Body composition and body mass index measures from 8 to 18 years old in children with cystic fibrosis.

BACKGROUND: Historically, body mass index (BMI) >50th percentile has represented optimal nutritional status in children with cystic fibrosis (CF) due to its positive association with lung function. Body composition parameters including fat-free mass index (FFMI) have been suggested as a more physiological nutrition benchmark. AIMS: (1) describe changes in body composition with age and gender; (2) assess the correlation between measures of nutritional status (FFMI-z, FMI-z, BMI-z) and lung function (forced expiratory volume in one second predicted; FEV1pp). METHODS: This retrospective, mixed cross-sectional and serial measures study consisted of children with CF (8 to 18 years) attending Sydney Children's Hospital (2007-2020). FFMI and fat mass index (FMI) were taken from biennial dual energy x-ray absorptiometry (DXA) scans. Z-scores were derived using Well's reference population [1]. Repeated measures correlation analyses assessed correlations between FFMI-z, FMI-z, and BMI-z with FEV1pp. RESULTS: 339 DXA reports were analysed from 137 patients. There were slight downwards trends in BMI-z and FMI-z, and an upwards trend in FFMI-z with increasing age and across both genders. Females had higher FMI-z and FFMI-z than males from 12.5 years. There was a weak, positive correlation between FEV1pp and BMI-z (r = 0.14, p = 0.04), and FFMI-z (r = 0.25, p<0.001). FMI-z had no correlation with FEV1pp (r=-0.06, p = 0.41). CONCLUSION: Deficits in FFMI exist despite increasing trends with age. FFMI-z and BMI-z had a weak, positive correlation with FEV1pp. In contemporary cohorts, nutritional status (reflected by surrogate markers such as FFMI and BMI) may be less influential upon lung function than in previous decades. [1]: Wells, J.C., et al. Body-composition reference data for simple and reference techniques and a 4-component model: a new UK reference child. Am. J. Clin. Nutr.96, 1316-1326 (2012).

The impact of skin cancer prevention efforts in New South Wales, Australia: Generational trends in melanoma incidence and mortality.

BACKGROUND: In Australia, skin cancer awareness campaigns have focused on raising the awareness and consequences of skin cancer and highlighting the importance of utilising sun protection. METHODS: Trends in melanoma incidence and mortality have been explored elsewhere in Australia and this study sought to examine the trends in NSW. Anonymised incidence and mortality data for in situ and invasive melanoma from 1988 to 2014 were obtained from the NSW Cancer Registry. Trends of melanoma incidence and mortality were analysed using segmented regression to allow for changes over time. Birth cohort patterns were assessed using age-period-cohort models. RESULTS: Over the period, incidence of in situ melanoma increased in all age groups although the rates were lowest in those under 40 years of age. Incidence of invasive melanoma was either stable or decreased in people under 60, while it increased in those aged 60 and above, particularly in men. Age-period-cohort analysis revealed decreasing age-specific incidence of invasive melanoma under 40 years of age. Melanoma mortality over the period was stable or decreased in all groups except in men aged 60 or over. Overall, mortality rates generally declined or remained stable particularly in recent years. CONCLUSION: It is encouraging that rates of invasive melanoma are declining in the younger age cohorts - which could be attributed to both primary prevention efforts with individuals protecting their skin as well as early detection through self assessment and clinician performed skin checks. In addition, whilst it is important to monitor the increasing rates of in situ melanoma, the increase is likely due to early detection and treatment of melanoma that could have progressed to invasive melanoma and therefore detection whilst still in situ is an improved outcome. Overall, the results demonstrate the need to continue to improve the understanding of and compliance with primary skin cancer prevention measures in order to reduce population UVR exposure and overall melanoma incidence.

A Flow Cytometric Assay to Detect Functional Ganglionic Acetylcholine Receptor Antibodies by Immunomodulation in Autoimmune Autonomic Ganglionopathy.

