Molecular Profiling of Innate Immune Response Mechanisms in Ventilator-associated Pneumonia

Abstract

Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is used in hospitals for VAP diagnosis and guiding treatment options. Although BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their use 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. The metaproteome approach successfully allowed simultaneous detection of pathogen peptides in patients' ETA, which may have potential use in diagnosis. Our findings suggest that ETA may facilitate early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore provide its diagnosis and treatment. Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is used in hospitals for VAP diagnosis and guiding treatment options. Although BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their use 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. The metaproteome approach successfully allowed simultaneous detection of pathogen peptides in patients' ETA, which may have potential use in diagnosis. Our findings suggest that ETA may facilitate early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore provide its diagnosis and treatment. Ventilator-associated pneumonia (VAP) is the second most common hospital-acquired infection (HAI) in intensive care units (ICU), and is associated to 60% of all HAI-related deaths in the United States (1Niederman M.S. Hospital-acquired pneumonia, health care-associated pneumonia, ventilator-associated pneumonia, and ventilator-associated tracheobronchitis: definitions and challenges in trial design.Clin. Infect. Dis. 2010; 51: S12-S17Crossref PubMed Scopus (52) Google Scholar). It occurs at least 48 h after mechanical ventilation and accounts for more than 300,000 cases annually in the United States. (1Niederman M.S. Hospital-acquired pneumonia, health care-associated pneumonia, ventilator-associated pneumonia, and ventilator-associated tracheobronchitis: definitions and challenges in trial design.Clin. Infect. Dis. 2010; 51: S12-S17Crossref PubMed Scopus (52) Google Scholar, 2Koenig S.M. Truwit J.D. Ventilator-associated pneumonia: diagnosis, treatment, and prevention.Clin. Microbiol. Rev. 2006; 19: 637-657Crossref PubMed Scopus (247) Google Scholar). Mechanical ventilation can injure tracheal epithelia and promote environmental microbial colonization and migration from the upper to lower airways (3Mangram A.J. Sohn J. Zhou N. Hollingworth A.K. Ali-Osman F.R. Sucher J.F. Moyer M. Dzandu J.K. Trauma-associated pneumonia: time to redefine ventilator-associated pneumonia in trauma patients.Am. J. Surg. 2015; 210 (discussion 1061-1052): 1056-1061Abstract Full Text Full Text PDF PubMed Google Scholar). All ICU patients with prolonged hospitalization are at high risk of developing VAP, increasing the cost of hospital stay by $40,000 to $50,000 per patient (3Mangram A.J. Sohn J. Zhou N. Hollingworth A.K. Ali-Osman F.R. Sucher J.F. Moyer M. Dzandu J.K. Trauma-associated pneumonia: time to redefine ventilator-associated pneumonia in trauma patients.Am. J. Surg. 2015; 210 (discussion 1061-1052): 1056-1061Abstract Full Text Full Text PDF PubMed Google Scholar, 4Cocanour C.S. Ostrosky-Zeichner L. Peninger M. Garbade D. Tidemann T. Domonoske B.D. Li T. Allen S.J. Luther K.M. Cost of a ventilator-associated pneumonia in a shock trauma intensive care unit.Surg. Infect. (Larchmt). 2005; 6: 65-72Crossref PubMed Scopus (70) Google Scholar, 5Kornblith L.Z. Kutcher M.E. Callcut R.A. Redick B.J. Hu C.K. Cogbill T.H. Baker C.C. Shapiro M.L. Burlew C.C. Kaups K.L. DeMoya M.A. Haan J.M. Koontz C.H. Zolin S.J. Gordy S.D. Shatz D.V. Paul D.B. Cohen M.J. Western Trauma Association Study, G. (2013) Mechanical ventilation weaning and extubation after spinal cord injury: a Western Trauma Association multicenter study.J. Trauma Acute Care Surg. 2013; 75 (discussion): 1060-1070Crossref PubMed Scopus (1) Google Scholar). VAP diagnosis is largely based on clinical criteria such as fever, infiltrate on chest radiograph, leukocyte counts, and positive cultures from bronchoalveolar lavages (BAL) (2Koenig S.M. Truwit J.D. Ventilator-associated pneumonia: diagnosis, treatment, and prevention.Clin. Microbiol. Rev. 2006; 19: 637-657Crossref PubMed Scopus (247) Google Scholar, 6Rotstein C. Evans G. Born A. Grossman R. Light R.B. Magder S. McTaggart B. Weiss K. Zhanel G.G. Clinical practice guidelines for hospital-acquired pneumonia and ventilator-associated pneumonia in adults.Can. J. Infect. Dis. Med. Microbiol. 