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Clinical

A Transcriptomic Reporter Assay Employing Neutrophils to Measure Immunogenic Activity of Septic Patients Plasma - Dendritic cells, experiment I - GSE49753

Purpose

The wide array of molecules carried by plasma regulates critical immune functions and constitutes valuable biomarkers and therapeutic targets. In recent years the introduction of “systems approaches” has provided investigators with powerful means for assessing immune responses in patient samples on a global scale. However, while the use of genome-wide profiling technologies has become widespread, measuring the plasma proteome still presents considerable challenges. An alternative approach that consists in measuring transcriptome responses in reporter cells exposed in vitro to patient plasma has been successfully employed in a limited number of studies. Here we devised such a “Transcriptomic Reporter Assay” system to assess the immunogenicity of plasma from septic patients and evaluate its potential for biomarker discovery. Sepsis is a common, severe systemic infectious process for which physicians still lack efficient diagnostic or prognostic tools. Of the three different cell reporter systems tested, neutrophils were identified as the most capable “plasma sensor”. Compared to peripheral blood mononuclear cells and dendritic cell preparations neutrophils were best able to discriminate between plasma from septic and control subjects and responded by upregulating a robust immune transcriptional program. Additionally, the amplitude of the neutrophil transcriptomic response was shown to be associated with disease severity in two additional sets of patients. Overall, our results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for assessing immunopathogenic processes in a complex and severe condition such as sepsis.

Hypothesis

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Experimental Design

Monocytes were isolated from two healthy donors. Monocytes were cultured with interleukin 4 (IL4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate monocyte derived dendritic cells (MoDCs). Plasma samples were obtained from patients with culture-confirmed sepsis (n=12) and from uninfected controls (n=12). MoDCs were cultured for 6 h in medium alone, plasma from patients with sepsis, plasma from uninfected controls, and LPS using a final concentration of 20%. Transcriptional profiles were acquired using Illumina HumanHT12 V4 BeadChips.

Experimental Variables

Septic Plasma Exposure (2021 ICD-10-CM code* = Not Applicable)

Controls

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Methods

The data were preprocessed using R, which included the following steps: quantile normalization, flooring all intensities <10 to 10, log2 transformation, and PALO (present at least once) filter. PALO, which selects only transcripts with detection p-values < 0.01 in at least one sample, was performed to reduce background noise.

Additional Information

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Microarray
Illumina HumanHT-12 v4
40 Samples Loaded: 40
Human (Homo sapiens)
Dendritic Cells
Septic Plasma Exposure
RNA was extracted using the QIAGEN MicroRNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with the Agilent Bioanalyser.
Sample Set Spreadsheet
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Samples Preview
Sample ID !Sample Title Gender Cell type activators experiment
GSM1206418 H1-DC-LPS1 - dendritic cells LPS I
GSM1206419 H2-DC-LPS1 - dendritic cells LPS I
GSM1206420 H2-DC-M1 - dendritic cells Medium alone I
GSM1206421 H1-DC-M1 - dendritic cells Medium alone I
GSM1206422 H2-DC-Bp2 Male dendritic cells Septic plasma I
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File
sampleset4000070_sampleannotations_v1.csv
Sample Set Spreadsheet
sampleset4000070_sampleannotations_v1.csv
Sample Set Spreadsheet
sampleset4000070_sampleannotations_v1.csv
Sample Set Spreadsheet
Log2 Transformed
Raw Signal
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Samples Viewer / Editor

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Sample ID !Sample Title Gender Cell type Activators Experiment Donors Severity Age (years) Bacterial identification Day of blood collection Sample id
GSM1206418
H1-DC-LPS1
-
dendritic cells
LPS
I
H1
-
 
