C57BL/6 male mice were purchased from Charles River Laboratories (Fairfield, NJ). The TREM-1^–/– mice were generated by the trans-NIH Knockout Mouse Project (KOMP) and obtained from the KOMP Repository, University of California, Davis (12). All experiments utilized age-matched healthy mice aged 9-12 weeks, and only male mice were included in the study. Both male and female sex steroids have been documented to exert diverse immune-modulating functions in humoral and cell-mediated immune responses under normal and various disease conditions (24). Since males and females exhibit distinct circadian rhythms, which could potentially impact the study’s findings (25, 26), we have specifically used male mice in this study to exclude the effect of sexual dimorphism on circadian gene expression. Animals were housed in a temperature-controlled room with a 12-hour light/12-hour dark cycle and fed a standard Purina rodent chow diet. Mice were allowed to acclimate to the environment for at least 7 days before being used in experiments. All experiments were performed following the National Institutes of Health guidelines for experimental animals and were approved by the Institutional Animal Care and Use Committee (IACUC).

Wild-type male mice were subjected to sepsis induction through cecal ligation and puncture (CLP) (12). Anesthesia was administered via 2% isoflurane inhalation, followed by shaving and disinfection of the abdominal area with povidone-iodine. A 1.5-cm midline incision allowed exposure of the cecum, which was ligated with 4-0 silk suture 1 cm proximal to the distal cecal tip. Subsequently, a through-and-through puncture was performed using a 22-gauge needle, extruding a small amount of cecal content. The cecum was then repositioned in the abdominal cavity, and the incision was closed in layers. Sham-operated mice underwent laparotomy without cecal ligation or puncture. After 20 hours post-CLP, mice were euthanized through CO₂ asphyxiation, and serum samples were collected to assess eCIRP levels.

Recombinant mouse CIRP (rmCIRP, i.e., eCIRP) was prepared in-house, and quality control assays were performed as described previously (11). The quality of the purified protein was assessed by Ponceau staining and Western blotting. A functional assay was performed by assessing the TNF-α levels in macrophages after treatment with purified rmCIRP. The level of endotoxin in the purified protein was measured by a limulus amebocyte lysate assay kit (Lonza, Basel, Switzerland). Only the purified protein lots that were free from endotoxin were used for experiments. We performed these quality control assays for each purified protein lot.

Peritoneal macrophages (PerM) were isolated from adult male mice, with euthanasia performed through CO₂ asphyxiation. Cells from the peritoneal cavity were collected via peritoneal lavage using cold Hanks’ Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺, supplemented with 2% FBS. Total peritoneal cells were isolated by centrifugation at 300 × g for 10 minutes at 4°C and subsequently cultured in complete RPMI 1640 medium [RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin–streptomycin, and 25 mM HEPES] at 37°C in 5% CO₂. After 2 hours, nonadherent cells were removed, and adherent cells, predominantly macrophages, were cultured overnight before use. Immortalized mouse bone marrow-derived macrophages (iBMDMs) were procured from Applied Biological Materials (Cat# T0673; Richmond, Vancouver, Canada) and cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 2 mM glutamine, and 100 IU/ml penicillin-streptomycin. Cells were maintained in a humidified incubator with 5% CO₂ at 37°C. For eCIRP treatment, PerM and iBMDMs were seeded into plates and cultured overnight. The media were changed to Opti-MEM medium (Cat# 31985-070; Thermo Fisher Scientific, Waltham, MA) 2 hours prior to treatment, and rmCIRP was applied at doses of 0.2 and 1.0 µg/ml in the experiments.

