Murine macrophage RAW 264.7 cell line was purchased from the American Type Culture Collection (ATCC). Macrophage cultures were routinely cultured in DMEM media containing 1% streptomycin/penicillin and 10% fecal bovine serum. When cell densities reached 80-90% confluence, adherent macrophages were cultured in serum-free OPTI-MEM I medium and stimulated with recombinant human pro-DCD-C34S. The intracellular levels of LC3-I and lipidated LC3-II were respectively measured by Western blotting using specific antibodies.

The concentrations of cellular LC3-I and lipidated LC3-II in pro-DCD-stimulated murine macrophage-like RAW 264.7 cells were measured by Western blotting using rabbit anti-mouse LC3A/B monoclonal antibodies (Cat. # 12741, Cell Signaling) as previously described (10). Briefly, equal volume of cell-conditioned culture medium or murine/human serum were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% nonfat milk, the membranes were incubated with the appropriate antibodies (anti-pro-DCD, 1:1000; anti-LC3A/B, 1:1000) overnight. Subsequently, the membranes were incubated with secondary antibodies (mouse-anti-rabbit IgG-HRP, Cat.# sc-2357, Santa Cruz; or donkey-anti-rabbit IgG-HRP, Cat. # NA934, GH Healthcare), and the immune-reactive bands were visualized by chemiluminescence. The relative levels of specific proteins were determined using the UN-SCAN-IT Gel Analysis Software Version 7.1 (Silk Scientific Inc., Orem, UT, USA) and expressed in arbitrary units (AU).

The cDNA encoding human pro-dermcidin (residue 20-110) or a mutant with a Cys (C)→Ser (S) substitution at residue 34 (pro-DCD-C34S) was cloned into a pReceiver expression vector downstream of a T7 promoter with an N-histidine tag, and expressed in E. coli BL21 (DE3) pLysS cells as previously described (11, 12). The inclusion body-associated recombinant pro-DCD or pro-DCD-C34S protein was isolated by differential centrifugation and urea solubilization, followed by refolding in Tris buffer (pH 8.0) containing N-lauroylsarcosine. The recombinant pro-DCD and pro-DCD-C34S were further purified using histidine-affinity chromatography, followed by extensive Triton X-114 extractions to remove contaminating endotoxins. The recombinant pro-DCD or pro-DCD-C34S protein were tested for LPS content by the chromogenic Limulus amebocyte lysate assay (Endochrome; Charles River), and the endotoxin content was less than 0.01 U per microgram of recombinant protein.

Polyclonal antibodies were generated in Female New Zealand White Rabbits by the Covance Inc. (Princeton, NJ, USA) using recombinant human pro-DCD combined with Freund’s complete adjuvant following standard procedures. Blood samples were collected in 3-week cycles of immunization and bleeding, and the antibody titers were determined by direct pro-DCD ELISA. Total IgGs and pro-DCD antigen-binding IgGs were purified from the anti-pro-DCD rabbit serum using Protein A and pro-DCD-affinity column chromatography, respectively. Briefly, rabbit serum was pre-buffered with PBS and slowly loaded onto the Protein A/G Sepharose (Cat. # ab193262) column to allow sufficient binding of IgGs. After washing with 1×PBS to remove unbound serum components, the IgGs were eluted with acidic buffer (0.1 M glycine-HCl, pH 2.8), and then immediately dialyzed into 1×PBS buffer at 4°C overnight. For pro-DCD antigen-affinity purification, recombinant human pro-DCD was conjugated to cyanogen bromide (CNBr)-activated Sepharose4 agarose beads (Cat. # 17098101, GE Healthcare), which were then loaded into columns. After repeated washings with acid buffer (0.1 M Acetic/Sodium Acetate, 0.5 M NaCl, pH 4.0) and alkali buffer (0.1 M Tris-HCl, 0.5 NaCl, pH 8.0), anti-pro-DCD total IgGs were slowly loaded onto the column, and the flow-through fractions were collected. After repetitive washing with 1×PBS buffer, the pro-DCD-binding antibodies were eluted with acidic elution buffer, and immediately neutralized in 1×PBS solution.

A library of 13 synthetic peptides corresponding to various regions of human pro-DCD sequence was synthesized at the Genscript, and spotted (0. 1 μg in 2.5 μl) onto a nitrocellulose membrane (Thermo Scientific, Cat No. 88013). Subsequently, the membrane was probed with anti-pro-DCD rabbit serum or pro-DCD antigen affinity-purified IgGs, following a standard protocol as previously described (13, 14).

Murine Cytokine Antibody Arrays (Cat. No. M0308003, RayBiotech Inc., Norcross, GA, USA), which simultaneously detect 62 cytokines on one membrane, were used to measure relative cytokine concentrations in murine serum as described previously (13–15).

