Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H 2 O 2 , plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H 2 O 2 , greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H 2 O 2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniaeinduced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.DNA damage | Streptococcus pneumoniae | hydrogen peroxide | γH2AX | Ku80

Seasonal influenza virus infections may lead to debilitating disease, and account for significant fatalities annually worldwide. Most of these deaths are attributed to the complications of secondary bacterial pneumonia. Evidence is accumulating to support the notion that neutrophil extracellular traps (NETs) harbor several antibacterial proteins, and trap and kill bacteria. We have previously demonstrated the induction of NETs that contribute to lung tissue injury in severe influenza pneumonia. However, the role of these NETs in secondary bacterial pneumonia is unclear. In this study, we explored whether NETs induced during pulmonary influenza infection have functional significance against infections with Streptococcus pneumoniae and other bacterial and fungal species. Our findings revealed that NETs do not participate in killing of Streptococcus pneumoniae in vivo and in vitro. Dual viral and bacterial infection elevated the bacterial load compared to animals infected with bacteria alone. Concurrently, enhanced lung pathogenesis was observed in dual-infected mice compared to those challenged with influenza virus or bacteria alone. The intensified NETs in dual-infected mice often appeared as clusters that were frequently filled with partially degraded DNA, as evidenced by punctate histone protein staining. The severe pulmonary pathology and excessive NETs generation in dual infection correlated with exaggerated inflammation and damage to the alveolar-capillary barrier. NETs stimulation in vitro did not significantly alter the gene expression of several antimicrobial proteins, and these NETs did not exhibit any bactericidal activity. Fungicidal activity against Candida albicans was observed at similar levels both in presence or absence of NETs. These results substantiate that the NETs released by primary influenza infection do not protect against secondary bacterial infection, but may compromise lung function.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5 , ATG7 , and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial (mt)DNA-dependent induction of type I interferon (IFN-I). Here, we show that mice lacking the GTPase IRGM1 (IRGM homologue) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired execution of mitophagy with cell-specific consequences. In fibroblasts, mtDNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)–Stimulator of Interferon Genes (STING)-dependent IFN-I, whereas in macrophages, lysosomal TLR7 was activated. In vivo , Irgm1 −/− tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology were abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe Influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of two weeks in vivo. We show that influenza induces DNA damage both when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage, persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67 positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DSB repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.

MicroRNAs (miRNAs) 3 are small (ϳ22-nucleotide) noncoding RNAs that regulate gene expression by binding to partially complementary sites in the 3Ј-untranslated regions (UTRs) of specific mRNAs, thereby promoting degradation and/or repressing translation of target mRNAs. The human genome contains Ͼ2500 unique mature miRNAs (1). Because individual miRNAs typically have multiple targets, it is thought that Ͼ60% of all human genes may be subject to regulation by miRNAs (2). The promiscuity of miRNAs for target RNAs is expected to represent a fundamental mechanism of cross-talk and coordination among signaling networks in development, health, and disease.Among the signaling networks that have been shown in recent years to be regulated by miRNAs are the Toll-like receptors (TLRs) of the innate immune system. Multiple TLRs up-regulate miRNAs, including miR-155, miR-146, miR-21, miR-147, and miR-9, whereas activation of TLR4 by lipopolysaccharide (LPS) down-regulates a distinct set of miRNAs (3, 4). In turn, miRNAs "fine tune" the proinflammatory signaling output of TLR cascades by controlling the expression of TLR pathway members (3, 4). Thus, miR-146 suppresses signaling by multiple TLRs via targeting the common signaling hubs IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 (5), whereas miR-155 has complex effects, repressing the TLR adaptor myeloid differentiation primary response 88 (MyD88) (6, 7) and TAK1-binding protein 2 (8), an upstream activator of the mitogen-activated protein kinases, but also promoting cytokine expression through actions on other targets (3). Although virtually all known examples of TLR regulation by miRNAs operate through direct targeting of TLR pathway components, it is expected that miRNAs may also indirectly impact the innate immune response by regulating other networks that cross-talk with TLRs.miR-33a and miR-33b (present in primates but absent in rodents and lower species in which miR-33a is simply referred to as "miR-33") are now known to be master regulators of cho-

Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection.

Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.

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