Apolipoprotein H as a Crucial Immunomodulatory Factor in Sepsis

doi:10.21203/rs.3.rs-3332030/v1

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Research Article

Apolipoprotein H as a Crucial Immunomodulatory Factor in Sepsis

https://doi.org/10.21203/rs.3.rs-3332030/v1

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published 30 Jan, 2024

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Background.

Sepsis is a life-threatening acute organ dysfunction caused by a dysregulated immune response to infections. Apolipoprotein H (APOH) is an important plasma protein that regulates diverse biological processes. However, the role of APOH in the immunopathology of sepsis remains unidentified.

Methods.

APOH concentration was determined in pediatric patients with sepsis and healthy individuals. The effect of APOH on survival, organ injury, and inflammation were measured in an experimental sepsis model of cecal ligation and puncture (CLP). The possible mechanisms of APOH on anti-inflammation response were explored in PBMC, PM, BMDM and RAW 264.7 macrophages.

Results.

APOH concentration was significantly decreased in the patients with sepsis compared with those of healthy controls, and was negatively linked with severity and mortality of pediatric sepsis. Therapeutic administration of recombinant APOH protein reduced the mortality rate in mice with sepsis, alleviated organs injury and inhibited inflammation in mice with severe sepsis. Conversely, neutralizing APOH by anti-APOH monoclonal antibody led to an increased mortality rate, organs injury and inflammation in mice with nonsevere sepsis. Intriguingly, APOH had negligible influences on bacterial burden and the counts of neutrophils and macrophages in sepsis mice model, and bacterial phagocytosis and killing upon P. aeruginosa infection in PM, RAW 264.7 and PBMC cells. Mechanistic investigation in PM and RAW 264.7 cells indicated that APOH reshaped macrophage polarization by inhibiting M1 macrophage through suppression of toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway.

Conclusion.

This proof-of-concept study elucidated that APOH played an important role in the host defense response to sepsis and suggested a potential therapeutic role for APOH in the treatment of sepsis.

Sepsis

Apolipoprotein H

Macrophage polarization

TLR4/ NF-κB

Sepsis is defined as organ dysfunction syndrome with a high mortality rate caused by a dysregulated host response to infection, causing systemic multi-organ and multi-system damage (1–3). It has been estimated that 48.9 million incident sepsis cases and 11 million sepsis-related deaths worldwide in 2017 and its management and treatment is still a major challenge in medicine (4). The World Health Organization (WHO) declared that the improvement of sepsis prevention, recognition, and treatment is a global health priority (5). Despite numerous clinical trials, the development of potential drugs against sepsis has been particularly frustrating. Sepsis is considered to involve hyperinflammatory and immunosuppressive, which ultimately pertain to systemic inflammatory responses and deteriorating organ function (6). This result accounts for high mortality rates in patients afflicted with sepsis. Therefore, current efforts were focused on identification of immune regulators participating in disturbing host immune response, which may point toward novel therapeutic leads in sepsis.

The apolipoprotein H (APOH), also known as beta-2-glycoprotein I (β2GPI), with a molecular size of about 50 kDa, is a highly abundant plasma protein consisting of five domains (I-V) (7). APOH is mainly produced by liver cells and sever as a complement regulator in a variety of biological processes, including antiphospholipid syndrome (APS), procoagulant state, diabetes, and immune disorders (8–10). Alongside these, several studies supported that the APOH exerts a component of innate immunity through binding to viruses and bacteria (11, 12). Despite former researches have shown the role of APOH in multiple diseases, the correlation between the presence of APOH and the pathogenesis of sepsis remains unclear.

To examine the possible contribution of APOH in sepsis, we determined the level of circulating APOH in pediatric patients with sepsis. In addition, a clinically relevant model of cecal ligation and puncture (CLP)-induced sepsis, and multiple macrophages cells (including PBMC, PM, BMDM and RAW 264.7) were applied to investigate the role of APOH in host defense and explore the underlying molecular mechanism.

Study population

In pilot cohort, sixty-eight pediatric patients who met clinical criteria for International Pediatric Sepsis Conference 2005 (13) were recruited from Pediatric Intensive Care Unit (PICU) of Children’s Hospital of Chongqing Medical University between from December 2016 to October 2018. Sixty-eight sepsis patients were divided into two groups, 42 cases of survivors and 26 cases of nonsurvivors. Twenty-six healthy controls from healthy volunteers. Collected peripheral blood samples from each group were pooled into six samples for detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). In validation cohort, thirty-six septic pediatric patients were enrolled at admission to the PICU of Children’s Hospital of Chongqing Medical University between from January 2022 to March 2023. Peripheral blood was collected at the time of patient admission of 24 hours, 48 hours and 72 hours, and serum was isolated and stored at -80℃ until further analysis. The clinical data were recorded, including gender, age, white blood cells (WBC), procalcitonin (PCT) and C-reaction protein (CRP), Pediatric Sequential Organ Failure (pSOFA), Pediatric Critical Illness Score (PCIS) and systemic inflammatory response syndrome (SIRS) score at 24 hours on admission. Besides, microbiological findings, ventilation, use of adrenocortical hormone, renal replacement therapy, length of ICU stay, and mortality during the 28-day study period were also recorded. Sixty-nine healthy control blood samples were taken from healthy donors. These protocols were approved by the Clinical Research Ethics Committee of Institutional Review Board of Children’s Hospital of Chongqing Medical University (File No: ChiCTR-ROC-17011164 and (2022) Ethical Review Research No. 427) and informed consents were obtained from all participants according to the Declaration of Helsinki.