Autoimmune Autonomic Ganglionopathy (AAG) is an uncommon immune-mediated neurological disease that results in failure of autonomic function and is associated with autoantibodies directed against the ganglionic acetylcholine receptor (gnACHR). The antibodies are routinely detected by immunoprecipitation assays, such as radioimmunoassays (RIA), although these assays do not detect all patients with AAG and may yield false positive results. Autoantibodies against the gnACHR exert pathology by receptor modulation. Flow cytometric analysis is able to determine if this has occurred, in contrast to the assays in current use that rely on immunoprecipitation. Here, we describe the first high-throughput, non-radioactive flow cytometric assay to determine autoantibody mediated gnACHR immunomodulation. Previously identified gnACHR antibody seronegative and seropositive sera samples (RIA confirmed) were blinded and obtained from the Oxford Neuroimmunology group along with samples collected locally from patients with or without AAG. All samples were assessed for the ability to cause gnACHR immunomodulation utilizing the prototypical gnACHR expressing cell line, IMR-32. Decision limits were calculated from healthy controls, and Receiver Operating Characteristic (ROC) curves were constructed after unblinding all samples. One hundred and ninety serum samples were analyzed; all 182 expected negative samples (from healthy controls, autonomic disorders not thought to be AAG, other neurological disorders without autonomic dysfunction and patients with Systemic Lupus Erythematosus) were negative for immunomodulation (<18%), as were the RIA negative AAG and unconfirmed AAG samples. All RIA positive samples displayed significant immunomodulation. There were no false positive or negative samples. There was perfect qualitative concordance as compared to RIA, with an Area Under ROC of 1. Detection of Immunomodulation by flow cytometry for the identification of gnACHR autoantibodies offers excellent concordance with the gnACHR antibody RIA, and overcomes many of the shortcomings of immunoprecipitation assays by directly measuring the pathological effects of these autoantibodies at the cellular level. Further work is needed to determine the correlation between the degree of immunomodulation and disease severity.

Conspicuous animal signals avoid the cost of predation by being intermittent or novel: confirmation in the wild using hundreds of robotic prey.

Social animals are expected to face a trade-off between producing a signal that is detectible by mates and rivals, but not obvious to predators. This trade-off is fundamental for understanding the design of many animal signals, and is often the lens through which the evolution of alternative communication strategies is viewed. We have a reasonable working knowledge of how conspecifics detect signals under different conditions, but how predators exploit conspicuous communication of prey is complex and hard to predict. We quantified predation on 1566 robotic lizard prey that performed a conspicuous visual display, possessed a conspicuous ornament or remained cryptic. Attacks by free-ranging predators were consistent across two contrasting ecosystems and showed robotic prey that performed a conspicuous display were equally likely to be attacked as those that remained cryptic. Furthermore, predators avoided attacking robotic prey with a fixed, highly visible ornament that was novel at both locations. These data show that it is prey familiarity-not conspicuousness-that determine predation risk. These findings replicated across different predator-prey communities not only reveal how conspicuous signals might evolve in high predation environments, but could help resolve the paradox of aposematism and why some exotic species avoid predation when invading new areas.

Meta-analysis of prospective studies evaluating breast cancer detection and interval cancer rates for digital breast tomosynthesis versus mammography population screening.

