An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4

An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4

Cell Reports, Volume 9
Supplemental Information
An MHC II-Dependent Activation Loop
between Adipose Tissue Macrophages and CD4+
T cells Controls Obesity-Induced Inflammation
Kae Won Cho, David L. Morris, Jennifer L. DelProposto, Lynn Geletka, Brian Zamarron,
Gabriel Martinez-Santibanez, Kevin A. Meyer, Kanakadurga Singer, Robert W. O’Rourke,
and Carey N. Lumeng
Figures S1-S6
Figure S1 (Related to Figure 1)
Figure S1. Visualization of ATMs and CD4+ T cells in eWAT from CD11cmCherry mice (Related to Figure 1)
(A) Flow cytometry analysis of mCherry+ cells from eWAT of CD11c-mCherry
mice (B) Immunofluorescence images from eWAT from mice IP or IV injected
with CFSE-labeled CD4 cells. Scale bar = 50 µm. (C) Quantitation of
CFSE+CD3+CD4+ T cells were from spleen, lymph nodes, and visceral fat depots
(Epididymal and omentum). Percentage of total CD3+CD4+ lymphyocytes is
indicated.
Figure S2 (Related to Figure 2)
Figure S2. Deficiency of MHCII does not affect energy expenditure (Related
to Figure 2)
MHCII deficient mice (MHCIIKO) and wild-type control male mice (WT) were fed
with normal chow diet (ND) or high-fat diet (HFD) for 20 weeks and metabolic
cage studies were conducted. (A) Oxygen consumption (VO2) (B) Carbon
dioxide production (VCO2) (C) body length. Data are means ± SEM.
Figure S3 (Related to Figure 2)
Figure S3. MHC II deficient mice are protected from hepatic steatosis,
hyperinsulinnemia and adipose tissue inflammation during HFD-induced
obesity. (Related to Figure 2)
MHCIIKO (KO) and WT control male mice were fed ND or HFD for 20 weeks. (A)
Representative of H&E sections of livers and triglyceride content. Scale bar =
200 µm (B) Insulin and non-esterified free fatty acid (NEFA) levels in serum from
ND and HFD fed WT and KO mice after 6 hours of fasting. Expression of gene in
eWAT (C) and liver (D) from HFD fed WT and KO mice. (E) Circulating 7/4hi
monocyte levels in ND or HFD fed WT and KO mice. Data are means ± SEM.
*p<0.05; *** p<0.001.
Figure S4 (Related to Figure 4)
Figure S4. Reduced proinflammatory cytokine gene expression in adipose
tissue and liver from macrophage-specific MHCII deficient mice in
response to HFD. (Related to Figure 4)
Control mice and MMKO male mice were fed ND and HFD for 14 weeks.
Expression of genes associated inflammatory cytokines in eWAT (A) and liver (B)
from HFD fed CON and MMKO mice. Data are means ± SEM. *p<0.05, **
p<0.01, *** p<0.001 versus CON.
Figure S5 (Related to Figure 6)
Figure S5. Macrophage-specific MHCII deficiency does not affect spleen T
lymphocyte population, proliferation and Th1 polarization in response to
HFD. (Related to Figure 6)
(A) Gating strategy of Treg. (B) Representative flow cytometry plot of Treg in
spleen and eWAT from ND and HFD for 14 weeks. (C) Quantitation of CD3+
lymphocytes, CD4+, CD8+, Tconv, Treg, proliferating CD4+ and CD8+ T
lymphocytes, and IFNg expression in sorted CD4+ cells in spleen from HFD fed
CON and MMKO. Data are means ± SEM.
Figure S6 (Related to Figure 7)
Figure S6. Depletion of CD11c+ cells improves HFD-induced glucose
intolerance without changes of adiposity. (Related to Figure 7)
PBS or Diphteria Toxin (DT) was injected into CD11c-DTR bone marrow chimeric
mice fed ND or HFD for 8 weeks during the last 2 weeks of HFD feeding. (A)
Body weight (B) eWAT weight (C) Fasting glucose and insulin levels (D) GTT.
Data are means ± SEM. *p<0.05; ** p<0.01versus PBS.
Movies S1-S2
Movie S1. Dynamic interaction of CD4+ T cells with ATMs in the absence of
antigen. (Related to Figure 1)
CFSE-labeled CD4+ T cells from OT II mice were adoptively transferred to
CD11c-mcherry mice. 2 h post injection of BSA, CFSE-CD4+ T cell (green) and
CD11c-mCherry+ ATMs (red) were imaged in eWAT of an anesthetized mouse
using two-photon microscopy.
