To obtain SPLUNC1-knockout mice that allowed conditional and global disruption of the SPLUNC1 gene, we used the Cre/loxP and flp/FRT recombination systems to target exons 3 of SPLUNC1. A single loxp site was inserted before exon 3 and before exon 4, and an frt-flanked PGK neo cassette was inserted between exons 3 and 4 to serve as a positive selectable marker. The targeting construct was electroporated into embryonic stem (ES) cells and the germline SPLUNC1^loxp-neo chimeras were obtained by homologous recombination. Then, the FRT-flanked PGK neo cassette was deleted by crossing female SPLUNC1^loxp-neo mice with transgenic Flp male mice to obtain the homozygous mice for the floxed SPLUNC1 allele (SPLUNC1^flox/flox). To generate SPLUNC1^−/− mice, the SPLUNC1^flox/flox mice were mated with EIIα-Cre mice (purchased from the Jackson Laboratory, Bar Harbor, ME, USA).

To produce animals for experiments, heterozygous mice were crossed to generate wild-type littermate controls. All mice used in the present study were male, 8–12 weeks old, and housed under specific pathogen-free conditions. The present study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Experimental protocols were approved by the Animal Ethics Committee of Central South University.

Mice were randomly divided into four groups: WT control, KO control, WT-LPS, and KO-LPS. The LPS group was intraperitoneally injected with a single dose of 5 mg/kg body weight of LPS (Sigma, Carlsbad, CA, USA) which was dissolved in saline at a concentration of 1 mg/ml. Meanwhile, the mice in the control group were intraperitoneally injected with saline alone. The mice were euthanized at 24, 48, 72 and 96 h after LPS or saline administration.

The proteins were extracted from mouse lung tissues and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked using 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 and then incubated with primary antibodies: SPLUNC1 (R&D Systems, Minneapolis, MN, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight. After washing three times with TBST, the membranes were incubated with HRP-conjugated secondary antibody. Bands were detected with an electrochemiluminescence detection system.

Single cell suspensions were prepared by homogenization through a sterile stainless steel mesh from the spleens. Erythrocytes were depleted using RBC lysing buffer (Sigma), and splenocytes were washed with phosphate-buffered saline (PBS). Then, the cells were stained with anti-CD11b and anti-Gr-1 antibodies (eBioscience, San Diego, CA, USA) according to the manufacturer's protocol. Fluorescence was measured using a MoFlo™ XDP High-Performance Cell Sorter and data were analyzed by Summit 5.2 software (both from Beckman Coulter, Inc., Brea, CA, USA).

Reverse transcription reaction was performed using a Fermentas Revert Aid First Strand cDNA Synthesis kit (Fermentas, Burlington, ON, Canada), according to the manufacturer's instructions. qRT-PCR was performed using an iQ5 Multicolor Detection system (Bio-Rad, Hercules, CA, USA). The sequences of the primers were as follows: β-actin forward, 5′-TTTCCAGCCTTCCTTCTT-3′ and reverse, 5′-GGTCTTTACGGATGTCAACG-3′; IL-6 forward, 5′-CTGATGCTGGTGACAACCAC-3′ and reverse, 5′-CAGAATTGCCATTGCACAAC-3′; IL-8 forward, 5′-CGTCCCTGTGACACTCAAGA-3′ and reverse, 5′-GGAGCATCAGGATCCAAACA-3′; CCL-2 forward, 5′-ATGCAGTTAACGCCCCACTC-3′ and reverse, 5′-ACCCATTCCTTCTTGGGGTC-3′; CCL-3 forward, 5′-GCAACCAAGTCTTCTCAGCG-3′ and reverse, 5′-TTGGACCCAGGTCTCTTTGG-3′; CCL-5 forward, 5′-ACCATATGGCTCGGACACCA-3′ and reverse, 5′-GCGGTTCCTTCGAGTGACA-3′; CXCL1 forward, 5′-TGGCTGGGATTCACCTCAAG-3′ and reverse, 5′-CCGTTACTTGGGGACACCTT-3′; IL-1β forward, 5′-TGCCACCTTTTGACAGTGATG-3′ and reverse, 5′-AAGGTCCACGGGAAAGACAC-3′. The expression of mRNA was assessed by evaluated threshold cycle (CT) values. The CT values were normalized with the expression levels of β-actin and the relative amount of mRNA specific to each of the target genes was calculated using the 2^−ΔΔCt method (22–25). β-actin was used as an internal control.