INTRODUCTION: Breast cancer (BC) screening using digital breast tomosynthesis (DBT) has been shown to increase cancer detection compared with mammography; however, it is unknown whether DBT impacts interval cancer rate (ICR). METHODS: We systematically identified prospective DBT studies reporting data on screen-detected and interval BCs to perform a study-level meta-analysis of the comparative effect of DBT on ICR in population screening. Meta-analysis of cancer detection rate (CDR), ICR, and the differences between DBT and mammography in CDR and ICR pooled estimates, included random-effects. Sensitivity analysis examined whether study methods (imaging used, comparison group design, interval BC ascertainment) affected pooled estimates. RESULTS: Five eligible prospective (non-randomised) studies of DBT population screening reported on 129,969 DBT-screened participants and 227,882 mammography-only screens, including follow-up publications reporting interval BC data. Pooled CDR was 9.03/1000 (95% confidence interval [CI] 8.53-9.56) for DBT, and 5.95/1000 (95% CI 5.65-6.28) for mammography: the pooled difference in CDR was 3.15/1000 (95% CI 2.53-3.77), and was evident for the detection of invasive and in-situ malignancy. Pooled ICR was 1.56/1000 DBT screens (95% CI 1.22-2.00), and 1.75/1000 mammography screens (95% CI 1.46-2.11): the estimated pooled difference in ICR was -0.15/1000 (95% CI -0.59 to 0.29) and was not substantially altered in several sensitivity analyses. CONCLUSIONS: Meta-analysis shows consistent evidence that DBT significantly increased CDR compared with mammography screening; however, there was little difference between DBT and mammography in pooled ICR. This could suggest, but does not demonstrate, some over-detection. Meta-analysis using individual participant data, randomised trials and comparative studies quantifying cumulative detection and ICR over repeat DBT screen-rounds would provide valuable evidence to inform screening programs.

Chronic disease prevention programs offered by Aboriginal Community Controlled Health Services in New South Wales, Australia.

OBJECTIVES: To identify and describe chronic disease prevention programs offered by Aboriginal Community Controlled Health Services (ACCHSs) in New South Wales (NSW), Australia. METHODS: ACCHSs were identified through the Aboriginal Health and Medical Research Council of NSW website. Chronic disease programs were identified from the Facebook page and website of each ACCHS. Characteristics, including regions, target population, condition, health behaviour, modality and program frequency were extracted and summarised. RESULTS: We identified 128 chronic disease programs across 32 ACCHSs. Of these, 87 (68%) programs were broad in their scope, 20 (16%) targeted youth, three (2%) targeted Elders, 16 (12%) were for females only and five (4%) were for males only. Interventions included physical activity (77, 60%), diet and nutrition (74, 58%), smoking (70, 55%), and the Aboriginal and Torres Strait Islander Health Check (44, 34%), with 93 programs (73%) of ongoing duration. CONCLUSIONS: Chronic disease prevention programs address chronic conditions by promoting physical activity, diet and nutrition, smoking cessation and health screening. Most target the general Aboriginal community, a few target specific groups based on gender and age, and more than one-quarter are time-limited. Implications for public health: Chronic disease programs that are co-produced with specific groups, based on age and gender, may be needed.

Survival after Kidney Transplantation during Childhood and Adolescence.

BACKGROUND AND OBJECTIVES: Survival in pediatric kidney transplant recipients has improved over the past five decades, but changes in cause-specific mortality remain uncertain. The aim of this retrospective cohort study was to estimate the associations between transplant era and overall and cause-specific mortality for child and adolescent recipients of kidney transplants. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Data were obtained on all children and adolescents (aged <20 years) who received their first kidney transplant from 1970 to 2015 from the Australian and New Zealand Dialysis and Transplant Registry. Mortality rates were compared across eras using Cox regression, adjusted for confounders. RESULTS: A total of 1810 recipients (median age at transplantation 14 years, 58% male, 52% living donor) were followed for a median of 13.4 years. Of these, 431 (24%) died, 174 (40%) from cardiovascular causes, 74 (17%) from infection, 50 (12%) from cancer, and 133 (31%) from other causes. Survival rates improved over time, with 5-year survival rising from 85% for those first transplanted in 1970-1985 (95% confidence interval [95% CI], 81% to 88%) to 99% in 2005-2015 (95% CI, 98% to 100%). This was primarily because of reductions in deaths from cardiovascular causes (adjusted hazard ratio [aHR], 0.25; 95% CI, 0.08 to 0.68) and infections (aHR, 0.16; 95% CI, 0.04 to 0.70; both for 2005-2015 compared with 1970-1985). Compared with patients transplanted 1970-1985, mortality risk was 72% lower among those transplanted 2005-2015 (aHR, 0.28; 95% CI, 0.18 to 0.69), after adjusting for potential confounders. CONCLUSIONS: Survival after pediatric kidney transplantation has improved considerably over the past four decades, predominantly because of marked reductions in cardiovascular- and infection-related deaths.

