Articles in PresS. Am J Physiol Lung Cell Mol Physiol (May 8, 2015). doi:10.1152/ajplung.00061.2014
1
Alterations of lung microbiota in a mouse model of LPS-induced lung injury
2 3
Valeriy Poroyko1, Fanyong Meng2, Angelo Meliton2, Taras Afonyushkin2, Alexander
4
Ulanov3, Ekaterina Semenyuk4, Omar Latif5, Vera Tesic6, Anna A. Birukova2, Konstantin
5
G. Birukov2.
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
1
Department of Pediatrics, The University of Chicago, Chicago, Illinois 60637, USA Section of Pulmonary and Critical Medicine, Lung Injury Center, Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA 3 Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign, USA 4 Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153, USA 5 Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA 6 Department of Pathology, University of Chicago, Chicago, Illinois 60637, USA 2
Running title: Microbiome changes in LPS-induced ALI Abbreviations: ALI - acute lung injury, LPS - lipopolysaccharide, ARDS - acute respiratory distress syndrome, BAL - bronchoalveolar lavage, PBS - phosphate buffered saline, OTU - operational taxonomic units, PCR - polymerase chain reaction, QPCR quantitative polymerase chain reaction. Keyword: acute lung injury, LPS, microbiota, metabolic profiling Correspondence and requests for reprints should be addressed to: Valeriy Poroyko, PhD The University of Chicago Medical Center Department of Pediatrics KCBD, Room 4110 900 E.57th Street, Chicago, IL, 60637 Office: (773)702-8584 Fax: (773)702-6888 Email: [email protected] Supported in part by NIH grants 1R21AI099713-01, HL087823 and HL107920.
39
Copyright © 2015 by the American Physiological Society.
40
Abstract.
41
Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are
42
common responses to a variety of infectious and non-infectious insults. We used a mouse
43
model of ALI induced by intratracheal administration of sterile bacterial wall
44
lipopolysacharide (LPS) to investigate the changes in innate lung microbiota and study
45
microbial community reaction to lung inflammation and barrier dysfunction induced by
46
endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection
47
(n=6), another - sterile water (n=7). Bronchoalveolar lavage (BAL) was performed at 72h
48
after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-
49
tags (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was
50
increased 5-folds (p=0.03). The community complexity remained unchanged (Simpson
51
index, p=0.7), Shannon diversity index indicated the increase of community evenness in
52
response to ALI (p=0.07). Principal coordinate analysis and ANOSIM test (p=0.005)
53
revealed significant difference between microbiota of control and ALI groups. Bacteria
54
from families Xanthomonadaceae and Brucellaceae increased their abundance in ALI
55
group as determined by METASTATS test (p99.9%) community
211
coverage. The annotated sequences were attributed to 17 bacterial families with
212
abundance level above 1% in in LPS(-) and LPS(+) groups, of them 15 and 5 were
213
present in LPS(-) and LPS(+) groups respectively (Table 1).The lungs of mice from
214
LPS(-) group harboring microbiota dominated by bacteria from family
215
Alicyclobacillaceae which alone accounted for 55% of sequences. The remaining 14
216
families were present at relative abundances between 1-5%, of them, 7 families were
217
falling between 5% and 2% of abundance (Moraxellaceae, Enterobacteriaceae,
218
Xanthomonadaceae, unclassified Burkholderiales, Staphylococcaceae,
219
Carnobacteriaceae, Brucellaceae) and 7 more between 2% and 1%. qPCR analysis was
220
performed (36) to compare amount of bacterial DNA in the BAL of LPS(-) and LPS(+)
221
mice. The extracted DNA was normalized to 1 ml of BAL fluid prior reaction. On
222
average we detected 2.5 PCR cycles difference between mean Ct values from LPS treated
223
and control groups (p=0.03) (Fig. 2A). These data indicate an average 5-fold increase of
224
bacterial load in BAL samples from the experimental ALI mice. The analysis of 16S
225
RNA-tags indicated that the community complexity, measured by Gini-Simpson (1-λ)
226
diversity index, remained unchanged after LPS treatment (p=0.7) (Fig. 2B), while
227
Shannon diversity indices shift indicated the tendency to increase the community
228
evenness in response to ALI (p=0.07) (Fig. 2C). While the effect of ALI on both
229
measures was statistically insignificant, the community evenness exhibited stronger
230
change. Thus the analysis suggested that the observed bacterial growth is attributed to
231
species existing within the lung ecosystem before the ALI rather than new species
232
acquired post-trauma. A matrix based on Yue & Clayton measures of dissimilarity
233
between the community structures was used to differentiate samples by principal
234
coordinate analysis (PCoA) (Fig. 2D). The analysis indicated that 66% of total variation
235
in the dates could be explained by the principal coordinate PC1. In our study the PC1 was
236
collinear with LPS treatment and experimental ALI. ANOSIM test confirmed significant
237
separation between control and ALI groups at the level of p=0.005. The Metastats-based
238
comparison of LPS(-) and LPS(+) groups revealed significant (p
1
Alterations of lung microbiota in a mouse model of LPS-induced lung injury
2 3
Valeriy Poroyko1, Fanyong Meng2, Angelo Meliton2, Taras Afonyushkin2, Alexander
4
Ulanov3, Ekaterina Semenyuk4, Omar Latif5, Vera Tesic6, Anna A. Birukova2, Konstantin
