CD300ld promotes neutrophil bacterial phagocytosis in sepsis

Abstract

Sepsis is a life-threatening condition caused by a dysregulated immune response to infection. Neutrophils act as first line of defense against infection, but their function can become impaired in sepsis. CD300 antigen-like family member d (CD300ld), predominantly expressed on neutrophils, associates with Fc receptor common gamma-chain (FcRγ chain), a component vital for phagocytosis. In this study, we investigated the role of CD300ld in neutrophil phagocytosis. Our results demonstrate a marked decrease in CD300ld expression on neutrophils isolated from both septic mice and patients. CD300ld was positively correlated with bacterial phagocytosis in neutrophils. The transcriptomic analysis of CD300ld knock-out neutrophils revealed a downregulation of genes related to defense response to bacteria, suggesting that CD300ld is a key modulator of bacterial clearance. Stimulation of CD300ld with an agonist antibody in neutrophils led to the activation of Rac2, a key regulator of actin polymerization, facilitating the enhanced phagocytosis. Furthermore, CD300ld activation significantly enhanced the in vitro phagocytosis of Escherichia coli and Staphylococcus aureus by neutrophils. Septic mice adoptively transferred with CD300ld-activated neutrophils exhibited markedly reduced bacterial loads in the blood and peritoneum, decreased inflammatory cytokine levels, and alleviated organ injury. These findings highlight the critical role of CD300ld signaling in neutrophil-mediated bacterial clearance in sepsis and provide a solid foundation for future research aimed at developing novel immunotherapies against this deadly disease condition.

Sepsis-induced downregulation of CD300ld on neutrophils impairs bacterial phagocytosis. A CD300ld agonist antibody can restore this function, reducing bacterial burden, inflammation, and organ injury in sepsis.

1. Introduction

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection.¹ It poses a significant global health threat due to its high incidence and mortality rates. According to the Global Burden of Disease study, there are estimated 48.9 million incident cases of sepsis and 11.0 million sepsis-related deaths worldwide annually, accounting for 19.7% of all global deaths.² Bacteremia induces hyperinflammation by excessively activating immune cells, ultimately leading to fatal organ damage, septic shock, and death.¹ Therefore, efficient bacterial clearance is prerequisite for sepsis treatment. Current sepsis management primarily relies on the prompt administration of antibiotics; however, this approach faces significant challenges due to the rising prevalence of antibiotic-resistant bacteria.³ Consequently, novel treatment strategies are actively being explored as alternatives to antibody-based antimicrobial therapy. Regulating host immunity to enhance bacterial clearance during the early stages of infection represents a potential approach to address these limitations.

Neutrophils are crucial for the host defense against infection, acting as the primary line of defense in pathogen control, particularly during the acute phase of infection. The clearance of pathogens by neutrophil phagocytosis is a critical mechanism for alleviating sepsis, and impaired neutrophil phagocytic function is associated with adverse outcomes in sepsis.⁴ It has been shown that reduced phagocytic activity of neutrophils within the first 24 h post-admission was a negative prognostic factor for survival in sepsis patients.⁴ Conversely, enhancing neutrophil function has the potential to improve sepsis prognosis. Nevertheless, the regulatory mechanisms governing neutrophil phagocytosis in sepsis remain poorly understood.

The CD300 family of molecules modulates a broad array of immune cell processes through their activating or inhibitory receptor functions, and their discovery has opened new avenues of research. Recent studies have demonstrated the involvement of CD300 proteins in the pathogenesis of several diseases.^5–7 Additionally, this receptor family holds significant potential as therapeutic targets in infectious diseases, allergies, cancer, and other pathological conditions.^8–10 However, the specific function and role of CD300 antigen-like family member d (CD300ld) remain largely unexplored. One reason is that CD300ld is predominantly expressed on neutrophils,^8,11 whereas other CD300 family members are expressed on diverse immune cells such as macrophages, monocytes, and lymphocytes.^9,12 This narrow expression profile, particularly within neutrophils, cells primarily specialized for pathogen elimination, has limited the focus on CD300ld in the context of immune homeostasis.

CD300ld was initially studied for its interactions with adaptor molecules before its functional analysis, as it lacks intrinsic signaling motifs. Studies have shown that CD300ld is coupled with Fc receptor common gamma-chain (FcRγ chain). Experiments using mutant cells transiently transfected with CD300ld, as well as FcRγ chain knockout cells, demonstrated a positive correlation between CD300ld and FcRγ chain expression.^11,13 The FcRγ chain contains an immunoreceptor tyrosine-base activation motif (ITAM), which activates spleen tyrosine kinase (Syk).¹⁴ Thus, while CD300ld lacks intrinsic signaling motifs, studies have shown that it functions as an activating receptor through its association with the FcRγ chain.^9,13 Moreover, evidence suggests that FcRγ chain is involved in phagocytic signaling and activity,^15,16 indicating that CD300ld may be strongly involved in phagocytosis. Here, we aim to explore the critical role of neutrophil CD300ld on phagocytosis and pathogen clearance in sepsis. Our findings provide valuable insights into the potential of CD300ld as a therapeutic approach for enhancing phagocytosis and reducing the pathogenic burden in sepsis.