IRF5 controls both acute and chronic inflammation.

Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

Irf5 deficiency in macrophages promotes beneficial adipose tissue expansion and insulin sensitivity during obesity.

Accumulation of visceral adipose tissue correlates with elevated inflammation and increased risk of metabolic diseases. However, little is known about the molecular mechanisms that control its pathological expansion. Transcription factor interferon regulatory factor 5 (IRF5) has been implicated in polarizing macrophages towards an inflammatory phenotype. Here we demonstrate that mice lacking Irf5, when placed on a high-fat diet, show no difference in the growth of their epididymal white adipose tissue (epiWAT) but they show expansion of their subcutaneous white adipose tissue, as compared to wild-type (WT) mice on the same diet. EpiWAT from Irf5-deficient mice is marked by accumulation of alternatively activated macrophages, higher collagen deposition that restricts adipocyte size, and enhanced insulin sensitivity compared to epiWAT from WT mice. In obese individuals, IRF5 expression is negatively associated with insulin sensitivity and collagen deposition in visceral adipose tissue. Genome-wide analysis of gene expression in adipose tissue macrophages highlights the transforming growth factor ß1 (TGFB1) gene itself as a direct target of IRF5-mediated inhibition. This study uncovers a new function for IRF5 in controlling the relative mass of different adipose tissue depots and thus insulin sensitivity in obesity, and it suggests that inhibition of IRF5 may promote a healthy metabolic state during this condition.

IRF5:RelA interaction targets inflammatory genes in macrophages.

Interferon Regulatory Factor 5 (IRF5) plays a major role in setting up an inflammatory macrophage phenotype, but the molecular basis of its transcriptional activity is not fully understood. In this study, we conduct a comprehensive genome-wide analysis of IRF5 recruitment in macrophages stimulated with bacterial lipopolysaccharide and discover that IRF5 binds to regulatory elements of highly transcribed genes. Analysis of protein:DNA microarrays demonstrates that IRF5 recognizes the canonical IRF-binding (interferon-stimulated response element [ISRE]) motif in vitro. However, IRF5 binding in vivo appears to rely on its interactions with other proteins. IRF5 binds to a noncanonical composite PU.1:ISRE motif, and its recruitment is aided by RelA. Global gene expression analysis in macrophages deficient in IRF5 and RelA highlights the direct role of the RelA:IRF5 cistrome in regulation of a subset of key inflammatory genes. We map the RelA:IRF5 interaction domain and suggest that interfering with it would offer selective targeting of macrophage inflammatory activities.

Selective tumor necrosis factor receptor I blockade is antiinflammatory and reveals immunoregulatory role of tumor necrosis factor receptor II in collagen-induced arthritis.

OBJECTIVE: Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. METHODS: Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. RESULTS: Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. CONCLUSION: We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

Cross-species analysis reveals evolving and conserved features of the nuclear factor κB (NF-κB) proteins.

NF-κB is a key regulator of immune gene expression in metazoans. It is currently unclear what changes occurred in NF-κB during animal evolution and what features remained conserved. To address this question, we compared the biochemical and functional properties of NF-κB proteins derived from human and the starlet sea anemone (Nematostella vectensis) in 1) a high-throughput assay of in vitro preferences for DNA sequences, 2) ChIP analysis of in vivo recruitment to the promoters of target genes, 3) a LUMIER-assisted examination of interactions with cofactors, and 4) a transactivation assay. We observed a remarkable evolutionary conservation of the DNA binding preferences of the animal NF-κB orthologs. We also show that NF-κB dimerization properties, nuclear localization signals, and binding to cytosolic IκBs are conserved. Surprisingly, the Bcl3-type nuclear IκB proteins functionally pair up only with NF-κB derived from their own species. The basis of the differential NF-κB recognition by IκB subfamilies is discussed.