5
G. Birukov2.
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
1
Department of Pediatrics, The University of Chicago, Chicago, Illinois 60637, USA Section of Pulmonary and Critical Medicine, Lung Injury Center, Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA 3 Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign, USA 4 Department of Microbiology and Immunology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153, USA 5 Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA 6 Department of Pathology, University of Chicago, Chicago, Illinois 60637, USA 2
Running title: Microbiome changes in LPS-induced ALI Abbreviations: ALI - acute lung injury, LPS - lipopolysaccharide, ARDS - acute respiratory distress syndrome, BAL - bronchoalveolar lavage, PBS - phosphate buffered saline, OTU - operational taxonomic units, PCR - polymerase chain reaction, QPCR quantitative polymerase chain reaction. Keyword: acute lung injury, LPS, microbiota, metabolic profiling Correspondence and requests for reprints should be addressed to: Valeriy Poroyko, PhD The University of Chicago Medical Center Department of Pediatrics KCBD, Room 4110 900 E.57th Street, Chicago, IL, 60637 Office: (773)702-8584 Fax: (773)702-6888 Email: [email protected] Supported in part by NIH grants 1R21AI099713-01, HL087823 and HL107920.
39
Copyright © 2015 by the American Physiological Society.
40
Abstract.
41
Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are
42
common responses to a variety of infectious and non-infectious insults. We used a mouse
43
model of ALI induced by intratracheal administration of sterile bacterial wall
44
lipopolysacharide (LPS) to investigate the changes in innate lung microbiota and study
45
microbial community reaction to lung inflammation and barrier dysfunction induced by
46
endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection
47
(n=6), another - sterile water (n=7). Bronchoalveolar lavage (BAL) was performed at 72h
48
after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-
49
tags (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was
50
increased 5-folds (p=0.03). The community complexity remained unchanged (Simpson
51
index, p=0.7), Shannon diversity index indicated the increase of community evenness in
52
response to ALI (p=0.07). Principal coordinate analysis and ANOSIM test (p=0.005)
53
revealed significant difference between microbiota of control and ALI groups. Bacteria
54
from families Xanthomonadaceae and Brucellaceae increased their abundance in ALI
55
group as determined by METASTATS test (p99.9%) community
211
coverage. The annotated sequences were attributed to 17 bacterial families with
212
abundance level above 1% in in LPS(-) and LPS(+) groups, of them 15 and 5 were
213
present in LPS(-) and LPS(+) groups respectively (Table 1).The lungs of mice from
214
LPS(-) group harboring microbiota dominated by bacteria from family
215
Alicyclobacillaceae which alone accounted for 55% of sequences. The remaining 14
216
families were present at relative abundances between 1-5%, of them, 7 families were
217
falling between 5% and 2% of abundance (Moraxellaceae, Enterobacteriaceae,
218
Xanthomonadaceae, unclassified Burkholderiales, Staphylococcaceae,
219
Carnobacteriaceae, Brucellaceae) and 7 more between 2% and 1%. qPCR analysis was
220
performed (36) to compare amount of bacterial DNA in the BAL of LPS(-) and LPS(+)
221
mice. The extracted DNA was normalized to 1 ml of BAL fluid prior reaction. On
222
average we detected 2.5 PCR cycles difference between mean Ct values from LPS treated
223
and control groups (p=0.03) (Fig. 2A). These data indicate an average 5-fold increase of
224
bacterial load in BAL samples from the experimental ALI mice. The analysis of 16S
225
RNA-tags indicated that the community complexity, measured by Gini-Simpson (1-λ)
226
diversity index, remained unchanged after LPS treatment (p=0.7) (Fig. 2B), while
227
Shannon diversity indices shift indicated the tendency to increase the community
228
evenness in response to ALI (p=0.07) (Fig. 2C). While the effect of ALI on both
229
measures was statistically insignificant, the community evenness exhibited stronger
230
change. Thus the analysis suggested that the observed bacterial growth is attributed to
231
species existing within the lung ecosystem before the ALI rather than new species
232
acquired post-trauma. A matrix based on Yue & Clayton measures of dissimilarity
233
between the community structures was used to differentiate samples by principal
234
coordinate analysis (PCoA) (Fig. 2D). The analysis indicated that 66% of total variation
235
in the dates could be explained by the principal coordinate PC1. In our study the PC1 was
236
collinear with LPS treatment and experimental ALI. ANOSIM test confirmed significant
237
separation between control and ALI groups at the level of p=0.005. The Metastats-based
238
comparison of LPS(-) and LPS(+) groups revealed significant (p
