Innate immune activation in neonatal tracheal aspirates suggests endotoxin-driven inflammation

Abstract

Background: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates.

Methods: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay.

Results: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1β protein. Expression of S100A12 protein was localized to TA neutrophils.

Conclusion: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population.

Figure 1

Innate immune transcriptome of TA pellets. TA pellet mRNA (n = 25, except for CXCL8, for which n = 14) was analyzed by quantitative real-time PCR. mRNA levels of 82 genes are shown as mean ± SEM mRNA abundance and grouped by function as follows: (a) pattern recognition receptors, (b) intracellular signaling, (c) cytokine/chemokines, (d) cytokine/chemokine receptors, (e) anti-infective, and (f) other. All mRNA transcripts except for LBP were consistently detected in all subjects. Statistical analyses by one-way ANOVA, Bonferroni posttest, ^**P < 0.01, ^†P < 0.001. Colored stars and daggers signify statistically significant differences from the correspondingly colored bar within each graph. LBP, lipopolysaccharide binding protein; TA, tracheal aspirate.

Figure 2

S100A12 protein expression localizes to TA PMNs. Representative dot plot of TA cells stained with (a) fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) isotype control antibodies and (b) antibodies for cell-surface antigens FITC-CD66b (PMN) and intracellular PE-S100A12 by flow cytometry. Representative confocal fluorescent microscopy (100×) of TA cells stained with (c) isotype control antibodies, or (d) antibodies against CD66b (green) and S100A12 (red), both with DRAQ5 nuclear stain (blue). (d) A characteristic CD66b⁺/S100A12⁺ PMN. PMN, polymorphonuclear leukocyte; TA, tracheal aspirate.

Figure 3

Correlation of TA cytokine expression at mRNA and protein levels. (a) Cytokine transcription levels correlated with respective TA supernatant cytokine protein levels measured using Milliplex Cytokine 6-Plex assay (n = 43–46, 115 data points, Spearman r = 0.73, P < 0.001); (b) IL-10, TNF, IL-1β, IL-6, CXCL8, and CCL2 were all present at the protein level. TA supernatant cytokine protein levels measured using Milliplex Cytokine 6-Plex assay are shown in pg/ml (n = 43–46). IL, interleukin; TA, tracheal aspirate; TNF, tumor necrosis factor.

Figure 4

Endotoxin in TA supernatants correlates with transcription of innate immune genes. (a) Of 25 TA pellets assayed for mRNA transcripts, 24 had TA supernatants available for endotoxin testing via LAL assay. Endotoxin-positive TAs (n = 6) demonstrated greater mRNA expression of IL-6, IL-10, TNF, and SERPINE1 (P < 0.05, Student's t test) as compared with endotoxin-negative samples (n = 18). (b) The data in (a) are plotted as a ratio of expression in endotoxin-positive vs. endotoxin-negative (E+/E−) samples for transcripts with differences greater than twofold (^*P < 0.05). IL, interleukin; LAL, Limulus amoebocyte lysate; SERPINE1, serpin peptidase inhibitor, clade E member 1; TA, tracheal aspirate; TNF, tumor necrosis factor.

Figure 5

Endotoxin in TA supernatants correlates with postnatal DOL and cytokine concentrations. (a) TA supernatants from neonates with postnatal DOL ≥4 d (n = 12, mean GA = 32.5 ± 5.3 wk, solid bars) demonstrated higher levels of TNF, IL-1β, and CXCL8 than neonates with postnatal DOL <4 d (n = 27–29, mean GA = 26.3 ± 2.1 wk, open bars). (b) TNF, IL-1β, and CXCL8 protein levels shown in relation to DOL 0–3 (n = 12), 4–9 (n = 12–15), 10–19 (n = 10), and >20 (n = 9). TNF (square with dotted line), IL-1β (open circle with dashed line), and CXCL8 (solid circle with solid line) increased with DOL 0–3 and remained elevated through DOL >20. (c) The presence of detectable endotoxin was positively correlated with postnatal DOL (n = 53; Spearman r = 0.37, P < 0.01). (d) Endotoxin-positive TA supernatants (n = 18 to 19, mean GA = 25.8 ± 1.9 wk, solid bars) contained greater IL-10, TNF, and IL-1β concentrations than endotoxin-negative supernatants (n = 24–27, mean GA 29.5 ± 4.7 wk, open bars). (e) IL-10 levels increased significantly in endotoxin-positive (n = 2–7, solid circles) TAs from DOL 0–3 to DOL 4–9 compared to endotoxin-negative (n = 12–15, open circles) TAs. Mann–Whitney unpaired t test applied for all comparisons, ^*P < 0.05, ^**P < 0.01, and ^†P < 0.001. DOL, day of life, GA, gestational age; IL, interleukin; LAL, Limulus amoebocyte lysate; TA, tracheal aspirate; TNF, tumor necrosis factor.