2008; 19: 19-53Crossref PubMed Scopus (174) Google Scholar). With trauma patients, these symptoms are often nonspecific and lead to overestimation of true VAP episodes resulting in the prescription of inadequate broad-spectrum antibiotics (6Rotstein C. Evans G. Born A. Grossman R. Light R.B. Magder S. McTaggart B. Weiss K. Zhanel G.G. Clinical practice guidelines for hospital-acquired pneumonia and ventilator-associated pneumonia in adults.Can. J. Infect. Dis. Med. Microbiol. 2008; 19: 19-53Crossref PubMed Scopus (174) Google Scholar, 7Croce M.A. Swanson J.M. Magnotti L.J. Claridge J.A. Weinberg J.A. Wood G.C. Boucher B.A. Fabian T.C. The futility of the clinical pulmonary infection score in trauma patients.J. Trauma. 2006; 60 (discussion 527–528): 523-527Crossref PubMed Scopus (90) Google Scholar). A study involving 300 U.S. hospitals showed a 52% increase in antibiotic consumption rate in intensive care unit (ICU) compared with noncritical care (8Baggs J. Fridkin S.K. Pollack L.A. Srinivasan A. Jernigan J.A. Estimating National Trends in Inpatient Antibiotic Use Among US Hospitals From 2006 to 2012.JAMA Intern. Med. 2016; 176: 1639-1648Crossref PubMed Scopus (146) Google Scholar). In addition, VAP accounts for over half of the antibiotics used in the ICU, which may lead to multi-drug resistance (9Kalanuria A.A. Ziai W. Zai W. Mirski M. Ventilator-associated pneumonia in the ICU.Crit. Care. 2014; 18: 208Crossref PubMed Scopus (234) Google Scholar). Culture-based testing can reduce antibiotic misuse, but it is time-consuming and can delay diagnosis and treatment. Therefore, careful investigation of VAP pathogenesis is required to better understand host response to microbial dysbiosis and provide insights into the molecular mechanisms underlying the progression of infection. Previous studies have proposed several candidate biomarkers (interleukin-1β, interleukin-8, soluble triggering receptor expressed on myeloid cells type 1, C-reactive protein (CRP), procalcitonin, and the mid-region fragment of pro-adrenomedullin) to assist VAP diagnosis in serum, plasma or BAL (10Palazzo S.J. Simpson T. Schnapp L. Biomarkers for ventilator-associated pneumonia: review of the literature.Heart Lung. 2011; 40: 293-298Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 11Povoa P. Martin-Loeches I. Ramirez P. Bos L.D. Esperatti M. Silvestre J. Gili G. Goma G. Berlanga E. Espasa M. Goncalves E. Torres A. Artigas A. Biomarker kinetics in the prediction of VAP diagnosis: results from the BioVAP study.Ann. Intensive Care. 2016; 6: 32Crossref PubMed Scopus (32) Google Scholar, 12Luyt C.E. Combes A. Reynaud C. Hekimian G. Nieszkowska A. Tonnellier M. Aubry A. Trouillet J.L. Bernard M. Chastre J. Usefulness of procalcitonin for the diagnosis of ventilator-associated pneumonia.Intensive Care Med. 2008; 34: 1434-1440Crossref PubMed Scopus (115) Google Scholar, 13Conway Morris A. Kefala K. Wilkinson T.S. Moncayo-Nieto O.L. Dhaliwal K. Farrell L. Walsh T.S. Mackenzie S.J. Swann D.G. Andrews P.J. Anderson N. Govan J.R. Laurenson I.F. Reid H. Davidson D.J. Haslett C. Sallenave J.M. Simpson A.J. Diagnostic importance of pulmonary and in ventilator-associated 2010; PubMed Scopus Google Scholar). and have been successfully in BAL for VAP diagnosis Morris A. Kefala K. Wilkinson T.S. Moncayo-Nieto O.L. Dhaliwal K. Farrell L. Walsh T.S. Mackenzie S.J. Swann D.G. Andrews P.J. Anderson N. Govan J.R. Laurenson I.F. Reid H. Davidson D.J. Haslett C. Sallenave J.M. Simpson A.J. Diagnostic importance of pulmonary and in ventilator-associated 2010; PubMed Scopus Google Scholar). most of these are and have and VAP detection C.E. Combes A. Reynaud C. Hekimian G. Nieszkowska A. Tonnellier M. Aubry A. Trouillet J.L. Bernard M. Chastre J. Usefulness of procalcitonin for the diagnosis of ventilator-associated pneumonia.Intensive Care Med. 2008; 34: 1434-1440Crossref PubMed Scopus (115) Google Scholar, R. G. J. D. B. and diagnostic and in ventilator-associated PubMed Google Scholar). potential of and the for on VAP BAL has been a to study pulmonary A.K. lavage in the diagnosis of ventilator-associated pneumonia: to or that is the Surg. 2011; PubMed Google Scholar, S.J. Schnapp of bronchoalveolar lavage in patients with ventilator-associated 2013; PubMed Scopus Google Scholar). studies have the use of BAL for microbial and VAP infection S.J. Schnapp of bronchoalveolar lavage in patients with ventilator-associated 