M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-β production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-β expression, and p21 knockdown in human monocytes corroborated its role in IFN-β regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.
Gorjana Rackov, Enrique Hernández-Jiménez, Rahman Shokri, Lorena Carmona-Rodríguez, Santos Mañes, Melchor Álvarez-Mon, Eduardo López-Collazo, Carlos Martínez-A, Dimitrios Balomenos
Submitter: Muhammad Ashfaq-Khan | firstname.lastname@example.org
Authors: Muhammad Ashfaq-Khan (1) and Detlef Schuppan (2)
(1) Institute of Translational Immunology and Research Centre for Immunotherapy, University Medical Centre, Johannes Gutenberg University, Mainz, Germany and (2) Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA
Published September 28, 2016
The p21 protein is mainly known as a key regulator of several phases of the cell cycle by inhibiting the cyclin dependent kinases Cdk2, Cdk3, Cdk4, and Cdk6 (1).
In a recently published paper in JCI (2) Gurjana et al studied a novel function of p21 demonstrating that it mediates reprogramming of macrophages via modulating the balance between active p65-p50 and inhibitory p50-50 NF-κB pathways. Thus p21 deficiency failed to attenuate macrophage IFN-β production, a marker of an activated NF-κB pathway, and also prevented macrophages from attaining an M2-like hyporesponsive state.
While these findings are novel and relevant, the authors did not provide data on the subcellular localization status of p21 protein nor discussed its important role. Thus the intracellular fate and function of the p21 protein correlates with its divergent roles in the nucleus or the cytoplasm (3). Inside the nucleus, where it causes cell cycle arrest, e.g. in response to DNA damage by blocking the transition between the G1 and S phase (4), p21 is largely controlled on the transcriptional level by p53 (5-7). In contrast, in the cytoplasm p21 displays anti-apoptotic activities ( 3) via binding to procaspase 3, an activity which blocks its proteolytic cleavage and activation to caspase 3 that represents the main execution pathway of apoptosis. The result is inhibition of both cellular FAS-receptor mediated extrinsic and intrinsic, cytochrome-C mediated mitochondrial apoptosis (7-10).
This dual function of p21 has previously been shown to be operative in macrophages. Thus the Th2 T cell and M2 macrophage polarizing cytokine IL-4 attenuates M-CSF-dependent macrophage proliferation via inducing p21 in a STAT6-dependent manner which is followed by cell cycle arrest by p21 nuclear activity (11). In contrast, U937 monocytic-macrophage cells differentiated with vitamin D3 and also U937 cells stably transfected with 21 cDNA express cytoplasmic p21 which drives apoptosis by forming a physical complex with pro-apoptotic signal-regulating kinase 1 (ASK1), in addition to its ability to bind to procaspase 3 (12). Moreover, atherosclerotic lesions of p21-/-/apoE-/- mice showed a more stable phenotype, with increased apoptosis and no change in intimal macrophage proliferation and plaque formation compared to lesions of p21+/+/ apoE-/- mice (13). It is tempting to speculate that the expression of p21 in this model of atherosclerosis is predominantly cytoplasmic, since macrophages in the intimal lesions of p21-/-/apoE-/- mice exhibited prominent apoptosis.
In addition, the authors did not provide data on a physical interaction of p21 protein with the studied components of the NF-kB pathway. They did also not investigate its previously well-defined anti-apoptotic role in the context of their current paper. Furthermore, they did not study the effect of p21 protein on these NF-kB pathway components in monocytes vs macrophages which may be different. This more differentiated approach would be needed, since the specific function of both p21 protein and the studied components of the NF-kB pathway depend on their location inside the cell (3). We therefore believe that depending on the in vitro and in vivo settings it is possible that repeated exposure of sublethal doses of LPS that were employed to generate LPS tolerant macrophages the intracellular distribution of p21 protein may have undergone significant changes from cytoplasmic to nuclear or vice versa. This distinction is also important for potential therapeutic implications when either a dampening or an enhancement of macrophage responses is desired, and where a pharmacological agent would need to be delivered primarily to the cytoplasm or to the nucleus. Examples for the former are sepsis and for the latter myeloid diseases of immunodeficiency
In summary, further studies will need to answer these questions to support or relate the authors’ claim on the role of p21 and its interaction with the NF-kB pathway in macrophage reprogramming/polarization and tolerization.
2. Rackov, G. et al. 2016. p21 mediates macrophage reprogramming through regulation of p50-p50 NF-kappaB and IFN-beta. J Clin Invest 126:3089-3103.
3. Cmielova, J., and Rezacova, M. 2011. p21Cip1/Waf1 protein and its function based on a subcellular localization [corrected]. J Cell Biochem 112:3502-3506.
4. Solozobova, V., Rolletschek, A., and Blattner, C. 2009. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage. BMC Cell Biol 10:46.
5. Brugarolas, J., Moberg, K., Boyd, S.D., Taya, Y., Jacks, T., and Lees, J.A. 1999. Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation. Proc Natl Acad Sci U S A 96:1002-1007.
6. Ju, Z., Choudhury, A.R., and Rudolph, K.L. 2007. A dual role of p21 in stem cell aging. Ann N Y Acad Sci 1100:333-344.
7. Cazzalini, O., Scovassi, A.I., Savio, M., Stivala, L.A., and Prosperi, E. 2010. Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response. Mutat Res 704:12-20.
