Reversing Post-Infectious Epigenetic-Mediated Immune Suppression

Abstract

The immune response must balance the pro-inflammatory, cell-mediated cytotoxicity with the anti-inflammatory and wound repair response. Epigenetic mechanisms mediate this balance and limit host immunity from inducing exuberant collateral damage to host tissue after severe and chronic infections. However, following treatment for these infections, including sepsis, pneumonia, hepatitis B, hepatitis C, HIV, tuberculosis (TB) or schistosomiasis, detrimental epigenetic scars persist, and result in long-lasting immune suppression. This is hypothesized to be one of the contributing mechanisms explaining why survivors of infection have increased all-cause mortality and increased rates of unrelated secondary infections. The mechanisms that induce epigenetic-mediated immune suppression have been demonstrated in-vitro and in animal models. Modulation of the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR), nuclear factor of activated T cells (NFAT) or nuclear receptor (NR4A) pathways is able to block or reverse the development of detrimental epigenetic scars. Similarly, drugs that directly modify epigenetic enzymes, such as those that inhibit histone deacetylases (HDAC) inhibitors, DNA hypomethylating agents or modifiers of the Nucleosome Remodeling and DNA methylation (NuRD) complex or Polycomb Repressive Complex (PRC) have demonstrated capacity to restore host immunity in the setting of cancer-, LCMV- or murine sepsis-induced epigenetic-mediated immune suppression. A third clinically feasible strategy for reversing detrimental epigenetic scars includes bioengineering approaches to either directly reverse the detrimental epigenetic marks or to modify the epigenetic enzymes or transcription factors that induce detrimental epigenetic scars. Each of these approaches, alone or in combination, have ablated or reversed detrimental epigenetic marks in in-vitro or in animal models; translational studies are now required to evaluate clinical applicability.

Keywords: bioengineering; chronic infections; epigenetics; immune exhaustion; tolerance.

Figure 1

Signaling cascade and transcription factors that mediate epigenetic changes that inhibit host immunity. In T cells, protein kinase Lck and ZAP-70 initiate a signaling cascade that result in activation of PLCγ1 and production of InsP3 (IP3), a second messenger, binding to the InSP3 receptor on the ER leading to release of Ca²⁺ from the ER. The reduction of Ca²⁺ activates STIM, which recruits SOCE such as ORAI in the plasma membrane. Opening of ORAI channels in the plasma membrane results in sustained Ca²⁺ influx and activation of several Ca²⁺ regulated enzymes such as serine/threonine phosphatase calcineurin, which dephosphorylates NFAT enabling its translocation to the nucleus where it binds to promoters of effector genes including Il2. NFAT requires AP-1 generated through another second messenger DAG activation of PKCΦ and RAS/MAPK/ERK1 pathways. Lck also mediates activation of PI3K activating AKT and mTOR, which govern the phosphorylation of FOXO1. Phosphorylated FOXO1 is transported out of the nucleus and exists in complex with 14-3-3 in the cytoplasm. In exhausted T cells, either through activation of inhibitory receptors such as PD-1, CTL4, a dephosphorylating protein SHP1/2 is activated, which dephosphorylates Lck and ZAP70, suppressing the subsequent signaling cascades. SHP2 inhibits among others RAS, AKT, PI3K and even the TCR-MHCII microcluster, thus weakening or abrogating the effector signals at multiple levels (red inhibition arrows). This leads to widespread change in the cellular TF landscape. SHP1/2 activate BATF3, a TF, due to non-availability of AP-1 to partner with NFAT. Partnerless NFAT, alone leads to transcription of inhibitory genes and receptors including pdcd1, which is also transcribed by increased nuclear retention of unphosphorylated FOXO1 in the nucleus, in absence of a PI3K/AKT/mTOR activation. Unpartnered NFAT transcribes, Nr4A and TOX1/2, which further contribute to inhibitory signaling by increasing transcription of Pdcd1. NFAT homodimer transcribes inhibitory genes Grail3/Erg3. TOX leads to transcription of genes such as Id3, Nr4a1, Klrg1, Helios. Many of these genes and TF lead to epigenetic modifications, which further contribute to exhausted phenotype. TF, Ikaros (Helios family) can directly bind to the Il2 promoter and recruit NuRD, which has HDAC and deacetylates Il2 leading to its transcriptional repression. Deacetylation is usually followed by recruitment of PRC, which through EZH2 can further add to closing of chromatin by adding methylation marks at H3K27, as seen at the Ifng locus. Another, NAD: NADH+ dependent deacetylase, SIRT can directly deacetylate NF-κB to decreases IL1 transcription. Lck, LCK proto-oncogene, Src family tyrosine kinase; ZAP-70, zeta chain of T cell receptor associated protein kinase 70; PLC, Phospholipase C; IP3/InsP₃, inositol 1,4,5-trisphosphate; ER, Endoplasmic reticulum; STIM, stromal interaction molecule 1; SOCE, Store-operated calcium entry; ORAI, ORAI calcium release-activated calcium modulator; NFAT, Nuclear factor of activated T-cells; IL, Interleukin; AP-1, Activator protein1; DAG, Di-Acyl Glycerol; PKCΦ, Protein kinase C; MAPK, Mitogen-Activated Protein Kinase; FOXO1, Forkhead box protein O1; 14-3-3, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (encoded by YWHAQ); PD-1/Pdcd, programmed cell death 1; CTL4, Cytotoxic T-Lymphocyte Associated Protein 4; SHP, Src homology 2 domain-containing tyrosine phosphatase 2; PI3K, phosphatidylinositol 3-kinase; AKT, Protein kinase B; mTOR, mammalian target of rapamycin; MHC, Major Histocompatibility complex; TF, Transcription factors; BATF, Basic Leucine Zipper ATF-Like Transcription Factor; Nr4A, Nuclear Receptor Subfamily 4 Group A Member 1; TOX, Thymocyte Selection Associated High Mobility Group Box; Erg, ETS transcription factor ERG; Id3, Inhibitor Of DNA Binding 3; Klrg1, Killer Cell Lectin Like Receptor G1; NuRD, Nucleosome and DNA Remodeling complex; PRC, Polycomb Repressive Complex; EZH2, Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit; H3K27me3, H3 lysine 27 trimethylation; Ac, Acetylation; Me, Methylation. Created with BioRender.com.