2013; PubMed Scopus Google Scholar, Anderson J. P. T. S. R. B. S. Walsh T.S. Morris A. Simpson A.J. of of pneumonia to reduce antibiotic use study for a 2016; PubMed Scopus Google Scholar, Morris A. N. P. S. Walsh T.S. K. Simpson A.J. R. can patients with ventilator-associated PubMed Scopus Google Scholar, A. C. Trouillet J.L. N. M. C. Chastre J. and of ventilator-associated Full Text Full Text PDF PubMed Scopus Google Scholar). (ETA) is as a for and has been for cultures in VAP diagnosis M.L. M. J. J. A.A. S. J.A. P. J.L. of With Hospital-acquired and Ventilator-associated Clinical by the of and the Infect. Dis. 2016; PubMed Scopus Google Scholar). the molecular of ETA has not been as as BAL to understand host responses to infection. We that in ETA is to more longitudinal molecular of host response and to microbial early infection. We that this mechanistic insights into VAP We used a approach and on longitudinal ETA and BAL collected from intubated patients for this and were from or The used in this study were and the and were mechanical ventilation at the ICU trauma at were for this A from patient or a The clinical for collection by the and the Western All procedures to the in the of and the of and with positive clinical symptoms 48 h of in or pulmonary on chest and a positive BAL for or were with VAP. Clinical diagnosis of VAP by BAL microbial were to A of to as of a as of more than potential The chest on all the patients in the The patients with of clinical symptoms were as ETA collected at the of positive clinical BAL collected as of procedures and used for microbial cultures to in clinical diagnosis. 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M. of units for and by the 2011; PubMed Scopus Google Scholar). the were to and the were and with The protein after and protein can be by the dynamic of ETA and BAL of high such as and at of infection, and the as a of the BAL The were with at The resulting peptides were and with with The peptides were and at LC–MS/MS the of were in and The of peptides after from to The in the of from ETA and BAL to and were to LC–MS/MS a to an of peptides from on a a from to and to A at a rate of The were to for to for to to for to for for and at for were over a of to the at by of for with of a 60 time and dynamic of 60 with of to were at a of in the E. on were and after and of to were with the a protein on the and The and were to and The rate to based on the in used to the rate with a of and a of for peptides and microbial a of common VAP of and from of the and protein protein were used as and in this study were with for pathogen peptides were compared with peptides to for that be of peptides common and VAP were to peptides to and were for from the were R. A. A. P. S. L. P. B. of 18: PubMed Scopus Google for their microbial and to of and were by in VAP and ETA to from the The used to and were the from of BAL or ETA were to The were in by A of ETA and BAL were and used as were the conducted on an with a all ETA and BAL, simultaneous for the amino acids, The were All the were in and and were used this The as a of in and of acids, and with and with and The longitudinal ETA and BAL from 16 intubated patients VAP patients, and as in the VAP to longitudinal ETA were collected per in the up to ETA were collected per patient of ETA and BAL of were not patients of not All were for processing and protein were In VAP patients, in of or VAP positive were for of these compared with from with of in of were for with were from the and the were to by M.J. C. P.J. by is it and it J. 2011; PubMed Scopus Google Scholar). protein or of The of and were the were not and therefore to the The were the The were VAP positive to to and to or with or were as J. B.J. for and candidate PubMed Scopus Google Scholar). and A. K. P. M. K. R. B. S. S. L. B. M. K. M. J. M. G. L. H. P. The 2016; PubMed Google Scholar, A. J. J. S. in 2014; PubMed Scopus Google Scholar). The and for ETA were into and the with to in VAP The by to positive or of and as The score the increase or in of positive or The and were for ETA and BAL D. Med. 2015; PubMed Google Scholar). Our study of 16 trauma patients intubated up to 15 of these patients symptoms of pneumonia and not of pulmonary infection The clinical and antibiotic are in and of in VAP than patients A of patients patients and VAP patients were antibiotics at antibiotics were to the and VAP In VAP patients, antibiotic at the time of not better compared