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Abbreviations: Arg1, arginase1; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; IRF, interferon regulatory
factor; TNF, tumour necrosis factor
320 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
Regulatory role of M2 peritoneal macrophage
contrast, cells of the M2 phenotype typically produce IL- In this study, we found that resident peritoneal macro-
10 and IL-1 receptor antagonist (IL-1Ra) and have high phages possess the same ability as bone-marrow-derived
levels of scavenger, mannose and galactose receptors.9 macrophages, i.e. polarization to the M1 and M2 pheno-
M1 macrophages have been shown to have strong anti- types, and that each phenotype produced a specific cyto-
bacterial and anti-tumour effects.7,8 In contrast, M2 kine profile. We also found that M2-polarized peritoneal
macrophages are thought to have immunoregulatory macrophages produced regulatory cytokines and could
functions and to be involved in parasite containment and suppress T-cell proliferation in vitro. Based on our data,
promotion of tissue remodelling and tumour progres- we suggest that resident peritoneal macrophages could be
sion.1 Recent studies showed that there are two key tran- used in adoptive transfer therapy for autoimmune/inflam-
scriptional regulators, interferon regulatory factor 5 matory diseases.
(IRF5) and IRF4 that polarize macrophages to the M1
and M2 phenotypes, respectively. IRF5 expression drives
Materials and methods
M1 macrophage polarization by directly inducing the
expression of pro-inflammatory cytokines, such as IL-6,
Mice
IL-12 and IL-23, while repressing the transcription of
anti-inflammatory cytokines such as IL-10.10,11 In con- Female C57BL/6J mice (6–7 weeks of age) were purchased
trast, IRF4 has been shown to be a crucial mediator of from Japan SLC (Hamamatsu, Japan). Mice were housed in
M2 macrophage polarization.12,13 Jumonji domain con- an animal facility under a 12-hr light/dark cycle and were
taining-3, an upstream effector of IRF4-induced M2 given standard chow and water ad libitum. Mice weighing
polarization, and IRF4 are critically involved in IL-4- 18–22 g at 7–9 weeks of age were used for this study.
dependent induction of M2 marker genes, such as Arg1,
Ym1 and Fizz113; however, the molecular mechanisms
Isolation of peritoneal macrophages, bone-marrow-
underlying the induction of M2 macrophage polarization
derived macrophages, and CD4+ T cells
by IRF4 are unknown.
Interestingly, the phenotype of polarized M1 and M2 The method used to isolate peritoneal macrophages from
macrophages can be reversed in vitro and in vivo.14,15 mice has been described previously.18 Briefly, mice were
Cytokines and growth factors are involved in the repro- killed, and 5 ml of ice-cold PBS was injected into the
gramming of M1 and M2 macrophages. Interferon-c abdomen. The fluid was withdrawn, centrifuged (350 g
(IFN-c) induces M1 macrophages, whereas stimulation of for 5 min at 4°), and the cell pellet was resuspended in
macrophages with IL-4 or IL-13 induces M2 macro- 1 ml of Dulbecco’s modified Eagle’s medium supple-
phages.16,17 In these studies, molecular and functional mented with 2% penicillin-streptomycin and 10% bovine
analyses of murine macrophages were performed using calf serum. These peritoneal exudate cells were cultured
bone-marrow-derived macrophages. on Petri dishes (> 4 hr at 37°), non-adherent cells were
If we could stimulate macrophages obtained from our removed, and the adherent cells were detached by diges-
body to become tumoricidal macrophages (M1 polarized) tion with trypsin (05%).
or immune-regulatory macrophages (M2 polarized) ex vivo To isolate bone-marrow-derived macrophages, pelvic
using molecular biological methods and introduce them and femoral bones were dissected, and all the tissue
back into the body, these macrophages may be of therapeu- remaining on the bones was removed. The end of each
tic value for targeting cancer or inflammation. Although bone was cut off, and the bone marrow was expelled.
the removal of macrophages from bone marrow or spleen Cells from bone marrow were cultured for 7 days with
is invasive, it is relatively safe and easy to collect peritoneal 10 ng/ml macrophage colony-stimulating factor. Adherent
macrophages from ascites, especially for patients with cells were detached by digestion with trypsin (05%).