Experimental animals

All animal experiments were performed in accordance with guidelines from the Chongqing Experimental Animal Center and the Animal Committee of Children’s Hospital (CHCMU-IACUC20221227002).

C57BL/6 mice were purchased from Chongqing Medical University and were bred in a controlled environment (20–24°C and 12 hours light/dark cycle) with free access to standard chow and water. A model of polymicrobial sepsis was established through cecal ligation puncture (CLP) (14). Prior to surgery, mice were anesthetized with pentobarbital sodium (75 mg/kg body weight) intraperitoneally. About 1cm incision was performed in the midline abdomen after skin disinfection and the cecum was exposed, ligatured at the end 60% (severe CLP, 20–40% survival) or 40% (nonsevere CLP, 60–80% survival) and punctured with 21-gauge needle. Then the cecum was placed back into the abdominal cavity, and the incisions was closed. Normal saline (0.05 ml/g body weight) was injected intraperitoneally for resuscitation, and water and lab chow were reinstated after operation. Survival was monitored every day from day 2 to day 14 after operation. A humane endpoint was used for the CLP mice.

In vivo administration of recombinant mouse APOH

Mice were divided into two groups: CLP + 0.1% bovine serum albumin (BSA) and CLP + recombinant murine apolipoprotein H (rAPOH). Mice in the rAPOH group were injected with 20 µg of murine rAPOH (R&D systems, 6575-AH-050, USA) dissolved in 100 µl of phosphate buffer solution (PBS) intraperitoneally, whereas those in BSA group were received 0.1%BSA in 100 µl PBS instead.

Antibody-mediated blockade of APOH

In order to neutralize the activity of APOH in nonsevere CLP, 10 µg of anti-mouse APOH neutralizing monoclonal antibody (Proteintech, Monoclonal Mouse IgG1, 66074-1-Ig, China) dissolved in 100 µl PBS was injected in CLP + anti-APOH (anti-APOH), whereas another group mice were injected mouse IgG1 antibody as control (IgG control).

Enzyme-linked immunosorbent assay (ELISA)

The human concentrations of APOH were quantified using commercial ELISA kits (Ruixin biotech, RX103075, China,). The levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) (Neobioscience, EMC102a.96, China), Interleukin (IL)-1β (Neobioscience, EMC001b.96, China), IL-6 (Neobioscience, EMC004.96, China) and IL-10 (Neobioscience, EMC005.96, China) in serum and peritoneal lavage fluids (PLF) were detected using the commercially available ELISA kits.

Histopathology

Lungs, livers, and kidneys tissues were harvested and the hematoxylin and eosin (H&E) were used to assess pathology changes. Fresh samples were washed with clod PBS and fixed in 4% paraformaldehyde. Then the tissues were dehydrated, embedded in paraffin, cut into 4 µm thickness slices and stained routinely. The pathology scores of lungs, livers, and kidneys were performed according to the following categories respectively: lung histological alterations including edema, congestion, interstitial inflammation, and inflammatory cell infiltration, and livers, and kidneys histological alterations including the numbers of thrombi and abscesses, inflammation, and necrosis (15, 16).

Bacterial colony-forming units

Dilutions of peripheral blood, PLF, liver, lung, or kidney of mice were plated on blood-agar plates overnight at 37℃. Colony-forming unit (CFU) counts were then counted after 24 hours culture (15) .

Isolation of peritoneal macrophages, bone marrow-derived macrophages and peripheral blood mononuclear cell

Mice were intraperitoneally injected with thioglycollate fluid culture medium to recruit macrophage. To isolate peritoneal macrophages (PMs), peritoneal lavage was performed with 5 ml of PBS. The peritoneal lavage fluid (PLF) was centrifuged at 500 ×g for 10 min at 4°C and, then the erythrocytes were lysed by red blood cells (RBC) lysis buffer (TIANGEN, RT122-02, China,) for 5 minutes at room temperature. The sediments were obtained through 500 ×g for 10 minutes at 4°C and washed with PBS. Then cells were harvested by plastic adherence.