2. Materials and methods

2.1. Mice

Wild-type (WT) male 8 to 12-wk-old WT C57BL/6 mice were purchased from Charles River (Charles River, Wilmington, MA). Mice were housed in a temperature-controlled room on 12-h light cycles and provided standard laboratory chow and water. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Feinstein Institutes for Medical Research.

2.2. Polymicrobial sepsis

Mice were anesthetized with isoflurane (2%) in oxygen, and sepsis was induced via cecal ligation and puncture (CLP).¹⁷ A 1.5 cm midline abdominal incision was made to expose the cecum, which was ligated 1 cm proximal to its end using a 4-0 silk suture.

The cecum then was perforated with a single through-and-through puncture using a 22-gauge needle, positioned midway between the ligation and the tip of the cecum. A small amount of fecal material was gently extruded from the perforation sites to confirm the patency of punctures. The cecum, along with the feculent material, were returned to the abdomen, and the abdominal wound was closed in two layers. Sham mice underwent identical abdominal incisions but without ligation and puncture of the cecum.

2.3. Isolation and purification of bone marrow-derived neutrophils

Bone marrow-derived neutrophils (BMDNs) were isolated from WT mice.¹⁸ Briefly, bone marrow cells from femurs and tibias were flushed out with RPMI 1640 medium using a 25-gauge needle. Cells were filtered through a sterile 70 μm nylon filter and resuspended the cells at 1 × 10⁸ cells/mL in phosphate buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS). BMDNs were purified by immunomagnetic negative selection using the EasySep mouse neutrophil enrichment kit (Cat. No. 19762, STEMCELL Technologies, Vancouver, BC, Canada).

2.4. Stimulation of BMDNs by cross-linking of anti-CD300ld ab

BMDNs were seeded into 96-well plates coated with 5 μg/ml anti-CD300ld Ab (TX69, Cat. No HM1147, Hycult Biotech, Uden, the Netherlands) or 5 μg/ml purified Rat IgG2a, κ Isotype control Ab (RTK2758, Cat. No 400502, BioLegend). BMDNs were then collected at the specified time points following stimulation for subsequent analysis.

2.5. Isolation of leukocyte from the blood and lungs

Blood samples were obtained via cardiac puncture and red blood cells (RBC) were lysed using RBC lysis buffer (BD Biosciences) before collecting the remaining cells. Lung tissues were minced into small pieces and incubated with 1 mg/ml collagenase type I (Cat. No LS004197, Worthington Biochemical, Lakewood, NJ, United States of America) at 37 °C for 30 min with periodic agitation. Digested tissue was then crushed, filtered through a 100 μm cell strainer (Corning, NY, United States of America), and treated with RBC lysis buffer (BD Biosciences) before collecting the cells. The isolated cells were stained with Abs and analyzed by flow cytometry.

2.6. Flow cytometry

Cell suspensions were incubated with a combination of monoclonal fluorescently conjugated Abs: Ly6G-Alexa Fluor 488 (1A8, Cat. No 127626, BioLegend, San Diego, CA, USA), Ly6C-BV605 (HK1.4, Cat. No 128036, BioLegend), CD300ld-PE (TX69, Cat. No 139605, BioLegend), CD3ε-APC/Fire 750 (17A2. BioLegend), and CD45-PerCP/Cy5.5 (I3/2.3, Cat. No 147706, BioLegend). To analyze the intracellular phosphorylated Syk and phosphoinositide 3-kinase (PI3K) (p-Syk, p-PI3K), BMDNs were stimulated with anti-CD300ld Ab or IgG for 10 min. Cells were then fixed to stop stimulation by adding IC fixation buffer (Thermo Fisher Scientific). The cells were permeabilized with 90% ice-cold methanol and incubated for 30 min, followed by staining with p-Syk-APC (moch1ct, Cat. No 17-9014-42, Thermo Fisher Scientific) and p-PI3K-PE Abs (PI3KY458-1A11, Cat. No MA5-28027, Thermo Fisher Scientific). TruStain FcX PLUS (Cat. No 156604, BioLegend) was used to prevent nonspecific antibody binding and the cell viability was determined using a Zombie Aqua Fixable Viability Kit (BioLegend). The absolute number of cells was calculated by using Precision Count Beads (BioLegend). Flow cytometric analysis was performed on a FACSymphony (BD Biosciences) and data were processed using FlowJo software (BD Biosciences).

2.7. RNA isolation and real-time quantitative PCR

Total RNA was extracted from blood neutrophils isolated using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL Technologies). RNA extraction was performed following the manufacturer's instructions with the Illustra RNAspin Mini RNA Isolation kit (Cytiva, Marlborough, MA, United States of America). cDNA was synthesized using M-MLV reverse transcriptase (Thermo Fisher Scientific). Quantitative real-time PCR (qPCR) was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR thermocycler (Thermo Fisher Scientific) for detecting CD300ld mRNA expression. Mouse β-actin served as an endogenous control to normalize mRNA levels using the comparative Ct method. The primer sequences were: β-Actin, forward 5′-CGTGAAAAGATGACCCAGATCA-3′ and reverse 5′-TGGTACGACCAGAGGCATACAG-3′; CD300ld, forward 5′-CGCTGAGAACACTGGCAAGGAA-3′ and reverse 5′-CCACAAAGACCATCAGCAGGAAG-3′.

