288 | Nature | Vol 579 | 12 March 2020
Article
Skewing monocytic MDSC differentiation
Further query of the GSEA-derived data indicated that gene sets related
to macrophage or myeloid differentiation and activation were upregu-
lated in monocytic MDSCs from the lungs of LLC mice treated with
low-dose AET (Fig. 3a). Moreover, DAVID pathway analysis reveals that
the NF-κB and PPAR signalling pathways were significantly downregu-
lated and upregulated, respectively (Extended Data Fig. 7a). Consist-
ent with differential regulation of these pathways occurring during
the monocyte-to-macrophage differentiation^25 , there is a preferential
increase in transcription factors associated with monocytic differen-
tiation that are mainly macrophage-related (EGR1, EGR2, MAFB, MAF
and PPARγ)^25 ,^26 , and not in those that are dendritic-cell-related (SPIB,
RELB, STAT3 and FOXP1)^26 ,^27 (Fig. 3b). The related factors have also
been validated in vitro and in vivo by quantitative PCR and western
blot analyses (Extended Data Fig. 7b, c). In vitro epigenetic treatment of
splenic monocytic MDSCs from mice bearing LLC tumours decreased
the percentage and absolute number of monocytic MDSCs significantly,
whereas those of macrophages increased significantly (Fig. 3c)—imply-
ing that low-dose AET might promote the differentiation of monocytic
MDSCs into macrophages.
Characterization using Immunological Genome Project (ImmGen)
criteria further defined the resulting populations in the lung premeta-
static microenvironment^28. Our top 200 upregulated genes in mono-
cytic MDSCs from the lungs of mice treated with low-dose AET map
specifically to lung interstitial macrophages (Fig. 3d, Supplementary
Table 2). Additionally, flow cytometry shows that lung interstitial mac-
rophages (CD11b+CD11c+CD64highMHC-II+CD24−)^29 increase in mice
treated with low-dose AET (Extended Data Fig. 8a, b). This observation is
confirmed by adoptively transferring 5 × 10^6 CD45.1+ monocytic MDSCsRegulation of macrophage
differentiationRegulation of macrophage
activationM-MDSCs PMN-MDSCs MacrophagesMock
LD-AETP = 0.002P < 0.001P < 0.0010204060Percentage of total cellsLung CD103+dendritic cellsLung CD11b+CD24+dendritic cells
Lung interstitialmacrophagesLung alveolar
macrophages
Lung monocytes-50510–50510Fold change over mock(logtransformed) 2P < 0.001 P < 0.001
P < 0.001P < 0.001aSPIBRELBSTAT3FOXP1
MAFBPPARγ
MAFEGR1EGR2Representative transcriptional factors
related to monocyte differentiation1
0
–1
–22Fold change over mock(logtransformed) 2bcdeGR1CD11b
MHC-IIF4/80MockLD-AETMDSCs MacrophagesMockLD-AETCD45.1CD45P = 0.005Mock LD-AET051510Percentage of CD45.1+ cellsInterstitial macrophagesLLC tumour-bearing
CD45.2 miceLLC tumour-bearing
CD45.1 miceIsolate bone-
marrow cellsTumourresection Sorted cells
tail vein injection36 h FACS
analysisMockLD-AETCD45.2 miceCD45.2 miceSort M-MDSCsNES
1.73
1.62
1.45
1.43
1.38
1.36
1.31
1.31
Cd741.605Gpr1161.245Thbs11.016Havcr20.79Lbp0.733Muc5b0.608Il1rl10.606
Il4ra0.528Tnip20.464Rora0.413Hspd10.397
Il330.381Slc7a20.378Wnt5a0.127
Il40.096
Bpi0.087Stap10.074Nr1h30.052Spaca30.016
Il13–0.062Shpk–0.066Snca–0.16Il31ra–0.182Zc3h12a3.0152.2251.4010.9960.2810.2740.1610.1490.1440.1120.094–0.065–0.136–0.142–0.149–0.161–0.227–0.37–0.4 –0.347
Pf4C1qcInhba
Csf1PrkcaCasp8
CalcaId2
Gata2 Ptpn2Trib1Rb1Il34Lif
Zbtb46InhaRipk1FaddHcls13.02.0
1.0 0
–0.25–0.51.51.0
0.5 0
–0.52 (log transformed)Fold change Fold change(log(^2) transformed)
0.7
0.60.5
0.4
0.3
0.20.1
0
0.7
0.60.5
0.40.3
0.20.1
0
0.8
FDR = 0.029 FDR = 0.042
Enrichment score Enrichment score
Regulation of macrophage
derived from cell differentiation
Regulation of
macrophage activation
Regulation of
macrophage differentiation
Positive regulation of
myeloid leukocyte differentiation
Macrophage differentiation
Macrophage activation
Positive regulation of
myeloid cell differentiation
Regulation of myeloid
leukocyte differentiation
105
104
103
0
0103104105
105
104
103
0
0103104105
104
103
102
101
(^00101102103104)
104
103
102
101
(^00101102103104)
104
103
102
101
(^00101102103104)
104
103
102
101
(^00101102103104)
0.51
0.12
Fig. 3 | Low-dose AET skews monocytic MDSCs towards an interstitial
macrophage-like population in the lung premetastatic microenvironment.
a, GSEA analysis revealed that macrophage or myeloid differentiation and
activation gene sets were upregulated (left) in monocytic MDSCs from the
lungs of LLC mice treated with low-dose AET. Representative upregulated
GSEA plots with core-enriched genes (middle and right). NES, normalized
enrichment score. Colour gradation is representative of log 2 -transformed fold
change over mock (n = 3 biological replicates). Gene-set enrichment P values,
NES values and FDR values reported are calculated with 1,000 permutations in
the GSEA software. FDR q < 0.25 was deemed significant. Gpr116 is also known
as Adgrf5. b, Significant changes of representative transcription factors (FDR-
adjusted P < 0.05) associated with monocytic differentiation (n = 3 biological
replic ate s). c, FACS showing the differentiation of sorted monocytic MDSCs
in vitro. Splenic monocytic MDSCs from LLC mice were cultured for 3 days with
tumour-conditioned medium (n = 3 biological replicates). d, The top 200
significantly upregulated genes (FDR-adjusted P < 0.05) were mapped back to
the reference ImmGen populations. The following populations from naive mice
were used: lung CD103+ dendritic cells (ImmGen code, DC_103+11b-_Lu);
CD11b+CD24+ lung dendritic cells (DC_103-11b+24+_Lu); lung interstitial
macrophages (MF_11c-11b+Lu); lung alveolar macrophages (MF Alv_Lu); and
lung monocytes (Mo_Lu). The expressions of these 200 genes in 6 populations
were transformed by zero-mean normalization. Two-sided Mann–Whitney U-
test. n = 3 biological replicates. e, FACS showing that low-dose AET can skew the
differentiation of transferred monocytic MDSCs towards an interstitial
macrophage-like phenotype in vivo. Purified 5 × 10^6 CD 45.1+ monocytic MDSCs
from bone marrow (day 0) were adoptively transferred into CD45.2+ recipient
mice within 24 h of resection (n = 3 in each group) in the LLC model. Mice
received low-dose AET or vehicle treatment for 36 h. In b, c and e, two-sample,
two-sided t-tests were used. All bars show mean ± s.e.m.