Mouse Bone marrow-derived macrophages (BMDMs) were isolated from femur and tibia of C57BL/6 mice and differentiated into macrophage by 50 ng/ml recombinant Granulocyte macrophage colony-stimulating factor (GM-CSF) (Medchemexpress, HY-P7361, USA) in DMEM (Gibco, Life Technologies, Germany).

Peripheral blood mononuclear cells (PBMCs) were isolated from blood buffy using a Lympholyte®-H (Cedarlane, CL5020-R, Canada) through density gradient. The PBMCs were washed twice with 1640 (Gibco, Life Technologies, Germany) containing 2% fetal bovine serum (FBS) and incubated in an erythrocyte lysis buffer for 5 minutes at room temperature. Subsequently, PBMCs suspended in RPMI 1640 medium containing 10% FBS and differentiated into macrophage by 20 ng/ml recombinant GM-CSF.

In vitro administration of recombinant APOH

RAW 264.7 macrophages were obtained from Institute of Children's Hospital Affiliated to Chongqing Medical University. PMs, BMDM, PBMC and RAW 264.7 macrophages were cultured in DMEM or 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO₂ incubator at 37℃. Cells were preincubated with rAPOH 30 minutes (20 ng/ml) and treated with ultrapurified lipopolysaccharide (LPS) (100 ng/ml) from Escherichia coli O55:B5 (Abmole, M9524, American) for 6 hours.

Flow cytometry experiments

To ascertain whether any observable differences in the abundance of infiltrating leukocytes after CLP within PLF between the rAPOH and BSA groups could be observed. Peritoneal macrophages were collected and incubated accordingly with monoclonal antibodies against CD16/32 (Biolegend,101302, USA), CD11b (Biolegend, 101212, USA), isotypic CD11b (Biolegend, 400611, USA), Ly6G (Biolegend, USA, 127605), isotypic Ly6G (Biolegend,400505, USA), F4/80 (Biolegend, 123110, USA) and isotypic F4/80 (Biolegend, 400507, USA).

In vitro experiment, RAW 264.7 and PMs cells were preincubated with rAPOH 30 minutes and treated with LPS 6 hours. Then cells were adjusted to 10⁶/µl, and 100 µl specimens and 1µl F4/80, CD11b, CD86 (Biolegend,105013, USA), and CD206 (Biolegend, 141709, USA) antibodies were added and incubated at room temperature for 30 minutes in the dark and were examined by flow cytometry (FACSVerse™, BD Biosciences).

Phagocytosis function assay

PMs, PBMC and RAW 264.7 cells were seeded in 24-well plates and were incubated with rAPOH for 20 hours. Cells were infected with fluorescein isothiocyanate–labeled Pseudomonas aeruginosa (P.a) at a multiplicity of infection (MOI) of 100 for 30 minutes at 37℃ and washed and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China, C1005) and TRITC Phalloidin (Solarbio China, CA1610), followed by visualization using confocal laser scanning microscopy (Nikon C2+).

Bacterial killing assays

For determination of bacterial killing by macrophages, PMs and RAW 264.7 macrophages (1x10⁵ cells) were preincubated with recombinant mouse APOH 20 h and infected with Pseudomonas aeruginosa (P. a) (MOI,1:100) at 37°C incubator for 30 minutes at 70 revolutions per minute (RPM), and then cells were washed with buffer containing gentamycin (200 ug/ml) for 15 minutes at 4°C to remove extracellular bacteria and washed 3 times in PBS. Then cells were lysed by sterile double distilled water for 20 minutes at room temperature. Live intracellular bacteria were calculated to quantitate bacterial killing by culture of lysates at 37°C for 2 hours. Lysates were cultured on blood-agar plates overnight at 37℃ and CFU counts were counted. Killing rate= (number of CFUs = 0 hour - number of CFUs = 2 hours)/ number CFUs = 0 hour was calculated.

RNA extraction and quantitative real-time PCR

Total RNAs were extracted from cells using the RNA Isolation Kit (Beyotime, R0027, China) and reversely transcribed to cDNA by using ABScript III RT Master Mix for qPCR with gDNA remover kit (ABclonal, RK20429, China) from 1 µg RNA. The Real-time quantitative PCR (qPCR) was performed by using SYBR Green (ABclonal, Rk21203, China) to detect gene expression on BIO-RAD CFX Connect™ Real-Time System (Bio-Rad, USA). The primer sequences are listed in Additional file 2: Supplemental Table 2 and the results were calculated by the 2−^△△Cq normalized to GAPGH.