cancerous or inflammatory peritonitis. Indeed, a large FACS sorting (BD Bioscience, San Jose, CA) was per-
number of macrophages exist in the peritoneal cavity; how- formed to obtain F4/80-positive and CD11c-negative cells.
ever, whether peritoneal macrophages can be polarized to Then, the harvested cells (05 9 106 to 1 9 106) were
M1 and M2 phenotypes has not yet been fully addressed. cultured in six-well plates containing complete RPMI-
Interestingly, Hunter et al.18 showed that intraperi- 1640 with 10% fetal bovine serum for 24 hr at 37°.
toneal injection of murine peritoneal macrophages differ- CD4+ T cells were isolated from the spleens of wild-
entiated into the M2 phenotype reduced the severity of type mice. Spleens were dissected from the abdominal
experimental colitis in mice. They suggested that IL-10 cavity and passed through a 40-lm nylon filter. Red cell
production from M2-polarized macrophages partly con- lysis buffer was used to remove red blood cells. A single
tributed to the attenuation of colitis. However, the pro- splenic cell suspension was obtained, and CD4+ T cells
files of the other cytokines and factors produced by these were isolated by a magnetic cell separation (MACS) tech-
cells and the functional effects on T cells were not fully nique using the CD4+ T-cell isolation kit II (Miltenyi
addressed. Biotec, Bisley, UK).
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 321
S. Oishi et al.
322 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
Regulatory role of M2 peritoneal macrophage
* P < 0·05
(a) Peritoneal BM-derived (b) Peritoneal BM-derived
Irf5 Irf5 iNOS iNOS
Relative to GAPDH
Relative to GAPDH
0·8 * 0·08 * *
0·4 0·04
0 0
IFN-γ – + – + IFN-γ – + – +
Relative to GAPDH
Relative to GAPDH
12 10
6 5
0 0
IFN-γ – + – + IFN-γ – + – +
Figure 1. Induction of the M1 phenotype in peritoneal and bone-marrow-derived macrophages by stimulation with interferon-c (IFN-c). (a)
RT-PCR assay for the expression of Irf5 mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. (b) RT-
PCR assay for the expression of iNOS mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. (c) RT-PCR
assay for the expression of Il12 mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c.(d) RT-PCR assay for
the expression of Tnfa mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. Data are representative of
five independent experiments. Error bars represent SD. *P < 005 (Student’s t-test).
peritoneal macrophages possess the ability to differentiate and M2 phenotypes with or without LPS stimulation
into M1-polarized macrophages following stimulation in vitro (Fig. 3a,b). M2-polarized but not M1-polarized
with IFN-c ex vivo. peritoneal macrophages strongly induced T helper type 2
(Th2) cytokines, including IL-4 and IL-13, after stimula-
tion with LPS for 24 hr. Unexpectedly, M2-polarized
Peritoneal macrophages differentiate into the M2
peritoneal macrophages expressed significantly higher Ifng
subtype after stimulation with IL-4/IL-13
than M1-polarized peritoneal macrophages after stimula-
We next investigated whether resident peritoneal macro- tion with LPS. Il12a, Il17a, and Tnfa were not up-regu-
phages could differentiate into the M2 subtype following lated in M2-polarized or M1-polarized peritoneal
stimulation with IL-4 and IL-13 ex vivo. Irf4, a master macrophages after stimulation with LPS. The mRNA
regulator of the M2 phenotype, was strongly up-regulated expression of Il6 was suppressed in M2-polarized peri-
in both peritoneal macrophages and bone-marrow- toneal macrophages compared with the levels in M1-
derived macrophages isolated from wild-type mice polarized peritoneal macrophages after stimulation with
(Fig. 2a). To monitor M2 status, we evaluated the mRNA LPS. These data clearly showed that M2-polarized peri-
expression level of arginase1 (Arg1) and Fizz1, two mark- toneal macrophages predominantly up-regulate Th1 and
ers of the M2 phenotype. Stimulation with IL-4 and IL- Th2 cytokines but not Th17 and pro-inflammatory
13 induced the expression of Arg1 (Fig. 2b) and Fizz1 cytokines after stimulation with LPS.