Lungs of the differentially treated mice were fixed with 4% paraformaldehyde, and embedded in paraffin. Subsequently, the lung tissues were sliced into 4-µm sections, and then stained with hematoxylin and eosin to observe the inflammatory response and pathological changes using an Olympus BX51 microscope (Olympus, Tokyo, Japan). The degree of histologic lung injury was assessed based on the following criteria: neutrophil infiltration, alveolar congestion, interstitial edema, necrosis and atelectasis. The severity of injury was evaluated via a numerical rating system that ranges from 0 to 4 (score): no injury, 0; injury to 25% of the field, 1; injury to 50% of the field, 2; injury to 75% of the field, 3; and diffuse injury, 4. The mean score was then analyzed for comparison between groups.

IHC was carried out using the peroxidase anti-peroxidase technique following a microwave antigen retrieval procedure. Anti-CD11b (MAB1124; 1:300), anti-Gr-1 (MAB1037; 1:300) (both from R&D Systems) was overlaid on the tissue sections and incubated overnight at 4°C. Secondary antibody incubation (Santa Cruz Biotechnology, Inc.) was performed at room temperature for 30 min. Color reaction was developed using 3,3′-diaminobenzidine tetrachloride (DAB) chromogen solution. All slides were counterstained with hematoxylin. Positive control slides were included in every experiment in addition to the internal positive controls. The specificity of the antibody was determined with matched IgG isotype antibody as a negative control.

Immunostaining was evaluated by two investigators in a blinded manner in an effort to provide a consensus on staining patterns by light microscopy (Olympus). CD11b or Gr-1 staining was assessed according to the methods described by Hara and Okayasu (26) with minor modifications. Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were randomly chosen, and >1,000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, <30% of tissue stained positive; 2+, between 30 and 60% stained positive; 3+, >60% stained positive. The minimum score when summed (extension + intensity) was, therefore, 0, and the maximum, 6. A combined staining score (extension + intensity) of ≤2 was considered to be a negative staining (low staining); a score between 3 and 4 was considered to be a moderate staining; whereas a score between 5 and 6 was considered to be a strong staining. An optimal cut-off level was identified as follows: a staining index score of 0–2 was used to define tumors with negative expression and 3–7 indicated positive expression of these two proteins. Agreement between the two evaluators was 95%, and all scoring discrepancies were resolved by discussion between the two evaluators.

The data obtained in the present study are expressed as the mean values ± standard deviation (SD) from at least three independent experiments using triplicate samples for the individual treatments. Statistical analyses were performed using two-way ANOVA or Fisher's exact test (Prism 6; GraphPad Software, San Diego, CA, USA). A P-value of <0.05 was considered to indicate a statistically significant difference.

To identify the role of SPLUNC1 in vivo, we generated SPLUNC1-knockout mice. In this procedure, Cre/loxp and Flp/FRT recombination systems were used to target exon 3 of the mouse SPLUNC1 gene (Fig. 1A). The genomic DNA was extracted from the mouse tail and used as a template for PCR analysis with primer P1 and P2 to identify genotypes (Fig. 1B). SPLUNC1 protein expression in wild-type and SPLUNC1^−/− mice was shown by western blot analysis (Fig. 1C). A specific SPLUNC1 band was detected in the SPLUNC1^+/+ and SPLUNC1^+/− lung, while no SPLUNC1 protein was detected in the lung of the SPLUNC1^−/− mice. Then, we observed that knockout of SPLUNC1 did not cause embryonic death, and there was no obvious phenotype difference between the wild-type and SPLUNC1^−/− mice. Similar results were reported by Gally et al (13) that SPLUNC1^−/− mice were viable, fertile, and largely indistinguishable from WT littermates in general appearance, body weight, locomotion and overt behavior.

MDSCs commonly express the markers CD11b and Gr-1 in mice; such immature myeloid cells are present in the bone marrow of healthy mice. However, they differentiate into mature myeloid cells and accumulate in the spleen under acute inflammation conditions. In the present study, we first measured the percentage of CD11b⁺Gr-1⁺ MDSCs in the bone marrow of the mice at different time points after LPS administration. The number of CD11b⁺Gr-1⁺ MDSCs in the WT group was reduced from 34.92±8.48 to 13.61±1.33% and in the KO group, from 37.65±8.06 to 12.81±5.92% (P<0.05; Fig. 2A) after LPS injection for 24 h, and then it rapidly recovered to the base level after injection for 48 h. Meanwhile, the number of these cells gradually increased at 72 and 96 h post LPS injection; it reached the highest level both in the WT and KO groups (70.98±2.84% in WT vs. 68.92±3.24% in KO mice at 96 h). Although the percentage of CD11b⁺ cells in KO mice was higher than that in WT mice at 24 h after LPS induction (P<0.05; Fig. 2B), no significant differences were observed between WT and KO groups at the different time-points of 0, 24, 48, 72 and 96 h after LPS injection (P>0.05; Fig. 2B). These results indicated that knockout of SPLUNC1 had no influence on the production of MDSCs in the bone marrow of the mice with ALI.