2.8. Pathohistological analysis of lung injury

Lung tissues were fixed in 10% formalin, sectioned into 5-μm-thick slices, and stained with hematoxylin and eosin (HE). Histological assessment of lung injury was performed using the scoring system of the American Thoracic Society.¹⁹ Scores, ranged from 0 to 1, were assigned based on the following features: Neutrophils in the alveolar space, neutrophils in the interstitial space, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening. The average score per field was calculated at ×400 original magnification.

2.9. Rac2 pull-down assay

The assay was performed by using the Rac2 Activation Assay Kit (Cat. No STA-401-2, Cell Biolabs, Inc., San Diego, CA, United States of America) according to the manufacturer's instructions. Briefly, 1.45 × 10⁷ BMDNs were stimulated with anti-CD300ld Ab or IgG for 4 h. BMDNs were collected, and proteins were extracted using the lysis buffer from the assay kit. The lysis buffer contained protease inhibitors (Thermo Fisher Scientific). Proteins from 5 × 10⁵ BMDNs were used to assess total Rac2 and β-actin. Proteins from 1.4 × 10⁷ BMDNs were subjected to a pull-down assay, and Guanosine Triphosphate (GTP)-bound Rac2 in the cell lysate was pulled down by the agarose beads. Following the pull-down assay, samples were eluted and run on NuPAGE 4% to 12% Bis-Tris gels for electrophoresis (Thermo Fisher Scientific). Rac2 immunoblot positive control was used from the assay kit. Gels were transferred into nitrocellulose membranes (Thermo Fisher Scientific), blocked with 0.1% casein, and finally reacted with primary Abs against Rac2 contained in the kit or β-actin Ab (AC-15, Sigma-Aldrich). The blots that reacted with Rac2 or β-actin Abs were incubated with secondary Abs labeled with an infrared dye (Li-Cor Biosciences, Lincoln, NE). The protein bands were detected using an Odyssey Clx imaging system (Li-Cor Biosciences) and quantified with Image Studio 5.2 software (Li-Cor Biosciences).

2.10. Phagocytosis assay

BMDNs (4 × 10⁵) were stimulated with anti-CD300ld Ab or IgG for 4 h before the assay. A bacterial phagocytosis assay was performed with pHrodo Green Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) BioParticles (Thermo Fisher Scientific) without the opsonization step. BMDNs with BioParticles were incubated at 37 °C for 30 min, and phagocytosis efficiency was evaluated by flow cytometry.

2.11. Bacterial culture

Blood was collected by cardiac puncture. Peritoneal lavage fluids were collected after the injection of 1 ml of sterile normal saline into the peritoneal cavity. The blood samples were serially diluted at 1:10, 1:100, and 1:1,000 in sterile normal saline. The peritoneal lavage fluid samples were serially diluted at 1:1,000 and 1:10,000 in sterile normal saline. A total of 100 μL of each diluted sample was plated on trypticase soy agar plates with 5% sheep blood (Thermo Fisher Scientific). The colonies on the plates were counted after the plates were incubated at 37 °C for 24 h. The colony forming units (CFU) of the plates were counted with ImageJ software, opting in the threshold, binary, and watershed tools.²⁰ The colony-forming unit per milliliter was then calculated by multiplying the observed colony counts by the respective dilution factor and by 10 to adjust to ml.

2.12. Bulk RNA-Seq data analysis

Neutrophil gene expression data from human septic patients (GSE186054) and CD300ld knock-out (KO) mice (GSE199602) were obtained from the Gene Expression Omnibus public database. The gene expression data retrieval, processing, and quantification were performed using ikra (ver. 2.0.1),²¹ an automated RNA-seq analysis pipeline for both human and mouse data. The pipeline retrieve FASTQ files via the fasterq-dump program in the SRA tool kit (ver. 2.10.9), performs read quality control and trimming with Trim Galore (ver. 0.6.7),²² using Cutadapt v 3.4,²³ and quantifies transcript using Salmon (ver. 1.4.0).²⁴ GENCODE Release 37 (GRCh38) was used for human data and GENCODE Release M26 (GRCm39) was used for mouse data. The quantified transcript-level scaled Transcripts Per Million (TPM) was summarized into a gene-level scaled TPM by using the R package tximport v.1.6.0. These tools were used with default parameters. Downstream analysis, including gene ontology (GO) enrichment, scatter plot, and volcano plot, was performed with integrated Differential Expression and Pathway analysis (iDEP v2.01), an integrated web application for GO analysis of RNA-Seq data with default parameters.²⁵ Differentially expressed genes were extracted with a false discovery rate (FDR) cutoff of 0.1 and minimum fold-change of 2.

2.13. Adoptive transfer of CD300ld-activated neutrophils into mice

BMDNs from WT mice were stimulated with anti-CD300ld Ab or IgG for 4 h. Mice were given 1.2 × 10⁶ BMDNs with PBS or only PBS intraperitoneally immediately after surgery. Blood samples and peritoneal lavage fluids were collected 20 h after CLP.

