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Clinical

A Transcriptomic Reporter Assay Employing Neutrophils to Measure Immunogenic Activity of Septic Patients Plasma - Neutrophils, experiment II -GSE49756

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

Polymorphonuclear neutrophils (PMNs) were isolated a healthy donor. Plasma samples were obtained from a first set of patients with culture-confirmed sepsis (n=29) and from uninfected controls (n=17). PMNs 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
49 Samples Loaded: 49
Human (Homo sapiens)
Neutrophils
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
GSM1206538 H3-LPS1 - neutrophil LPS II
GSM1206539 H3-Med1 - neutrophil Medium alone II
GSM1206540 H3-Med2 - neutrophil Medium alone II
GSM1206541 H3-Ot9 Female neutrophil Septic plasma II
GSM1206542 H3-BP13 Female neutrophil Septic plasma II
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File
sampleset4000073_sampleannotations_v1.csv
Sample Set Spreadsheet
sampleset4000073_sampleannotations_v1.csv
Sample Set Spreadsheet
Log2 Transformed
Raw Signal
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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 Severity V1
GSM1206538
H3-LPS1
-
neutrophil
LPS
II
H2
-
 
-
 
LPS1
 
GSM1206539
H3-Med1
-
neutrophil
Medium alone
II
H2
-
 
-
 
Med1
 
GSM1206540
H3-Med2
-
neutrophil
Medium alone
II
H2
-
 
-
 
Med2
 
GSM1206541
H3-Ot9
Female
neutrophil
Septic plasma
II
H2
Non-severe**
71
Sphingomonas spp.
1
Ot9
Non-severe
GSM1206542
H3-BP13
Female
neutrophil
Septic plasma
II
H2
Non-severe
40
Burkholderia pseudomallei
3
Bp13
Non-severe
GSM1206543
H3-BP7
Male
neutrophil
Septic plasma
II
H2
Severe
39
Burkholderia pseudomallei
5
Bp7
Severe
GSM1206544
H3-BP14
Male
neutrophil
Septic plasma
II
H2
Severe
87
Burkholderia pseudomallei
1
Bp14
Severe
GSM1206545
H3-BP25
Male
neutrophil
Septic plasma
II
H2
Severe
59
Burkholderia pseudomallei
2
Bp25
Severe
GSM1206546
H3-Ot13
Female
neutrophil
Septic plasma
II
H2
Non-severe**
79
Escherichia coli
1
Ot13
Non-severe
GSM1206547
H3-BP24
Male
neutrophil
Septic plasma
II
H2
Severe
42
Burkholderia pseudomallei
1
Bp24
Severe
GSM1206548
H3-Ot12
Female
neutrophil
Septic plasma
II
H2
Non-severe
77
Streptococcus pyrogenes
1
Ot12
Non-severe
GSM1206549
H3-Ot20
Male
neutrophil
Septic plasma
II
H2
Severe
85
Escherichia coli
1
Ot20
Severe
GSM1206550
H3-BP12
Male
neutrophil
Septic plasma
II
H2
Severe
72
Burkholderia pseudomallei
3
Bp12
Severe
GSM1206551
H3-Ot15
Male
neutrophil
Septic plasma
II
H2
Non-severe**
79
Sphingobacterium spp.
1
Ot15
Non-severe
GSM1206552
H3-Ot24
Female
neutrophil
Septic plasma
II
H2
Severe
77
Escherichia coli
2
Ot24
Severe
GSM1206553
H3-BP17
Female
neutrophil
Septic plasma
II
H2
Severe
43
Burkholderia pseudomallei
2
Bp17
Severe
GSM1206554
H3-BP10
Male
neutrophil
Septic plasma
II
H2
Severe
69
Burkholderia pseudomallei
4
Bp10
Severe
GSM1206555
H3-Ot10
Female
neutrophil
Septic plasma
II
H2
Non-severe
64
Escherichia coli
1
Ot10
Non-severe
GSM1206556
H3-BP11
Male
neutrophil
Septic plasma
II
H2
Non-severe
71
Burkholderia pseudomallei
3
Bp11
Non-severe
GSM1206557
H3-BP20
Male
neutrophil
Septic plasma
II
H2
Severe
65
Burkholderia pseudomallei
1
Bp20
Severe
GSM1206558
H3-Ot8
Male
neutrophil
Septic plasma
II
H2
Severe
74
Citrobacter freundii
5
Ot8
Severe
GSM1206559
H3-Ot22
Male
neutrophil
Septic plasma
II
H2
Severe
55
Staphylococcus aureus
1
Ot22
Severe
GSM1206560
H3-Ot11
Female
neutrophil
Septic plasma
II
H2
Non-severe
87
Enterobacter cloacae
1
Ot11
Non-severe
GSM1206561
H3-BP26
Female
neutrophil
Septic plasma
II
H2
Severe
44
Burkholderia pseudomallei
1
Bp26
Severe
GSM1206562
H3-Ot7
Male
neutrophil
Septic plasma
II
H2
Non-severe
44
Staphylococcus aureus
3
Ot7
Non-severe
GSM1206563
H3-Ot19
Female
neutrophil
Septic plasma
II
H2
Severe
76
Klebsiella pneumoniae
2
Ot19
Severe
GSM1206564
H3-BP9
Female
neutrophil
Septic plasma
II
H2
Non-severe
65
Burkholderia pseudomallei
4
Bp9
Non-severe
GSM1206565
H3-BP8
Male
neutrophil
Septic plasma
II
H2
Non-severe
69
Burkholderia pseudomallei
1
Bp8
Non-severe
GSM1206566
H3-Ot18
Female
neutrophil
Septic plasma
II
H2
Severe
40
Enterococcus spp.
1
Ot18
Severe
GSM1206567
H3-BP22
Male
neutrophil
Septic plasma
II
H2
Severe
63
Burkholderia pseudomallei
1
Bp22
Severe
GSM1206568
H3-BP19
Male
neutrophil
Septic plasma
II
H2
Non-severe
69
Burkholderia pseudomallei
4
Bp19
Non-severe
GSM1206569
H3-Ot26
Male
neutrophil
Septic plasma
II
H2
Severe
88
Escherichia coli
1
Ot26
Severe
GSM1206570
H3-HC17
Female
neutrophil
Uninfected plasma
II
H2
-
45
-
1
HC17
 
