1) p53 activation through nutlin-3a treatment suppressed M2 macrophage polarization by downregulating M2 marker genes like c-MYC, IRF4 and FIZZ1. 2) Loss of p53 increased M2 macrophage polarization both in vitro and in vivo by increasing the expression of M2 marker genes. 3) p53 was found to suppress M2 macrophage polarization by directly binding to the promoter region of c-MYC gene, reducing its expression and influencing the expression of downstream M2 genes.

1. L Li*, DSW Ng, W-C Mah, FF Almeida, SA Rahmat, VK Rao, SC Leow, F Laudisi, MT Peh, AM
Goh, JSY Lim, GD Wright, A Mortellaro, R Taneja, F Ginhoux, CG Lee, PK Moore and DP Lane*
Commentator: Dr. Yao Chang
Speaker: Yi-Zhen Wu
Date: 2015.11.04
1
A unique role for p53 in the regulation of M2 macrophage polarization

2. 2
Macrophage
Phagocytosis
Antigen presentation
Inflammation
Innate immunity Adaptive immunity

3. 3
Front. Immunol., 16 November 2011
Macrophage subtypes
1. parasite containment
2. Wound healing
3. signatures is production of enzyme Arginase-1
1. help killing pathogens
2. secrete pro-inflammatory cytokines
and chemokines
Dynamic changePolarization

4. 4
p53 in M1 macrophage
These results indicate that p53 inhibits innate immune responses through downregulating STAT-1
and proinflammatory cytokines.

5. Transcriptional factor p53
5

6. 6
p53 network
Journal of Cell Science 2003 116: 4077-4085; doi: 10.1242/jcs.00739

7. Specific Aim
Although a role for p53 in M1 macrophage function has been suggested,
there is little information relating to its effect in M2 macrophages.
7
To investigate the role of p53 in M2 macrophage polarization

8. Does the polarization of M2 macrophage activate the p53 ？
8

9. Polarization of macrophages to the M1 or M2 subtype increased the
expression of p53 and its downstream markers including MDM2 and p21
9
M1 polarization inducer : LPS/IFN-γ
M2 polarization inducer : IL-4/IL-13
Nutlin-3a , which inhibits the p53-MDM2 interaction
Journal of Cell Science 2003 116: 4077-4085; doi: 10.1242/jcs.00739

10. M2 polarization activated PI3K /AKT,
which phosphorylates MDM2 and increased p53 ubiquitination
10
PI3k
inhibitor
Journal of Cell Science 2003 116: 4077-
4085; doi: 10.1242/jcs.00739

11. 11
proteasome
inhibitor
M2 polarization activated PI3K /AKT,
which phosphorylates MDM2 and increased p53 ubiquitination

12. Does p53 regulate polarization of M2 macrophage in vitro ?
12

13. Nutlin-3a reduced the expression of M2 genes
13
M2 marker gene: c-MYC,IRF4,FIZZ1

14. 14
Nutlin-3a reduced the expression of M2 genes
M2 marker gene: c-MYC,IRF4,FIZZ1

15. • p53 transcriptional activity also increase in both M1- and M2-polarized
macrophages and minimal ubiquitination of p53 occurred in M1 macrophages
and this is largely unaffected by nutlin-3a,nutlin-3b or LY294002
• Activation of p53 by nutlin-3a reduce the expression of M2 gene.
15
Summary 1

16. Does the activation of p53 in M2 macrophages result in a macrophage
phenotype intermediate between M1 and M2 ?
16

17. 17
Nutlin-3a induced activation of p53 under M2-polarizing conditions
thwarts the establishment of the M2 subtype
CD80high/CD206low/DECTIN-1low
CD206high/DECTIN-1high/ CD80low

18. 18
Nutlin-3a induced p53 activation on M2 macrophage impair
the functional consequences of M2 activation

19. Summary 2
• Nutlin-3a induced p53 activation in M2 polarization macrophage impairs
not only M2 gene expression but also the functional consequences of
M2 activation.
19

20. Loss of p53 increased the expression of several M2 genes
20
M2 marker gene: c-MYC,IRF4,FIZZ1

21. 21
Loss of p53 increased the functional consequences of M2 activation

22. Trp53R172H Mutant mice
• Trp53R172H affects the overall structure of the p53 DNA-binding domain
• p53 accumulated in mutant cells, it did not cause translation of downstream target proteins.
22

23. 23
Loss of p53 transactivation in mutant cells was associated with
upregulation of the expression of several key M2 markers
M2 marker gene: c-MYC,IRF4,FIZZ1

24. • Loss of p53 increased the expression of several M2 genes, increased arginase
activity and enhanced proliferation of M2 macrophages.
• Loss of p53 transactivation in mutant cells was associated with upregulation
of the expression of several key M2 markers
Summary 3
24

25. What is the molecular mechanism of p53 in regulating M2 polarization ?
25

26. 26
c-MYC
• p53 activation downregulates c-myc expression through binding to the c-myc promoter
• The M2 polarization requires the transcription factor c-MYC
p53
C-MYC
M2
polarization