(Fig. 2c) in both peritoneal and bone-marrow-derived
macrophages. These data clearly showed that resident
M2-polarized peritoneal macrophages predominantly
peritoneal macrophages possess the ability to differentiate
produce regulatory cytokines
into M2-polarized macrophages following stimulation
with IL-4 and IL-13 ex vivo. We next investigated the mRNA expression of anti-inflam-
matory cytokines in M1- and M2-polarized macrophages.
Tgfb mRNA expression was significantly higher in M2-
M2-polarized but not M1-polarized peritoneal
polarized peritoneal macrophages than in M1-polarized
macrophages predominantly express Th1 and Th2
peritoneal macrophages (Fig. 4a). However, there was no
cytokines but not Th17 cytokines
significant difference in Tgfb mRNA expression between
We next investigated the expression of inflammatory M0-status peritoneal macrophages and M2-polarized peri-
cytokines in peritoneal macrophages polarized to the M1 toneal macrophages. The expression of Il10 mRNA was
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 323
S. Oishi et al.
4·0
Th2 cytokine production is increased in T cells co-
cultured with M2-polarized peritoneal macrophages
We next evaluated whether the cytokine profile was
0
IL-4 + IL-13 – + – + altered in CD4+ T cells co-cultured with M1- and M2-
polarized peritoneal macrophages. The purity of CD4+ T
Figure 2. Induction of the M2 phenotype in peritoneal and bone- cells re-isolated after co-culture with macrophages was
marrow-derived macrophages by stimulation with interleukin-4 confirmed using flow cytometric analyses (Fig. 6a).
(IL-4)/IL-13. (a) RT-PCR assay for the expression of Irf4 mRNA in
Interestingly, CD4+ T cells co-cultured with M2-polar-
isolated peritoneal and bone-marrow-derived macrophages stimulated
ized peritoneal macrophages strongly expressed Th2 cytoki-
with IL-4/IL-13. (b) RT-PCR assay for the expression of Arg1 mRNA
nes, including Il4 and Il13, compared with the levels in
in isolated peritoneal and bone-marrow-derived macrophages stimu-
lated with IL-4/IL-13. (c) RT-PCR assay for the expression of Fizz1 CD4+ T cells co-cultured with M1-polarized peritoneal
mRNA in isolated peritoneal and bone-marrow-derived macrophages macrophages (Fig. 6b). However, the expression levels of
stimulated with IL-4/IL-13. Data are representative of five independent regulatory cytokines, such as Tgfb and Il10, were not
experiments. Error bars represent SD. *P < 005 (Student’s t-test). increased in CD4+ T cells co-cultured with M2-polarized
peritoneal macrophages compared with the levels in CD4+
T cells co-cultured with M1-polarized peritoneal macro-
significantly higher in M2-polarized peritoneal macro- phages (Fig. 6b). These results indicate that M2-polarized
phages than in M0-status and M1-polarized peritoneal peritoneal macrophages induce CD4+ T cells to produce
macrophages after stimulation with LPS (Fig. 4a). To assess Th2 cytokines but not regulatory cytokines.
IL-10 protein level, an ELISA was performed. Production
of IL-10 was significantly higher in M2-polarized macro-
Discussion
phages than in M1-polarized peritoneal macrophages after
stimulation with LPS (Fig. 4b). However, there was no sig- Based on the findings described in this report, we drew
nificant difference in IL-10 production between M0-status the following conclusions concerning the immunological
324 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
Regulatory role of M2 peritoneal macrophage
* P < 0·05
(a) LPS (–)
* * *
45
*
40
35
Relative to GAPDH
30
25
20
15
10
0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
* P < 0·05
(b) LPS (+)
160 * * * * *
140 * *
120
Relative to GAPDH
100
80
60
40
20
0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
Tnfα Ifn γ Il4 Il6 Il12a Il13 Il17a
Figure 3. The expression of inflammatory cytokines in peritoneal M1- and M2-polarized macrophages. Total RNA was isolated from the M1-
and M2-polarized macrophages after stimulation without lipopolysaccharide (LPS) (a) or with LPS (b) and then analysed by RT-PCR to detect
inflammatory cytokines, including Tnfa, Ifng, Il4, Il6, Il12a and Il17a. Cytokine mRNA expression levels were normalized to GAPDH. Data are
representative of five independent experiments. Error bars represent SD. *P < 005 (Student’s t-test).
