At the intersection of DNA damage and immune responses

Abstract

DNA damage occurs on exposure to genotoxic agents and during physiological DNA transactions. DNA double-strand breaks (DSBs) are particularly dangerous lesions that activate DNA damage response (DDR) kinases, leading to initiation of a canonical DDR (cDDR). This response includes activation of cell cycle checkpoints and engagement of pathways that repair the DNA DSBs to maintain genomic integrity. In adaptive immune cells, programmed DNA DSBs are generated at precise genomic locations during the assembly and diversification of lymphocyte antigen receptor genes. In innate immune cells, the production of genotoxic agents, such as reactive nitrogen molecules, in response to pathogens can also cause genomic DNA DSBs. These DSBs in adaptive and innate immune cells activate the cDDR. However, recent studies have demonstrated that they also activate non-canonical DDRs (ncDDRs) that regulate cell type-specific processes that are important for innate and adaptive immune responses. Here, we review these ncDDRs and discuss how they integrate with other signals during immune system development and function.

Fig. 1:|. V(D)J recombination.

aThe initial step of the V(D)J recombination reaction is the synapsis of two gene segments (variable (V) to joining (J) recombination is shown) and their flanking recombination signal sequences (RSSs; triangles) with recombination-activating gene 1 (RAG1) and RAG2 proteins, which recognize the RSSs. b | DNA cleavage by RAG proteins leads to the generation of a blunt signal end and a hairpin-sealed coding end at each RAG-mediated DNA double-strand break (DSB). c | The two coding ends are then joined to form a coding join that completes the second exon of the antigen receptor gene. This joining is imprecise, with a gain and/or loss of nucleotides at the join leading to only one-third of completed antigen receptor genes being in-frame and productive. The signal ends are joined somewhat precisely to form a signal join. Binding of KU70, KU80 and MRE11–RAD50–NBS1 (MRN) to RAG DSBs activates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) kinases, respectively. ATM and DNA-PKcs initiate a canonical DNA damage response (cDDR) to promote RAG DSB repair. ATM also activates non-canonical DNA damage response (ncDDR) signalling, which regulates B cell developmental programmes. NHEJ, non-homologous end joining.

Fig. 2:|. B cell development.

B cell development proceeds in the bone marrow through several discrete steps. From a standpoint of DNA double-strand break (DSB) generation and potential non-canonical DNA damage response (ncDDR) signalling, these stages can be generally divided into the pro-B cell stage in which Igh gene assembly occurs in all cells, the pre-B cell stage in which Igh gene assembly occurs in all cells and the immature B cell stage in which receptor editing occurs in some cells. DNA DSBs are also generated in mature B cells once they are activated and initiate class switch recombination (CSR). AID, activation-induced deaminase; APE1, apurinic/apyrimidinic endonuclease 1; BCR, B cell receptor; RAG, recombination-activating gene; UNG, uracil-DNA glycosylase.

Fig. 3:|. Large pre-B cell to small pre-B cell transition.a

In large pre-B cells, IL-7 receptor (IL-7R) signalling activates AKT and the Janus kinase (JAK)–signal transducer and activator of transcription 5 (STAT5) pathway. This inhibits Iglk gene transcription and accessibility, inhibits recombination-activating gene (RAG) expression by causing degradation of forkhead box protein O1 (FOXO1) and promotes proliferation and survival of large pre-B cells, in part, through inhibition of the apoptosis regulator BAX by PIM1-mediated and AKT-mediated phosphorylation of BCL-2-associated agonist of cell death (BAD). b | In small pre-B cells, loss of IL-7R signalling leads to increased activity of tyrosine protein kinase SYK and B cell linker protein (BLNK), which augments signalling through the pre-B cell receptor (pre-BCR), leading to the induction of interferon regulatory factor 4 (IRF4) expression and induction of Iglk gene transcription and accessibility. Loss of AKT signals results in FOXO1-driven expression of Rag1 and Rag2 and loss of IL-7R-mediated proliferative and survival signals. Pre-BCR signals can also drive proliferation, although not as strongly as IL-7R signals. c | The initiation of Iglk gene rearrangement and the generation of RAG double-strand breaks (DSBs) leads to the activation of ataxia telangiectasia mutated (ATM), which initiates both a canonical DNA damage response (cDDR) and a non-canonical DNA damage response (ncDDR). The ncDDR is mediated, in part, by the activation of a nuclear factor-κB1 (NF-κB1)-dependent and NF-κB2-dependent genetic programme. The induction of SPIC represses Iglk gene transcription and antagonizes pre-BCR signals by inhibiting SYK and BLNK expression. This ncDDR genetic programme also promotes survival through the induction of PIM2, which phosphorylates BAD to inhibit BAX. p53 and PIM2 also enforce the G1 to S checkpoint. ATM activation leads to reduced Rag expression through inhibition of growth arrest and DNA damage-inducible protein GADD45α expression and through direct phosphorylation and inhibition of RAG2. RAG DSBs also trigger an ncDDR that inhibits the production of IL-7 by local stromal cells. Collectively, these ncDDR pathways prevent the generation of additional RAG DSBs, promote survival and block proliferation in cells with existing RAG DSBs. P, phosphorylation.