with of antibiotic on the of mechanical antibiotic treatment as per BAL and clinical diagnosis. on BAL the most common VAP pathogen patients S. and patients S. all patients in study were a of their on the patient proteome and metabolome not We a of in ETA all patients and time We compared ETA and BAL collected on the and and in ETA and BAL collected on the of BAL proteome of this and to The were neutrophil degranulation, of and degranulation, of protein and suggest of associated to host in ETA and of these showed that ETA and BAL as most of the the upper of and the lower of The for the to and were in proteome BAL and of the and study on VAP patients and compared the of time in VAP patients and compared ETA proteome in VAP and The patients in study were intubated for of time and developed infection at We longitudinal to clinical of such as VAP and protein for showed of of and VAP patients that at the time of ETA of and VAP patients The ETA proteome in VAP patients the of progression of VAP infection from VAP positive and In VAP patients, a of and were in the and VAP positive ETA, with time of to of were all ETA, proteome in longitudinal The ETA were used to the and VAP positive in VAP were compared with and on all VAP positive and Acute response of by and are and of are were by the and from which of and leukocyte and as most and were to be in compared with and early of leukocyte mediated in response to VAP and VAP positive to the neutrophil response to and leukocyte and were to reactive oxygen species and neutrophil mediated response and in VAP response infection, compared ETA VAP and the in the longitudinal in VAP compared with of VAP with time in showed increase of and in the VAP and were not in VAP positive but were in all their in protein of these in VAP positive may pathogen and of A and were in VAP ETA the were to of the with neutrophil and to of all of host in VAP of 48 were to The of to to pathogen and most R. 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A of for and 2010; PubMed Scopus Google Scholar). their potential in early VAP infection, to neutrophil mediated pathogen Longitudinal by LC–MS/MS of the key of neutrophil and were by were in to compared with and VAP positive to compared with the of neutrophil and may as early detection to the showed of in ETA and BAL as the of were the of for of upper of and lower of The showed of and the of the ETA more with that at the time of ETA of and VAP patients were and as infection from VAP positive to The of with may an of of of infection We of several at or VAP or in were in and may patient responses infection. of VAP positive and ETA an increase in and a in VAP infection The high of amino and in VAP positive ETA may have from the activity of neutrophil and of and its may reactive oxygen species by or neutrophil In VAP positive ETA, a to increase of as of and and their of on as a in the of to in pneumonia A. M. M.A. C. L. P. A. M. C. C. R. W. B. P. of and in patients with 18: PubMed Scopus Google Scholar). We and VAP infection. of plasma have been in VAP patients D. S. K. S. M. for of in patients.J. Full Text Full Text PDF PubMed Scopus Google Scholar). We a of microbial peptides to to VAP in ETA and BAL of 15 patients and peptides were associated to to and to In ETA, peptides of were in VAP patients compared with peptides with in peptides were in BAL, of which were in BAL Although and of microbial peptides in ETA and BAL their with VAP associated with and were in ETA from VAP and however, peptides were in VAP in to were more in VAP patients most of the days, as by their peptides were at least to VAP diagnosis in most of the patients the of the microbial peptides at species and peptides to of which and is associated with were to and and peptides species for and for peptides are associated with of which is to We peptides to of these peptides are associated with and of VAP of which are associated to to and to The study VAP mediated host in ETA and BAL of 16 intubated is the characterization of the ETA proteome and We in ETA compared with in We a increase in BAL compared with studies S.J. Schnapp of bronchoalveolar lavage in patients with ventilator-associated 2013; PubMed Scopus Google Scholar, J. S. Schnapp of the bronchoalveolar lavage 2008; PubMed Scopus Google Scholar). of a proteome and invasive, ETA has been in of BAL and bronchoalveolar lavage diagnostic in ventilator-associated Microbiol. 