Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.

Eosinophils are predominantly known for their contribution to allergy. Here, we have examined the function and regulation of gastrointestinal eosinophils in the steady-state and during infection with or We find that eosinophils are recruited to sites of infection, directly encounter live bacteria, and activate a signature transcriptional program; this applies also to human gastrointestinal eosinophils in humanized mice. The genetic or anti-IL-5-mediated depletion of eosinophils results in improved control of the infection, increased inflammation, and more pronounced Th1 responses. Eosinophils control Th1 responses via the IFN-γ-dependent up-regulation of PD-L1. Furthermore, we find that the conditional loss of IFN-γR in eosinophils phenocopies the effects of eosinophil depletion. Eosinophils further possess bactericidal properties that require their degranulation and the deployment of extracellular traps. Our results highlight two novel functions of this elusive cell type and link it to gastrointestinal homeostasis and anti-bacterial defense.

SummaryTo improve replication fidelity, mismatch repair (MMR) must detect non-Watson-Crick base pairs and direct their repair to the nascent DNA strand. Eukaryotic MMR in vitro requires pre-existing strand discontinuities for initiation; consequently, it has been postulated that MMR in vivo initiates at Okazaki fragment termini in the lagging strand and at nicks generated in the leading strand by the mismatch-activated MLH1/PMS2 endonuclease. We now show that a single ribonucleotide in the vicinity of a mismatch can act as an initiation site for MMR in human cell extracts and that MMR activation in this system is dependent on RNase H2. As loss of RNase H2 in S.cerevisiae results in a mild MMR defect that is reflected in increased mutagenesis, MMR in vivo might also initiate at RNase H2-generated nicks. We therefore propose that ribonucleotides misincoporated during DNA replication serve as physiological markers of the nascent DNA strand.

The gastric lamina propria is largely uncharted immunological territory. Here we describe the evolution and composition of the gastric, small intestinal, and colonic lamina propria mononuclear phagocyte system during the steady state and infection with the gastric pathogen Helicobacter pylori. We show that monocytes, CXCR1 macrophages, and CD11b dendritic cells are recruited to the infected stomach in a CCR2-dependent manner. All three populations, but not BATF3-dependent CD103 DCs, sample red fluorescent protein (RFP)Helicobacter pylori (H. pylori). Mice reconstituted with human hematopoietic stem cells recapitulate several features of the myeloid cell-H. pylori interaction. The differentiation in and/or recruitment to gastrointestinal, lung, and lymphoid tissues of CD11b DCs requires NLRP3, but not apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC) or caspase-1, during steady-state and chronic infection. NLRP3 mice fail to generate Treg responses to H. pylori and control the infection more effectively than wild-type mice. The results demonstrate a non-canonical inflammasome-independent function of NLRP3 in DC development and immune regulation.

The depletion of eosinophils represents an efficient strategy to alleviate allergic asthma, but the consequences of prolonged eosinophil deficiency for human health remain poorly understood. We show here that the ablation of eosinophils severely compromises antitumor immunity in syngeneic and genetic models of colorectal cancer (CRC), which can be attributed to defective Th1 and CD8+ T cell responses. The specific loss of GM-CSF signaling or IRF5 expression in the eosinophil compartment phenocopies the loss of the entire lineage. GM-CSF activates IRF5 in vitro and in vivo and can be administered recombinantly to improve tumor immunity. IL-10 counterregulates IRF5 activation by GM-CSF. CRC patients whose tumors are infiltrated by large numbers of eosinophils also exhibit robust CD8 T cell infiltrates and have a better prognosis than patients with eosinophillow tumors. The combined results demonstrate a critical role of eosinophils in tumor control in CRC and introduce the GM-CSF–IRF5 axis as a critical driver of the antitumor activities of this versatile cell type.

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The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103 + dendritic cells (DCs). We show here that CD103 + DCs are strictly required for gastric Th1 responses to H . pylori and for H . pylori infection control. A similar dependence of type 1 immunity on CD103 + DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3 + effector and regulatory T-cells to target tissues and in their local expansion.

DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.