by Daanish Sheikh
INTRODUCTION
Although it is well established that the adaptive immune system can mount an improved immune response upon reinfection with most pathogens, newer insight into innate immune system components suggest that macrophages can be “trained” to also elicit a stronger second immune response, known as immune training. However, the opposite effect, a reduced immune response, has also been observed when monocytes are pre-stimulated with bacterial cell wall component lipopolysaccharide (LPS) [1]; this phenomenon has been termed immune tolerance. Much like how exhausted CD8+ T cells following chronic infections are unable to produce cytokines at optimal levels, tolerized myeloid cells seem to produce fewer cytokines and have diminished phagocytic activity. Monocyte derived macrophage impairment characteristic of immune tolerance has also been linked with immune paralysis following sepsis [2] and in patients recovering from tuberculosis (TB) infection [3-4]. Both TB and sepsis survivors have an increased risk of later secondary infection and an increased risk of mortality [5-6]. Long lasting epigenetic changes are presumed to be responsible for the immune suppression that is observed in these patients [7]. These epigenetic changes are induced by metabolic shifts that upregulate glycolysis, the citric acid cycle, and oxidative phosphorylation, which in turn are activated via the cellular metabolism regulating pathways governed by PI3K, Akt, and mTOR. Pathogen-associated molecular patterns (PAMPs) are recognized by macrophage cell surface pattern recognition receptors whose downstream pathways include these metabolism regulating enzymes. The most well characterized PAMP in endotoxin tolerance involves LPS, which activates the macrophage via cell surface Toll-like receptor 4 (TLR4). PAMP-mediated immune activation also induces epigenetic remodeling of various inflammatory response genes and tolerizing genes. This paper will explore the epigenetic and metabolic shifts that are characteristic of immune tolerance or lead to its development, including the metabolic rheostats by which said long-term epigenetic shifts are mediated.
Myeloid Tolerance and TLR4 Pathway Mediation
We begin with a brief overview of the pathways downstream of TLR4 and then explore how the pathway varies in immune tolerant cells. Resembling conditions present in sepsis, long term exposure to LPS can result in myeloid immune tolerance; in fact, this model remains the most well characterized example. In LPS tolerance models, immune activation is initially induced via TLR4 exposure to LPS and is transiently characterized by increased energy metabolism via the mTOR and NFAT pathways. TLR4 mediated immune activation occurs via two pathways; first, activation initially results in the production of pro-inflammatory cytokines such as TNF, IL-1, IL-12, and CCL3 via a kinase cascade that includes myeloid differentiation factor 88 (MyD88), various interleukin receptor associated kinases (IRAKs 1,2, and 4), TNF receptor associated factor 6 (TRAF6), and transcription factors NF-κB and AP-1. Meanwhile via a second pathway, the initial activation of TLR4 also upregulates the secretion of interferon ß (IFNß) via a pathway including Toll/IL-1 receptor domain-containing adaptor (TRIF), IKKi kinase, TANK-binding kinase (TBK1), and transcription factor IRF3 [8]. IFNß secretion from this second pathway increases expression of interferon-inducible cytokines that are essential in an immune response [9] (Figure 1). Meanwhile, upregulation of the mTOR and NFAT pathways causes shifts in cellular metabolism that emphasize glycolysis, the TCA cycle, and the electron transport chain, allowing greater energy metabolism by the cell to meet the high energy demands of immune activation [10].
Figure 1: Simplified depiction of the TLR4 pathway and two of its downstream transcription factors
Myeloid immune tolerance is characterized by diminished expression of TLR4, decreased interaction of MyD88 and TRIF with TLR4, and diminished NF-κB and AP-1 signaling. As such, proinflammatory cytokine production is significantly reduced [11]. Upregulation of the mTOR energy metabolism pathways during immune activation is catalyzed via association of upstream kinase PI3K with the TLR4; this pathway is fairly well characterized but summarized here for clarity. PI3K, upon activation via association with active TLR4 and MyD88, phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to form PIP3, which in turn associates with phosphate dependent kinase-1 (PDK-1) to activate protein kinase B (PKB or Akt). Phosphorylated Akt activates the mTOR protein resulting in the aforementioned downstream upregulated metabolic effects [12]. However, diminished expression of TLR4 and MyD88 binding in myeloid tolerance terminates this metabolic upregulation, further exacerbating tolerance.
