Damage- and pathogen-associated molecular patterns play differential roles in late mortality after critical illness

Abstract

Multiple organ failure (MOF) is the leading cause of late mortality and morbidity in patients who are admitted to intensive care units (ICUs). However, there is an epidemiologic discrepancy in the mechanism of underlying immunologic derangement dependent on etiology between sepsis and trauma patients in MOF. We hypothesized that damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), while both involved in the development of MOF, contribute differently to the systemic innate immune derangement and coagulopathic changes. We found that DAMPs not only produce weaker innate immune activation than counterpart PAMPs, but also induce less TLR signal desensitization, contribute to less innate immune cell death, and propagate more robust systemic coagulopathic effects than PAMPs. This differential contribution to MOF provides further insight into the contributing factors to late mortality in critically ill trauma and sepsis patients. These findings will help to better prognosticate patients at risk of MOF and may provide future therapeutic molecular targets in this disease process.

Keywords: Cell Biology; Cellular immune response; Immunology; Innate immunity; Thrombosis.

Figure 1. Comparison between the innate immune stimulation by DAMPs and PAMPs.

Necrotic fibroblast supernatants (DAMPs) and necrotic gram-negative bacteria supernatants (PAMPs) generated by sonication were used as innate immune stimuli. (A–D) Macrophage cell line RAW264.7 was stimulated with (A and B) DAMPs or (C and D) PAMPs at the indicated concentration. After first overnight incubation, macrophage culture supernatants were collected (×1), followed by washing once and replenishing with fresh culture media. This procedure was repeated once (×2) or twice (×3). (E) TLR4-NF-κB-SEAP reporter cell line was stimulated with the DAMPs or PAMPs. The level of TLR4-mediated NF-κB activation was determined by measuring the amounts of secreted SEAP in reporter cell culture supernatants. (F and G) RAW264.7 cells were incubated with purified PAMPs (LPS) or DAMPs (HS, HMGB1), followed by washing and replenishing with fresh culture media. The amounts of TNF-α and IL-10 released from the cells after first (×1) and second (×2) incubation were measured. *P < 0.01 (between indicated groups; Dunnett’s multiple-comparisons test and paired t test).

Figure 2. Macrophage cell death induction and growth inhibition by PAMPs, but not DAMPs.

RAW264.7 cells were stimulated with PBS control, (A and F) necrotic gram-negative bacteria supernatants (PAMPs), (B and F) necrotic fibroblast supernatants (DAMPs), (C, E, and F) LPS, or (D, E, and F) HS at indicated concentration. Fresh complete media supplemented with the stimuli were replenished every day for 1 to 2 days. After 3 days after first stimulation, (A and B) cell growth inhibition (MTT) and cell death (C and D) trypan blue staining and (E) annexin V/7-AAD staining were determined. (F) After first stimulation with the stimuli, cells were stained with propidium iodide, followed by cell cycle analysis using flow cytometry. *P < 0.05 (vs. untreated or PBS; Dunnett’s multiple-comparisons test).

Figure 3. Local injection of PAMPs, but not DAMPs, induces systemic inflammation and organ damage in mice.

Mice were injected intraperitoneally with PBS control, necrotic fibroblast supernatants (DAMPs) (1500 μg), or necrotic gram-negative bacteria supernatants (PAMPs) (1500 μg). (A–F) Plasma was collected at 24 and/or 48 hours after injection. Plasma markers of (A and B) systemic inflammation (TNF-α and IL-6), (C) liver injury (ALT), (D) kidney dysfunction (creatinine), (E) DAMP generation (exDNA), and (F) thrombosis (TATc) were determined. (G) Mortality was monitored every day. n = 5. *P < 0.05 (vs. PBS). ^#P < 0.05 (between indicated groups; Kruskal-Wallis test and log-rank test).

Figure 4. Single systemic injection of PAMPs and DAMPs differentially induces mortality and organ damage in mice.