Movie S2. Stable interaction of CD4+ T cells with ATMs in the presence of
antigen. (Related to Figure 2)
CFSE-labeled CD4+ T cells from OT II mice were adoptively transferred to
CD11c-mcherry mice. 2 h post injection of OVA, CFSE-CD4+ T cell (green) and
CD11c-mCherry+ ATMs (red) were imaged in eWAT of an anesthetized mouse
using two-photon microscopy
Tables S1-S2
Table S1. Sequences for RT-PCR primers used in this study (Related to
Experimental Procedures)
Gene
Arbp
Ccl2
AdipoQ
NOS2
Arg1
tbet
gata3
pparg2
Il6
Il10
leptin
foxp3
itgax
RORg
ifng
Il2
Il12
cd4
GLUT4
tnfa
Il4
Primer
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Sequence
5’-AGA TTC GGG ATA TGC TGT TGG C-3’
5’-TCG GGT CCT AGA CCA GTG TTC-3’
5’-TTA AAA ACC TGG ATC GGA ACC AA-3’
5’-GCA TTA GCT TCA GAT TTA CGG GT-3’
5’-GCA GGC ATC CCA GGA CAT C-3’
5’-GCG ATA CAT ATA AGC GGC TTC T-3’
5’-CGC AGC TGG GCT GTA CAA A-3’
5’-TGA TGT TTG CTT CGG ACA TCA-3’
5’-CTC CAA GCC AAA GTC CTT AGA G-3’
5’-AGG AGC TGT CAT TAG GGA CAT C-3’
5’-GGT GTC TGG GAA GCT GAG AG-3’
5’-GAA GGA CAG GAA TGG GAA CA-3’
5’-GCA GGT CAC ACG CCT CCT-3’
5’-GAT TCC TTG GCG CTC AGA GA-3’
5’-GGA AGA CCA CTC GCA TTC CTT-3’
5’-TCG CAC TTT GGT ATT CTT GGA G-3’
5’-TAG TCC TTC CTA CCC CAA TTT CC-3’
5’-TTG GTC CTT AGC CAC TCC TTC-3’
5’-GCT CTT ACT GAC TGG CAT GAG-3’
5’-CGC AGC TCT AGG AGC ATG TG-3’
5’-GAG ACC CCT GTG TCG GTT C-3’
5’-CTG CGT GTG TGA AAT GTC ATT G-3’
5’-CCC ATC CCC AGG AGT CTT G-3’
5’-ACC ATG ACT AGG GGC ACT GTA-3’
5’-CTG GAT AGC CTT TCT TCT GCT G-3’
5’-GCA CAC TGT GTC CGA ACT C-3’
5’-GAG GAA GTC CAT GTG GGA GA-3’
5’-TCC TAA CCA GCA CCA CTT CC-3’
5’-ATG AAC GCT ACA CAC TGC ATC-3’
5’-CCA TCC TTT TGC CAG TTC CTC-3’
5’-TGA GCA GGA TGG AGA ATT ACA GG-3’
5’-GTC CAA GTT CAT CTT CTA GGC AC-3’
5’-GGA AGC ACG GCA GCA GAA TA-3’
5’-AAC TTG AGG GAG AAG TAG GAA TGG-3’
5’-CAA GCG CCT AAG AGA GAT GG-3’
5’-CAC CTG TGC AAG AAG CAG AG-3’
5’-GTG ACT GGA ACA CTG GTC CTA-3’
5’-CCA GCC ACG TTG CAT TGT AG-3’
5’-ACG GCA TGG ATC TCA AAG AC-3’
5’-AGA TAG CAA ATC GGC TGA CG-3’
5’-GGT CTC AAC CCC CAG CTA GT-3’
5’-GCC GAT GAT CTC TCT CAA GTG AT-3’
Table S2. Flow cytometry antibodies used in this study (Related to
Experimental Procedures)
Antibody
BrdU
CD3e
CD4
CD8e
CD11b
CD11c
CD115
CD44
CD45
CD45.1
CD45.2
CD62L
CD64
F4/80
Foxp3
Ki-67
Ly-6C
Ly-6G
MHC I-A/I-E
Clone
PRB-1
145-2C11
RM4-5
53-6.7
M1/70
N418
AFS98
1M7
30-F11
A20
104
MEL-14
X54-5/7.1
BM8
FJK-16s
SolA15
HK1.4
1A8
M5/114.15.2
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