Metabolic and Epigenetic Modification in Myeloid Tolerance
Some of the causes of myeloid immune tolerance can be attributed to pathways that branch off the TLR4 activation pathway. For instance, PI3K, the kinase implicated in upregulating metabolism, is also implicated in pathways that result in production of anti-inflammatory cytokines [13]. p50, a subunit of NF-κB, is essential for the development of immune tolerance by homodimerization rather than the canonical heterodimerization with p65, another subunit [14]. Other transcription factors that are commonly observed in resting macrophages can also be characteristic of tolerized macrophages. Transcriptional repressor Bcl-6 inhibits the production of inflammatory gene products by regulating the TLR4 transcriptome via association with transcription factor Pu.1. Pu.1 and NF-κB association with inflammatory gene promoters upregulate transcription of inflammatory genes by conferring a tri-methylation to the 4th lysine residue of the H3 histone protein in chromatin (H3K4me3) near those genes [25]. Because Bcl-6 recruits histone deacetylases and histone demethylases to the same site, the same inflammatory genes are downregulated in its presence [24]. Other prominent transcription factors responsible for myeloid tolerance include RelB (a subunit of NF-κB) and high mobility group box 1 protein (HMGB1). RelB complexes with H3K9 methyltransferase G9a to form heterochromatin at the IL-1ß promoter, inhibiting the transcription of IL-1ß, while HMGB1 binds to the TNF-α promoter and recruits RelB complex assembly to downregulate TNF-α transcription [26-27]. It seems clear that transcription factor regulators of myeloid cell activity are highly varied and impact individual gene products allowing a significantly greater degree of control over specific effects of myeloid tolerance as opposed to the widely ranging effects of regulating the TLR4 pathway upstream.
However, over a longer period of stimulation, metabolic shifts in NFAT and mTOR also induce the epigenetic changes that eventually lead to immune tolerance [15-16]. There are three primary links between these metabolic and epigenetic aspects of the cell: Sirtuin1 (Sirt1), alpha-ketoglutarate dependent dioxygenases (αKG-DD), and NuRD10. Activation of Sirt1 occurs at increased concentrations of NAD+, and high dosage LPS exposure drives myeloid cells to upregulate a de novo NAD+ synthesis pathway [17]. Sirt1 is part of the NAD-dependent deacetylase sirtuin family and silences several proinflammatory genes including Tnf and Il1b. Sirt1 also deacetylates RELA, the gene that codes for NF-κB subunit p65 while also binding to RelB, another subunit [18-20]. This SIRT1-RelB complex then serves to assemble a repressor complex that via chromatin methylation later results in endotoxin tolerance18. The second rheostat αKG-DD is a group of epigenetic enzymes whose activity is balanced by αKG and succinate levels in the cell; succinate, as well as various other TCA metabolites including malate, itaconate, fumarate, and 2-hydroxyglutarate, inhibit the activity of these enzymes [21-22]. However, further elucidation of the timing, duration, and relevant metabolites of said TCA cycle shifts to induce epigenetic remodeling by these enzymes is needed. Finally, the NuRD complex is responsible for histone deacetylation and DNA hypermethylation that prevent T-cells from targeting native cells or macrophages from excess inflammation [23]. Upon immune activation, large quantities of reactive oxygen species (ROS) are produced via the electron transport chain; mice with cancer induced immune exhaustion have large quantities of ROS and similar epigenetic changes as to what is induced by NuRD. High levels of mitochondrial ROS also seem to be associated with CD8+ T cell immune exhaustion during TB infection which leads to immune activation via the same Toll-like receptors as LPS [20].
Figure 2: Epigenetic rheostats that link metabolic shifts in the Krebs cycle to immune suppression
CONCLUSION
Although immune tolerance acts as a suppressive mechanism to prevent an excessive inflammatory response, immune tolerance increases the likelihood of secondary infections months following the initial immune challenge; as observed by the long-term morbidity and mortality following chronic infections such as TB and sepsis. Further research considering the possible rescue of immune tolerance phenotypes when cells are exposed to drugs modulating the three aforementioned rheostats should be undertaken to explore the clinical possibilities of reversing immune tolerance and thereby restoring optimal immune function after chronic infection.
BIBLIOGRAPHY
1. Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of Lipopolysaccharide-Induced Signal Transduction in Endotoxin-Tolerized Mouse Macrophages: Dysregulation of Cytokine, Chemokine, and Toll-Like Receptor 2 and 4 Gene Expression. J Immunol. 2000;164(11):5564-5574. doi:10.4049/jimmunol.164.11.5564
2. Cross D, Drury R, Hill J, Pollard AJ. Epigenetics in Sepsis: Understanding Its Role in Endothelial Dysfunction, Immunosuppression, and Potential Therapeutics. Front Immunol. 2019;10:1363. doi:10.3389/fimmu.2019.01363
3. Waitt CJ, Banda P, Glennie S, et al. Monocyte unresponsiveness and impaired IL1β, TNFα and IL7 production are associated with a poor outcome in Malawian adults with pulmonary tuberculosis. BMC Infect Dis. 2015;15:513. doi:10.1186/s12879-015-1274-4
4. DiNardo AR, Rajapakshe K, Nishiguchi T, et al. DNA hypermethylation during tuberculosis dampens host immune responsiveness. J Clin Invest. 130(6):3113-3123. doi:10.1172/JCI134622
5. Wang T, Derhovanessian A, De Cruz S, Belperio JA, Deng JC, Hoo GS. Subsequent Infections in Survivors of Sepsis: Epidemiology and Outcomes. J Intensive Care Med. 2014;29(2):87-95. doi:10.1177/0885066612467162
6. Miller TL, Wilson FA, Pang JW, et al. Mortality Hazard and Survival After Tuberculosis Treatment. Am J Public Health. 2015;105(5):930-937. doi:10.2105/AJPH.2014.302431
7. DiNardo AR, Netea MG, Musher DM. Postinfectious Epigenetic Immune Modifications — A Double-Edged Sword. N Engl J Med. 2021;384(3):261-270. doi:10.1056/NEJMra2028358
8. Biswas SK, Tergaonkar V. Myeloid differentiation factor 88-independent Toll-like receptor pathway: Sustaining inflammation or promoting tolerance? Int J Biochem Cell Biol. 2007;39(9):1582-1592. doi:10.1016/j.biocel.2007.04.021
9. IFNB1 interferon beta 1 [Homo sapiens (human)] - Gene - NCBI. Accessed February 22, 2022. https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSearch=3456
10. Abhimanyu null, Ontiveros CO, Guerra-Resendez RS, et al. Reversing Post-Infectious Epigenetic-Mediated Immune Suppression. Front Immunol. 2021;12:688132. doi:10.3389/fimmu.2021.688132
11. Fan H, Cook JA. Molecular mechanisms of endotoxin tolerance. J Endotoxin Res. 2004;10(2):71-84. doi:10.1179/096805104225003997
12. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th ed. Garland Science; 2002.
