSF1 composed of activated plasmid, while the transcriptional activity of the mutant SGT1 was significantly decreased. EMSA showed that HSF1 activation significantly increased the binding of HSF1 to the SGT1 promoter region. These results suggest that HSF1 can bind the HSE in the SGT1 promoter region to promote the transcription expression of SGT1.

Protein interaction prediction from database String (https://string-db.org/) suggested that there may be interactions between SGT1 and NLRP3. To prove this hypothesis, we extracted primary peritoneal macrophages from mice and stimulated them with LPS+ATP for immunofluorescence co-localization. As shown in Figure 6 , SGT1 and NLRP3 were found to co-locate in the cytoplasm. Immunoprecipitation showed that SGT1 protein in the cytoplasm could be precipitated by an anti-NLRP3 antibody. Similarly, anti-SGT1 antibodies precipitate the NLRP3 protein in the cytoplasm successfully. The expression of SGT1 was increased and the expression of NLRP3 was decreased after HSF1 overexpression. In contrast, the expression of SGT1 was reduced and the expression of NLRP3 was increased after HSF1 knockout.

SGT1 can interact with a variety of proteins such as certain SCF ubiquitin ligases (23), so we hypothesize that SGT1 may be involved in the ubiquitination of NLRP3 inflammasome. As shown in Figure 6 , the ubiquitination of NLRP3 was significantly increased after SGT1 and HSF1 overexpression. However, in the primary peritoneal macrophages of the HSF1^−/− group, the ubiquitination of NLRP3 was significantly reduced compared with the HSF1^+/+ group. These results suggest that HSF1 may inhibit the activation of NLRP3 inflammasome through SGT1-mediated ubiquitination of NLRP3.

The serum of septic patients was collected to further clarify NLRP3 inflammasome activation. The serum levels of IL-1β and IL-18 from septic patients and healthy controls (HCs) were detected. As shown in Figure 7 , compared with HCs, septic patients had a higher expression of IL-1β and IL-18. Further detection of TRAF3 and SGT1 in the serum found that the expressions of TRAF3 and SGT1 were significantly lower in septic patients than in HCs ( Figure 7 ). These results suggest that decreased expression of TRAF3 and SGT1 as well as increased expression of IL-1β and IL-18 may be associated with the poor prognosis of patients with clinical sepsis, and this result is also consistent with our previous observation on the overall animal and cellular levels.