2014; PubMed Scopus Google Scholar). Our study that ETA is diverse and in in and that it is an to study infection. In VAP patients, of and in the VAP infection, leading to pathogen processing neutrophil in The of and may extracellular and migration of J.D. in and 2013; PubMed Scopus Google Scholar, M. in and Rev. PubMed Scopus Google Scholar). of pathogen and in ETA may be to with D. M. M. I. K. M. T. is a protein that the 2005; PubMed Google Scholar). In of neutrophil and with and activity M.A. N. P. of and migration to the in 2008; PubMed Google were VAP infection. the increase in such as and A. K. L. H. H. C. A. J. The neutrophil by the or of into in and to in 2013; PubMed Scopus Google Scholar, S. N. of neutrophil and with of neutrophil 2013; PubMed Scopus Google all and neutrophil to promote pathogen Li and an increase in plasma with the and progression of VAP infection and with of in with J. Med. 2016; PubMed Scopus Google Scholar). infection, of these neutrophil may have on of The high of amino as in ETA suggest of neutrophil on has been L.A. in the of but in the of PubMed Scopus Google Scholar). The are key of in such as VAP extracellular in and Rev. 18: PubMed Scopus Google Scholar). In and neutrophil VAP which may promote and of neutrophil extracellular The of in VAP pathogenesis has been in BAL of patients C. R. C. extracellular are in the of patients with ventilator-associated Care. PubMed Scopus Google Scholar). a pathogen infection, studies have that can be to the N. a in 2013; PubMed Scopus Google Scholar). the of of in 2016; Full Text Full Text PDF PubMed Scopus Google their high at VAP infection may have in as a of neutrophil on ETA investigation a of neutrophil with neutrophil and migration the of infection. of and as a host response VAP. of and by the of neutrophil in early host response to VAP. have to and to of at from 48 to in pneumonia M. Reid C. M.J. S. C.E. and C-reactive protein in patients with pneumonia or of or 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. S. M. J.M. The Diagnostic of for in with Acute 2016; Scopus Google Scholar). The and of and at VAP positive is and We for such as interleukin-8, soluble triggering receptor expressed on myeloid cells type 1, procalcitonin, and the mid-region fragment of in ETA from VAP and We in ETA of VAP and patients of these were and VAP. We the of protein and in VAP ETA The of protein and and by and D. J. I. A. is required for neutrophil extracellular for 2011; PubMed Scopus Google Scholar, H. A. E. S. K. M. C. A. J. is from the by reactive oxygen Med. 2015; PubMed Scopus Google Scholar, E. P. A. R. a to the of 2016; Google Scholar). have of to the and of is characteristic of neutrophil and has been in pulmonary extracellular in pulmonary of a 2016; PubMed Scopus Google Scholar). Our is the investigation of longitudinal ETA which has host of pathogen processing and of VAP In a and BAL a clinical for VAP diagnosis to VAP Our candidate peptides at and species for VAP Along with clinical pathogen detection has a in VAP diagnosis and antibiotic treatment patients were on antibiotics of and the of the ventilation Our study on VAP ETA and BAL, the collection of ventilation not to antibiotic treatment not antibiotic treatment with of pathogen the of peptides in to VAP infection. The peptides were in most patients' ETA over with at least to VAP diagnosis. microbial compared with host the of this studies to the and of these microbial peptides and their use in VAP diagnosis. is of the most common in care and has been to of infection and mortality. of stay and antibiotic treatment, as as such as or may to microbial dysbiosis and of these to the host may be at the pulmonary interface. Our study has on ETA to a molecular of upper airways VAP and We have that ETA is reflective of a rich and diverse In VAP, an early of associated with an early host response to infection. We for VAP pathogen peptides in ETA, and peptides with In the of VAP patients, these pathogen signatures were at least to than the BAL diagnosis. Although from BAL, ETA may be an for and clinical diagnosis of pneumonia in intubated The have been to the with The for and host are in We the patients, their and the at for their and this We for this with bronchoalveolar lavage C-reactive protein extracellular neutrophil endotracheal aspirates hospital infection Intensive Care neutrophil extracellular of A reactive oxygen species membrane protein ventilator-associated

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