Western blotting analysis

Proteins were harvested from cells by using radio immunoprecipitation assay (RIPA) lysis buffer (MCE, HY-K1001, USA), supplemented with 1% phenylmethanesulfonylfluoride (PMSF) (Beyotime, ST506, China), 1% proteases inhibitor (MCE, HY-K0010, USA) and 1% phosphatase inhibitors (MCE, HY-K0021, USA). The protein concentration was measured with NanoDrop one (Thermo Fisher). The proteins were separated by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels (EpiZyme Biotechnology, PG112, China) electrophoresis, and then were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010, USA). The membranes were blocked in NcmBlot blocking buffer (New Cell & Molecular Biotech, P30500, China) for 20 minutes and incubated with corresponding primary antibodies overnight at 4℃.The following primary antibody were used: anti-TLR4 (Proteintech, 66350-1-Ig, China), anti-TNF receptor associated factor 6 (TRAF6) (Zen-Bio, 380803, China), anti-Myd88 (Zen-Bio, 340629, China), anti- Jun N-terminal Kinase (JNK) (Abcam, USA,ab124956), anti-phospho-c Jun N-terminal kinase (p-JNK) (Abcam, ab179461, USA), anti-nuclear factor kappa-B (NF-κB) p65 (Zen-Bio, R25149, China), anti-phospho-p65 (Cell Signaling Technology, 3033T, USA), anti-IL-1β (Proteintech, 26048-1-AP, China), anti-TNF-α (Proteintech, 17590-1-AP, China), Inducible Nitric Oxide Synthase (iNOS) (Zen-Bio, 340688, China ), Arginase (Arg-1) (Zen-Bio, R380744, China).Then the brands were incubated with secondary antibodies: goat anti-rabbit HRP-conjugated polyclonal antibody (Hua bio, ET1601-4, China) and goat anti-mouse HRP-conjugated polyclonal antibody (Proteintech,66009-1-Ig, China) at the room temperature for 1 hour separately, and were visualized by Bio-Rad ChemiDoc™ Touch Imaging System (Bio-Rad, California, USA).

Statistical Analysis

Data were presented as mean ± standard deviation (SD). Groups difference were performed using t test (Mann-Whitney U test) or one-way ANOVA (Tukey’s multiple comparisons test). Log-rank (Mantel-Cox) tests were performed for survival studies and Non-parametric Spearman's Rank Correlation Coefficient was used to analyze correlations. Receiving operating characteristic (ROC) curves were constructed to analyze the discriminative power of APOH between healthy control and sepsis patients, and then the area under the curve (AUC) was determined with its 95% confidence interval (CI). All statistical tests were done using GraphPad Prism version 6.02 (GraphPad Software, San Diego, CA). P-values of < 0.05 were considered statistically significant.

APOH concentration is significantly decreased in patients with sepsis

In present study, our group using LC-MS/MS to search for candidate biomarkers of sepsis in pilot cohort (Data not shown) and using ELISA to validate the expression of APOH in validation cohort. Finally, a murine model of CLP-induced sepsis to verify the therapeutical effect of APOH (Fig. 1a). Characteristics of patients with sepsis and healthy controls in pilot cohort were shown in Additional file 1: Supplemental Table 1. We found that the level of APOH was lower in sepsis patients than healthy control (Fig. 1b), and the septic nonsurvivors had decreased expression of APOH than survivors (Fig. 1c). Additionally, we used AUC to analyze the diagnostic value of APOH levels between patients with sepsis and healthy control, and the predictive value between septic survivors and nonsurvivors. The AUC of APOH on diagnosing sepsis was 0.94 ([95% CI] 0.849–1.040, p < 0.001, Fig. 1d), and prognosing death was 0.88 ([95% CI] 0.702–1.076, p < 0.005, Fig. 1e). Subsequently, APOH expression levels were further validated in a novel patient cohort. Thirty-six patients with sepsis and sixty-nine healthy control individuals were recruited in this study. Characteristics of patients with sepsis and healthy controls were shown in Table 1. As expected, the serum concentrations of APOH were significantly reduced in patients with sepsis with respect to healthy control subjects (Fig. 1f). In addition, the levels of APOH were observed to diminish throughout the subsequent progression of sepsis (Fig. 1g). To further evaluate the role of APOH levels in predicting 28-day mortality in sepsis patients, the AUC of APOH, CRP, PCT and SOFA scores were studied. The AUC for APOH was 0.644 ([95% CI] 0.423–0.866, p = 0.270), higher than the AUC for PCT (AUC = 0.538, [95% CI] 0.285–0.791, p = 0.772), and PSOFA (AUC = 0.571, [95% CI]0.285–0.85, p = 0.772), but slightly lower than the CRP (AUC = 0.698 [95% CI]0.496–0.899, p = 0.135) (Fig. 1h). Furthermore, the AUC combination of APOH and CRP (AUC = 0.759, [95% CI]0.575–0.944, p = 0.05) was higher than APOH combination with PCT (AUC = 0.673, [95% CI]0.436–0.910, p = 0.192) and PSOFA (AUC = 0.623, [95% CI]0.363–0.884, p = 0.35) (Fig. 1i).