IRF5 is a specific marker of inflammatory macrophages in vivo.

Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Both functions are accomplished by a pivotal network of different macrophage subtypes, including proinflammatory M1 and anti-inflammatory M2 macrophages. Previously, our laboratory identified the transcription factor interferon regulatory factor 5 (IRF5) as the master regulator of the M1 macrophage polarisation. IRF5 was found to be highly expressed in human M1 compared to M2 macrophages. Furthermore, IRF5 dictates the expression of proinflammatory genes such as IL12b and IL23a whilst repressing anti-inflammatory genes like IL10. Here we show that murine bone marrow derived macrophages differentiated in vitro with GM-CSF are also characterised by high levels of IRF5 mRNA and protein and express proinflammatory cytokines upon LPS stimulation. These macrophages display characteristic expression of M1-marker MHC II but lack the M2-marker CD206. Significantly, we develop intracellular staining of IRF5- expressing macrophages and utilise it to recapitulate the in vitro results in an in vivo model of antigen-induced arthritis, emphasising their physiological relevance. Thus, we establish the species-invariant role of IRF5 in controlling the inflammatory macrophage phenotype both in vitro and in in vivo.

IL-17 receptor adaptor protein Act1/CIKS plays an evolutionarily conserved role in antiviral signaling.

Double-stranded RNA-induced antiviral gene expression in mammalian cells requires activation of IFN regulatory factor 3 (IRF3). In this study, we show that the IL-17R adaptor protein Act1/CIKS is involved in this process. Small interfering RNA-mediated knockdown of Act1 in primary human skin fibroblasts specifically attenuates expression of IFN-ß and IFN-stimulated antiviral genes induced by a synthetic viral mimic, polyinosinic-polycytidylic acid. Ectopic expression of Act1 potentiates the IRF3-driven expression of a synthetic reporter construct as well as the induction of antiviral genes. We demonstrate that this effect is dependent on the ability of Act1 to functionally and physically interact with IκB kinase ε (IKKε), a known IRF3 kinase, and IRF3: 1) Act1 binds IKKε and IRF3; 2) Act1-induced IRF3 activation can be blocked specifically by coexpression of a catalytically inactive mutant of IKKε; and 3) mutants of IRF3, either lacking the C terminus or mutated at the key phosphorylation sites, important for its activation by IKKε, do not support Act1-dependent IRF3 activation. We also show that a zebrafish Act1 protein is able to trigger antiviral gene expression in human cells, which suggests an evolutionarily conserved function of vertebrate Act1 in the host defense against viruses. On the whole, our study demonstrates that Act1 is a component of antiviral signaling.

FOXO3 as a new IKK-ε-controlled check-point of regulation of IFN-ß expression.

Cell survival transcription factor FOXO3 has been recently implicated in moderating pro-inflammatory cytokine production by dendritic cells (DCs), but the molecular mechanisms are unclear. It was suggested that FOXO3 could antagonize NF-κB activity, while IKK-ß was demonstrated to inactivate FOXO3, suggesting a cross-talk between the two pathways. Therefore, FOXO3 activity must be tightly regulated to allow for an appropriate inflammatory response. Here, we show that in human monocyte-derived DCs (MDDCs), FOXO3 is able to antagonize signaling intermediates downstream of the Toll-like receptor (TLR) 4, such as NF-κB and interferon regulatory factors (IRFs), resulting in inhibition of interferon (IFN)-ß expression. We also demonstrate that the activity of FOXO3 itself is regulated by IKK-ε, a kinase involved in IFN-ß production, which phosphorylates and inactivates FOXO3 in response to TLR4 agonists. Thus, we identify FOXO3 as a new IKK-ε-controlled check-point of IRF activation and regulation of IFN-ß expression, providing new insight into the role of FOXO3 in immune response control.

IL-17 boosts proinflammatory outcome of antiviral response in human cells.