-
 
LPS
GSM1206419
H2-DC-LPS1
-
dendritic cells
LPS
I
H2
-
 
-
 
LPS
GSM1206420
H2-DC-M1
-
dendritic cells
Medium alone
I
H2
-
 
-
 
M1
GSM1206421
H1-DC-M1
-
dendritic cells
Medium alone
I
H1
-
 
-
 
M1
GSM1206422
H2-DC-Bp2
Male
dendritic cells
Septic plasma
I
H2
Severe
50
Burkholderia pseudomallei
22
Bp2
GSM1206423
H1-DC-Ot6
Female
dendritic cells
Septic plasma
I
H1
Severe
70
Escherichia coli (ESBL)
30
Ot6
GSM1206424
H2-DC-Bp6
Male
dendritic cells
Septic plasma
I
H2
Severe
44
Burkholderia pseudomallei
4
Bp6
GSM1206425
H1-DC-Ot4
Female
dendritic cells
Septic plasma
I
H1
Severe
74
Klebsiella pneumoniae
1
Ot4
GSM1206426
H2-DC-Ot4
Female
dendritic cells
Septic plasma
I
H2
Severe
74
Klebsiella pneumoniae
1
Ot4
GSM1206427
H2-DC-Bp1
Male
dendritic cells
Septic plasma
I
H2
Severe
57
Burkholderia pseudomallei
1
Bp1
GSM1206428
H2-DC-Bp4
Male
dendritic cells
Septic plasma
I
H2
Severe
66
Burkholderia pseudomallei
3
Bp4
GSM1206429
H1-DC-Ot3
Female
dendritic cells
Septic plasma
I
H1
Severe
54
Enterococus faecium
0
Ot3
GSM1206430
H2-DC-Ot3
Female
dendritic cells
Septic plasma
I
H2
Severe
54
Enterococus faecium
0
Ot3
GSM1206431
H1-DC-Ot2
Female
dendritic cells
Septic plasma
I
H1
Severe
60
Streptococcus pnumoniae
0
Ot2
GSM1206432
H1-DC-Bp2
Male
dendritic cells
Septic plasma
I
H1
Severe
50
Burkholderia pseudomallei
22
Bp2
GSM1206433
H2-DC-Ot1
Male
dendritic cells
Septic plasma
I
H2
Severe
24
Streptococcus pnumoniae
0
Ot1
GSM1206434
H1-DC-Bp5
Male
dendritic cells
Septic plasma
I
H1
Non-severe
85
Burkholderia pseudomallei
3
Bp5
GSM1206435
H2-DC-Bp5
Male
dendritic cells
Septic plasma
I
H2
Non-severe
85
Burkholderia pseudomallei
3
Bp5
GSM1206436
H2-DC-Ot2
Female
dendritic cells
Septic plasma
I
H2
Severe
60
Streptococcus pnumoniae
0
Ot2
GSM1206437
H2-DC-Ot5
Female
dendritic cells
Septic plasma
I
H2
Severe
77
Pseudomonas aeruginosa
0
Ot5
GSM1206438
H1-DC-Bp3
Male
dendritic cells
Septic plasma
I
H1
Severe
72
Burkholderia pseudomallei
2
Bp3
GSM1206439
H1-DC-Ot1
Male
dendritic cells
Septic plasma
I
H1
Severe
24
Streptococcus pnumoniae
0
Ot1
GSM1206440
H1-DC-Bp6
Male
dendritic cells
Septic plasma
I
H1
Severe
44
Burkholderia pseudomallei
4
Bp6
GSM1206441
H2-DC-Bp3
Male
dendritic cells
Septic plasma
I
H2
Severe
72
Burkholderia pseudomallei
2
Bp3
GSM1206442
H1-DC-Bp1
Male
dendritic cells
Septic plasma
I
H1
Severe
57
Burkholderia pseudomallei
1
Bp1
GSM1206443
H2-DC-Ot6
Female
dendritic cells
Septic plasma
I
H2
Severe
70
Escherichia coli (ESBL)
30
Ot6
GSM1206444
H1-DC-Bp4
Male
dendritic cells
Septic plasma
I
H1
Severe
66
Burkholderia pseudomallei
3
Bp4
GSM1206445
H1-DC-Ot5
Female
dendritic cells
Septic plasma
I
H1
Severe
77
Pseudomonas aeruginosa
0
Ot5
GSM1206446
H2-DC-HC2
Male
dendritic cells
Uninfected plasma
I
H2
-
60
-
 
HC2
GSM1206447
H2-DC-HC4
Female
dendritic cells
Uninfected plasma
I
H2
-
64
-
 
HC4
GSM1206448
H1-DC-HC5
Male
dendritic cells
Uninfected plasma
I
H1
-
56
-
 
HC5
GSM1206449
H1-DC-HC3
Female
dendritic cells
Uninfected plasma
I
H1
-
57
-
 
HC3
GSM1206450
H1-DC-HC1
Male
dendritic cells
Uninfected plasma
I
H1
-
59
-
 
HC1
GSM1206451
H2-DC-HC3
Female
dendritic cells
Uninfected plasma
I
H2
-
57
-
 
HC3
GSM1206452
H1-DC-HC2
Male
dendritic cells
Uninfected plasma
I
H1
-
60
-
 
HC2
GSM1206453
H1-DC-HC4
Female
dendritic cells
Uninfected plasma
I
H1
-
64
-
 
HC4
GSM1206454
H2-DC-HC5
Male
dendritic cells
Uninfected plasma
I
H2
-
56
-
 
HC5
GSM1206455
H2-DC-HC6
Male
dendritic cells
Uninfected plasma
I
H2
-
56
-
 
HC6
GSM1206456
H1-DC-HC6
Male
dendritic cells
Uninfected plasma
I
H1
-
56
-
 
HC6
GSM1206457
H2-DC-HC1
Male
dendritic cells
Uninfected plasma
I
H2
-
59
-
 
HC1

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