2.14. Colorimetric enzymatic assays and enzyme-linked immunosorbent assay (ELISA)

Serum levels of organ injury markers, including alanine transaminase (ALT), aspartate transferase (AST), and lactate dehydrogenase (LDH), were measured using colorimetric enzymatic assays (MedTest Dx, MI, United States of America) according to the manufacturer's protocol. Serum levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα) and interleukin 6 (IL-6), were quantified using mouse-specific ELISA kits (BD Biosciences).

2.15. Statistical analysis

Statistical analysis was conducted using Prism 10 (GraphPad Software, San Diego, CA), with P values <0.05 considered statistically significant. The results are presented as mean values with standard error of the mean (SEM). One-way ANOVA was used for comparison among multiple groups, with significance determined by the Tukey post hoc test. All experiments were performed in triplicate or more.

3. Results

3.1. Neutrophil CD300ld expression is decreased in sepsis

We first examined CD300ld expression on different types of immune cells under normal conditions and found that CD300ld was predominantly expressed on neutrophils (Fig. S1), which is consistent with a recent study.⁸ Next, we induced sepsis in WT mice by CLP and collected blood and lungs at 4 and 24 h post-surgery to assess CD300ld expression in neutrophils. The frequency of CD300ld⁺ neutrophils and median fluorescence intensity (MFI) of CD300ld on neutrophils were significantly decreased in both the blood (Fig. 1a to c) and lungs (Fig. 1d to f) of CLP mice compared to sham mice. Moreover, CD300ld expression on BMDNs was also significantly reduced in CLP mice (Fig. S2). Furthermore, the number of neutrophils in peripheral tissues expressing higher CD300ld levels than BMDNs was significantly reduced at 4 and 24 h post-surgery, suggesting that the observed reduction in the frequency of CD300ld⁺ neutrophils in the blood and lungs was not merely due to an influx of low CD300ld-expressing neutrophils from the bone marrow (Fig. S3). To assess whether this reduction was due to decreased gene expression, we examined CD300ld mRNA levels in neutrophils from septic mice. The mRNA expression of CD300ld in neutrophils was significantly lower in the blood of 4 h post-CLP mice relative to sham controls (Fig. 1g), suggesting that the de novo synthesis of CD300ld is inhibited during sepsis. To further extend these findings, we analyzed the transcriptomic data from neutrophils isolated from septic patients, where we observed a similar decrease in CD300ld gene expression compared to healthy controls (Fig. 1h). Collectively, these results indicate that neutrophil CD300ld expression is decreased during sepsis in both mice and humans.

Fig. 1.

Neutrophil CD300ld expression is significantly decreased in sepsis. Sepsis was induced in mice using CLP. Neutrophils from blood (a to c) and lungs (d to f) were harvested at 4 or 24 h after surgery to determine CD300ld expression by flow cytometry and real-time PCR. (a, d) Representative gating strategy of flow cytometry plots for detecting live neutrophils (CD45⁺ Ly6G⁺) and histograms showing CD300ld expression. (b, e) Percentage and (c, f) MFI of CD300ld expression on neutrophils in CLP and sham were shown. Data represent the mean ± SEM (n = 5 to 7/group). (g) mRNA levels of blood neutrophils were assessed 4 h after surgery. Data represent the mean ± SEM (n = 6/group). (h) Volcano plots showing gene expression of human CD300ld in CD14⁻CD15⁺ CD16⁺ neutrophils from sepsis patients and healthy controls (GSE186054). Experiments were performed twice, and all data were analyzed. The groups were compared by Student's t-test or one-way ANOVA followed by a Tukey's multiple comparisons. *P < 0.05 vs Sham.

3.2. CD300ld is positively associated with bacterial phagocytosis by neutrophils

We aim to investigate the role of neutrophil CD300ld by first analyzing transcriptomic datasets from WT and CD300ld KO neutrophils. GO enrichment analysis revealed that the most downregulated pathway in CD300ld KO neutrophils compared to WT neutrophils, was the defense response to bacteria (Fig. 2a). Additionally, the gene expression of Rac2, a critical mediator of neutrophil phagocytosis by facilitating cytoskeletal rearrangement, was downregulated in CD300ld KO neutrophils compared to WT neutrophils (Fig. 2b). To further explore the role of neutrophil CD300ld in bacterial phagocytosis, a critical mechanism for pathogen clearance during sepsis, neutrophils were cultured with E. coli, and the relationship between CD300ld expression and phagocytic activity was assessed. Notably, the frequency of bacterial phagocytosis was significantly higher in the CD300ld^higher neutrophil population compared to the CD300ld^lower population (Fig. 2c and d). These data demonstrate that CD300ld expression in neutrophils is positively associated with bacterial phagocytosis, and CD300ld deficiency leads to the downregulation of key pathways and mediators essential for this process.

Fig. 2.