function of M1- and M2-polarized murine peritoneal mesenchymal stromal cells for Crohn’s disease has been
macrophages. (i) Similar to bone-marrow-derived macro- reported.23 Because M1 phenotype macrophages also have
phages, resident peritoneal macrophages possess the abil- strong anti-tumour effects7,8 and M2 phenotype macro-
ity to differentiate into M1- and M2-polarized phages have anti-inflammatory effects,1 macrophages
macrophages ex vivo, (ii) M2-polarized but not M1-polar- could be developed as a new cell-based therapy for cancer
ized macrophages predominantly express Th1 and Th2 and/or inflammatory diseases. However, most basic stud-
cytokines, (iii) M2-polarized but not M1-polarized ies on the regulation of macrophage polarization have
macrophages predominantly produce anti-inflammatory used bone-marrow-derived macrophages. Removing
cytokines, (iv) M2-polarized peritoneal macrophages sup- macrophages from the bone marrow of patients for
press T-cell proliferation ex vivo, (v) CD4+ T cells co-cul- immunotherapy is invasive, but removing macrophages
tured with M2-polarized peritoneal macrophages from the ascites in the peritoneal cavity, in which a large
predominantly express Th2 cytokines but not regulatory number of macrophages exist, seems less invasive. For
cytokines. example, a large number of macrophages extracted from
Several basic and clinical immunotherapy studies have the ascites of patients with cancer or inflammatory dis-
been performed with adoptive autologous cells, such as T eases could be differentiated into an anti-tumour pheno-
cells, natural killer cells and dendritic cells, targeting type for the treatment of cancer or into an anti-
cancer.19–22 Immunotherapy studies advance not only the inflammatory phenotype for the treatment of inflamma-
field of cancer but also the field of inflammatory disease. tory diseases, and then these cells could be transferred
For example, a clinical trial of immunotherapy using back into patients; hence, peritoneal macrophages could
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 325
S. Oishi et al.
* P < 0·05
(a) Tgf β Il10 *
* *
Relative to GAPDH
Relative to GAPDH
20 15 *
15
10
10
5 5
0 0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
* P < 0·05
(b) IL-10 IL-10
* * * *
40 250
30 200
pg/ml
pg/ml
150
20
100
10
50
0 0
M0 M1 M2 M0 M1 M2
LPS (–) LPS (+)
Figure 4. The expression of regulatory cytokines in peritoneal M1- and M2-polarized macrophages. (a) Total RNA was isolated from M1- and
M2-polarized macrophages stimulated without or with lipopolysaccharide (LPS) and then analysed by RT-PCR to detect the anti-inflammatory
cytokines Tgfb and Il10. Cytokine mRNA expression levels were normalized to GAPDH. Data are representative of five independent experiments.
Error bars represent SD. (b) M1- and M2-polarized peritoneal macrophages (2 9 106) were cultured for 24 hr without or with LPS. The culture
supernatants were then analysed by ELISA to detect interleukin-10 (IL-10). Data are representative of five independent experiments. Error bars
represent SD. *P < 005 (Student’s t-test).
be of therapeutic value for cancer or inflammatory dis- proliferation than M0-status or M1-polarized peritoneal
eases. macrophages. Therefore, we believe that peritoneal
Interestingly, a recent study showed that intraperitoneal macrophages polarized to an M2 phenotype ex vivo have
injection of M2-polarized peritoneal macrophages stronger anti-inflammatory effects than M0 or M1 pheno-
reduced the severity of experimental colitis in mice in type macrophages. In our study, no significant difference
part through an IL-10-dependent mechanism.18 However, was seen in the Tgfb mRNA expression between M0 and
in this study, the anti-inflammatory effects of other M1 macrophages. However, the Tgfb mRNA expression
cytokines (besides IL-10) produced by these cells and the was higher in M0 than in M1 macrophages. In addition,
functional effects of these cells on pathogenic T cells were the protein level of IL-10 was significantly higher in M0
not clear. In our study, the expression of both Il10 and than in M1 macrophages. Hence, our observations sup-
Tgfb mRNA was significantly higher in M2-polarized port the data of Liu et al.24 that M0 macrophages have
peritoneal macrophages than in M1-polarized peritoneal the ability to suppress inflammation.
