• Lipids play an important role in dermatitis as inflammatory
mediators and as cell signaling molecules, particularly
phospholipids and free fatty acids.
• Alterations in the lipid composition can lead to diminished
barrier function of the skin and are associated with diseases like
atopic dermatitis.
• SHARPIN-deficient cpdm mice develop a chronic dermatitis with
similarities to atopic dermatitis in humans and present changes in
their epidermis lipids profiles.
• To gain knowledge of the localization of these changes, we
used Matrix-Assisted Laser Desorption Ionization MALDI MSI (mass
spectrometry imaging) to localize discriminative lipids from cpdm
mice compared to wild-type littermates.
• These lipids may be of diagnostic and clinical utility in
atopic dermatitis in humans and domestic animals.
Introduction
Methods
Figure 2. Detection of free fatty acids in cpdm and WT samples.
(A) MSI of WT and cpdm skin samples of 5 weeks old mice showing
relative intensities of lipid ions in skin cross sections.
Docosatetraenoic acid and araquidonic acid results appear increased
in cpdm samples compared to WT, which is agreement with
MRM-profiling results. (B) Distribution of free fatty acids in the
epidermis by MRM-profiling. Free fatty acids identified by carbon
chain length and degree of unsaturation were analyzed in negative
ion mode by SIM. The relative amounts of arachidonic acid (20:4)
and docosatetraenoic acid (22:4) were increased in cpdm mice. Bars
represent the means + SE of the samples. *significantly different
at p < 0.05 following multiple comparison correction.
Cholesterol sulfate
Variations in skin lipid content in a mouse model of dermatitis
analyzed by Mass Spectrometry Imaging
Jackeline Franco1,2, Christina Ferreira2, Bindesh Shrestha3,
John P. Sundberg4, Harm HogenEsch1,5 * 1 Department of Comparative
Pathobiology, Purdue University, West Lafayette, IN; 2 Metabolite
Profiling Facility, Bindley Bioscience Center, Purdue University,
West Lafayette, IN; 3 Waters Corporation, Beverly, MA; 4 The
Jackson Laboratory, Bar Harbor, ME; 5 Purdue Institute of
Inflammation, Immunology and Infectious Diseases, Purdue
University, West Lafayette, IN. *[email protected]
Phospholipid
• Mass spectrometry imaging identifies highly discriminative
lipids in a mouse model of dermatitis.
• MS imaging was successful at providing the spatial location of
lipids. • MS imaging results were in agreement with results from an
agnostic
(i.e. comprehensive and not dependent on database ID)
mass-spectrometry strategy for biomarker discovery termed
multiple-reaction monitoring (MRM)-profiling.
• The identification and location of these lipid changes may
represent a molecular tool to assess inflammation and therapeutic
outcomes of skin barrier improvement treatments.
Conclusions
Acknowledgments
These studies were supported in part by grants from the National
Institutes of Health (AR049288), the Purdue Institute for
Inflammation, Immunology and Infectious Diseases, Waters
corporation and COLCIENCIAS, Colombia. The authors thank Dr.
Fangjia Lu for his helpful assistance.
B A
Free Fatty Acids
Figure 3. Detection of cholesterol sulfate in WT and cpdm
samples. (A) MSI of WT and cpdm skin samples of 8 weeks old mice
showing cholesterol sulfate (red) restricted to the epidermis and
ubiquitous PI(38:4) (green). (B) H&E stained cross section of
WT and cpdm skin samples. (C) Relative amounts of cholesterol
sulfate monitored by MRM-profiling of the epidermis of 8 week old
mice. The relative amounts of cholesterol sulfate were decreased in
cpdm mice. Bars represent the means of 18 samples *P = 0.0058
Sharpincpdm WT
Mass Spectrometry Imaging (MSI) workflow
Epidermis
Dermis
*
Snap frozen in carboxymethylcellulose
10µm cross section in glass slides
Skin samples
5 and 8 week old females
WT/Cpdm (n=4)
MSI data was compared with data acquired by MRM-profiling using
ESI-MS
MSI A Multivariate statistical analysis MRM methods
Screening (n=36)
Free Fatty Acids (FFA) Phospholipids (PC, PE, PS, PI)
Discovery Testing set (n=15)
Analysis
Lipid extraction by Bligh and Dyer
method
Epidermis samples
8 week old females WT/Cpdm N=36
MR
M-P
rofi
ling:
Mas
s sp
ectr
om
etry
ap
pro
ach
A A
B
Linoleic acid (18:2)
5 weeks old
Epidermis
Dermis
Araquidonic Acid (20:4)
Epidermis
Dermis
5 weeks old
Epidermis
Dermis
Docosahexaenoic acid (22:6)
5 weeks old
Epidermis
Dermis
Docosatetraenoic acid (22:4)
5 weeks old
PE(36:1)/PC (33:1)/pPC(34:0) [M-H]- m/z 745.8
5 weeks old
Figure 4. Detection of phospholipid in WT and cpdm samples. (A)
MSI of WT and cpdm skin samples of 8 week old mice showing [M-H]-
m/z 745.8 with tentative attribution of PE(36:1)/PC
(33:1)/pPC(34:0) increased in cpdm sample compared to WT. (B)
Relative amounts of [M-H]- m/z 745.8 monitored by MRM-profiling of
the epidermis 8 week old mice. Bars represent the mean of the 18
samples *P
