COPD disease severity and innate immune response to pathogen-associated molecular patterns

Figures & data

Table 1 Subject characteristics

Variable	N=28	(%)	Variable	N=28	(%)
Mean age, years (SD)	69.3	(±9.8)	Medication use
Race/ethnicity			Short-acting B-agonist	28	100
White	26	92.9	Short-acting anticholinergic	22	78.6
African American	2	7.1	Long-acting B-agonist	19	67.9
Current smoker	5	17.9	Long-acting anticholinergic	5	17.9
Chronic bronchitisa	15	53.6	Inhaled steroidd	22	78.6
Chronic respiratory symptoms			Theophylline	2	7.1
Dyspnea	26	92.9	Medications used to treat in last 12 months
Cough	13	46.4
Wheeze	17	69.7	≥1 Prednisone course	8	28.6
Sputum production	9	32.1	≥1 Antibiotic course	8	28.6
Mean pack-years (SD)	52.0	(±30.9)	≥1 Prednisone or antibiotic	13	46.4
Mean FEV1 in liters (SD)b	1.6	(±0.5)	COPD health care utilization in last 12 months
Mean FEV1 % predictedb	45.6	(±13.3)
GOLD stage			ED visits	6	21.4
II – Moderate	12	42.9	Hospitalizations	1	3.6
III – Severe	12	42.9	Hospitalization or ED visit	6	21.4
IV – Very severe	4	14.3
Home oxygen usec	7	25

Notes:

a N=1 missing.

b Postbronchodilator except for one patient with missing value for which the prebronchodilator value was used.

c As reported by the patient.

d Flunisolide, triamcinolone, and mometasone.

Abbreviations: SD, standard deviation; GOLD, Global Initiative for Chronic Obstructive Lung Disease; ED, Emergency Department.

Figure 1 Hierarchical clustering of cytokine-PAMP combinations by subject.

Notes: In the heatmap, each column corresponds to an individual patient. The presence or absence (Y/N) of chronic bronchitis or severe COPD (FEV₁<50%) is indicated at the top for each patient. Each row corresponds to cytokine production in response to a specific PAMP. Several cytokines formed distinct clusters in response to multiple PAMPs (IL-1RA, G-CSF, MCP-1, IL-10, IL-8) and have been highlighted. Note the aggregation of multiple subjects with severe COPD in the “low responder” group.
Abbreviations: PAMP, pathogen-associated molecular pattern; IL, interleukin; G-CSF, granulocyte-colony stimulating factor.

Table 2 Differences in IL-10 levels by COPD disease severity after stimulation of whole blood by PAMPs

PAMP	COPD severity				P-value*
	FEV1 <50 (N=16)		FEV1 50–80 (N=12)
	Median	IQR	Median	IQR
Media	0.24	(0.2–0.5)	0.2	(0.2–0.2)	0.6
Pam3Cys (10 ng/mL)	31.3	(7.8–108.7)	52.6	(20.9–100.3)	0.6
Pam3Cys (100 ng/mL)	57.8	(13.7–160.0)	84.1	(54.5–145.0)	0.5
Zymosan (10 μg/mL)	5.2	(2.2–9.2)	7.4	(5.8–20.9)	0.05
Zymosan (100 μg/mL)	34.9	(22.6–55.2)	67.3	(39.6–104.2)	0.007
FSL-1 (10 ng/mL)	9.9	(1.5–33.3)	35.1	(12.4–45.3)	0.06
FSL-1 (100 ng/mL)	40.5	(18.1–98.5)	80.2	(46.6–119.1)	0.08
O111:B4 (0.1 ng/mL)	1.4	(0.7–3.0)	2.1	(0.8–5.1)	0.4
O111:B4 (1 ng/mL)	10.6	(3.2–21.1)	16.6	(12.5–24.0)	0.2
O111:B4 (10 ng/mL)	18.2	(7.0–31.8)	29.1	(24.3–45.8)	0.05
Re595UP (0.01 ng/mL)	2.7	(0.2–9.6)	9.1	(6.8–12.1)	0.009
Re595UP (0.1 ng/mL)	29.0	(15.2–52.6)	49.9	(34.6–74.6)	0.05
Re595UP (1 ng/mL)	58.7	(32.5–83.9)	79.5	(57.5–108.0)	0.2
Flagellin (100 ng/mL)	0.9	(0.9–1.3)	0.9	(0.9–0.9)	0.4
R848 (0.1 μM)	0.6	(0.2–1.6)	0.2	(0.2–0.3)	0.1
R848 (1 μM)	347.2	(168.2–574.3)	440.1	(329.3–673.0)	0.3

Note:

* Wilcoxon rank sum test (Mann–Whitney).

