Molecular profiling of innate immune response mechanisms in ventilator-associated pneumonia

Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is utilized in hospitals for VAP diagnosis and guiding treatment options. While BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their utility has not been thoroughly explored. Longitudinal ETA and BAL were collected from 16 intubated patients up to 15 days, of which 11 developed VAP. We conducted a comprehensive LC-MS/MS based proteome and metabolome characterization of longitudinal ETA and BAL to detect host and pathogen responses to VAP infection. We discovered a diverse ETA proteome of the upper airways reflective of a rich and dynamic host-microbe interface. Prior to VAP diagnosis by microbial cultures from BAL, patient ETA presented characteristic signatures of reactive oxygen species and neutrophil degranulation, indicative of neutrophil mediated pathogen processing as a key host response to the VAP infection. Along with an increase in amino acids, this is suggestive of extracellular membrane degradation resulting from proteolytic activity of neutrophil proteases. Days prior to VAP diagnosis, detection of pathogen peptides with species level specificity in ETA may increase specificity over culture-based diagnosis. Our findings suggest that ETA may provide early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore facilitate its diagnosis and treatment. Graphical abstract


Introduction
shown variable sensitivity and specificity towards VAP detection (12,14). This raises potential 81 issues of misdiagnosis and emphasizes the need for further research on specific VAP biomarkers. 82 BAL has been a widely accepted matrix to study pulmonary infections (15,16). Many studies have 83 demonstrated the utility of BAL for microbial culture, 16S rDNA analysis and determining host-84 response against VAP infection (16)(17)(18)(19). Endotracheal aspirate (ETA) is regarded as a source for 85 non-invasive respiratory sampling and recently has been recommended for semi quantitative 86 cultures in VAP diagnosis (20). However, the molecular composition of ETA has not been 87 explored as extensively as BAL to understand host responses to infection. We hypothesize that 88 reduced invasiveness involved in ETA sampling is permissive to more frequent longitudinal 89 molecular snapshots of host immune response and changes to microbial flora during early 90 infection. We anticipate that this enhanced granularity provides valuable mechanistic insights into 91 VAP pathogenesis. We used a multi-disciplinary approach integrating proteomics and quantitative (1 hour, 37 °C). The proteins were digested with Trypsin Gold overnight at 37°C (Promega,140 Madison, WI). The resulting peptides were de-salted using C18 SPE cartridges (Waters,Milford,141 MA) and eluted with 70% acetonitrile (ACN) with 0.1% trifluoroacetic acid (v/v) (Waters). The 142 eluted peptides were vacuum-dried and frozen at -20 °C until LC-MS/MS analysis. On the day of 143 analysis, the samples were reconstituted in 0.1% formic acid. Sample preparation and data 144 acquisition were randomized separately in order to minimize bias. 145 LC-MS/MS analysis was performed using a nanoAcquity ultra performance liquid 146 chromatography system (Waters, Milford, MA) coupled to an Orbitrap Fusion Lumos Tribrid mass 147 spectrometer (Thermo Fisher, San Jose, CA). One μg of peptides from each sample was separated 148 on a BEH C18, 1.7 μM, 0.1 x 100 mm column (Waters, Milford, MA) using a 83.5 minute gradient 149 from 3 to 90% solvent B (ACN, 0.1% FA) and 97 to 10% solvent A (Water, 0.1% FA) at a flow 150 rate of 0.5 μL/min. The following gradient conditions were employed: 3 to 7% B for 1 minute, 7 151 to 25% B for 1 to 72 minutes, 25 to 45% B for 10 minutes, 45 to 90% B for 0.5 minutes, 90% B 152 for 0.5 minute and column equilibration at 3% B for 10 minutes. MS spectra were acquired over a 153 scan range of m/z 380 to 2000 using the orbitrap at 120,000 resolution followed by quadrupole 154 isolation (width 1.6 TH) of precursor ions for data-dependent higher-energy collisional on Proteome Discover Version 2.1.1.21 (Thermo Scientific) using the following parameters: 162 trypsin rules, maximum 2 missed cleavages, fixed cysteine carbamidomethylation (+57.021 Da) 163 and variable methionine oxidation (+15.995 Da). The precursor and product ion mass tolerances 164 were set to 10 ppm and 0.5 Da, respectively. The target false discovery rate (FDR) was set to 1%. 165 Peptide quantitation was performed using label-free quantitation based on precursor ion area under 166 the curve (AUC). 167 For microbial proteomics, we curated a list of common VAP pathogens comprised of gram-168 positive, gram-negative bacteria and yeast (Supplemental Table S1 proteomics and metabolomics data was tested using the Shapiro-Wilk test. Both datasets were not 207 normally distributed (p>0.05) and therefore subjected to non-parametric analysis using the 208 Wilcoxon rank-sum test. The p-values were adjusted using the Benjamini-Hochberg post hoc test.