Cellular RNA extraction was performed using the RNAspin mini-isolation kit (Cat# 25050072; Cytiva, Buckinghamshire, UK). For PCR Array analysis, cDNA was synthesized utilizing the RT first Strand kit (Cat# 330401; Qiagen, Germantown, MD). The impact of eCIRP on a panel of circadian genes was assessed through the mouse circadian rhythms RT2 Profiler PCR Array (Cat# 330231 PAMM-153ZA; Qiagen), comprising 84 circadian-related genes. Data analysis was carried out using an online spreadsheet-based tool provided by Qiagen. In the case of relative quantitative RT-PCR, cDNA synthesis employed MLV reverse transcriptase (Cat# 28025013; Thermo Fisher Scientific). PCR reactions were executed in a final volume of 20 μl, containing 0.08 μM of each forward and reverse primer ( Table 1 ), cDNA, water, and SYBR Green master mix (Cat# 4368708; Thermo Fisher Scientific). Amplification and analysis were performed using a StepOnePlus real-time PCR machine (Thermo Fisher Scientific). Mouse β-actin mRNA served as an internal control for amplification, and relative gene expression levels were determined using the ^ΔΔCT method. The mRNA expression was presented as fold change compared to the control group.

Serum concentrations of eCIRP were assessed using an ELISA kit obtained from American Research Products (Cat# EM6504; Waltham, MA). The analysis of cytokine levels in cell culture supernatants, specifically TNF-α and IL-6, was conducted through ELISA kits procured from BD Biosciences (Cat# 558534 and 555240; San Jose, CA). All ELISA assays adhered to the protocols outlined by the respective manufacturers.

CRISPR Activation Plasmid products facilitate the identification and upregulation of specific genes through a D10A and N863A deactivated Cas9 (dCas9) nuclease fused to a VP64 activation domain, paired with sgRNA (MS2). This sgRNA is engineered to bind the MS2-P65-HSF1 fusion protein (27), forming a synergistic activation mediator (SAM) transcription activation system. SAM complex binds to a specific site upstream of the transcriptional start site (TSS) of the target gene, recruiting transcription factors and thereby activating endogenous transcription (27). The mouse BMAL2 CRISPR activation plasmid and the control CRISPR activation plasmid were procured from Santa Cruz Biotechnology (Cat# sc-435462-ACT and sc-437275; Dallas, TX). iBMDMs were seeded into 12-well plates in complete DMEM medium without antibiotics and cultured to 50-70% confluency. CRISPR BMAL2 activation and control plasmids were transfected into the cells using UltraCruz transfection reagents (Cat# sc-395739; Santa Cruz Biotechnology), followed by a 24-hour incubation period. Subsequently, cells were collected by centrifugation, and total RNA was extracted for the analysis of BMAL2 expression.

The promoter region of mouse CD274 (PD-L1) was identified by analyzing the 2000-nucleotide sequence upstream from the transcription start site of CD274 (NC_000085.7). Additionally, the sequence was retrieved from the Eukaryotic Promoter Database with the Promoter ID: CD274_1 (28). Structural modeling of the CD274 promoter was carried out using the 3DNA tool (29). This tool employs all possible canonical and non-canonical base pair geometries, relying on sequence-specific base pair step and base pair rigid body parameters, as well as template coordinates to generate a 3-dimensional structure model. The BMAL2 structure was modeled using the ITasser (Iterative Threading ASSEmbly Refinement) tool (30), which utilizes a threading approach to identify templates and model structures based on maximum percentage identity, sequence coverage, and confidence. The protein structure underwent further refinement through repeated relaxations, employing short molecular dynamics simulations for both mild (0.6 ps) and aggressive (0.8 ps) relaxations with a 4-fs time step after structural perturbations. Model refinement resulted in improved parameters, including enhanced Rama-favored residues and a decrease in poor rotamers. Additionally, a model for the scramble DNA sequence was generated.

The E-box motif of the BMAL2 transcription factor was identified within the CD274 (PD-L1) promoter. Subsequently, the BMAL2 protein was docked onto both the CD274 promoter and a scramble DNA sequence using the HDock tool (31) to create protein-DNA complexes. HDock employs a Fast Fourier Transform (FFT) based translational search algorithm, optimized through iterative knowledge-based scoring functions. Interactions between BMAL2 and the CD274 promoter, as well as the scramble DNA, were computed using the PDBePISA tool (32). To facilitate structure visualization, the BMAL2-CD274 promoter complex was rendered using PyMOL and Chimera tools (33).

