Poster created for Colorado State University Celebrate Undergraduate Research and Creativity Showcase, an annual gathering at CSU to honor outstanding student researchers and creators. Review of work completed in lab of Dr. Alan Schenkel in the 2017-2018 school year.

1. Role of Clots and Macrophages in the Progression of Idiopathic
Pulmonary Fibrosis
Jordon Aragon, Cole Mills, Moriah Glennon, Kadi Horn, Catie Pearson, Nikko Garcia, Dr. Alan Schenkel PhD.
Colorado State University: Department of Microbiology, Immunology and Pathology
Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal
disease, killing about 30,000 Americans per year. Currently, the only
cure is a lung transplant, though there are some new drugs in
testing. Currently, it is believed that alveolar epithelial cells die, and
the alveolar structure is damaged. Attempts to repair the damage
lead to scar tissue (fibrosis). But little is known about what happens
between the damage and fibrosis steps.
Our hypothesis is that after alveoli structures become damaged,
bleeding occurs leaving blood clots in the alveolar spaces.
Macrophages in the lung start to eat the red blood cells to clean up
the alveoli. They also make repair cytokine signals, like TGF-β. Due
to the chronic nature of IPF, the alveoli continue to bleed so the
macrophages keep eating the red blood cells and continuously
produce the cytokines, IL-10 and TGF-β which leads to scar tissue
formation and fibrosis. It is not known how long macrophages live
after eating blood clots or what signals they might make.
Mouse bone marrow and bronchoalveolar lavage macrophages
were cultured with blood clots. We found that macrophages only
phagocytosed old blood clots (2-3 days old) and accumulated
fluorescent proteins, which may be Ym1, a marker of alternatively
activated macrophages. We are using quantitative digital droplet
PCR (ddPCR) to examine all the kinetics of several possible repair
signals like CD163 (used for hemoglobin phagocytosis), Ym1, IL-10,
IL-17A, IL-4, and TGF-β at several time points after incubation with
blood clots in different types of macrophages. We are also going to
study macrophage function and health after long-term exposure to
clots.
Abstract
This experiment shows that macrophages harvested from vivo,
and exposed to RBC’s produce cytokines that promote tissue repair
and regeneration.
● TGF-β was seen in cells that were exposed to clot but only on
the first day.
● IL10 was also detected only in cells that were exposed to the
blood clots as well.
● YM1 was seen in cells only in the 7th
day of testing.
● IL4 was seen in day 7 cells with clots.
● Arginase 1 was also only expressed in only the controls of the
day 1 and day 7.
● Mouse macrophages were harvested from mouse bone marrow
and via bronchoalveolar lavage and cultivated in 2-3 day old
clots.
● RNA was extracted from the macrophages and then made into
cDNA.
● cDNA was then analyzed with digital droplet PCR.
Digital droplet polymerase chain reaction (ddPCR) uses oils to
conduct the PCR process. DNA is replicated inside of very small
droplets which are then fed through a device that can read
fluorescence levels that indicate how much test DNA was being
replicated by the macrophages.
Methods and Materials
From this experiment sever conclusions can be drawn:
● Arg 1 presence in the day 1 and day 7 controls is a little
paradoxical due to Arg1 signalling for mediation of inflammation
○ due to Arg1 gene is normally seen as an inflammatory
cytokine that is expressed by M2 macrophages.
○ More testing will be necessary
● IL4 is normally seen in macrophages trying to induce an
inflammatory response.
○ More testing will be necessary.
● The detection of TGF-β and IL10 shows that we are
successfully causing a phenotypic switch.
● The detection of Ym1:
○ Correlated with IPF in mice
○ Is a fluorescent molecule that fluoresces when inside clots.
○ Because it was only detected in the 7 day samples, this
supports our hypothesis that YM1 crystals only occur in a
chronic environment, where macrophages prefer old clots.
Conclusions
Results
Future Directions
Future directions for experimentation are:
● Testing the cytokine response of interstitial macrophages.
● Testing if the fluorescence may be caused by a RBC digestion by
product rather than YM1.
● Focus our investigation on whole transcriptome sequencing to
see what is upregulated when the macrophages are exposed to
RBCs.
References and Acknowledgements
● Neil Ahluwalia; Barry S. Shea; Andrew M. Tager; Am J Respir Crit Care Med 190:867-878
(2014)S.Y. Tan & W. Weninger Nature Immunol. 17:1335-1336 (2016).
● Schenkel, Alan R. et al. “Different Susceptibilities of PECAM-Deficient Mouse Strains to
Spontaneous Idiopathic Pneumonitis.” Experimental and molecular pathology 81.1
(2006): 23–30. PMC. Web. 29 Mar. 201
Acknowledgements:
Dr. Alan Schenkel, Wilusz Lab, John Anderson, Nicholas Garcia, Catie Pearson
Possible YM1 Crystals in macrophages
Fluorescence in Macrophages