GSM1206571
H3-HC13
Male
neutrophil
Uninfected plasma
II
H2
-
49
-
1
HC13
 
GSM1206572
H3-HC18
Female
neutrophil
Uninfected plasma
II
H2
-
48
-
1
HC18
 
GSM1206573
H3-HC24
Male
neutrophil
Uninfected plasma
II
H2
-
51
-
1
HC24
 
GSM1206574
H3-HC12
Female
neutrophil
Uninfected plasma
II
H2
-
40
-
1
HC12
 
GSM1206575
H3-HC14
Male
neutrophil
Uninfected plasma
II
H2
-
54
-
1
HC14
 
GSM1206576
H3-HC21
Male
neutrophil
Uninfected plasma
II
H2
-
35
-
1
HC21
 
GSM1206577
H3-HC16
Female
neutrophil
Uninfected plasma
II
H2
-
50
-
1
HC16
 
GSM1206578
H3-HC8
Male
neutrophil
Uninfected plasma
II
H2
-
59
-
1
HC8
 
GSM1206579
H3-HC20
Male
neutrophil
Uninfected plasma
II
H2
-
47
-
1
HC20
 
GSM1206580
H3-HC15
Female
neutrophil
Uninfected plasma
II
H2
-
35
-
1
HC15
 
GSM1206581
H3-HC22
Female
neutrophil
Uninfected plasma
II
H2
-
55
-
1
HC22
 
GSM1206582
H3-HC7
Male
neutrophil
Uninfected plasma
II
H2
-
49
-
1
HC7
 
GSM1206583
H3-HC25
Male
neutrophil
Uninfected plasma
II
H2
-
51
-
1
HC25
 
GSM1206584
H3-HC10
Female
neutrophil
Uninfected plasma
II
H2
-
39
 
1
HC10
 
GSM1206585
H3-HC11
Male
neutrophil
Uninfected plasma
II
H2
-
39
 
1
HC11
 
GSM1206586
H3-HC23
Female
neutrophil
Uninfected plasma
II
H2
-
38
-
1
HC23
 

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