Abbreviations: IL, interleukin; PAMP, pathogen-associated molecular pattern; IQR, inter-quartile range.

Table 3 Differences in G-CSF levels by COPD disease severity after stimulation of whole blood by PAMPs

PAMP	COPD severity				P-value*
	FEV1 <50 (N=16)		FEV1 50–80 (N=12)
	Median	IQR	Median	IQR
Media	1.5	(0.5–4.7)	1.5	(0.5–3.5)	0.7
Pam3Cys (10 ng/mL)	9.0	(4.6–18.5)	16.9	(5.4–37.7)	0.2
Pam3Cys (100 ng/mL)	16.4	(5.4–25.6)	28.1	(11.8–49.0)	0.08
Zymosan (10 μg/mL)	6.5	(1.1–10.6)	8.3	(5.5–16.1)	0.1
Zymosan (100 μg/mL)	68.0	(38.2–93.5)	134.8	(67.0–174.7)	0.02
FSL-1 (10 ng/mL)	1.4	(0.5–4.8)	4.6	(1.9–5.7)	0.1
FSL-1 (100 ng/mL)	3.3	(0.5–6.2)	8.1	(4.3–12.9)	0.005
O111:B4 (0.1 ng/mL)	2.7	(0.5–4.6)	2.5	(0.8–10.3)	0.7
O111:B4 (1 ng/mL)	13.1	(6.4–71.8)	34.7	(24.1–54.0)	0.1
O111:B4 (10 ng/mL)	22.2	(17.6–101.4)	74.1	(60.7–149.7)	0.04
Re595UP (0.01 ng/mL)	6.7	(1.1–12.4)	14.3	(5.8–18.8)	0.09
Re595UP (0.1 ng/mL)	77.1	(48.9–107.3)	130.3	(86.9–198.4)	0.04
Re595UP (1 ng/mL)	216.9	(176.9–411.4)	332.0	(227.3–492.5)	0.1
Flagellin (100 ng/mL)	1.6	(0.5–3.4)	1.7	(0.5–3.0)	0.9
R848 (0.1 μM)	1.2	(0.5–3.9)	1.5	(0.5–2.7)	0.96
R848 (1 μM)	20.8	(13.0–35.3)	33.2	(16.5–54.1)	0.3

Note:

* Wilcoxon rank sum test (Mann–Whitney).

Abbreviations: PAMP, pathogen-associated molecular pattern; IQR, inter-quartile range.

Table 4 Differences in IL-1β levels by COPD disease severity after stimulation of whole blood by PAMPs

PAMP	COPD severity				P-value*
	FEV1 <50 (N=16)		FEV1 50–80 (N=12)
	Median	IQR	Median	IQR
Media	1.4	(1.1–2.1)	1.1	(0.9–3.6)	0.5
Pam3Cys (10 ng/mL)	56.7	(32.9–85.4)	99.4	(68.4–171.0)	0.02
Pam3Cys (100 ng/mL)	68.9	(35.1–150.5)	157.6	(90.8–331.8)	0.02
Zymosan (10 μg/mL)	54.4	(20.6–122.0)	145.0	(70.7–178.9)	0.005
Zymosan (100 μg/mL)	1,048.3	(593.9–2,114.1)	1,717.0	(1,516.2–2,261.6)	0.1
FSL-1 (10 ng/mL)	8.4	(3.0–20.8)	15.3	(4.2–39.3)	0.3
FSL-1 (100 ng/mL)	15.1	(6.8–27.5)	23.4	(10.5–59.4)	0.1
O111:B4 (0.1 ng/mL)	39.4	(9.9–97.66)	41.6	(18.5–204.1)	0.2
O111:B4 (1 ng/mL)	644.2	(187.3–2,218.1)	1,112.9	(983.8–2,335.1)	0.2
O111:B4 (10 ng/mL)	1,899.4	(1,214.7–6,574.6)	3,989.2	(2,526.2–6,145.9)	0.2
Re595UP (0.01 ng/mL)	126.4	(39.6–202.4)	252.2	(106.7–499.1)	0.02
Re595UP (0.1 ng/mL)	1,630.2	(596.1–2,555.2)	2,375.7	(2,171.4–4,362.5)	0.06
Re595UP (1 ng/mL)	5,139.8	(3,230.8–10,174.4)	8,745.4	(5,673.4–10,901.3)	0.2
Flagellin (100 ng/mL)	3.0	(1.1–17.8)	2.1	(1.5–19.4)	0.8
R848 (0.1 μM)	18.0	(8.5–33.8)	31.9	(11.8–59.9)	0.3
R848 (1 μM)	5,685.6	(3,641.2–7,468.2)	5,204.5	(4,169.0–8,544.3)	0.9

Note:

* Wilcoxon rank sum test (Mann–Whitney).