Recombinant pro-DCD-C34S was pegylated using methyl-PEG₂₄-NHS ester (MW of 1214.39 Daltons, Cat.# 22687, Thermo Scientific, Rockford, IL, USA) according to manufacturer’s instructions. Briefly, methyl-PEG₂₄-NHS ester was dissolved in DMSO (5.0 mg/ml) and mixed with a solution of pro-DCD-C34S at a 20-fold molar excess to the protein, ensuring the final DMSO concentration was below 10%. After incubating the mixture on ice for two hours, the solution was dialyzed overnight at 4° C in 1×PBS buffer to remove any unreacted PEG NHS ester.

Every effort was made to minimize the number of animals used in this study in accordance with the ARRIVE guidelines developed by the British National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Additionally, all experiments were conducted following the International Expert Consensus Initiative for Improvement of Animal Modeling in Sepsis - Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) (16). This includes practices such as randomizing animals within each experimental group, implementing delayed therapeutic interventions (with agents like pegylated pro-DCD-C34S) (17), establishing specific criteria for euthanizing moribund septic animals (e.g., labored breathing, minimized response to human touch, and immobility), and administering fluid resuscitation and antibiotics (18). This study received administrative approval from the IACUC of the Feinstein Institutes for Medical Research (FIMR, Protocol # 2017–038 Term II; Date of Approval, February 22, 2021).

Adult male and female Balb/C mice (7–8 weeks old, 20–25 g body weight) were purchased from Charles River Laboratories (Wilmington, MA), and acclimated for at least 5–7 days before usage. To induce experimental sepsis, Balb/C mice underwent a surgical procedure known as “cecal ligation and puncture” (CLP) as previously described (13, 14, 19). Prior to CLP surgery, all animals received a buprenorphine injection (0.05 mg/kg, subcutaneously) to manage immediate surgical pain, because repetitive use of buprenorphine in the CLP model could paradoxically elevate sepsis surrogate markers and increase animal lethality (20, 21). Approximately 30 min post CLP surgery, all experimental animals were subcutaneously injected with a dose of imipenem/cilastatin (0.5 mg/mouse) (Primaxin, Merck & Co., Inc.) to avoid their potential adverse effects on the therapeutic efficacy of pro-DCD-C34S or derivatives. Animals were randomly assigned to control vehicle and experimental groups, and pro-DCD, pro-DCD-C34S, or pegylated pro-DCD-C34S was intraperitoneally injected to septic mice at various time points post-CLP. Animal survival was monitored for two weeks to ensure no late death occurred. To elucidate the potential protective mechanisms of peg-pro-DCD-C34S, a separate group of Balb/C mice underwent CLP and received peg-pro-DCD-C34S (0.2 mg/kg) at 2 h and 20 h post-CLP. At 24 h post CLP, animals were euthanized to collect blood and measure: i) serum levels of various cytokines and chemokines using murine Cytokine Antibody Arrays; ii) markers of liver injury using specific colorimetric enzymatic assays; and iii) blood bacterial colony forming units as previously described (13).

Male and female mice were not mixed in the same experiments to avoid potential influences of female hormones on the host immune response to infections. Instead, all experiments were initially conducted using male mice and subsequently replicated in age-matched female mice in separate studies to assess potential gender-specific differences. When no gender-specific differences were observed, the results from both male and female mice were pooled to reduce the number of animals used while ensuring robust and reliable conclusions.

Blood samples were collected at 24 h post-CLP following intraperitoneal administrations of pegylated pro-DCD-C34S (0.2 mg/kg) at 2 h and 20 h post-CLP. The samples were centrifuged at 3000 x g for 10 min to collect serum, and serum levels of liver injury markers such as aspartate aminotransferase (AST, Cat. No. 7561) and alanine aminotransferase (ALT, Cat. No. 7526) were measured using specific colorimetric enzymatic assays (Pointe Scientific, Canton, MI) according to manufacturer’s instructions as previously described (13, 22).

The liver samples were collected at 6 h post CLP, fixed in 10% buffered formalin, and embedded in paraffin. The paraffin-embedded tissues were sectioned into 5-μm slices, stained with hematoxylin-eosin, and examined under light microscopy. Liver parenchymal injury was assessed in a blinded fashion by summing three different Suzuki scores (ranging from 0-4) for sinusoidal congestion, hepatocyte cytoplasmic vacuolization, and parenchymal necrosis as previously described (12, 14). The parameter scores were calculated using a weighted equation with a maximum score of 100 per field and then averaged to determine the final liver injury score for each experimental group.