To examine the direct interaction between recombinant human BMAL2 and PD-L1 promoter, surface plasmon resonance (SPR), OpenSPR (Nicoya, kitchener, Ontario), was performed between BMAL2 and PD-L1 promoter. Recombinant human BMAL2 protein (Cat# H00056938-Q01-25 µg; Novus Biologicals, Centennial, CO) was immobilized on high-sensitivity carboxyl sensor and the DNA oligo with the predicted sequence of BMAL binding site was injected as an analyte at concentration of 50 nM, 100 nM and 200 nM. Briefly, the carboxyl sensor was first cleaned by injection 150 µl of 10 mM HCL, followed by injection of 150 µl of the mixture of 1 aliquot of EDC and 1 aliquot of NHS to activate the sensor surface. An aliquot of 200 µl of 50 µg/ml of the ligand diluted in 10 mM sodium acetate (pH 5.5) was injected at 20 µL/min into flow cell-channel-2 of the sensor for immobilization. The flow cell channel-1 was used as a control to evaluate nonspecific binding. The binding analysis were performed at a flow rate of 30 μl per minute at 20°C. To evaluate the binding, the analyte was injected into flow cells-channel-1 and channel-2, and the real-time interaction data were analyzed by TraceDrawer software (Nicoya). The signals from the control channel (channel-1) were subtracted from the channel coated with the ligand (channel-2) for all samples. The data were globally fitted for 1:1 binding.

Data are presented as mean ± SEM. Statistical comparisons among multiple groups were conducted using one-way ANOVA followed by Tukey’s test. For comparisons between two groups, an unpaired two-tailed Student’s t-test was employed. A significance level of P<0.05 was considered statistically significant.

We examined the serum levels of eCIRP in septic mice and observed a significant elevation 20 hours after CLP compared to sham mice ( Figure 1A ). To investigate the induction of macrophage endotoxin tolerance by eCIRP, we pre-treated macrophages with varying doses of eCIRP for 24 hours. Subsequently, the media was replaced, and the cells were stimulated with LPS for 5 hours. Following this, the levels of TNF-α and IL-6 in the culture supernatants were evaluated. Notably, macrophages pre-treated with eCIRP exhibited a decreased release of TNF-α and IL-6 upon LPS challenge compared to cells pre-treated with PBS ( Figures 1B, C ). This observation suggests that pre-treatment with eCIRP induced macrophages to develop tolerance to LPS responsiveness. Interestingly, the magnitude of macrophage LPS tolerance was more pronounced in cells pre-treated with an increased dose of eCIRP compared to those treated with a lower dose. This implies a dose-dependent relationship, where the intensity of macrophage LPS tolerance correlates with the escalating doses of eCIRP. Higher doses of eCIRP resulted in more intense tolerance. Subsequently, we assessed the expression of genes commonly examined as markers of immune suppression in macrophages, including PD-L1, IL-10, and signal transducer and activator of transcription 3 (STAT3). Our data revealed a significant, dose-dependent increase in the expression of these genes upon eCIRP treatment ( Figures 1D–F ). These findings suggest that eCIRP promotes macrophage endotoxin tolerance.

Given the recognized role of TREM-1 as a receptor for eCIRP-mediated function (12), we assessed TREM-1 expression in macrophages after eCIRP treatment. Our findings demonstrated a substantial increase in TREM-1 expression in macrophages treated with eCIRP compared to controls ( Figure 2A ). The involvement of TREM-1 in eCIRP-mediated immune tolerance remains elusive. To address this, we pre-treated TREM-1 knockout macrophages with either PBS or eCIRP for 24 hours. Subsequently, the media were replaced, and the cells were subjected to LPS stimulation for 5 hours. The culture supernatants were then collected to evaluate TNF-α release. Strikingly, our data revealed that TREM-1 knockout macrophages pre-treated with eCIRP did not exhibit any noticeable decrease in TNF-α release compared to PBS pre-treated cells ( Figure 2B ). This suggests that the deficiency of TREM-1 could attenuate macrophage endotoxin tolerance. Additionally, we identified that WT macrophages treated with eCIRP exhibited a significant increase in the expression of immune suppression markers, including PD-L1, IL-10, and STAT3 ( Figures 2C–E ). Intriguingly, the expression of these genes was significantly reduced in TREM-1 knockout macrophages compared to eCIRP-treated WT macrophages ( Figures 2C–E ). These findings imply that eCIRP induces macrophage endotoxin tolerance through TREM-1, highlighting TREM-1 as a crucial mediator in eCIRP-induced macrophage endotoxin tolerance.