CD300ld expression is positively correlated with bacterial phagocytosis in neutrophils. (a) GO enrichment analysis (biological process) derived from bulk RNA-seq using a public database (GSE199602). A lollipop plot displays the top 10 most significantly downregulated GO terms in neutrophils from CD300ld KO mice compared to WT mice. The dots' size and color represent the number of genes and -log₁₀ (FDR), respectively. The length of the bar indicates fold enrichment. The y axis represents the GO terms for the biological function. (b) Scatter plots of RNA-seq data showing gene expression in neutrophils from WT and CD300ld KO mice. Green (down in CD300ld KO) and red (up in CD300ld KO) plots show genes with at least a 2-fold difference in gene expression, and FDR is <0.1. (c) Representative gating strategy of flow cytometry histogram and plots, showing the phagocytosis of CD300ld-higher and -lower neutrophils. (d) Percentages of phagocytosis of CD300ld^higher and CD300ld^lower neutrophils. Data represent the mean ± SEM (n = 5/group). The experiments were performed twice, and all data were analyzed. The groups were compared by Student's t-test. *P < 0.05 vs CD300ld^lower.

3.3. Phagocytic signaling is induced by CD300ld activation in neutrophils

We further explored the intracellular signaling pathways activated in neutrophils upon CD300ld activation. It has been known that anti-CD300ld Ab (clone: TX-69) acts as an agonist by cross-linking CD300ld.^8,13,26 Neutrophils were stimulated with either anti-CD300ld Ab or IgG isotype control, and intracellular signaling was assessed. Treatment with anti-CD300ld Ab significantly increased phosphorylated Syk, a known downstream effector of CD300ld, compared to PBS and IgG control, confirming that the antibody successfully activated CD300ld in neutrophils (Fig. 3a to c). Additionally, we found a significant increase in phosphorylated PI3K, a downstream target of Syk, in neutrophils treated with anti-CD300ld Ab compared to PBS and IgG control (Fig. 3d to f). To specifically evaluate whether CD300ld activation promotes phagocytic signaling in neutrophils, we assessed Rac2 activity upon anti-CD300ld Ab treatment. GTP-Rac2, an activated form of Rac2, was significantly increased in response to anti-CD300ld Ab treatment compared to IgG control (Fig. 3g and h; Fig. S4). These results indicate that CD300ld activation leads to the activation of intracellular signaling pathways that promote phagocytic function in neutrophils.

Fig. 3.

CD300ld promotes neutrophil phagocytosis through the PI3K-Rac2 axis. BMDNs were stimulated by cross-linking of CD300ld using anti-CD300ld Ab for 10 min. Representative flow cytometric (a) histograms, (b) percentages, and (c) MFI of Syk phosphorylation in BMDNs were shown. Representative flow cytometric (d) histograms, (e) percentages, and (f) MFI of PI3K phosphorylation in BMDNs. Data represent the mean ± SEM (n = 6/group). BMDNs were stimulated by cross-linking of CD300ld with specific antibody for 4 h. Then, GTP-Rac2 levels were assessed by a GTP-Rac2 pull-down assay. (g) Representative blots and (h) the corresponding bar diagram were shown. Data represent the mean ± SEM (n = 5/group). The experiment was performed at least twice. The groups were compared by Student's t-test. *P < 0.05 vs IgG, ^#P < 0.05 vs PBS.

3.4. Bacterial phagocytosis is promoted by CD300ld activation in neutrophils

We next evaluated the impact of CD300ld activation on neutrophil-mediated bacterial phagocytosis. Neutrophils were treated with anti-CD300ld Ab or IgG isotype control and then cultured with E. coli to assess phagocytic activity. Treatment with anti-CD300ld Ab significantly increased the engulfment of E. coli by neutrophils compared to the IgG control (Fig. 4a to c), indicating that CD300ld stimulation promotes E. coli phagocytosis. Similarly, anti-CD300ld Ab treatment significantly increased the phagocytosis of S. aureus by neutrophils compared to IgG control (Fig. 4d to f). These data indicate that CD300ld activation enhances the phagocytosis of both Gram-negative and Gram-positive bacteria in neutrophils.

Fig. 4.

CD300ld facilitates neutrophil bacterial phagocytosis. BMDNs were stimulated by cross-linking of CD300ld using anti-CD300ld Ab for 4 h. Neutrophil bacterial phagocytosis was assessed by flow cytometry using pHrodo (a to c) E. coli or (d to f) S. aureus BioParticles. (a, d) Representative gating strategy of flow cytometry plots and histograms, (b, e) percentages, and (c, f) MFI of phagocytosis were shown. Data represent the mean ± SEM (n = 7 to 8/group). The experiment was performed at least 3 times and all data were analyzed. The groups were compared by Student's t-test. *P < 0.05 vs IgG.