Figure 2

Signaling cascade and transcription factors that mediate epigenetic changes that inhibit host immunity in myeloid cells. TLR4 recognizes LPS, and engages the MyD88-TRIF pathway to induce the TFs: NF-κB, AP-1, IRF3, IRF5 which leads to the induction of pro-inflammatory genes such as TNFA, IL1B, IL6, COX2 etc. IRF3 induces the production of IFNβ and TGFβ, which adds to the IFN signaling and induces STAT1 leading to transcription of CCL5, CXCL10 and IRF7. Overwhelming LPS stimulation as seen in sepsis, leads to lesser production and engagement of TLR4 and its pathway components, with over-inflammation leading to production of inhibitory molecules such as IRAK-M, A20, Pellino-3, SHIP, which inhibit various parts of the LPS-TLR4 signaling cascade, leading to a tolerized phenotype. Epigenetically, multiple mechanisms have been shown to lead to and maintenance of the tolerized phenotype. Guided by TFs such as NF-κB and its isoform RelB, which can recruit HDACs (SIRT included) either alone or in a repressome complex, usually with a chromatin modifier such as SWI/SNF results in deacetylation of histones, followed by addition of repressive methylation (H3K9, H3K3) by DNMT such as SMYD5 (in the NCOR-HDAC3 repressome), or KMT such as G9a bound to HMGB1 (can recruit H1 and HP1) to close the chromatin and suppress gene expression. Lineage TFs such as PU.1 provide good example of this assembly of the SWI/SNF complex containing BRG1 which can recruit HAT (p300) to acetylate H4K, HMT (MLL1/2/3) to add permissive H3K4 and demethylase such as JMJD3 to remove repressive H3K27 to activate inflammatory genes upon LPS stimulation. The same PU.1 when bound to co-repressor BCL6 can induce tolerance by losing the SWI/SNF complex and recruitment of NuRD, which recruits HDAC3 to remove acetylation and induce de novo methylation via DNMT1/3B to close the chromatin and thus shutting down inflammatory gene transcription in tolerance. Created with BioRender.com. TLR4, Toll-like receptor 4; LPS, Bacterial Lipopolysaccharide; MyD88, myeloid differentiation factor 88; TRIF, TIR-domain containing adapter-inducing interferon; TF, Transcription factor; STAT1, signal transducer and activator of transcription; IRAK, interleukin-1 receptor-associated kinase; TRAF6, TNF Receptor Associated Factor 6; SHIP; SH2 domain-containing inositol phosphatase 1; IKK, Ikappa B Kinase; TBK, TANK-binding kinase 1; MAPK, Mitogen-Activated Protein Kinase; I-KB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; IRF, Interferon regulatory factors; SWI/SNF, SWItch/Sucrose Non-Fermentable; BRG1, Brahma-related gene-1; HDAC, Histone deacetylase; H1, H1.1 Linker Histone; HP1,Heterochromatin protein-1; HMGB1, High Mobility Group Box 1; DNMT, DNA methyltransferase; MLL, mixed lineage leukemia (lysine methyl transferase); JMJD, Jumonji domain containing protein; BCL6, B-cell lymphoma 6; MBD3, Methyl-CpG Binding Domain Protein 3.

Figure 3

Metabolic intermediates of the TCA cycle guide epigenetic changes that inhibit host immunity. The TCA cycle metabolites act as co-factor for major epigenetic enzymes that shape the epigenomic landscape post infection via three overlapping and redundant major metabolic-epigenetic rheostats (RST). RST1: NAD+: NADH-SIRT: Dependent on the level of NAD+ in the cell, Sirtuins, which are histone deacetylases, can remove acetyl groups and lead to immune suppression; RST2: Succinate-αKG-αKGDD: Dependent on the levels of α−ketoglutarate and succinate (which along with fumarate, malate and Itaconate acts as inhibitors of KGDDs), leads to the activation/inhibition of a family of enzyme dioxygenases which regulate the DNA methylation levels by methylating (via DNMTs) and demethylate (via KDM, JMJD and TET) the DNA; RST3: ROS-PAC1-NuRD: guided by the activation of the ETC, which leads to electron leak and induction of ROS and activation of NuRD, which is multiprotein complex guiding DNA methylation and chromatin accessibility. The main enzymes of the TCA cycle and the ETC are shown, along with the drugs that can be used to block specific enzymes and help with reversing epigenetic mediated Immune suppression. NAD, Nicotinamide adenine dinucleotide; SIRT, Sirtuins; αKGDD, α−ketoglutarate-dependent-dioxygenases; DNMT, DNA methyl transferase; KDM, Histone demethylase; JMJD, Jumonji domain containing protein; ROS, reactive oxygen species; PAC1, Phosphatase of activated cells 1; NuRD, Nucleosome Remodeling and DNA methylation complex; TCA, Tri-carboxylic acid cycle; ETC, Electron transport chain. Created with BioRender.com.