Table 1

Characteristics of pediatric patients with sepsis and healthy controls
Characteristics	Sepsis patients (n = 36)	healthy controls (n = 69)	P value
Sex (male/female)	16/20	41/28	0.144
Age (years)	2.54(0.88–6.86)	6.58(4.88–8.09)	0.004
WBC (10⁹/L)	7.94(4.21–13.24)	NA
CRP (mg/L)	65.23(16.57–99.96)	NA
Procalcitonin (ng/ml)	2.2(0.40–9.080)	NA
Infection site (Number of patients)
Respiratory	31	NA
Abdominal	4	NA
Vascular	0	NA
Urinary	1	NA
Bacteremia	1	NA
Isolates (Number of patients)
Gram positive	0	NA
Gram negative	9	NA
Fungus	0	NA
Virus	4	NA
Miscellaneous	13	NA
Other	10	NA
pSOFA score	4 (4–7)	NA
PCIS score	79(74–86)	NA
SIRS score	3(2–4)	NA
ICU stay(days)	7 (4-10.75)	NA
Ventilation	24/36	NA
use of adrenocortical hormone	13/36	NA
Renal replacement therapy	10/36	NA
Died/survived	6/36	NA
NOTE: Data are expressed as median (interquartile range) unless otherwise indicated. WBC: white blood cells; CRP: C-reaction protein; PCT: procalcitonin; SOFA: sequential organ failure assessment; ICU: intensive care unit; NA: not applicable.

Administration of recombinant murine APOH protein protects septic mice

To show the variation of APOH expression during the progression of sepsis, the serum levels of APOH were detected in CLP-induced polymicrobial sepsis. The concentration of APOH was significantly decreased in CLP mice than sham mice throughout the subsequent progression of sepsis (Fig. 2a). Obviously, CLP mice exhibited increased level of IL-6 than Sham mice (Fig. 2b), and had a significant negative correlation between APOH expression (Fig. 2c). Besides, we found that APOH levels in the lung, liver, and kidney were decreased in CLP group than Sham group (Fig. 2d-e). For the induction of severe sepsis resulting in survival rates of 20%-40%, the cecum is ligated (indicated by dotted green line) at 60% distance between distal pole and the base of the cecum (yellow dotted line) (Fig. 3a). To further confirm the protective role of APOH in the pathogenesis of CLP mice, the effects doses (5–20 µg) of recombinant murine APOH on severe sepsis were assessed in the model of CLP (Fig. 3b). The result showed that supplementation of rAPOH (20 µg) significantly improved mice survival than the BSA-treated (Fig. 3c). The increased survival of rAPOH mice may be related to decrease tissue inflammation and organ injury, because there were ameliorated levels of tissue inflammation and injuries in lung, liver, and kidney (Fig. 3d-e). Moreover, significantly reduced inflammatory cytokines, including TNFα, IL-1β and IL-6, but increased IL-10 level, were observed in samples of serum and PLF from rAPOH-treated septic mice than BSA control (Fig. 3f-g).

Anti-APOH antibody worsens mice from nonsevere sepsis

In addition, nonsevere CLP was established through ligating cecum (indicated by dotted green line) at 40% distance between distal pole and the base of the cecum (yellow dotted line) (Fig. 4a). We investigated the potential detrimental effects doses (5–10 µg) of blocking APOH using a murine anti-APOH antibody in vivo (Fig. 4b). The survival rate of septic mice in anti-APOH group was significantly lower compared with septic mice treated with IgG control (Fig. 4c). Lung, liver, and kidney injuries were significantly aggravated in septic mice treated with anti-APOH antibody after CLP as compared with control animals (Fig. 4d-e). Likewise, Blockade of APOH after CLP also significantly decreased the levels of serum and peritoneal inflammatory cytokines, including TNFα, IL-1β and IL-6, but decreased IL-10 level after nonsevere sepsis (Fig. 4f-g). These results showing that blockade of APOH could exacerbate sepsis.

APOH has minimal influence in bacterial phagocytosis and killing upon P. aeruginosa infection

The decreased inflammation levels in rAPOH mice could not be explained by improved bacterial clearance, because there was no statistical difference in local or systemic bacterial CFU levels in the septic mice treated with recombinant APOH compared with mice treated with BSA control (Fig. 5a). Furthermore, we used the flow cytometry experiments to detect the proportions of different cell types of infiltrating leukocytes within PLF between rAPOH and BSA groups, but we found that the proportions of macrophages exhibited an increasing trend after APOH intervention (Fig. 5b-c). To investigate whether APOH modulates the intrinsic antibacterial functions of macrophages, we preincubated PMs and RAW 264.7 macrophages for 20 hours with recombinant APOH before exposure to P. aeruginosa. However, preincubation with APOH did not have direct effects on the killing capacities (Fig. 5d-e) and bacterial uptake (Fig. 5f-g) in macrophages upon P. aeruginosa infection.