3.5. CD300ld-activated neutrophils attenuate bacterial burden, inflammation, and organ injury in sepsis

We investigated the effects of neutrophil CD300ld in sepsis. Mice were subjected to sepsis and intraperitoneally injected with neutrophils pretreated with either anti-CD300ld Ab or an IgG isotype control, rather than administering the antibody directly. This approach was chosen to specifically assess the role of neutrophil CD300ld while avoiding potential effects on other cell populations that express CD300ld. Twenty hours after the surgery, the blood, peritoneal lavage, and lungs were collected (Fig. 5a). Septic mice that received IgG-treated neutrophils exhibited a significantly higher bacterial load in both the blood (Fig. 5b and c) and peritoneal lavage (Fig. 5d and e) compared to sham mice. In contrast, administration of anti-CD300ld Ab-treated neutrophils into septic mice resulted in a significantly reduced bacterial burden in both the blood (Fig. 5b and c) and peritoneal lavage (Fig. 5d and e) compared to the IgG-treated neutrophil administrated septic mice. Blood levels of TNFα, IL-6, ALT, AST, and LDH were significantly elevated in septic mice injected with IgG-treated neutrophils, while those markers were significantly decreased in septic mice injected with anti-CD300ld Ab-treated neutrophils (Fig. 5f to j). Histological analysis of the lungs further revealed severe injury in CLP mice injected with IgG-treated neutrophils (Fig. 5k and l). However, treatment with anti-CD300ld antibody-treated neutrophils significantly mitigated lung injury following CLP (Fig. 5k and l). In summary, our findings indicate that CD300ld-activated neutrophils, with enhanced phagocytic capabilities, reduce bacterial burden, thereby alleviating inflammation and protecting against organ injury in sepsis (Fig. 6).

Fig. 5.

CD300ld signaling reduces bacterial load by enhancing neutrophil phagocytosis, thereby alleviating organ injury in sepsis. (a) BMDNs were treated with anti-CD300ld Ab to induce cross-linking of CD300ld or treated with IgG as control. BMDNs were stimulated with anti-CD300ld Ab for 4 h. Then, they were adoptively transferred into the murine peritoneal cavity immediately after CLP surgery. Sham mice received only PBS after surgery. Blood and lungs were harvested at 20 h after surgery. (b, c) Blood and (d, e) peritoneal lavage fluids were collected to evaluate the bacterial load (CFU/ml). Data represent the mean ± SEM (n = 7 to 9/group). The experiment was performed at least 3 times and all data were analyzed. The groups were compared by ANOVA was followed by a Tukey's multiple comparisons test to compare multiple groups. The levels of (f) TNFα, (g) IL-6, (h) ALT, (i) AST, and (j) LDH in blood were evaluated using specific colorimetric enzymatic assays or ELISA. Data represent the mean ± SEM (n = 9/group). The experiment was performed 3 times, and all data were analyzed. The groups were compared by ANOVA was followed by a Tukey's multiple comparisons test to compare multiple groups. (k) Representative histological H&E images were shown at ×200 original magnification. Scale bars: 100 μm. (l) Lung injury scores were calculated from 0 to 1 based on alveolar and interstitial neutrophil infiltration, hyalinization, protein filling in the airspaces, and wall thickening. Data represent the mean ± SEM (n = 5/group). The groups were compared by ANOVA was followed by a Tukey's multiple comparisons test to compare multiple groups. *P < 0.05 vs Sham, ^#P < 0.05 vs IgG-CLP.

Fig. 6.

Summary of the findings. CD300ld is predominantly expressed on the surface of neutrophils and plays a crucial role in promoting the phagocytic clearance of bacteria, including E. coli and S. aureus, by neutrophils. Our investigation into the underlying mechanism revealed that CD300ld facilitates the activation of Syk, which subsequently triggers downstream signaling through PI3K phosphorylation, leading to the activation of Rac2, a small GTPase protein belonging to the Rho family of GTPases. Rac2 activation enhances the neutrophils' ability to clear bacteria through phagocytosis. However, our data demonstrate that sepsis results in a reduction of CD300ld expression on neutrophils, impairing Rac2 activation and, consequently, bacterial phagocytosis. Therefore, preserving CD300ld expression may offer a novel therapeutic approach for treating sepsis.

4. Discussion

Neutrophil phagocytosis is a critical function for preventing sepsis-related death through the direct killing of pathogens. Understanding the regulatory mechanisms that govern neutrophil's phagocytic function is vital for improving sepsis outcomes. Previous studies have shown that enhancing phagocytosis in neutrophils by modulating specific mediators can improve sepsis prognosis,^27–31 suggesting that neutrophil phagocytic capacity is impaired during sepsis. However, the molecular mechanisms underlying this dysregulation remain unexplored. In the present study, we demonstrated that CD300ld plays a pivotal role in promoting neutrophil phagocytosis and identified a key downstream signaling pathway involved in this process. We also observed a significant downregulation of CD300ld expression in neutrophils from both septic patients and mice. Furthermore, neutrophils with enhanced phagocytic activity via the CD300ld signaling significantly ameliorated inflammation and organ damage during the acute phase of sepsis. These findings highlight CD300ld as a potential therapeutic target for sepsis, where regulating its expression or activity may enhance neutrophil phagocytosis and improve sepsis outcomes.

CD300ld has primarily been studied for its interaction with adaptor molecules. Among the potential adapter proteins for CD300ld, including DAP10 and DAP12, FcRγ chain has been regarded as the primary adapter for CD300ld in both humans and mice.^11,13,26,32 However, functional studies on CD300ld remain limited. While previous research has shown CD300ld-mediated TNFα and IL-6 release in neutrophils,^13,26 its role in neutrophil phagocytosis has not been explored. Bacterial antibiotic resistance poses a major challenge to current sepsis treatment strategies. Predictive statistical modeling estimated that, in 2019, there were 4.95 million deaths associated with antibiotic-resistant bacteria, including 1.27 million deaths directly attributable to bacterial antibiotic resistance, making it one of the leading causes of death globally.³ These statistics underscore the urgent need for alternative approaches to bacterial control in sepsis. Enhancing neutrophil phagocytosis via CD300ld may provide a promising strategy to address this problem.