We conducted a PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP. The analysis indicated an augmentation in the expression of circadian genes. Specifically, the core circadian genes, including BMAL2, CRY1, and PER2, exhibited a marked upregulation in peritoneal macrophages treated with eCIRP ( Figure 3A ). Notably, the expression of another BAML isotype, BMAL1, was less increased in these cells, prompting our focus on BMAL2 and its role in eCIRP-induced macrophage immune tolerance. Validation through qPCR confirmed a significant upregulation of these genes (BMAL2, CRY1, and PER2) in peritoneal macrophages stimulated with eCIRP compared to controls ( Figures 3B–D ). Further investigation involved evaluating the expression of circadian genes (BMAL2, CRY1, and PER2) in both WT and TREM-1 knock-out macrophages treated with eCIRP. Interestingly, the expression levels of BMAL2, CRY1, and PER2 in eCIRP-treated macrophages lacking TREM-1 were notably lower than those in eCIRP-treated WT macrophages ( Figure 3E ). These results indicate that eCIRP, increased in sepsis, enhances the expression of circadian genes through the mediation of TREM-1.

We analyzed the human scRNA-seq data of healthy control (HC) and septic patients obtained from the GEO database (GSE167363). We found that the gene expression of ARNTL2 (BMAL2) in the monocytes of septic patients was markedly higher than healthy controls ( Figures 4A, B ). Furthermore, we found that the gene expression of CD274 (PD-L1) in the monocytes of septic patients was markedly higher than healthy controls ( Figures 4A, B ). Having established that eCIRP induces both BMAL2 expression and macrophage endotoxin tolerance, we aimed to elucidate a potential positive correlation between them. To explore whether heightened BMAL2 expression as occur in eCIRP-treated condition could indeed enhance macrophage endotoxin tolerance, we activated BMAL2 expression in macrophages by transfecting them with either a control plasmid or an activation plasmid targeting BMAL2 expression. Following transfection, macrophages treated with the BMAL2 activation plasmid exhibited a significant increase in BMAL2 expression compared to those transfected with the control plasmid ( Figure 4C ). Subsequent treatment of these cells with LPS revealed that macrophages transfected with the BMAL2 activation plasmid displayed decreased TNF-α release and increased PD-L1 expression compared to control plasmid-treated cells ( Figures 4D, E ). These findings suggest that BMAL2 acts as a positive regulator of macrophage endotoxin tolerance.

The promoter region of mouse CD274 (PD-L1) was identified by analyzing the 2000-nucleotide sequence upstream from the transcription start site of CD274 (NC_000085.7). Using in silico model we determined the binding of mouse and human BAML2 protein at the promoter region of mouse and human PD-L1 gene by the free energy of binding upon complex formation (ΔⁱG). ΔⁱG of BMAL2-PD-L1 promoter on mouse and human were -29.8 and -24.0 Kcal/mol, respectively ( Figures 5A, C ; Table 2 ). In comparison with BMAL1, BMAL2 showed stronger binding to PD-L1 promoter by the decrease in the free energy of binding upon complex formation, ΔⁱG ( Table 3 ). Similarly, we also found that the mouse and human BMAL2 could bind to the E-box motif on the PD-L1 promoter with the free energy of binding upon complex formation ΔⁱG at -23.6 Kcal/mol in mouse and 14.9 Kcal/mol in human, respectively ( Figures 5B, D ; Table 2 ). We also experimentally validated the binding of human BMAL2 and human putative promoter sequence of PD-L1 by BIAcore assay revealing a strong binding between these two molecules at a K_D value of 2.09 x 10^-7 M ( Figure 5E ). These data reveal that BMAL2 positively regulate PD-L1 expression by binding to the PD-L1 promoter. Taken together, eCIRP increases BMAL2 expression in macrophages via TREM-1, ultimately leading to endotoxin tolerance ( Figure 6 ).