Seminar 1104

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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 ChangSpeaker: Yi-Zhen Wu

Date: 2015.11.04

A unique role for p53 in the regulation of M2 macrophage polarization

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Macrophage

Phagocytosis

Antigen presentationInflammation

Innate immunity Adaptive immunity

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Front. Immunol., 16 November 2011

Macrophage subtypes

1. parasite containment2. Wound healing 3. signatures is production of enzyme Arginase-1

1. help killing pathogens2. secrete pro-inflammatory cytokines

and chemokines

Dynamic changePolarization

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p53 in M1 macrophage

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

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Transcriptional factor p53

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p53 network

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

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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.

To investigate the role of p53 in M2 macrophage polarization

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Does the polarization of M2 macrophage activate the p53 ？

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Polarization of macrophages to the M1 or M2 subtype increased theexpression of p53 and its downstream markers including MDM2 and p21

M1 polarization inducer : LPS/IFN-γM2 polarization inducer : IL-4/IL-13Nutlin-3a , which inhibits the p53-MDM2 interaction Journal of Cell Science 2003 116: 4077-4085; doi: 10.1242/jcs.00739

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M2 polarization activated PI3K /AKT, which phosphorylates MDM2 and increased p53 ubiquitination

PI3k inhibitor

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

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proteasome inhibitor

M2 polarization activated PI3K /AKT, which phosphorylates MDM2 and increased p53 ubiquitination

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Does p53 regulate polarization of M2 macrophage in vitro ?

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Nutlin-3a reduced the expression of M2 genes

M2 marker gene: c-MYC,IRF4,FIZZ1

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• 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.

Summary 1

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

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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

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

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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.

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Loss of p53 increased the expression of several M2 genes


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Loss of p53 increased the functional consequences of M2 activation

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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.

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Loss of p53 transactivation in mutant cells was associated with upregulation of the expression of several key M2 markers


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• 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

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

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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

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Expression of c-MYC was downregulated by nutlin-3a

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


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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

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• 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

p53

C-MYC

M2 polarization

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

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

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IL4C also increased the number of viable peritoneal macrophagesto 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.

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• 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

Previous Studies

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Does p53 mediate M2 polarization during LPS tolerancein macrophages?

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Activating p53 with nutlin-3a reduced the M2-mediated process of LPS tolerance in macrophages

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

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Effect of nutlin-3a on plasma TNFα & IL6

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

Summary 5

p53M2

polarizationinflammationLPS

tolerance

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PI3K

AKT

p53

P

LY294002

Nutlin-3a

IL4 / IL13

p53

p53 M2 gene

Conclusions

MDM2

P

10058F4P

c-MYC

M2 gene

p53

M2 polarization Activate p53

Activatec-MYC

PI3K

AKTP

MDM2P

P

STAT6

STAT6

c-MYCP c-MYC

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• 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.

Discussion 1

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• 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).

Discussion 2

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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.

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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

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

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•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

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

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Previous Studies

v

v

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The molecular mechanism of action of p53 inregulating M2 polarization is unknown

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

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

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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

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10058F4 reduced the expression of M2 markers in p53R172H/R172H macrophages does not require a functional p53 to be effective

c-MYC inhibitor

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IL4 / IL13

C-MYC

M2 gene

M2 macrophage

P

c-MYC

P

STAT6

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

Health & Medicine

Seminar 1104