APOH downregulates inflammatory cytokine responses through inhibiting M1 polarization in the macrophages

To determine underlying mechanisms by which rAPOH mice exhibited lower levels of inflammation than BSA control. We examined the effects of APOH on the expression of cytokines when cells were challenged with LPS. As shown in Fig. 6, preincubation with recombinant APOH significantly reduced the mRNA and protein levels of TNF-a and IL-1β compared with the LPS alone group in PMs, BMDM, PBMC and RAW 264.7 cells (Fig. 6a-l). The inhibitory effect of APOH on inflammatory cytokines may be linked to the switch between M1 and M2 subtypes. To test this possibility, we evaluated the ability of APOH to regulate macrophages polarization in PMs and RAW 264.7 cells. Although no differences in the proportion of CD206 cells and protein level of Arg-1 were noted, preincubation with recombinant APOH down-regulated the proportion of CD86 cells and the protein level of iNOS (Fig. 7a-l). These data indicated that pretreatment of APOH could inhibit the M1 type and promote the M2 type polarization in macrophages to a certain degree.

APOH regulates macrophage polarization via the TLR4-mediated NF-κB signaling pathway

As a pattern recognition receptor, TLR4 plays a critical role in the macrophage-mediated inflammatory response (17, 18). Our results showed that the mRNA levels of TLR4/ NF-κB pathway were upregulated on PM and RAW 264.7 cells following LPS stimulation, an effect that was blocked by APOH pretreatment (Fig. 8a and j). Additionally, pretreatment with APOH also inhibited the proteins levels of TLR4/ NF-κB pathway in PMs (Fig. 8b-i) and RAW 264.7(Fig. 8k-r) cells. These findings indicated that APOH suppressed M1 macrophage polarization by the TLR4/NF-κB pathway, which is contributed to anti-inflammatory role of APOH in sepsis.

In the current study, we identified APOH to be centrally involved in the pathogenesis of sepsis. To our knowledge, APOH has never been evaluated in the pediatric patients with sepsis. Firstly, we found that circulating APOH levels were declined in pediatric patients with sepsis and remained low levels during the disease process than healthy control. Then, we found that the diagnostic and prognostic AUC of APOH was 0.94 and 0.88, suggesting that detection of APOH may be contributory to the diagnosis and prognosis of sepsis. Moreover, APOH was found to be an independent predictor of 28-day mortality on day of ICU admission to some extent than PCT and PSOFA. As treatment of sepsis, we demonstrate that APOH administration could improving survival rates in septic mice through decreasing the expressions inflammatory cytokine of TNF-α, IL-1β and IL-6, and alleviating the tissues injuries. Furthermore, neutralization of the APOH activity with antibody against APOH exacerbated mortality rate in a model of nonsevere sepsis. Finally, in vitro experiments, we revealed that APOH pretreatment could efficiently inhibit M1 polarization through down-regulating TLR4/NF-κB pathway upon LPS challenge not inhibiting bacterial phagocytosis and killing capacities.

It has been suggested that APOH could serve as a immunoregulation factor after binding to specific surfaces such as apoptotic cells and bacteria (19). After the conformational change of APOH, monocytes can clear APOH, resulting in decreased APOH serum level (11, 12). In a pilot cohort, the decreased relative abundance of APOH were found of sepsis patients than healthy controls. In addition, septic nonsurvivors had lower APOH than septic survivors. Likewise, similar results were verified in another new validation cohort. We revealed dramatically decreased APOH levels in pediatric patients with sepsis compared to healthy controls. Moreover, the expression levels of APOH at 48 and 72 hours after admission were significantly lower than those at 24 hours. It's worth noting that mortality in pilot cohort was higher than validation cohort. We suspected that the possibilities for that were the emphasis on the collection of nonsurvival patients to determine the disparity between nonsurvival patients and survival patients; Additionally, the time interval and the therapeutical regimen in the two cohorts were distinct.

Like our study, El-Assaad et al demonstrated a similar drop in total β2GPI (APOH) levels in male septic patients than normal male controls, but they found no difference in the levels of APOH between female septic patients and female non-septic controls in their study. In terms of sex-specific difference, they using an infection model of septicemia in both male and female wild type (WT) and β2GPI deficient (β2GPI ^−/−) mice challenged with Escherichia coli (E. coli) intravenously to further explore this phenomenon and suggested that the decreased β2GPI level in male was due to β2GPI being functionally utilized as part of the immune systems protective response against E. coli (12). Therefore, these fundings suggested that APOH may mediate an important role in modulating sepsis pathophysiology. In addition, we demonstrated, for the first time, that the insufficiency of APOH was closely related to deterioration in pediatric patient wellbeing, suggesting that continuous detection of APOH may be contributory to predict 28-day mortality on day of ICU admission. Although the AUC of APOH was lower than that of CRP in predicting 28-day mortality, it was superior to PCT and pSOFA.

