multispectral imaging reveals unique macrophage profiles … · 2018-11-15 · multispectral...
TRANSCRIPT
Introduction
• Spectral imaging microscopy is a powerful technique that enables quantification
and identification of distinct intrahepatic macrophage phenotypes in situ in human
FFPE liver tissue
• The multiplex staining panel and imaging analysis software algorithms that we have
developed allows these elusive cells to be studied in the context of intact hepatic
architecture.
• The unique macrophage profiles that we have identified will be correlated with
patient outcomes.
Table 1. Antibodies used to identify intrahepatic macrophages using this platform
Macrophage: Parent cell markers
Tissue resident Kupffer cells CD68+
Tissue remodeling / Pro-fibrotic CD163+
Systemic monocytes Mac387+
Markers analyzed by level of expression on parent cells
Classical (pro-inflammatory) macrophages CD14++/CD16-
Intermediate macrophages CD14++/CD16+
Non-classical (anti-inflammatory) macrophages CD14+/CD16++
Acknowledgments
Results
Intrahepatic macrophages greatly impact the composition of the hepatic
microenvironment, host immune response and development of fibrosis.
Studies of human intrahepatic macrophages can be challenging for several
reasons:
(i) they are difficult to isolate from human liver tissue
(ii) they become activated and change their phenotype when isolated or
manipulated
(iii) in vitro and mouse model systems of HCV infection or fatty liver disease do
not closely mimic the long-term, chronic infections that are observed in humans
We are using spectral imaging microscopy with advanced imaging analysis
software programs to analyze intrahepatic macrophages in situ in human liver
biopsies.
This platform is optimized for multiplex immunofluorescence staining of formalin-
fixed paraffin-embedded tissues and does not compromise the hepatic architecture.
This approach will allow us to gain an in-depth understanding of how variations in
human hepatic macrophage profiles affect the host immune response and
development of hepatic fibrosis.
Methods
ConclusionsFigure 1. Multiplex images obtained from staining FFPE liver biopsies from control patients compared to
patients with three common chronic liver diseases (NASH, AIH and HCV). Biopsies were stained with the
macrophage multiplex panel (CD68, CD163, Mac387, CD14, CD16, and DAPI) and representative images (20X)
were acquired. Panel A: Fluorescent multispectral image after staining with the multiplex panel. Panel B: The
multiplex images were then analyzed with Visiopharm phenotyping applications and each color represents a
unique cellular phenotype. Panel C: The t-SNE plots highlight the unique profiles of macrophages that were
identified in the livers of patients with chronic liver diseases versus controls. Panel D: Shows differences in the
numbers of Mac387+ macrophages in the portal tracts and lobules in patients with either NASH, AIH or HCV,
compared to control patients. Results shown are representative of three different patients with similar hepatitis
activity scores (i.e., MHAI) and fibrosis stages (using the Ishak criteria). NASH: Nonalcoholic steatohepatitis, AIH:
Autoimmune hepatitis; HCV: hepatitis C virus, P tract: Portal tract, tSNE: t-distributed Stochastic Neighbor
Embedding (tSNE) algorithm.
Results
OPAL- 6 Color Multiplex IHC
CD68 MAC87 CD16
CD14 CD163
Contact Information
Moody Endowment Research Award
UT Systems Rising STARs Award
Perkin Elmer: Kevin Mottershead
Visiopharm: Dr. Ben Freiberg and Dr. Alex Villa
University of Michigan: Dr. Arvind Rao
Rice University: Santhoshi Krishnan
Multispectral Imaging Reveals Unique Macrophage Profiles
Associated with Type of Liver Disease and Fibrosis StageOmar A. Saldarriaga1, Adam L. Booth1, Jared K. Burks, Netanya S. Utay2, MinKyung
Yi3, Monique Ferguson4, Laura Beretta5 and Heather L. Stevenson1. 1Department of Pathology, University of Texas Medical Branch, Galveston, TX; 2University of Texas Health Science Center at Houston,
Houston, TX; 3University of Texas Medical Branch, Galveston, TX; 4University of Texas MD Anderson Cancer Center, Houston, TX
Heather Stevenson-Lerner, M.D., PhD
Assistant Professor, UTMB, Dept. of Pathology
[email protected]; Office: 409-772-8554
Omar A. Saldarriaga, DVM., PhD
Research Scientist, UTMB, Dept. of Pathology
[email protected];Office: 409-747-0275
Adam L. Booth, MD
Chief Resident, PGY3, UTMB, Dept. of Pathology
[email protected], Office: 903-918-1790
Figure 2. Differences in intrahepatic macrophage profiles in HCV+ patients (genotype 1a+)
with minimal fibrosis or cirrhosis versus controls. We obtained low power images of two different
patient’s liver biopsies after staining with Masson’s trichrome (blue = fibrosis). Both patients are
males of similar age that report being infected with HCV approximately 20 years ago. Why did these
patients respond so differently to chronic hepatitis C? The first patient's biopsy (A) shows minimal
fibrosis (Ishak stage: 1/6) and the second patient (B) has cirrhosis with large areas of parenchymal
extinction. We used the multiplex macrophage panel to determine differences in the intrahepatic
macrophage profiles in the HCV+ patients when compared to controls. (C) t-SNE unsupervised
analysis of macrophage markers highlights the unique populations identified in these patients.
Panel A Panel B Panel C Panel D
Fig. 1 Control
0
2
4
6
8
10
12
14
P Tract Lobule
MA
C3
87
+
CE
LL
S (%
)
Non-alcoholic Steatohepatitis (NASH)
0
2
4
6
8
10
12
14
P Tract Lobule
MA
C3
87
+ C
EL
LS
(%
)
Autoimmune Hepatitis (AIH)
0
2
4
6
8
10
12
14
P Tract Lobule
MA
C3
87
+ C
EL
LS
(%
)
Chronic Viral Hepatitis (HCV)
0
2
4
6
8
10
12
14
P Tract Lobule
MA
C3
87
+ C
EL
LS
(%
)
B.
t-S
NE
t-SNE 1
t-S
NE
t-S
NE
t-SNE 1 t-SNE 1
A.
t-SNE 1
t-S
NE
CD68 - Opal 520 CD163 - Opal 650 Mac387- Opal 690 CD16 - Opal 620
CD14 - Opal 540 Macrophage phenotypes in NASH
t-SNE 1
t-S
NE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
CD68 (520)
Mac387 (690)
CD16 (620)
CD14 (540)
CD163 (650)
-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00
B.
Figure 3. Unsupervised analysis shows multiple macrophage phenotypes in FFPE liver
biopsies obtained from patients with NASH after staining with the macrophage panel. (A) t-
SNE plots use dimensional reduction to facilitate visualization of macrophage marker expression.
Cells with similar properties appear close together in a two-dimensional map and red (or “hot”)
markers show cells with relatively more expression of that specific marker when compared to blue
(or “cold”) markers, which indicate absent or minimal expression. (B) The phenotypic matrix
algorithm identified 16 distinct macrophage phenotypes (#: 1-16) in one multiplex image obtained
from a patient with NASH. Other cell populations, which likely include hepatocytes, lymphocytes
and epithelial cells, appear blue in the phenotype matrix map (#: 17-25).
Control
HCV+ Minimal
fibrosis
HCV+ Cirrhosis
Fig. 2. HCV+ minimal fibrosis HCV+ Cirrhosis t-SNE Plot
A. B. C.Spectral Imaging Library
Tyramide signal amplification
A.
@DrHSLovesLiver
@ALBoothMD
@UTMB_Pathology