Escherichia coli BL21 (DE) strain was inoculated into Luria Bertani Broth (LB broth), incubated at 37°C until the culture reached the mid-log phase. Pro-DCD or peg-pro-DCD-C34S was dissolved in 1 × PBS buffer at various dilutions and mixed with the E. coli suspension in 1 × PBS. The mixture was incubated at 37°C for 4 h in sterile test tubes as previously described (1, 23). After incubation, the bacterial suspension was serially diluted in sterile 1 × PBS solution and spread onto LB agar plates. Following incubation 37°C for 16–24 h, the number of colony-forming units (CFUs) on each plate was counted to determine whether pro-DCD or its derivatives reduced the CFU count of the E. coli suspension.

Murine macrophage-like RAW 264.7 cells stably transfected with GFP-LC3 were stimulated with pro-DCD (1.0 µg/ml) for 16 h, and the formation of GFP-LC3 punctate structures was examined under a fluorescence microscope as previously described (10). The ratio between the 18-kD cytosolic LC3-I and 16-kD lipidated LC3-II was determined by Western blotting analysis following previously described methods (10).

All data were first assessed for normality using the Shapiro-Wilk test before applying the appropriate statistical analyses. The Student’s t test was employed to compare two independent experimental groups. For comparison among multiple groups with non-normal (skewed) distribution, the Kruskal-Wallis ANOVA test followed by Dunn’s post hoc test was used to evaluate statistical differences. The Kaplan-Meier method, along with the nonparametric log-rank test, was used to compare mortality rates between different groups. Statistical significance was defined as a P value less than 0.05.

To understand the extracellular role of pro-DCD in sepsis, we synthetized thirteen peptides corresponding to different regions of human pro-DCD ( Figure 1A ) and used them to profile the epitopes of rabbit polyclonal antibodies that we raised against human pro-DCD ( Figure 1B ). As predicted, immunoblotting of recombinant pro-DCD with pre-immune rabbit serum did not reveal any immune-reactive band ( Figure 1B ), indicating that these rabbits did not produce pro-DCD-reactive autoantibodies. However, Western blotting with anti-pro-DCD serum revealed a strong band corresponding to the molecular weight of recombinant pro-DCD with an N-terminal 6×His tag (11, 12) ( Figure 1B ). Consistently, our home-made pro-DCD protein was recognized by some commercial polyclonal antibodies (ab175519) raised against a unique peptide sequence (residue 96-110) of human pro-DCD ( Supplementary Figure S1 ).

To obtain pro-DCD antigen-affinity purified total IgGs (“A-IgGs”), we first subjected anti-pro-DCD rabbit serum to Protein-A-affinity chromatography to obtain total IgGs (“pAbs”, Figure 1D , Top Panel), followed by pro-DCD-antigen-affinity chromatography to isolate the antigen-binding IgGs (“A-IgGs”) ( Figure 1D , Top Panel). Dot blotting analyses demonstrated that these anti-pro-DCD pAbs were reactive with recombinant pro-DCD at various dilutions, as well as several synthetic peptides (e.g., P1, P2, P3, P5, P8, P11 and P13) corresponding to different regions of pro-DCD ( Figure 1D ), confirming their specificity for human pro-DCD. Additionally, the epitope profiles of pro-DCD antigen-affinity purified IgGs (“A-IgGs”) were consistent with those of the crude anti-pro-DCD serum ( Figure 1D ), validating the specificity of our home-made anti-pro-DCD pAbs to multiple pro-DCD peptides, including epitopes corresponding to residue 97–110 of pro-DCD (i.e., “P13”, Figure 1D ). Despite some homology between two short peptides of pro-DCD (residue 65-80 or “P9”) and the acute phase protein serum amyloid A (SAA, residue 36-51, Figure 1C), our anti-pro-DCD pAbs did not react with the P9 peptide, suggesting that our home-made anti-pro-DCD pAbs were unlikely to cross-react with human SAAs.

To explore the role of pro-DCD in experimental sepsis, we assessed the impact of antigen affinity-purified anti-pro-DCD IgGs on sepsis-induced systemic inflammation and tissue injury. Notably, pro-DCD-specific IgGs (“A-IgGs”) significantly elevated sepsis-induced systemic accumulation of G-CSF, IL-6 and MCP-1 ( Figure 2A ), three surrogate markers of experimental (24, 25) and clinical sepsis (26). Consistently, these anti-pro-DCD antibodies exacerbated sepsis-induced liver injury ( Figures 2B, C ) and appeared to dose-dependently increase sepsis-induced animal lethality ( Figure 2D ). Collectively, these findings have suggested a potentially beneficial role of pro-DCD in an animal model of experimental sepsis.