macrophages. Furthermore, Th1 and Th2 cytokines were Previous studies showed that M1-polarized bone-mar-
also significantly higher in M2-polarized peritoneal row-derived macrophages predominantly produce Th1
macrophages than in M1-polarized peritoneal macro- cytokines, including IFN-c, and pro-inflammatory cytoki-
phages. Liu et al. showed that adoptive intravenous trans- nes, including TNF-a, IL-6 and IL-12.7,8 However, unex-
fer of freshly isolated murine peritoneal cells pectedly, our study showed that peritoneal macrophages
(macrophages and B cells) attenuated colitis.24 Interest- polarized into the M1 phenotype ex vivo produced negli-
ingly, in that study, although peritoneal macrophages gible amounts of Th1 and pro-inflammatory cytokines.
were not polarized into the M1 or M2 phenotype ex vivo Indeed, macrophages isolated from various tissues mani-
before adoptive cell transfer, IL-10 and transforming fest marked differences in functional activities.25–28 These
growth factor-b levels were significantly increased in peritoneal macrophages are remarkably distinct from
macrophages isolated from the colonic tissue of colitis those in other tissues for several reasons.28,29 For exam-
mice after adoptive transfer. In our study, polarization ple, Zhu et al.30 demonstrated that peritoneal macro-
into M2 macrophages ex vivo induced significantly higher phages have higher iNOS and IL-12 expression than do
Il10 and Tgfb expression than that observed in M1 splenic macrophages. However, our data suggested that
macrophages. In addition, M2-polarized peritoneal polarization into the M1 phenotype ex vivo induced lower
macrophages more effectively suppressed T-cell production of Th1 and pro-inflammatory cytokines than
326 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
Regulatory role of M2 peritoneal macrophage
*
102
400 000 *
CD4
101
300 000
100
T-cell number
F4/80
100 000
(b) * P < 0·05
2 * * *
1·8
0 1·6
Relative to GAPDH
T T+M0 T+M1 T+M2 1·4
1·2
CD3/CD28 (+)
1
Figure 5. Suppression of T-cell proliferation by M2-polarized peri- 0·8
toneal macrophages. (a) CD4+ T cells isolated from the spleen of 0·6
wild-type mice were stimulated with anti-CD3/CD28 beads and were 0·4
either cultured alone (T cells only) or with untreated peritoneal 0·2
macrophages (M0), interferon-c (IFN-c)-treated peritoneal macro- 0
1
2
M
M
phages (M1), or interleukin-4 (IL-4)/IL-13-treated peritoneal macro-
4/
4/
4/
4/
4/
4/
4/
4/
D
D
phages (M2) at a ratio of 1 : 10 (macrophages: CD4+ T-cells). Cells
C
C
Il4 Il13 Tgfβ Il10
were collected after 4 days and analysed by the MTT assay. Bars
show the mean SD from five separate cultures per condi-
Figure 6. Anti-inflammatory and T helper type 2 (Th2) cytokine
tion.*P < 005 (Student’s t-test).
mRNA expression in CD4+ T cells co-cultured with M1- and M2-
polarized peritoneal macrophages. (a) CD4+ T cells isolated from the
spleen of wild-type (WT) mice were stimulated with anti-CD3/CD28
that observed in M0-status macrophages. Therefore, beads. These cells were co-cultured with M1- or M2-polarized peri-
adoptive transfer of these M1-polarized peritoneal macro- toneal macrophages at a ratio of 1 : 10 (macrophages: CD4+ T cells).
phages may not provide anti-bacterial or anti-tumour After co-culturing, the floating cells were collected and stained for
effects in vivo. In contrast, polarization to the M2 pheno- CD4 and F4/80 and analysed by flow cytometry. (b) Total RNA was
type ex vivo induced much higher production of regula- isolated from CD4+ T cells co-cultured with M1- and M2-polarized
tory cytokines, suggesting that adoptive transfer of these peritoneal macrophages and then analysed by RT-PCR to detect the
M2-polarized macrophages may be well suited for the T helper type 2 cytokines, Il4 and Il13, and the anti-inflammatory
treatment of inflammatory diseases. cytokines, Tgfb and Il10. Cytokine mRNA expression levels were nor-
malized to GAPDH. Data are representative of five independent
It is not yet clear whether, after intraperitoneal or
experiments. Error bars represent SD. *P < 005 (Student’s t-test).
