Karen J. Mackenzie

cGAS surveillance of micronuclei links genome instability to innate immunity

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

RNA:DNA hybrids are a novel molecular pattern sensed by TLR9.

The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen‐associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral‐derived sequences efficiently induce pro‐inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88‐dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease.

Inhibition of eosinophil migration by lactoferrin

Eosinophilic granulocytes are innate effector cells that are important in immune responses against helminth parasitic infections and contribute towards the pathology associated with allergic inflammatory conditions, including allergic rhinitis and asthma. Their recruitment to inflammatory sites occurs in response to chemotactic and activation signals, such as eotaxin and interleukin‐5, and is a tightly controlled process. However, the mechanisms that counterbalance these positive chemoattractive processes, thereby preventing excessive eosinophil infiltration, have received little attention. Here, we show that, lactoferrin (LTF), a pleiotropic 80‐kDa glycoprotein with iron‐binding properties, acts as a powerful inhibitor of eosinophil migration. Irrespective of its source (milk or neutrophil derived), LTF inhibits eotaxin‐stimulated eosinophil migration with no effects on eosinophil viability. Transferrin, a closely related cationic glycoprotein, failed to produce an analogous effect. Furthermore, the iron‐saturation status of LTF did not influence the observed inhibitory effect on migration, proving that LTF exerts its effect on eosinophil chemotaxis independent of its iron‐chelating activity. These results highlight LTF as one of the few molecules reported to negatively regulate eosinophil migration. Thus, through its ability to inhibit eosinophil migration, LTF has potential as an effective therapeutic in the control of eosinophil infiltration in atopic inflammatory conditions.

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response.

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2bA174T/A174T knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders.

Cathelicidin Host Defence Peptide Augments Clearance of Pulmonary Pseudomonas aeruginosa Infection by Its Influence on Neutrophil Function In Vivo

Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

How evidence based is the management of two common sports injuries in a sports injury clinic

Objectives: To examine the diagnosis and management of adults attending a sports injury clinic, to establish to what extent the management of the two most common injuries treated at this clinic is evidence based, and to explore factors that affect management. Methods: A retrospective examination of 100 random case notes extracted age, sex, sport, type and site of injury, treatment, and outcome. Systematic literature reviews examined the extent and quality of scientific evidence for the management of the two most commonly presenting injuries. A clinical attachment period and practitioner interviews allowed recognition of factors impinging on management decisions. Results: Patellofemoral pain syndrome (PFPS; 10% of all injuries) and Achilles tendinopathy (6% of all injuries) were the most commonly presenting injuries. The mean (SD) number of treatments used for PFPS was 2.8 (0.9). The mean number of treatments used for Achilles tendinopathy was 3.7 (1.0). Clinicians reported that personal experience formed the basis of management plans in 44% of PFPS cases and 59% of Achilles tendinopathy cases, and that primary research evidence only accounted for 24% of management plans in PFPS and 14% in Achilles tendinopathy. Practitioners were unaware of literature supporting over 50% of the treatment modalities they used. However, clinicians were often using evidence based treatments, unaware of the supporting research data. Conclusions: This study highlights a lack of evidence base, a lack of knowledge of the research evidence, and a lack of management based on the current evidence that is available for these conditions. Practitioners practised evidence based medicine in under 50% of cases.

Novel insights into immune and inflammatory responses to respiratory viruses

Viral lower respiratory tract infection (LRTI) can lead to severe disease at all ages, but with the exception of influenza vaccination, prevention is not available for most respiratory viruses, hence, effective, disease-limiting therapy is urgently required. To enable the development of novel effective therapeutic approaches, we need to improve understanding of the pathological mechanisms of viral LRTI. Here, we will discuss recently gained new insight into early, innate immune and inflammatory responses to respiratory viruses by airway epithelial cells and mucosal immune cells. Following virus recognition, these cells generate a range of mediators, including innate interferons, proinflammatory cytokines, and growth and differentiation factors which have pivotal roles in effective virus control, and the development of inflammation and disease in viral LRTI.

Combination peptide immunotherapy based on T‐cell epitope mapping reduces allergen‐specific IgE and eosinophilia in allergic airway inflammation

Peptide immunotherapy using soluble peptides containing allergen‐derived immunodominant T‐cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323–339) have been unable to demonstrate therapeutic effects in ovalbumin‐induced allergic airway inflammation. We have previously shown that intravenous application of p323–339 can effectively tolerise p323–339‐reactive T cells in a non‐allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323–339 immunotherapy in a C57BL/6 model of ovalbumin‐induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin‐responsive T‐cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin‐reactive T‐cell lines were generated from ovalbumin‐immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin‐induced allergic airway inflammation. Peptide immunotherapy using the 323–339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T‐cell epitope in ovalbumin was identified within the 263–278 sequence. Combination peptide immunotherapy, using the 323–339 and 263–278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin‐specific IgE, with apparent reductions in interleukin‐5 and interleukin‐13. Characterization of the T‐cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

Viral respiratory tract infections and asthma in early life: cause and effect?

Interactions between viral respiratory tract infections in infancy and childhood, and asthma development and exacerbation, are complex and intriguing. This review aims to unravel some of these complexities. Does severe respiratory viral infection early in life predispose to later asthma development, or is it indicative of a predisposition to allergic respiratory disease? How could variables such as age and severity of viral infection affect the interaction between respiratory viral infections and asthma? How could respiratory viral infection drive allergic sensitization? Here, we review the evidence surrounding these questions, and discuss current and future research and therapeutic approaches targeting the interplay between viral respiratory tract infection and asthma.