To further investigate the role of APOH in the pathogenesis of sepsis, we established a clinically relevant murine model of sepsis induced by CLP. Consequently, we found that the CLP mice had lower serum level of APOH in the progression of disease and had decreased APOH expressions in lung, liver, and kidney tissues than Sham mice. This was consistent with what we have observed clinically. Subsequently, rAPOH supplementation increased survival and decreased tissues inflammation and injuries in mice after severe CLP. Furthermore, when neutralization of APOH resulted in exacerbated mortality rate, increased systemic inflammation and tissues injuries in a model of nonsevere sepsis. In line with a previous study, they showed that β2GPI(APOH) deficiency mice (β2GPI^−/−) developed severe septicemia earlier than WT mice (male β2GPI^−/−:71.4% vs male WT:28.6%, had a severity score of ≥ 3) within 24 hours following an intravenous injection of E. coli (12). Another similarity came from Zhou et al that showed β2GPI ^−/− (APOH) mice had significantly higher levels of inflammatory cytokines than WT mice at post LPS challenge and the total β2GPI levels were significantly decreased in WT mice after LPS injection compared to saline control (20). Notably, above evidences suggested that APOH was dispensable for controlling bacterial infection in CLP-induced polymicrobial sepsis and targeting APOH might represent a promising novel therapeutic approach for the treatment of sepsis.

To delineate the molecular mechanisms of the protective effect of on CLP mice, we assessed the influence of APOH on bacterial CFU levels and the abundance of infiltrating leukocytes types in the septic mice treated with recombinant APOH compared with mice treated with BSA control. We found no statistical difference in bacterial CFU levels, but macrophage abundance tended to increase after APOH intervention. Phagocytic cells, especially, macrophages, at the site of infection are an essential component of the inflammatory response that aims to clear invading bacteria (21, 22). At present study, we failed to find direct effects on phagocytosis and intracellular killing of live P. aeruginosa by macrophages after preincubation with recombinant APOH. The data of Nilsson et al found that APOH was not able to kill bacteria directly, but peptides from domain V had antibacterial activities against streptococcus pyogenes (23). However, Balasubramanian et al showed APOH could facilitate phagocytosis, by binding to phosphatidylserine (PS)-containing vesicles or apoptotic cells, which promoted engulfment by phagocytes (24). A difference between these studies, we have considered the possibility that the multiple functions of APOH might be mediated by binding with different partners in a cell specific or disease-specific manner.

Macrophages are deliberated to be polarized into M1 phenotype to release inflammatory cytokines in the early stage of infection. In order to counteract the excessive inflammatory response, macrophages polarize to M2 phenotype to facilitate wound healing (25). In fact, we noticed that administration with APOH could decrease the pro-inflammatory mediators TNF-α and IL-1β in PM, BMDMs, PBMC and RAW 264.7 cells, which led us to further explore the effect of APOH on macrophages polarization. As expected, we found that preincubation with recombinant APOH decreased M1 polarization but not increased M2 polarization obviously. The TLR4 signaling pathway is involved in the release of inflammatory mediators (such as IL-1, IL-6, TNF-α and iNOS by activating the NF-κB signaling pathway in macrophages (26, 27). Thus, we explored whether TLR4/NF-κB signaling pathway was involving in APOH mediated macrophages polarization. Indeed, APOH decreased the LPS-induced upregulation of TLR4/NF-κB pathway. Above all, these finds together suggested that APOH exerted protective role in sepsis by suppressing M1 polarization, at least partially, via the suppression of TLR4/NF-κB signaling.

The current study has limitations. Firstly, our research was a single-center study with relatively small number of patients with sepsis, and the AUC of APOH was lower than that of CRP in predicting 28-day mortality of septic patients and significant difference was no found than PCT and PSOFA, suggesting that a larger-size clinical trial and incremental further studies were required to investigate the diagnostic value of APOH and to investigate whether the combination of APOH with other risk biomarkers might provide additional prognostic value in septic patients, such as IL-26 (28), IL-8 and soluble tumor necrosis factor receptor-1 (29). Secondly, further investigation using APOH knockout mice is required to elucidate the underlying molecular mechanisms by which APOH amplifying local and systemic inflammation during sepsis. Lastly, in validation cohort, it should be mentioned that the ages in the healthy control group were significantly older than sepsis group, because obtaining samples from children under 3 years was difficult.

In summary, we here firstly establish that pediatric septic patients have a stable and sustained reduction of serum APOH levels, which is correlated with disease severity and mortality. More importantly, recombinant APOH has therapeutic capacity in sepsis mice model by reshaping macrophage polarization and through inhibiting the intracellular activation of TLR4/NF-κB signaling pathway. Our data provide new insight into the APOH driven regulation of anti-inflammatory function by macrophages, which might offer a novel therapeutic target for the treatment of sepsis.