intravenous adoptive transfer of M1- or M2-polarized
peritoneal macrophages, these cells can selectively migrate
to lesion sites. However, Hunter et al.18 showed that fluo- Therefore, these findings suggest that both intraperi-
rescence-labelled macrophages transferred intravenously toneally and intravenously adoptively transferred M2-
were detected in the spleen, mesenteric lymph nodes, cae- polarized peritoneal macrophages could migrate to the
cum and colon of naive mice. In addition, Liu et al. target organs and directly suppress T-cell proliferation at
showed that intraperitoneally transferred fluorescence- the site of inflammation. In addition, our observations
labelled macrophages were detected in the mesenteric showed that these M2-polarized macrophages could
lymph nodes and colons of colitic mice.24 These observa- induce CD4+ T cells to express Th2 cytokines. Therefore,
tions suggest that adoptively transferred peritoneal adoptive transfer of M2-polarized peritoneal macrophages
macrophages can migrate to most of the organs where may be more suitable for diseases involving Th1-mediated
inflammation exists. Our observations not only showed inflammation, such as Crohn’s disease, rather than Th2-
that M2-polarized macrophages produce humoral anti- mediated inflammation, such as ulcerative colitis.
inflammatory cytokines but also that direct inhibition of In conclusion, our findings showed that murine resi-
T-cell proliferation depends on cell–cell contact. dent peritoneal macrophages possess the same ability as
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 327
S. Oishi et al.
bone-marrow-derived macrophages, i.e. polarization to 8 Benoit M, Desnues B, Mege JL. Macrophage polarization in bacterial infections. J
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have different cytokine production patterns. M1-polarized tion. Eur J Immunol 2007; 37:14–6.
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13 El Chartouni C, Schwarzfischer L, Rehli M. Interleukin-4 induced interferon regulatory
inflammatory diseases based on the use of endogenous factor (Irf) 4 participates in the regulation of alternative macrophage priming. Immuno-
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This work was supported by a Grant-in-Aid for Scientific cited tumor infiltrating macrophages and dendritic cells towards tumor rejection. Can-
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Research (C) 25460948 from the Ministry of Education, 16 Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization:
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acknowledge the late Dr Masato Uchijima and the late cytes. Trends Immunol 2002; 23:549–55.
17 Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23–35.
Kunio Tsujimura for their excellent technical support. 18 Hunter MM, Wang A, Parhar KS, Johnston MJ, Van Rooijen N, Beck PL, McKay DM,
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Author contributions 19 Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing
the T cell response. Nat Rev Immunol 2012; 12:269–81.
SO designed and performed experiments and analysed the 20 Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer
data, RT, ST, and ST designed and performed experiments. 2012; 12:265–77.
21 Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer
MI, YH, KT, TN, SS, TH, SO, TF, and HM analysed the treatment. Nat Rev Cancer 2012; 12:237–51.
data and contributed to manuscript preparation. KS 22 Dahlberg CI, Sarhan D, Chrobok M, Duru AD, Alici E. Natural killer cell-based thera-
designed the study and experiments; acquired, analysed, pies targeting cancer: possible strategies to gain and sustain anti-tumor activity. Front
Immunol 2015; 6:605.
and interpreted the data; and prepared the manuscript. 23 Forbes GM, Sturm MJ, Leong RW, Sparrow MP, Segarajasingam D, Cummins AG,
et al. A phase 2 study of allogeneic mesenchymal stromal cells for luminal Crohn’s dis-
ease refractory to biologic therapy. Clin Gastroenterol Hepatol 2014; 12:64–71.
Disclosures 24 Liu T, Ren J, Wang W, Wei XW, Shen GB, Liu YT, et al. Treatment of dextran sodium
sulfate-induced experimental colitis by adoptive transfer of peritoneal cells. Sci Rep
The authors have no financial conflicts of interest to 2015; 5:16760.
report. 25 Davies LC, Taylor PR. Tissue-resident macrophages: then and now. Immunology 2015;
144:541–8.
26 Lepay DA, Steinman RM, Nathan CF, Murray HW, Cohn ZA. Liver macrophages in
murine listeriosis. Cell-mediated immunity is correlated with an influx of macrophages
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