209
The following sample groups were compared: VAP positive to Baseline, pre-VAP to Baseline and 210 post-VAP to Baseline. Proteins or metabolites with p < 0.05 or adj. p < 0.05 were considered as 211 significantly different. Gene Ontology (GO) annotation was performed using ToppFun (23).

212
Pathway analysis was performed using Reactome and Ingenuity Pathway Analysis (IPA,QIAGEN 213 Inc.) (24, 25). The p-values and fold-changes for ETA proteins were input into IPA and mapped 214 against the human Ingenuity Knowledgebase with default values to uncover enriched pathways in 215 VAP patients. The activation z-score was calculated by IPA software to determine positive or 216 negative enrichment of pathways, diseases and biological functions as categories. The score 217 predicts the increase or decrease in form of positive or negative z-score, respectively. The 218 proteomics and metabolomics data were further assessed for similarity between ETA and BAL 219 matrices using Bland-Altman analysis (26).

221
Study cohort 222 Our study cohort was composed of 16 trauma patients intubated up to 15 days. Eleven of these 223 patients exhibited symptoms of pneumonia (VAP patients) and five did not present any signs of 224 pulmonary infection (control patients). The clinical annotation and antibiotic regimens are 225 described in Table 1 and Figure 1A. Duration of intubation was longer in VAP (≥ 7 days) than 226 control patients (≤ 5 days). A total of 8 patients including 3 control patients and 5 VAP patients 227 were given broad spectrum antibiotics at intubation, whereas no antibiotics were given to the 228 remaining 2 controls and 6 VAP patients. In VAP patients, antibiotic prophylaxis at the time of 229 intubation did not show any better protection compared to no antibiotics; also, there was no clear-  Figure 1B). As all patients in our study cohort were administered a standard-of-care 235 regimen of antibiotics, their impact on the patient proteome and metabolome was not evaluated.

236
ETA proteome reveals neutrophil mediated response in VAP 237 We identified a total of 3067 unique proteins in ETA collections across all patients and time points. 238 We compared patient-matched ETA and BAL collected on the same day and identified 1811 and 239 1097 unique proteins in ETA and BAL collected on the same day. Of these, 975 proteins 240 represented 88.9% of BAL proteome of this cohort and mapped to 187 significant reactome 241 pathways. The top 10 mapped pathways were neutrophil degranulation, innate immune system, 242 immune system, complement cascade, regulation of complement cascade, platelet activation, 243 signaling and aggregation, platelet degranulation, regulation of insulin-like growth factor 244 transport, post-translational protein phosphorylation and hemostasis (Supplemental Table S2).

245
These suggest enrichment of proteins associated to host immunity in both ETA and BAL. Further,

246
Bland-Altman comparison of these shared proteins showed that there was no significant bias 247 between ETA and BAL as most of the data sits between the 95% confidence interval upper limit 248 of 4.1 and the lower limit of -4.4. Figure 2B). The GO terms for the biological processes related 249 to innate and humoral immunity were similarly enriched across both fluids ( Figure 2C). This 250 suggests similarities in proteome composition between BAL and ETA.

251
Due to the relative ease-of-access and increased sampling availability, we focused our study on 252 ETA. Since intubation was variable across VAP patients and controls, we compared the first day 253 of intubation (Baseline) against subsequent time points in VAP patients, and also compared 254 Baseline ETA proteome in VAP and control patients.  Table S3). GO terms above were further verified 274 by the biological functions and diseases from IPA, which reported degranulation of neutrophils, 275 granulocytes, and phagocytes, leukocyte migration, inflammation, apoptosis, and necrosis as most 276 enriched (Supplemental Table S4). These pathways and processes were also observed to be  Table S5). Absence of these proteins in VAP positive may 294 imply pathogen binding and clearance. Both isoform 2 of nucleoside diphosphate kinase A (NME-295 1) and CRP were detected in VAP ETA only (Table 3). To gain further insight, the significant 296 differentially abundant proteins were mapped to Reactome pathways ( Table 4). Two of the 297 pathways with low p-values (p < 6.6E-14), neutrophil degranulation (11 proteins) and innate  (27), hemoglobin subunit delta (HBD), peroxiredoxin-1 308 (PRDX1), peroxiredoxin-2 (PRDX2), peroxiredoxin-6 (PRDX6) and erythrocyte band 7 integral 309 membrane protein (STOM) mapped to tissue injury (Table 2, 4).