13. Learn CA, Boger MS, Li L, McCall CE. The Phosphatidylinositol 3-Kinase Pathway Selectively Controls sIL-1RA Not Interleukin-1β Production in the Septic Leukocytes *. J Biol Chem. 2001;276(23):20234-20239. doi:10.1074/jbc.M100316200
14. Bohuslav J, Kravchenko VV, Parry GC, et al. Regulation of an essential innate immune response by the p50 subunit of NF-kappaB. J Clin Invest. 1998;102(9):1645-1652. doi:10.1172/JCI3877
15. Saeed S, Quintin J, Kerstens HHD, et al. Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity. Science. 2014;345(6204):1251086-1251086. doi:10.1126/science.1251086
16. Zheng L, Leung ETY, Wong HK, et al. Unraveling methylation changes of host macrophages in Mycobacterium tuberculosis infection. Tuberc Edinb Scotl. 2016;98:139-148. doi:10.1016/j.tube.2016.03.003
17. Frontiers | Switch of NAD Salvage to de novo Biosynthesis Sustains SIRT1-RelB-Dependent Inflammatory Tolerance | Immunology. Accessed February 22, 2022. https://www.frontiersin.org/articles/10.3389/fimmu.2019.02358/full
18. NAD+-dependent SIRT1 Deacetylase Participates in Epigenetic Reprogramming during Endotoxin Tolerance* | Elsevier Enhanced Reader. doi:10.1074/jbc.M110.196790
19. NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response* | Elsevier Enhanced Reader. doi:10.1074/jbc.M112.362343
20. Liu TF, Yoza BK, El Gazzar M, Vachharajani VT, McCall CE. NAD+-dependent SIRT1 Deacetylase Participates in Epigenetic Reprogramming during Endotoxin Tolerance. J Biol Chem. 2011;286(11):9856-9864. doi:10.1074/jbc.M110.196790
21. Xu W, Yang H, Liu Y, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell. 2011;19(1):17-30. doi:10.1016/j.ccr.2010.12.014
22. Domínguez-Andrés J, Novakovic B, Li Y, et al. The Itaconate Pathway Is a Central Regulatory Node Linking Innate Immune Tolerance and Trained Immunity. Cell Metab. 2019;29(1):211-220.e5. doi:10.1016/j.cmet.2018.09.003
23. Xue Y, Wong J, Moreno GT, Young MK, Côté J, Wang W. NURD, a Novel Complex with Both ATP-Dependent Chromatin-Remodeling and Histone Deacetylase Activities. Mol Cell. 1998;2(6):851-861. doi:10.1016/S1097-2765(00)80299-3
24. Barish GD, Yu RT, Karunasiri M, et al. Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response. Genes Dev. 2010;24(24):2760-2765. doi:10.1101/gad.1998010
25. Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome | PNAS. Accessed March 26, 2022. https://www.pnas.org/doi/10.1073/pnas.0401866101
26. Chen X, El Gazzar M, Yoza BK, McCall CE. The NF-kappaB factor RelB and histone H3 lysine methyltransferase G9a directly interact to generate epigenetic silencing in endotoxin tolerance. J Biol Chem. 2009;284(41):27857-27865. doi:10.1074/jbc.M109.000950
27. Gazzar ME, Yoza BK, Chen X, Garcia BA, Young NL, McCall CE. Chromatin-Specific Remodeling by HMGB1 and Linker Histone H1 Silences Proinflammatory Genes during Endotoxin Tolerance. Mol Cell Biol. 2009;29(7):1959-1971. doi:10.1128/MCB.01862-08
28.Images created with Biorender