Excessive inflammation during bacterial and viral infections is destructive to the host and involves elevated production of proinflammatory cytokines. It is especially deleterious in organs with space constraints such as lung and the CNS. Indeed, a number of viruses that infect lungs, such as avian influenza virus, SARS-associated coronavirus, and respiratory syncytial virus, elicit a very high level of proinflammatory cytokines; however, it is unclear what triggers their production. In this study, we show that IL-17 commonly produced during viral infection specifically augments a proinflammatory response by directly synergizing with antiviral signaling. Costimulation of primary human fibroblasts with IL-17 greatly enhanced respiratory syncytial virus-induced or synthetic dsRNA-based viral mimic polyinosinic:polycytidylic acid-induced expression of proinflammatory genes without affecting expression of IFN-ß-stimulated or IFN-stimulated genes. Knockdown of expression of known mediators of the antiviral signaling pathway revealed that the IL-17-poly(I:C) synergy depends on the presence of the transcriptional factors RelA and IFN regulatory factor 3 and IκB kinases. Moreover, this synergy was blocked by an IκB kinase inhibitor, BAY 11-7082. These findings shed light on the molecular mechanisms behind IL-17-dependent immunopathology observed in viral infections.

Lineage specific methylation of the Elf5 promoter in mammary epithelial cells.

Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E-twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the Elf5 promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether Elf5 promoter methylation plays a role in lineage commitment during mammary development. An increase in Elf5 expression was associated with decreasing Elf5 promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased Elf5 expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the Elf5 promoter is methylated and silenced in the basal, stem cell-containing population relative to luminal cells. These results demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages.

Evolution of vertebrate immunity: sequence and functional analysis of the SEFIR domain family member Act1.

SEF/IL-17R/CIKS/ACT1 homology (SEFIR) domain containing proteins, which include the IL-17 receptors and an adaptor protein Act1, have essential roles in vertebrate immunity. However, the molecular mechanisms of Act1 function remain largely unexplored. In this article, we employed an evolutionary analysis to discover novel structural and functional properties of Act1. Firstly, we have found that the previously identified helix-loop-helix and Ufd2-box domains in human Act1 have relatively recent evolutionary origins in higher vertebrates. Zebrafish Act1, which lacks these domains, is unable to induce JNK phosphorylation and activate cytokine expression when expressed in human cells. Secondly, we have established that Act1-like proteins contain DEATH-domains in basal animals, such as Hydra and primitive chordates, but lack this domain in vertebrates. Finally, we have shown that Act1-TRAF6 interactions are conserved throughout vertebrate evolution: Act1 derived from zebrafish can bind to TRAF6 and activate NF-κB in human cells. Moreover, we have identified a novel highly conserved motif at the amino-terminus of Act1, which is critical for binding to TRAF6 and activating NF-κB-dependent gene expression. We propose a model of evolutionary changes in Act1-mediated signalling, which contributes to a better understanding of evolution of the vertebrate immunity.

IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

IRF5 is required for late-phase TNF secretion by human dendritic cells.

Spatially and temporally controlled expression of inflammatory mediators is critical for an appropriate immune response. In this study, we define the role for interferon regulatory factor 5 (IRF5) in secretion of tumor necrosis factor (TNF) by human dendritic cells (DCs). We demonstrate that DCs but not macrophages have high levels of IRF5 protein, and that IRF5 is responsible for the late-phase expression of TNF, which is absent in macrophages. Sustained TNF secretion is essential for robust T-cell activation by DCs. Systematic bioinformatic and biochemical analyses of the TNF gene locus map 2 sites of IRF5 recruitment: 5' upstream and 3' downstream of the TNF gene. Remarkably, while IRF5 can directly bind to DNA in the upstream region, its recruitment to the downstream region depends on the protein-protein interactions with NF-kappaB RelA. This study provides new insights into diverse molecular mechanisms employed by IRF5 to regulate gene expression and implicates RelA-IRF5 interactions as a putative target for cell-specific modulation of TNF expression.