APOH: Apolipoprotein H; CLP: Cecal ligation and puncture; TLR4: Toll-like receptor 4; NF-κB: Nuclear factor-κB; WHO: World Health Organization; β2GPI: Beta-2-glycoprotein I; APS: Antiphospholipid syndrome; PICU: Pediatric Intensive Care Unit; WBC: White blood cells; PCT: Procalcitonin; CRP: C-reaction protein; pSOFA: Pediatric Sequential Organ Failure; PCIS: Pediatric Critical Illness Score; SIRS: systemic inflammatory response syndrome; BSA: bovine serum albumin; rAPOH: Recombinant murine apolipoprotein H; PBS: phosphate buffer solution; TNF-α: tumor necrosis factor-alpha; IL-1β: Interleukin (IL)-1β; PLF: peritoneal lavage fluids; HE: hematoxylin and eosin; CFU: Colony-forming unit; PMs: peritoneal macrophages; RBC: red blood cells; BMDMs: Bone marrow-derived macrophages; GM-CSF: Granulocyte macrophage colony-stimulating factor; PBMCs: Peripheral blood mononuclear cells; FBS: fetal bovine serum; LPS: lipopolysaccharide; P.a: Pseudomonas aeruginosa; DAPI: 4′,6-diamidino-2-phenylindole; RPM: revolutions per minute; TRAF6: TNF receptor associated factor 6; JNK: Jun N-terminal Kinase; iNOS: Inducible Nitric Oxide Synthase; Arg-1: Arginase; SD: deviation; ROC: Receiving operating characteristic; AUC: area under the curve; CI: confidence interval; iTRAQ: isobaric tags for relative and absolute quantification; PRM: parallel reaction monitoring; WT: wild type; E. coli: Escherichia coli; PS: phosphatidylserine

Supplementary Information

The online version contains supplementary material.

Additional file 1. Patients’ characteristics for pilot cohort.

Additional file 2. Primer sequences List.

Acknowledgements

Not applicable.

Author contributions

ZCY and CXX contributed equally to this work. ZZ and XF contributed to the conception and design. ZCY and CXX performed the experiments, analyzed data, and wrote the manuscript. RL participated in the animal experiments. DDP helped to revise the picture. BJ helped in acquisition of clinic data. All authors read and approved the final manuscript.

Funding

This work was supported by the Key Program of Chongqing Clinical Medical Research (Grant No. NCRCCHD-2021-KP-03).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were approved by the Clinical Research Ethics Committee of Institutional Review Board of Children’s Hospital of Chongqing Medical University (File No: ChiCTR-ROC-17011164 and (2022) Ethical Review Research No. 427). All animal experiments were conducted under the rules approved by the Ethics Committee of Chongqing Experimental Animal Center and the Animal Committee of Children’s Hospital (CHCMU-IACUC20221227002).

Consent for publication

All listed authors consent to the submission, and all data are used with the consent of the person generating the data.

Competing interests

The authors are preparing the application for Chinese patent that using of APOH in the diagnosis and potential treatment for pediatric sepsis.

Author details

¹ Department of Critical Care Medicine, Children’s Hospital of Chongqing Medical University, Chongqing, China. ² National Clinical Research Center for Child Health and Disorders, Chongqing, China. ³ Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, China. ⁴Chongqing Key Laboratory of Pediatrics, Chongqing, China.⁵Molecular Biology Laboratory of Respiratory Disease, Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, China.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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No competing interests reported.

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SupplementalTable1.docx
Additional file 1. Patients’ characteristics for pilot cohort.
SupplementalTable2.docx
Additional file 2. Primer sequences List.

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Apolipoprotein H as a Crucial Immunomodulatory Factor in Sepsis

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Journal Publication

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Abstract

Background.

Methods.

Results.

Conclusion.

Figures

Introduction

Materials and methods

Study population

Experimental animals

In vivo administration of recombinant mouse APOH

Antibody-mediated blockade of APOH

Enzyme-linked immunosorbent assay (ELISA)

Histopathology

Bacterial colony-forming units

Isolation of peritoneal macrophages, bone marrow-derived macrophages and peripheral blood mononuclear cell

In vitro administration of recombinant APOH

Flow cytometry experiments

Phagocytosis function assay

Bacterial killing assays

RNA extraction and quantitative real-time PCR

Western blotting analysis

Statistical Analysis

Results

APOH concentration is significantly decreased in patients with sepsis

Administration of recombinant murine APOH protein protects septic mice

Anti-APOH antibody worsens mice from nonsevere sepsis

APOH downregulates inflammatory cytokine responses through inhibiting M1 polarization in the macrophages

APOH regulates macrophage polarization via the TLR4-mediated NF-κB signaling pathway

DISCUSSION

Abbreviations

Declarations

Publisher's Note

References

Additional Declarations

Supplementary Files

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