Genotoxic stress signalling as a driver of macrophage diversity

Abstract

Tissue macrophages arise from yolk sac, fetal liver and hematopoietic progenitors and adopt diverse transcriptional programs and phenotypes, instructed by their microenvironment. In chronic inflammation, such as in chronic infections, autoimmunity, or cancer, tissue microenvironments change dramatically thus imprinting new programs on tissue macrophages. While stress is a known driver of carcinogenesis in epithelial cells, emerging evidence suggests that macrophage responses to genotoxic stress are embedded in their 'physiologic' immune and tissue healing programs and in most cases do not lead to myeloid malignancies. The role of genotoxic stress as an instructor of macrophage-mediated immune defense and tissue remodeling is only beginning to be understood. Here, we review the evidence showing that genotoxic stress, which macrophages and their precursors face upon encountering inflammatory and/or growth signals, instructs their transcriptional programs, by activating non-canonical, cell-type specific DNA Damage Response (DDR)-driven signaling pathways. We propose that immune-cell specific, DDR-instructed programs are crucial for tissue homeostasis as well as for the maintenance and resolution of inflammatory responses in infection, cancer, autoinflammatory and autoimmune microenvironments.

Keywords: ATM; ATR; DNA damage; chronic inflammation; innate immunity; macrophage programs.

Figure 1. FIGURE 1: The DNA Damage Response.

There are two major arms to the DDR, one designed to deal with double strand breaks (DSBs), which are sensed by the MRN (Mre11, Rad50 and Nbs1) complex, alerting the DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and ATM (ataxia-telangiectasia mutated) kinases, which further phosphorylate Chk2 (checkpoint kinase 2), and the other one sensing single strand breaks (SSBs) and replication stress through RPA (Replication protein A), activating the ATR (Ataxia telangiectasia and Rad3 related)-Chk1 (checkpoint kinase 1) axis and both arms phosphorylating γH2AX (phosphorylated H2A histone family member X) and 53BP1 (p53-binding protein 1). Major regulators of cell fate such as p53 and cell cycle regulators (for instance cell division cycle 25 phosphatase (CDC25)) get activated and these events, based on the severity of the damage lead to either DNA repair, cell cycle arrest to allow the cell to repair this damage. If damage is substantial and beyond repair, the cell will either go into senescence (permanent cell cycle arrest) or apoptosis (cell death). Alternatively, these pathways can activate non-canonical programs that drive the cell into re-programming and differentiation.

Figure 2. FIGURE 2: RAG introduce programmed dsDNA breaks during the assembly and diversification of lymphocyte antigen receptor gene may promote cellular fitness of cytotoxic lymphocytes.

(A) RAG (recombination-activating genes)-induced dsDNA breaks activate DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and ATM (ataxia-telangiectasia mutated), initiating a canonical DDR that leads to double strand break repair by NHEJ (non-homologous end joining), while ATM signaling additionally induces a non-canonical DDR that regulates cell type-specific, developmental programs in B cells in the bone marrow. (B) In mature B cells, antigen stimulation and co-stimulatory signals induce AID (activation-induced deaminase) – dependent breaks that activate ATM, initiating a non-canonical DDR that promotes activated B cell receptor diversification in germinal centers, employing several repair pathways, such as mismatch repair (MMR). (C) RAG-induced dsDNA breaks upregulate the expression of DDR-related genes, such as Atm (encoding ataxia telangextasia mutated), Chek2 (encoding checkpoint kinase 2) and Prkdc (encoding DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)) in NK and CD8 T cells, which may increase their ability to deal with genomic instability upon virus-induced proliferation and cellular stress, thus facilitating their population expansion and cytotoxic effector functions.

Figure 3. FIGURE 3: ATM regulates macrophage responses to MCSF, LPS and IFN-γ.

(A) MCSF (macrophage colony stimulating factor) induces macrophage proliferation and differentiation, while activating type I interferon (IFN) responsive genes as well as potentially introducing genotoxic stress sensed by the MRE complex. This in turn downregulates ROS (reactive oxygen species) and activates downstream players of the DDR (such as ATM), thus suppressing genomic instability and promoting population expansion. Activated ATM in turn suppresses ssDNA accumulation in the cytoplasm and thus cGAS (Cyclic GMP-AMP synthase) - STING (Stimulator Of Interferon Response cGAMP Interactor)-induced IFNβ activation and interferon responsive genes. (B) At steady state MCSF upregulates type I IFN responsive genes by activating DNA-PK, STING and subsequently IRF3, while ATM itself suppresses the type I IFN response. Upon irradiation, increased expression of type I IFN responsive genes requires ATM-mediated IRF1 activation. Upon irradiation or following stimulation with IFNγ, ROS-induced ATM activation additionally leads to IRF5 upregulation, thus further activating type 1 IFN responses. (IRF-Interferon Regulatory Factor, ISG-Interferon Signature Genes). (C) Bacterial infection as well as stimulation with LPS and IFN-γ in BMDMs cause genotoxic stress, through the generation of ROS and reactive nitrogen species (RNS), thus activating ATM and DNA-PKcs in a type I IFN dependent manner. This leads to the upregulation of an antimicrobial macrophage genetic program as well as inflammasome activation.

Figure 4. FIGURE 4: Nucleic acid metabolic pathways control antiviral programs and autoinflammation and may have genome-protecting functions by suppressing cytoplasmic cGAS activation and type I interferon responses.

(A) Nucleic acid-metabolizing enzymes SAMHD1 (SAM domain and HD domain-containing protein 1), TREX1 (three prime repair exonuclease 1), RNASEH2A-C (Ribonuclease H2, Subunit A, C-Term), and ADAR (Adenosine Deaminase RNA Specific), control systemic type I interferon (IFN) levels by negatively regulating the cGAS-STING pathway. Unphosphorylated SAMHD1 restricts viral replication in myeloid cells either by depleting the dNTP pool or through its RNAse activity. This antiviral activity of SAMHD1 is suppressed upon SAMHD1 phosphorylation by cyclin A2/Cdk1 in cycling cells, while genotoxic stress that leads to p53 and p21 upregulation and subsequently CDK1/2 (cyclin-dependent kinase 1/2) suppression and SAMHD1 dephosphorylation increase this antiviral activity. (B) SAMHD1 promotes degradation of ssDNA at stalled replication forks by stimulating the exonuclease activity of MRE11 and thus activating ATR-CHK1 to alleviate replication stress. This prevents ssDNA accumulation in the cytosol where they would activate cGAS-STING leading to increased expression of type I interferons. TREX1 (three prime repair exonuclease 1) also promotes the removal of ssDNA molecules (formed due to replication stress) through RPA70 (Replication protein A 70 kDa DNA-binding subunit) and Rad51, thus preventing leakage of ssDNA to the cytosol and phosphorylation of IRF3. TREX1 also limits cGAS activation at micronuclei, by degrading micronuclear DNA. Replication stress, ssDNA and micronuclei promote the cGAS – STING – type I IFN activation pathway.

Figure 5. FIGURE 5: The DNA damage response may control multinucleated macrophage cell fitness.

(A) In osteoclasts RANK (Receptor Activator of NF-κB) signaling activates the NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) pathway promoting survival, an effect inhibited by ATM, leading to increased apoptosis. (B) Chronic bacterial infection and TLR2 ligands upregulate c-MYC and induce replication stress, which causes cytokinesis failure. ATR promotes growth, survival and genomic stability of polyploid macrophages in granulomatous microenvironments.