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To Induce or Not to Induce: Acrolein’s Effect on IL-1β-
dependent Regulation of Lung γδ T Cells
Tiger TengChristopher Fleming, Jun YanDuPont Manual High School
Louisville, KY
Introduction
• Lung Cancer– 2nd most common cancer among men and women– Majority of primary lung cancers are carcinomas
of the lung, derived from epithelial cells– Multiple medical reports indicate the leading
causes of lung cancers are from cigarette smoking.
Introduction
• Acrolein– A clear or yellow liquid that evaporates quickly– Strong, unpleasing smell– One of the toxic chemicals found in cigaretts (a
simple unsaturated aldehyde)
Introduction
• gd t-cells
Introduction (cont.)
• The purpose of this study is to see Acrolein induces IL-1b production and toxicity of gd t-cells. – Hypothetically, IL-1b will attach to gd t-cells proliferation of gd t-cells massive production of IL-17 inflammation of lungs NF-kb signaling one of the causes of COPD increase risk of cancer or cancer progression
Acrolein
ROS
Epithelial Cell
Inflammasome Activation
IL-1 Receptors
δ T-cells
V6
V4 ??Proliferation
IL-1 Production
+
+
IL-17 Inflammation1. Neutrophil
Infiltration2. CD8 Infiltration3. Increased
Chemoattractants Increase infiltrations of other cells
4. NF- Signaling
Increased cancer risk
Increase cancer progression
IL-17 Production
Macrophages
Fibroblast
Methods
• Enzyme-Linked Immunosorbent Assay (ELISA) assay IL-1β capture antibodies were used to coat the bottom of a 96-well plate a day before adding the standards and samples. Next day, IL-1β detection antibody was added. Avidin-horesradish peroxidase and TMB Substrate Solution were added creating a colorimetric correlation to the concentration of IL-1β.
Methods
• Carboxyfluorescein Succinimidyl Ester (CFSE) StainingCells were resuspended in prewarmed PBS/0.1%
BSA at a final concentration of 1x107 cells/ml. Added 1μl of 1mM stock Invitrogen Cell Trace CFSE solution and incubated the dye for 10 min at 37.5°C while shaking after 5 min to label cells evenly. After 10 minutes, the staining was quenched by the addition of 5 equal volumes of ice-cold FBS to the cells and incubated for 5 minutes on ice. Cells are then washed two more times with complete, cold RPMI.
Methods
• Flow CytometryAfter CFSE labeling, plate 2x106 lung cells per well of a
24-well plate. Added cytokines- IL-23 (5ng/ml) and IL-1β (1ng/ml). After 72 hours, GolgiPlug (1μg/ml) was added for 6 hours. The harvested cells were added into flow tubes to be stained with flourochrome labeled, monoclonal antibodies and read by the flow cytometer. The cells were stained with CD3 APC-Cy7, γδTCR APC, Vγ4 PE, IL-17A PE-Cy7, CD45 PERCP, and purified anti-mouse Vγ6 IgM with rat anti-IgM FITC secondary antibody (all antibodies purchases from Biolegend except anti-mouse Vγ6 IgM.
Results
WT IL-1r-/-0
16
32
48
64
80 **
% o
f V 6
T
cel
ls
68.9%
0 102
103
104
105
0
50K
100K
150K
200K
250K
C57Bl6 WT
28.4%
0 102
103
104
105
0
50K
100K
150K
200K
250K
IL-1r-/-
SSC
Vγ6
A
6.10% 19.1%
48.1%26.6%
0 102
103
104
105
0
102
103
104
105 16.5% 9.54%
16.6%57.3%
0 102
103
104
105
0
102
103
104
105
IL-1
7
Vγ6
C57Bl6 WT IL-1r-/-
B
WT IL-1r-/-0
5
10
15
20
25
*%
of
IL-1
7+ V
6
T c
ells
Decrease % of total Vγ6 and IL-17+ Vγ6 γδ T-cells in the lung of IL-1r -/- mice. (A) Quantifying V6 γδ T-cells and (B) IL-17+ V6 γδ T-cells in IL-1r-/- and B6 WT single cell lung suspension using flow cytometry. Bar graphs represent duplicate experiments. The error bars represent 95% confidence intervals, *P < 0.05 and **P < 0.01.