8. Coqueret, O. 2003. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 13:65-70.
9. Child, E.S., and Mann, D.J. 2006. The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability. Cell Cycle 5:1313-1319.
10. Van Le, H., Minn, A.J., and Massague, J. 2005. Cyclin-dependent kinase inhibitors uncouple cell cycle progression from mitochondrial apoptotic functions in DNA-damaged cancer cells. Journal of Biological Chemistry 280:32018-32025.
11. Arpa, L., Valledor, A.F., Lloberas, J., and Celada, A. 2009. IL-4 blocks M-CSF-dependent macrophage proliferation by inducing p21Waf1 in a STAT6-dependent way. Eur J Immunol 39:514-526.
12. Asada, M., Yamada, T., Ichijo, H., Delia, D., Miyazono, K., Fukumuro, K., and Mizutani, S. 1999. Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. EMBO J 18:1223-1234.
13. Merched, A.J., and Chan, L. 2004. Absence of p21Waf1/Cip1/Sdi1 modulates macrophage differentiation and inflammatory response and protects against atherosclerosis. Circulation 110:3830-3841.
Submitter: Dimitrios Balomenos | email@example.com
Authors: Dimitrios Balomenos
Centro Nacional de Biotecnologia/CSIC
Published September 28, 2016
We thank Drs. Ashfaq-Khan and Schuppan for their interest in our work, published in the Journal of Clinical Investigation, and for pointing out the novelty of our findings.
One of the major points that the authors of this E-letter raised is whether a connection exists between the subcellular localization of p21 and its effect in promoting M2 macrophage polarization. These authors claimed that our work did not provide data on the nuclear or the cytoplasmic localization of p21 during the diverse LPS treatments. On the contrary, however, we addressed this point in Figure 6B. We showed that after primary LPS treatment (tolerization) and at different time points after secondary LPS treatment, p21 was present in both cytoplasm and nucleus in control wt macrophages. These data did not allow us to draw conclusions of whether the cytoplasmic or nuclear p21 participate in the pathway of M1 to M2 skewing. In order to determine whether cytoplasmic or nuclear p21 is driving M2 macrophage generation, experimental models need to be designed that inhibit the nuclear translocation of p21. Such experiments were beyond the scope of our work, which focused on showing a role for p21 in M1 to M2 reprogramming and how p21 exerted this effect. Of course, knowing the exact pathway of how p21 affects this process is the next logical step and is currently examined in our laboratory.
Another argument that was presented was the possible role of p21 in apoptosis. The authors presented literature examples where p21 had an anti-apoptotic effect in macrophage cell lines and during monocyte differentiation. However, these models do not reflect the conditions that were employed in our experiments in which fully differentiated macrophages that do not proliferate were subjected to LPS tolerization.
In a previous study by our group we showed that LPS treatment did not have differential apoptosis effects on p21-/- and wt macrophages (1). It is also accepted that independently of its cell cycle or apoptosis inhibiting effects, p21 suppresses excessive NF-kB activation in LPS-stimulated macrophages (2). Furthermore, in contrast with the claim of Ashfaq-Khan and Schuppan that we did not examine a possible anti-apoptotic of p21, we performed experiments, which did not support an anti-apoptotic role for p21 during LPS tolerance. These data appear in Figure S8 (C) and do not show apoptosis differences between wt and p21-/- macrophages after LPS-tolerization and LPS-tolerization followed by secondary LPS treatment. Cell cycle analysis showed that all cells were in the G0/G1 state after PI staining and no hypodiploid peak, indicative of apoptosis, was detected.
With respect to the article of Merched and Chan (E-letter, ref 13) we clearly state in the discussion of our paper that the atherosclerosis model is entirely different from the endotoxin tolerance model, which we studied. Furthermore, the effect of the lack of p21 in protecting atherosclerosis has been challenged, since a posterior study with p21-/-/apoE-/- mice indicated that reduced atherosclerosis in this system was caused mainly by an effect of p21 deficiency on vascular smooth muscle cells rather than on macrophages (3). Moreover, another study linked p21 deficiency to increased rather than reduced atherosclerosis (4).
Finally, we never claimed a direct physical interaction of p21 with components of the NF-kB pathway; instead, we indicated in the discussion that p21 can be envisaged as a modulator of upstream events that control posttranslational modifications of p50 and its binding affinity to NF-kB binding sites.
We believe that our data, provide firm evidence that p21 is a key molecule in regulating the balance between inflammatory (M1) and hyporesponsive (M2) macrophage states. Uncovering the exact pathway that underlies the effect of p21 on M1to M2 reprogramming is the key step, which could define targets for sepsis treatment.
1. Trakala M et al. Regulation of macrophage activation and septic shock susceptibility via p21(WAF1/CIP1). Eur J Immunol. 2009; 39(3):810–819.
2. Lloberas J and Celada A. p21(waf1/CIP1), a CDK inhibitor and a negative feedback system that controls macrophage activation. Eur J Immunol. 2009; 39(3):691-694.
3. Kunieda T et al. Angiotensin II Induces Premature Senescence of Vascular Smooth Muscle Cells and Accelerates the Development of Atherosclerosis via a p21-Dependent Pathway. Circulation. 2006; 114(9):953–960.
4. Khanna AK. Enhanced susceptibility of cyclin kinase inhibitor p21 knockout mice to high fat diet induced atherosclerosis. J Biomed Sci. 2009; 16(1):66.