Fig. 4:|. ordered assembly of antigen receptor genes.

Antigen receptor gene assembly at several loci is ordered inter-allelically, such that one allele rearranges at a time. Once completed, the rearrangement is tested to determine whether it is productive before rearrangement at the alternative allele occurs. This inter-allelic ordering is critical for enforcing allelic exclusion. Antigen receptor gene assembly is also intra-allelically ordered. After a rearrangement is initiated on one allele, the initiation of additional rearrangements on the same allele must be prevented. Cleavage events at an antigen receptor loci mediated by recombination-activating gene (RAG) proteins are regulated by accessibility of the loci. However, once RAG double-strand breaks (DSBs) are made, the non-canonical DNA damage response (ncDDR) participates in enforcing both inter-allelic and intra-allelic ordering to prevent additional RAG DSBs from being generated until the initial rearrangement has been completed and tested to determine whether it encodes a functional antigen receptor gene. ATM, ataxia telangiectasia mutated.

Fig. 5:|. The toggle model.

Small pre-B cells have two signalling states; first, pre-B cell receptor (pre-BCR) signalling that promotes Igl gene rearrangement and, second, signalling via ataxia telangiectasia mutated (ATM), in response to double-strand breaks (DSBs) generated by recombination-activating gene (RAG) proteins, that activates a non-canonical DNA damage response (ncDDR). The ncDDR suppresses additional RAG DSBs until the Igl gene assembly is complete and is tested to determine whether it encodes a functional IgL chain. Small pre-B cells may iteratively toggle between these two signalling states as they undergo multiple rounds of Igl chain gene rearrangements in an ordered manner as they attempt to generate a functional IgL chain.

Fig. 6:|. Immunoglobulin class switch recombination.

Immunoglobulin class switch recombination (CSR) is initiated by transcription through the switch μ (Sμ) region and a downstream switch region to which switching will occur (shown here to be S_γ3). Activation-induced deaminase (AID) targets the non-template single-strand DNA (ssDNA), deaminating cytosine to form uridine. The proteins uracil-DNA glycosylase (UNG) and apurinic/apyrimidinic endonuclease 1 (APE1) of the base excision repair pathway then deglycosylate and excise the uridine to generate a single-strand nick. The same reaction occurs on the template strand, although less efficiently, and the formation of closely staggered nicks on the two DNA strands leads to a DNA double-strand break (DSB). Broken DNA ends from the two switch regions are then joined by either classical or alternative non-homologous end joining. CSR DSBs activate ataxia telangiectasia mutated (ATM), initiating a non-canonical DNA damage response (ncDDR) that regulates CSR and B cell differentiation. Phosphorylation of AID by ATM in response to CSR DSBs promotes the association of APE1 with AID, leading to more efficient nick formation. ATM activity promotes the association of the two switch regions with DSBs. ATM activates the kinase LKB1, which inhibits CREB-regulated transcription co-activator 2 (CRTC2), a transcriptional co-activator of cAMP-responsive element-binding protein 1 (CREB1), promoting a plasma cell genetic programme. cDDR, canonical DNA damage response; dsDNA, double-strand DNA; P, phosphorylation.

Fig. 7:|. Activation of the ncDDR in macrophages.

Macrophages activated by signals through Toll-like receptors (TLRs) and interferon receptors produce nitric oxide (NO), which causes genomic DNA double-strand breaks (DSBs) that activate ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), leading to a canonical DNA damage response (cDDR) and a non-canonical DNA damage response (ncDDR). The ncDDR promotes a broadly functional genetic programme in these cells and regulates inflammasome activation. Type I interferon signals are required for optimal cDDR and ncDDR activation. NOS2, nitric oxide synthase.