310
To identify early VAP response mechanisms, we evaluated ETA collected two days prior to 311 clinical diagnosis (pre-VAP). We identified 21 significantly differentially abundant proteins (p < 312 0.05) with a fold change >2 compared to Baseline ( Figure 3C, Table 5). Consistent with our prior  Figure 4D). Both proteins were significantly higher in pre-VAP (4.8-to 5-fold compared 322 to Baseline, adj. p < 0.044) and VAP positive (3.4 to 4-fold compared to Baseline, adj. p < 0.038), 323 highlighting the rapid initiation of neutrophil degranulation and may serve as early detection 324 markers.

VAP patients harbor metabolic signatures of oxidative stress 326
Similar to the proteomic analysis, Bland-Altman analysis showed no significant bias of metabolites 327 in ETA and BAL matrices as the majority of data were within the limits of agreement for 95% 328 confidence interval (mean difference of 0, upper limit of 4.7 and lower limit of -4.8) ( Figure 5A).

329
The unsupervised temporal clustering using median metabolite concentrations showed two distinct   Table 7, Supplemental Table 4). Of these, 40 peptides were linked to 4 gram-positive VAP species, 355 95 peptides to 10 gram-negative VAP species, and 2 peptides were linked to Candida albicans. 356 Out of 40 Gram-positive specific peptides, 35 were specific to Propinibacterium, Staphylococcus, 357 Streptococcus or Enterococcus genera. Furthermore, 59 peptides were specific to Gram-negative 358 genera and 21 were explicit to Escherichia coli, Enterobacter clocae, Klebsiella aerogenes, 359 Pseudomonas aeruginosa and Serratia marcescens. Our metaproteomic approach using species- The present study describes VAP mediated host responses in ETA and BAL of 16 intubated 370 patients. This is also the first detailed characterization of the ETA proteome and metabolome. We 371 detected 3067 unique proteins in ETA sampled longitudinally, compared to 1139 proteins in BAL. 372 We also observed >10% increase in unique BAL proteins compared to previous studies (16, 32).

373
Despite our observation of a 3-fold higher proteome diversity and although less invasive, ETA has 374 been historically overlooked in favor of BAL (33). Our study revealed that ETA is functionally 375 diverse and highly enriched in proteins involved in innate and adaptive immunity, suggesting that 376 it is an attractive source to study lung infection. In VAP patients, we observed up-regulation of 377 inflammation and neutrophil-mediated innate immunity in the respiratory tract during VAP 378 infection, leading to pathogen processing (Figure 7). Elevated levels of vinculin and myosins may 379 imply extracellular matrix (ECM) adhesion and migration of neutrophils ( Figure 7B) (34, 35).

380
Increased abundance of pathogen recognition molecules ficolin-1 and properdin in ETA may be 381 linked to complement system activation via interaction with bacterial polysaccharides (36). In 382 support of this, antimicrobial neutrophil proteins S100A8 and S100A9, with proinflammatory and 383 chemotactic activity (37), were highly elevated during VAP infection. Furthermore, the VAP-  Through meta-proteomics analysis, we demonstrated that ETA and BAL harbored pathogen 419 protein products specific to VAP pathogens. Since microbial peptide abundance was low, 420 quantitation was beyond the scope of this study. The utility of these species specific peptides in 421 VAP diagnosis will be explored in future.

422
Intubation is one of the most common interventions in critical care and has been linked to increased 423 susceptibility of lung infection and mortality. Intubation procedure, length of stay and 424 inappropriate antibiotic treatment, as well as pre-existing conditions such as compromised or 425 weakened immunity may contribute to microbial dysbiosis and development of pneumonia.

426
Ultimately, these alterations to the host environment may be reflected at the pulmonary interface.

427
Our study has focused on ETA to conduct a molecular survey of upper airways during VAP 428 development and progression. We have shown that ETA is reflective of a rich and diverse airway 429 proteome. In VAP, we identified an early upregulation of immune-modulatory proteins associated 430 with an early host response to infection. We also looked for VAP pathogen peptides in ETA, and 431 detected unique, species-specific peptides correlated with cultures. In the majority of VAP patients, 432 these distinctive pathogen signatures were present at least 1 to 2 days earlier than the BAL culture   Supplemental Table S1.  Tables  613  Table 1