Mice were i.v. injected with PBS control (n = 4), necrotic gram-negative bacteria supernatants (PAMPs) (n = 6, 600 μg), or necrotic fibroblast supernatants (DAMPs) (n = 5, 600 μg) in the presence or absence of heparin. (A) Mortality was monitored every day. (B–G) Plasma was collected at 2 and 48 hours after injection. Plasma levels of (B) TNF-α, (C) IL-6, (D) ALT, (E) creatinine, (F) exDNA, and (G) TATc were determined. *P < 0.05 (vs. PBS). ^#P < 0.05 (between indicated groups; Kruskal-Wallis test and log-rank test). NA, not applicable.

Figure 5. Necrotic cell supernatants, but not necrotic bacteria supernatants contain procoagulants.

Supernatants of sonicated normal fibroblasts, B16 melanoma cells, and PANC-02 pancreatic cancer cells were used as DAMPs. Supernatants of sonicated Pseudomonas aeruginosa were used as PAMPs. (A and B) Normal mouse plasma was stimulated with PAMPs or DAMP, followed by measuring clotting time. PBS and silica were used as negative and positive controls. (A) Clotting time of normal mouse plasma (50 μL) stimulated with DAMPs (1.25, 5, or 20 μg) from fibroblasts or PAMPs (10 μg) was measured using a coagulometer. (B) Normal mouse plasma (50 μL) was stimulated with DAMPs (5 μg) from fibroblasts, B16 cells, and PANC-02 cells. (C) TF (CD142) expression on the fibroblasts, B16 cells, and PANC-02 cells was determined using flow cytometry. (D and E) Large particle (3,000–20,000-g fraction), small particle (20,000–100,000-g fraction), and supernatant fractions were isolated from necrotic cell supernatants by differential centrifugations. (D) Normal mouse plasma (50 μL) was stimulated with unfractionated cell supernatants, large particle, small particle, and supernatant fraction (5 μg), followed by measuring clotting time. (E) RAW264.7 cells were stimulated overnight with the fractions (100 μg/mL). *P < 0.05 (vs. PBS; Dunnett’s multiple-comparisons test). ^#P < 0.05 (between indicated groups; Tukey’s multiple-comparisons test).

Figure 6. Multiple systemic injections of low procoagulative DAMPs gradually increase proinflammatory cytokines and organ damage markers in mice.

PANC-02 cells were sonicated, followed by harvesting DAMP-containing supernatants. Mice (n = 5) were i.v. injected every day for 1–5 days with DAMPs (150 μg or 600 μg). (A) Mortality was monitored every day. (B–G) Plasma was collected on days 0, 2, and 5. Plasma levels of (B) TNF-α, (C) IL-6, (D) ALT, (E) creatinine, (F) exDNA, and (G) TATc were determined. *P < 0.05 (vs. untreated). ^#P < 0.05 (between indicated groups; Kruskal-Wallis test and log-rank test).

Figure 7. Early presence of circulating procoagulative particles and TLR-activating DAMPs after sterile injury predicts late-onset multiple organ failure (MOF) and mortality in trauma patients.

(A–D) HEK-TLR4 and HEK-TLR9 reporter cells were stimulated overnight with serum (20% v/v) isolated from healthy persons (n = 5) or polytrauma patients without MOF and mortality (n = 7) or with MOF (n = 8) at multiple time points after injury. (A and B) Serum isolated day 1 after trauma. (C and D) Sera were isolated at peak TLR-activating time point after trauma. (E and F) 3,000–100,000-g particle fraction was purified from 500 μL plasma isolated from healthy persons (n = 5) or trauma patients at 1 day after injury. Trauma patients without MOF and mortality (n = 7) and trauma patients with MOF (n = 8) were included in this study. (E) Total amounts of 3,000–100,000-g particle fraction was measured by BCA protein assay. (F) Normal human plasma (50 μL) was stimulated with the particle fraction (15 μg), following by measuring plasma clotting time. *P < 0.05 (between indicated groups; Kruskal-Wallis test). NS, not significant.