Results0.108% 10.9%
88.8%0.215%
0 102
103
104
105
0
102
103
104
105
Media alone
0.00% 0.00%
98.9%1.08%
0 102
103
104
105
0
102
103
104
105
C57Bl6WT
IL-1r-/-
22.6% 13.6%
53.6%10.3%
0 102
103
104
105
0
102
103
104
105
IL-23
0.00% 1.98%
97.4%0.660%
0 102
103
104
105
0
102
103
104
105
10.5% 9.09%
59.8%20.6%
0 102
103
104
105
0
102
103
104
105
IL-1β32.9% 10.1%
26.2%30.8%
0 102
103
104
105
0
102
103
104
105
IL-23+IL-1β
0.00% 0.395%
98.3%1.32%
0 102
103
104
105
0
102
103
104
105 0.00% 0.881%
97.4%1.32%
0 102
103
104
105
0
102
103
104
105
IL-1
7
CFSE
Lung Vγ6 γδ T-cell proliferation and IL-17 production are dependent on IL-1β in vitro. Endogenous IL-1β leads to increased IL-17 production during 3-day culture. Exogenous IL-1β induces proliferation and IL-17 production by γδ T-cells. IL-23 combined with IL-1 has a synergistic effect on proliferation and IL-17 production.
ResultsA Lung Alone
7AAD
SSC
1μM Acro 5μM Acro 10μM Acro
BAcrolein cytotoxicity of total lung cells.(A) Gating on the live and dead cells, higher concentration of Acrolein results in higher percentage of dead cells. (B)There was no significant difference between concentrations of Acrolein in terms of % of live cells, however the decreasing trend of live cells is obvious. The error bars represent 95% confidence intervals, *P < 0.05 and **P < 0.01.
Results
A
γδTC
R
CD3 Acrolein cytotoxicity to δ T-cells of the lungs (A) Using flow cytometry to measure the percentage of live δ and T-cells. (B) Analyzing representative data, lung alone vs 10 M of Acrolein shows a significant decrease in the % of total live δ and αβ T-cells . The error bars represent 95% confidence intervals, *P < 0.05 and **P < 0.01.
B
Lung Alone 1μM Acro 5μM Acro 10μM Acro
Result
med Lu alone 0.01 0.1 1 5 10 Lu+ tu Lu+Tu+A 10-50
0
50
100
150
200
250
300 **
Acrolein Concentrations (M)Treatment
IL-1
(p
g/m
l)
Acrolein was able to induce IL-1 production at 10μM. There is a decrease in IL-1 production between lungs only and lungs treated with 0.1 M of Acrolein. The error bars represent 95% confidence intervals, *P < 0.05 and **P < 0.01.
Conclusion/Future Work
This study shows that IL-1r signaling is crucial for the maintenance and function of lung Vγ6 γδ T cells. Comparison data between WT and IL-1r-/-
mice suggests that Vγ6 are specifically regulated by IL-1β, unlike Vγ4 and
Vγ1(data not shown). As shown in figure 3, IL-1 induces proliferation and
IL-17 production of Vγ6 T-cell and has a synergistic effect with IL-23 in IL-
17 production. However, with only 5 M of acrolein, cytotoxicity to total
lung cells and γδ T-cells is prevalent. From figure 6, it seems acrolein
begins to induce IL-1β production starting at 10 M but not at lower
concentrations. In conclusion, we used flow cytometry to demonstrate that
IL-1 does regulate Vγ6 γδ T-cells in both proliferation and IL-17
production. However, further optimization is needed in order to determine
how acrolein affects IL-1β production in the lungs.
Acknowledgement
• Thanks to:– Tumor Immunobiology Program, James Graham
Brown Cancer Center, University of Louisville– Christopher Fleming, Jun Yan, and others
Questions?