To assess the therapeutic potential of pro-DCD, we generated recombinant pro-DCD corresponding to residue 20-110 (excluding the signal leader peptide, Figure 3A ) with an N-terminal 6×His tag as previously described (11, 12). Because recombinant pro-DCD could form dimers via disulfide bond at Cys (C) 34, we also created a pro-DCD variant with a Cys (C)→Ser (S) substitution at residue 34 (pro-DCD-C34S, Figure 3A ), which migrated as a 16-kDa monomer on SDS-PAGE even in the absence of a reducing agent, DTT ( Figure 3C ). To extend the half-life of pro-DCD-C34S, we chemically conjugated highly hydrophilic polyethylene glycol (PEG) chains to the Lys (K) residue of this peptide ( Figure 3B ). This modification is expected to shield pro-DCD-C34S from enzymatic degradation and prevent it from renal clearance. As anticipated, pegylated pro-DCD-C34S was not detected by Coomassie blue staining ( Figure 3C , Left Panel), because PEGylation of amine groups of Lys (K) residues obstructed its electrostatic interaction with the Coomassie Brilliant Blue G-250 dye. However, the pegylated pro-DCD-C34S was still detected by silver staining as larger molecules ranging 25 to 37 kDa ( Figure 3C , Right Panel), since silver nitrate could still interact with other residues such as the carboxylic acid groups of Asp (D) and Glu (E) or imidazole of His (H) of pro-DCD-C34S.

After extensive extraction with Triton X-114 to remove endotoxin contaminants, we evaluated the therapeutic efficacy of highly purified pro-DCD, pro-DCD-C34S, and pegylated pro-DCD-C34S in a murine model of experimental sepsis. Administration of recombinant pro-DCD at 2 h and 24 h post-CLP significantly improved animal survival rate, increasing it from 10% in the saline control to 60% in the pro-DCD-treatment group ( Figure 3D , Left Panel). When the 1^st dose was delayed to 24 h post-CLP, the pro-DCD-C34S analog showed a similar tend towards increasing animal survival at the same dose (0.5 mg/kg BW, Figure 3D , Middle Panel). Notably, pegylated pro-DCD-C34S provided a significant protection against lethal sepsis even when given in a delayed fashion (24 h post-CLP) at a dose (0.2 mg/kg) that was approximately 5-fold lower in molar concentration compared to pro-DCD-C34S ( Figure 3D , Right Panel), considering the almost two-fold difference in molecular weight between pro-DCD-C34S (~15 kDa) and PEG-pro-DCD-C34S (25–37 kDa, Figure 3C ). Collectively, these results indicate that both pro-DCD and pro-DCD-C34S derivatives offer significant protection against lethal sepsis.

To investigate the mechanisms underlying pro-DCD-mediated protection, we assessed the effects of pegylated pro-DCD-C34S on sepsis-induced systemic inflammation, tissue injury, and bacterial dissemination. Intraperitoneal administration of peg-pro-DCD-C34S significantly reduced sepsis-induced systemic accumulation of G-CSF, IL-6, KC, MCP-1, MIP-2, and sTNFRI ( Figure 4A ), indicating that pro-DCD derivatives confer protection against sepsis by mitigating systemic inflammation of key sepsis surrogate biomarkers of experimental (24, 25) and clinical sepsis (26). Additionally, peg-pro-DCD-C34S significantly attenuated sepsis-induced elevation of liver enzymes such as AST and ALT ( Figure 4B ), suggesting that pro-DCD derivatives provide protection against lethal sepsis partly by alleviating both inflammation and tissue injury.

To further elucidate the protective mechanism of pro-DCD, we also examined the impact of peg-pro-DCD-C34S on blood bacterial counts. As predicted, CLP led to a substantial increase in blood bacterial levels ( Figures 4C, D ), reflecting cecal bacterial spillage into the blood stream. However, treatment with peg-pro-DCD-C34S almost completely diminished blood bacterial counts ( Figures 4C, D ), suggesting that pro-DCD conferred protection against lethal sepsis partly by facilitating bacterial clearance.

To understand how pro-DCD facilitates bacterial elimination in septic animals, we incubated pro-DCD or peg-pro-DCD-C34S with E. coli for 3–4 hours and plated the mixture onto LB broth agar at serial dilutions to count the colony forming unit (CFU). At concentrations up to 200 µg/ml, neither pro-DCD nor peg-pro-DCD-C34S reduced CFU ( Figure 5A ), suggesting that pro-DCD or peg-pro-DCD-C34S could not directly kill bacteria in septic animals. To explore alternative mechanisms by which pro-DCD might enhance bacterial clearance, we examined its effect on the activation of microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3, LC3) in macrophage cultures. Pro-DCD stimulation led to the formation of LC3-positive cytoplasmic puncta ( Figure 5B ), and the conversion of LC3-I to the lipidated LC3-II form ( Figure 5C ). It raised an interesting possibility that pro-DCD may promote bacterial elimination by promoting LC3-associated phagocytosis and/or autophagy, two highly conserved processes that clear extracellular and intracellular bacteria by many phagocytes.