Abbreviations: IL, interleukin; PAMP, pathogen-associated molecular pattern; IQR, inter-quartile range; G-CSF, granulocyte-colony stimulating factor.

Figure 2 Heatmap showing the strength of tests of associations comparing cytokine levels for subjects with and without severe COPD or chronic bronchitis for each PAMP–cytokine level pair.

Notes: Each square corresponds to the P-value for one Student’s t-test comparing cytokine levels after stimulation with a PAMP between subjects with and without severe COPD (A) or with and without chronic bronchitis (B). For example, the square in the upper right corner shows the P-value for the Student’s t-test comparing MCP-1 levels in response to media (no PAMP) between those with and without severe COPD. *Student’s t-test comparing the mean log-transformed cytokine values for a given PAMP between subjects with and without severe COPD. **Student’s t-test comparing the mean log-transformed cytokine values for a given PAMP between subjects with and without chronic bronchitis.
Abbreviations: PAMP, pathogen-associated molecular pattern; IL, interleukin; TNF, tumor necrosis factor; G-CSF, granulocyte-colony stimulating factor.

Table 5 Differences in IL-1RA levels by history of chronic bronchitis after stimulation of whole blood by PAMPs

PAMP	Chronic bronchitis				P-value*
	Absent		Present
	Median	IQR	Median	IQR
Media	499.7	(362.3–850.2)	989.6	(724.19–1,867.8)	0.02
Pam3Cys (10 ng/mL)	9,932.6	(5,429.4–14,082.6)	14,498.2	(7,963.9–21,908.3)	0.1
Pam3Cys (100 ng/mL)	11,471.6	(7,097.9–14,264.4)	12,716.7	(10,558.9–24,427.1)	0.09
Zymosan (10 μg/mL)	3,537.4	(2,926.0–4,300.9)	5,600.1	(3,550.4–9,032.1)	0.07
Zymosan (100 μg/mL)	5,346.6	(4,220.5–7,067.5)	5,837.3	(4,883.5–9,432.4)	0.4
FSL-1 (10 ng/mL)	5,574.8	(3,624.7–8,590.2)	7,482.2	(5,495.5–10,352.0)	0.1
FSL-1 (100 ng/mL)	5,976.2	(4,260.6–8,199.0)	7,842.0	(6,015.5–10,928.5)	0.04
O111:B4 (0.1 ng/mL)	7,598.2	(6,415.30–10,359.6)	11,435.0	(8,248.4–17,339.9)	0.09
O111:B4 (1 ng/mL)	11,101.8	(9,875.0–22,459.4)	16,835.1	(12,185.0–61,835.1)	0.2
O111:B4 (10 ng/mL)	12,512.4	(10,372.8–18,585.8)	16,466.1	(12,491.4–68,162.9)	0.06
Re595UP (0.01 ng/mL)	7,274.5	(5,542.0–12,339.5)	11,127.0	(9,240.7–16,173.4)	0.06
Re595UP (0.1 ng/mL)	12,818.7	(9,913.3–19,854.3)	21,332.8	(15,122.4–70,865.1)	0.05
Re595UP (1 ng/mL)	17,055.9	(11,721.6–22,342.9)	22,217.7	(15,477.0–28,963.2)	0.09
Flagellin (100 ng/mL)	659.2	(453.7–2,331.7)	2,002.5	(832.4–6,063.6)	0.02
R848 (0.1 μM)	8,881.5	(5,901.2–12,211.4)	13,410.1	(10,165.5–18,372.0)	0.02
R848 (1 μM)	27,460.5	(13,651.8–176,870.7)	223,855.1	(20,682.0–223,855.1)	0.11

Note:

* Wilcoxon rank sum test (Mann–Whitney).

Abbreviations: IL, interleukin; PAMP, pathogen-associated molecular pattern; IQR, inter-quartile range.