We activated the CD300ld signaling cascade in neutrophils by cross-linking CD300ld with an agonist antibody. CD300ld associates with FcRγ chain, which contains an ITAM that mediates intracellular signal transduction.^9,13 Upon activation, ITAM triggers Syk phosphorylation through the binding of the SRC homology 2 (SH2) domain of Syk to phosphorylated tyrosine residues within ITAM.¹⁴ As anticipated, our results demonstrated that the cross-linking of CD300ld led to Syk phosphorylation, a hallmark of ITAM activation. These findings align with the previous reports showing that other ITAM-containing receptors, such as FcγRs, the T-cell receptor, and the B-cell receptor, are similarly activated through cross-linking.³³ Additionally, cross-linking has also been widely employed to study signal induction in other CD300 family members like CD300c and CD300e.^34,35 These findings confirm that the agonistic antibody effectively induces downstream signaling of neutrophil CD300ld, as demonstrated in our study.

Rac, a member of the Rho GTPase family, is a small GTP-binding protein involved in various cellular processes, including cytoskeleton organization, migration, transcription, and proliferation.³⁶ While Rho family GTPases regulate a wide range of cellular behaviors, their most prominent effects are seen in the modulation of the actin cytoskeleton.³⁷ In particular, Rac signaling promotes actin polymerization, facilitating the formation of the phagocytic cup that enables the cell membrane to engulf and internalize target materials, a process supported by cofilin and ARP2/3.^37–39 In neutrophils, Rac2 specifically regulates cofilin- and ARP2/3-mediated actin assembly,³⁹ which directly regulates phagocytosis.⁴⁰ Notably, a study has demonstrated that the efficiency of neutrophil phagocytosis of bacteria remained unchanged in Rac1-deficient neutrophils but was significantly impaired in Rac2-deficient neutrophils.⁴¹ Our findings clearly demonstrated that CD300ld stimulation induces GTP-bound Rac2, the active form of Rac2, in neutrophils. These findings strongly suggest that CD300ld activation enhances phagocytosis via Rac2-mediated signaling.

Both Syk and PI3K have been shown to play pivotal roles in phagocytosis, as demonstrated by experiments using knockout cells or inhibitors.^42–44 These findings are consistent with our results showing that CD300ld stimulation induces Syk and PI3K phosphorylation, accompanied by enhanced bacterial phagocytosis. Syk is activated via ITAM and functions upstream of PI3K.^45,46 PI3K synthesizes phosphatidylinositol-3,4,5-trisphosphate (PtdIns[3,4,5]P3), which is involved in various cellular processes, including cell survival, gene expression, metabolism, and cytoskeletal rearrangements,⁴⁴ and can directly bind to Rac.⁴⁷ Notably, Rac2 activation in human neutrophils is inhibited by PI3K inhibitors, such as wortmannin or LY294002.⁴⁸ Further studies are warranted to elucidate the detailed downstream signaling cascade of CD300ld in neutrophils especially in Rac2 activation and phagocytosis.

We have demonstrated that CD300ld plays a crucial role in the defense mechanism against bacterial infections through GO enrichment analysis using Cd300ld KO mice. However, we acknowledge that GO analysis alone may not fully capture the functional contributions of CD300ld in neutrophil-mediated immunity. To more comprehensively elucidate the role of CD300ld in bacterial phagocytosis, future studies employing functional assays, ideally using CD300ld inhibitors, will be necessary. Nonetheless, to the best of our knowledge, no commercially available inhibitors targeting CD300ld currently exist. Furthermore, the endogenous ligand for CD300ld has not yet been identified,^8,11,13,26 further supporting the use of antibody-mediated activation as a surrogate strategy for functional studies.

In the present study, we did not identify the ligand for CD300ld, similar to previous studies.^8,11,13,26 Many members of the CD300 family bind to phospholipids, and the immunoglobulin (Ig) domain of CD300ld shares 91% identity and 97% similarity at the amino acid level with that of CD300f,¹¹ whose ligand is known to be phosphatidylserine (PS).⁴⁹ Therefore, PS may be a potential ligand for CD300ld. PS is known to mediate efferocytosis—the phagocytic clearance of apoptotic cells—by signaling through receptors that recognize apoptotic markers. This suggests that PS-mediated efferocytosis could, at least in part, be mediated by CD300ld. Moreover, PS is also expressed on extracellular vesicles, indicating that the potential interaction between PS and CD300ld might occur even in the absence of apoptotic cells.⁵⁰ Further focused studies are necessary to identify the ligands of CD300ld and explore their functional implications.