To investigate whether SPLUNC1 affects the recruitment of MDSCs from the bone marrow to the spleen in acute inflammatory response, we next analyzed the proportion of splenetic CD11b⁺Gr-1⁺ MDSCs in the WT and KO groups. There was significant accumulation of CD11b⁺Gr-1⁺ MDSCs after LPS injection, increasing from 3.26±0.41% of all splenocytes at baseline to a peak of 11.46±2.97% by 48 h in the WT group and from 3.44±0.46% of all splenocytes at baseline to a peak of 19.04±4.53% by 96 h in the KO group (Fig. 2C). The percentage of CD11b⁺ cells in the KO mice was higher than that in the WT mice at the time-points of 48 h (4.79±0.49% in WT mice vs. 8.67±2.38% in KO mice) and 96 h (3.44±0.74% in WT mice vs. 9.10±1.62% in KO mice) after LPS injection (P<0.05; Fig. 2D). Although the number of Gr-1⁺ cells in the WT mice was higher than that in the KO mice 96 h (15.18±2.87% in WT mice vs. 6.97±2.41% in KO mice) post LPS injection, the percentage of CD11b⁺Gr-1⁺ MDSCs in the KO mice was higher than that in the WT mice at the time point of 72 h (10.58±0.79% in WT mice vs. 16.36±2.42% in KO mice) and 96 h (10.95±1.11% in WT mice vs. 19.04±4.53% in KO mice) after LPS injection (P<0.05; Fig. 2D). These data revealed that the presence of SPLUNC1 suppresses the recruitment of CD11b⁺Gr-1⁺ MDSCs to the spleen in LPS-induced ALI.

Granulocyte differentiation antigen 1 (Gr-1) is highly expressed in neutrophils and Gr-1⁺ cells play a vital role in host defense, functioning as immunoregulators in the immune system. CD11b⁺ cells, specifically express M-CSF receptor (CD115) or IL-4R (CD124), having immunosuppressive activity. CD11b⁺ cells are distributed throughout the body and act as sentinels for the immune system. The frequency of CD11b⁺ myeloid cells varies in different organs, ranging from ~48% of brain myeloid (microglia) cells, 30% of bone marrow and 6% of total nucleated cells in the gut and spleen. In mice, MDSCs are identified as cells that co-express the myeloid lineage differentiation antigens Gr-1 and CD11b, which have been shown to accumulate quickly in mouse spleen and possess immunosuppressive effect in response to LPS administration. In the present study, we aimed to investigate whether SPLUNC1 affects the recruitment of MDSCs from the bone marrow to the spleen in acute inflammation; thus, it was important to distinguish between CD11b⁺Gr-1⁺ cells from CD11b⁺ and Gr-1⁺ cells.

In the present study, we examined the protein expression of Gr-1 and CD11b in the mouse spleen in response to LPS injection. In the WT and KO groups, few Gr-1⁺ cells were observed in the splenic tissues at 0 and 24 h after LPS injection, but a marked increase in Gr-1⁺ cells was noted by 48 h, particularly 72 and 96 h after LPS injection (Fig. 3A). Similarly, CD11b exhibited low expression at 0 and 24 h in cells of splenic tissues after LPS injection, while 48 h post injection, the number of cells expressing CD11b gradually increased. However, statistically significant differences between the WT and KO groups were not detected by IHC at the different time points after LPS injection (Fig. 3B).

To investigate the role of SPLUNC1 in acute lung inflammation, WT and SPLUNC1^−/− mice were treated with LPS as described in Materials and methods. The severity of lung injury was evaluated by histopathological analysis using hematoxylin and eosin (H&E) staining. In the WT and KO groups, the lungs of the mice injected with LPS showed neutrophil infiltration, thickened alveolar walls and lung congestion, while no such pathological changes were found in the control group (LPS-0 h). Meanwhile, when treated with LPS, the SPLUNC1^−/− group appeared to have more severe pulmonary damage and infiltrated inflammatory cells compared with the WT mice after LPS treatment for 48 h (Fig. 4).

Furthermore, we compared the differential expression of these cytokines in the WT and SPLUNC1^−/− groups at different time points after LPS treatment. The mRNA expression levels of IL-6, CCL-2 and CCL-3 in the SPLUNC1^−/− group were higher than these levels in the WT group 48 h post LPS injection. The level of CXCL-1 mRNA expression in the SPLUNC1^−/− group was higher than the level in the WT group after LPS injection at 72 and 96 h (P<0.05; Fig. 5A). The SPLUNC1 expression levels were increased in the lung tissues of the WT mice after LPS injection (Fig. 5B). Taken together, these data demonstrated that SPLUNC1 deficiency upregulated ALI-related cytokines, which may promote early inflammation leading to more severe lung injury after LPS injection.