27. Expression of c-MYC was downregulated by nutlin-3a
27
c-MYC Inhibitor
• Previous studies have shown an association between p53
activation and downregulation of c-myc expression
• Chromatin immunoprecipitation assays indicate that p53 is
bound to the c-myc promoter in vivo.
• Suggest that p53 represses c-myc transcription through a
mechanism that involves histone deacetylation
M2 marker gene: c-MYC,IRF4,FIZZ1

28. 28
Treatment of M2-polarized cells with nutlin-3a
enhanced p53 recruitment to the c-MYC promoter
CHIP assay
Recruitment of p53 to four different loci of
the c-Myc gene promoter in M2-polarized
H3K9 acetylation enrichment at two loci of the
c-Myc gene promoter in M2-polarized
H3K9 acetylation

29. • p53 suppresses transcription of the c-MYC gene by binding to its promoter
region, triggering chromatin remodeling and influencing the expression of a
subset of M2 genes.
Summary 4
29
p53
C-MYC
M2
polarization

30. Dose endogenous p53 regulates IL4-elicited M2 functional phenotype
in peritoneal macrophages in vivo ?
30

31. Expression of M2 genes was increased in peritoneal macrophages from
p53-/- mice administered slow-releasing IL4
31

32. 32
IL4C also increased the number of viable peritoneal macrophages
to a greater extent in p53-/- than wild-type animals
as in vitro, endogenous p53 regulates
IL4-elicited M2 functional phenotype
in peritoneal macrophages in vivo.

33. • As M2-polarized macrophages are important for the development of tolerance to LPS
• Gene responses during endotoxin tolerance were similar to those found during M2 polarization,
including reduced production of proinflammatory mediators.
• LPS tolerance, defined as the reduced capacity of a cell to respond to LPS activation after an initial
exposure to this stimulus
33
Previous Studies

34. 34
Does p53 mediate M2 polarization during LPS tolerance
in macrophages?

35. Activating p53 with nutlin-3a reduced the M2-mediated
process of LPS tolerance in macrophages
35
medium
NT
M/L
(L+D)/L
(L+N)/L
medium(LPS)
medium(DMSO+LPS)
medium(Nut-3a+LPS)
LPS
LPS
24hr0hr
0hr
0hr
0hr
24hr

36. 36
Effect of nutlin-3a on plasma TNFα & IL6

37. • Overall, these data show that endogenous p53 regulates M2 polarization
during LPS tolerance in vitro and in vivo.
Summary 5
37
p53
M2
polarization
inflammationLPS
tolerance

38. degradation
PI3K
AKT
p53
P
LY294002
Nutlin-3a
IL4 / IL13
p53
p53 M2 gene
Conclusions
MDM2
P
10058F4
38
P
c-MYC
M2 gene
p53
M2 polarization Activate p53
Activate
c-MYC
PI3K
AKT
P
MDM2
P
P
STAT6
STAT6
c-MYC
P c-MYC

39. • In conclusion, p53 is the first transcription factor reported to suppress M2
macrophage polarization.
• We propose that manipulation of the p53 system provides an additional approach to
study the molecular basis of macrophage plasticity and a new therapeutic target
for small molecule p53 activators such as nutlin-3a.
39
Discussion 1

40. • This, coupled with the ability of p53 to regulate M2 marker expression, raises the
possibility that activating macrophage p53 may reduce the density and the
protumoral phenotype of M2-like tumor-associated macrophages (TAM).
40
Discussion 2

41. 41

42. 42
M2 Macrophage
• Tumor-associated macrophages (TAMs) are derived from circulating monocytes, which are
highly abundant within the tumor and provide a key link between inflammation and cancer. TAMs
develop a phenotype similar to M2-polarized macrophages and respond to recruitment to the
tumor microenvironment
• Many observations indicate that TAM express several M2-associated protumoural functions,
including promotion of angiogenesis, matrix remodelling and suppression of adaptive immunity.

43. 43
At an evolutionary scale of the development of macrophage-like immune cells, arginase-1 may
have been primarily a wound healing protein, transcriptionally induced by proteins of the TGF-
β family

44. • Principal Component Analysis (PCA) was used to gauge the extent
that samples differ significantly from each other and to identify
potential biological outliers
44

45. • 為什麼要看C-MYC
• The transcription factor c-Myc is essential for cellular proliferation
and is one of the most frequently activated oncogenes
45

46. 46
TNF-α & IL6 affect lung histology in LPS-tolerant animals

47. Previous Studies
47
v
v

48. 48
Nutlin-3a reduced the expression of M2 genes

49. The molecular mechanism of action of p53 in
regulating M2 polarization is unknown
49

50. Expression of M1 marker gene was also reduced by nutlin-3
50

51. 51

52. 52
Nutlin-3a did not affect the expression of M2 markers in macrophages
from p53-/-animals thereby confirming that this effect is p53-dependent

53. Why p21 was not an important role
• Activation of p53 using nutlin-3, PM2 or MO11 was equally effective in inhibiting
the expression of M2 markers in both wild-type and p21-/- cells
53

54. 10058F4 reduced the expression of M2 markers in p53R172H/R172H macrophages
does not require a functional p53 to be effective
54
c-MYC inhibitor

55. IL4 / IL13
55
C-MYC
M2 gene
M2 macrophage
P
c-MYC
P
STAT6

56. 56
10058F4 & Nutlin-3a suppressed a similar subset of
M2 transcriptome activation