We recognize that the role of CD300ld in bacterial clearance may not be limited to enhancing phagocytosis. Recent studies have shown that CD300ld on neutrophils positively regulates neutrophil extracellular trap (NET) formation.⁸ Additionally, degranulation and reactive oxygen species (ROS) production in neutrophils are positively regulated by receptors containing ITAM,^51,52 to which CD300ld is also linked via the FcRγ chain. These findings indicate that CD300ld may not only enhance bacterial engulfment via the Syk-PI3K-Rac2 signaling pathway, as demonstrated in our study, but also contribute to neutrophil bactericidal functions through complementary mechanisms, including NET formation, degranulation, and ROS production. Additionally, CD300ld activation may confer resistance to apoptosis in BMDNs,¹³ potentially increasing the number of effective and phagocytic BMDNs in circulation. In sepsis, neutrophils survive longer than their conventional half-life, and the proportion of aged neutrophils is elevated, although their CD300ld expression is decreased. It is possible that exogenously treated neutrophils with anti-CD300ld antibody may exhibit an extended half-life along with enhanced phagocytosis. These effector functions may work synergistically, contributing to the observed reduction in inflammatory markers, organ injury markers, and bacterial loads in septic mice receiving CD300ld-activated BMDNs. This strongly supports the notion that the adoptive transfer of these activated BMDNs could serve as a potential therapeutic strategy to improve survival in sepsis. Furthermore, these findings suggest that CD300ld may serve as an indicator of neutrophil activation, similar to CD11b.^53,54

Our findings demonstrate that CD300ld expression on neutrophils is significantly decreased in sepsis, suggesting that CD300ld-mediated activation may be insufficient under this condition. This notion is supported by our data demonstrating that adoptive transfer of neutrophils activated via CD300ld improves systemic outcomes in sepsis, including enhanced bacterial clearance. Furthermore, we observed that CD300ld-stimulated neutrophils more efficiently engulfed pHrodo E. coli than S. aureus. This suggests that bacterial engulfment via CD300ld activation is strain-dependent, with more efficient engulfment observed for E. coli. Given that E. coli is a major causative pathogen in septic peritonitis, such as in the CLP model, this observation aligns with our findings and further supports the physiological relevance of CD300ld-mediated neutrophil activation in sepsis.

The expression levels of cellular molecules in neutrophils can be altered within the individual cells and may also be influenced by the recruitment of neutrophils from the bone marrow, particularly under inflammatory conditions such as sepsis. CD300ld expression on neutrophils is influenced by their maturation state, with previous studies showing that neutrophil CD300ld expression is lower in the bone marrow than in peripheral blood.⁸ Nevertheless, we propose that the reduction of neutrophil CD300ld in the blood and lungs during sepsis is primarily due to decrease de novo synthesis because of the following reasons. If neutrophils with inherently low CD300ld expression were being recruited from the bone marrow during sepsis, we would expect to see two distinct peaks (high and low) in CD300ld expression, representing the coexistence of 2 populations. Instead, our data showed a gradual decline in CD300ld expression, with a single peak in the flow cytometry histograms at all time points, indicating no distinct subpopulations. Additionally, CD300ld expression was reduced not only in neutrophils within peripheral tissues but also in BMDNs during sepsis. Furthermore, neutrophil numbers in peripheral tissues expressing higher CD300ld than BMDNs were significantly reduced at 4 and 24 h post-CLP, further suggesting that the observed decline in CD300ld⁺ neutrophils in the blood and lungs was not merely due to an influx of low CD300ld-expressing neutrophils from the bone marrow. These findings support the notion that the decrease in neutrophil CD300ld during sepsis is mainly due to a reduction in de novo CD300ld synthesis within each neutrophil, alongside a potential influx of a subset with inherently low CD300ld expression.

In this study, we did not identify the specific cause of the reduction in CD300ld expression on neutrophils in sepsis. In vitro treatment of neutrophils with individual major inflammatory mediators, such as lipopolysaccharides (LPS), TNFα, or IFNγ, failed to induce a reduction in CD300ld expression (data not shown). These findings suggest that other unidentified mediators or multiple regulatory mechanisms, including cell–cell interactions or combinatorial cytokine effects, may contribute to the reduction of CD300ld expression in sepsis. Further studies are necessary to elucidate the precise molecular mechanisms underlying CD300ld downregulation in sepsis.

In summary, we identified a novel mechanism of bacterial clearance, CD300ld-mediated neutrophil bacterial phagocytosis in sepsis. CD300ld expression on neutrophils was significantly decreased during the acute phase of sepsis. CD300ld stimulation induced the Syk-PI3K-Rac2 signaling cascade and enhanced bacterial phagocytosis in neutrophils. The enhanced clearance of bacteria in septic mice resulted in reduced bacterial burden, inflammation, and organ injury. Therefore, CD300ld represents a promising therapeutic target in sepsis to promote neutrophil phagocytosis and modulate the inflammatory response.

Author contributions

Y.A., A.M., M.A., and P.W. designed the experiments. Y.A. conducted the experiments. Y.A., A.M., and M.A. analyzed the data. Y.A., A.M., and M.A. wrote the initial draft. P.W. reviewed and edited the manuscript. M.A. and P.W. supervised the project. P.W. and M.A. conceived the idea.

Supplementary material

Supplementary material is available at Journal of Leukocyte Biology online.

Funding

This work was supported by National Institutes of Health (NIH) grants R01GM129633, R35GM118337, and U01AI170018.

Conflicts of interest. The authors declare no conflict of interest.

Consent

Consent to participate is not applicable as it is not involving studies with human subjects.

Data availability

The data will be made available upon reasonable request.