IRF5 promotes influenza-induced inflammatory responses in human iPSC-derived myeloid cells and murine models.

Recognition of Influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activates innate immune cells, and regulates adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate mechanisms driving influenza-induced inflammation in humans. Interferon regulatory factor (IRF5) is a transcription factor that plays important roles in induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in Irf5-/- mice, but not impacting type 1 IFN production or virus replication. Using cytometry by time-of-flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human induced pluripotent stem cells (hIPSCs) with biallelic mutations in IRF5, demonstrating for the first time iPS-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of TLR7 and, possibly, RIG-I after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans, and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication.ImportanceThe inflammatory response to Influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene-I. Our data demonstrate the importance of IRF5 in influenza-induced inflammation, suggesting genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.This work was supported by The Wellcome Trust. This work was funded by a Wellcome 641 Trust Senior Research Fellowship to Ian Humphreys (207503/Z/17/Z); Medical Research 642 Council, United Kingdom (MR/L018942/1 and MRC Human Immunology Unit Core); 643 Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences 644 (CIFMS), China (grant number: 2018-I2M-2-002). The Wellcome Trust Sanger Institute was 645 the source of the Kolf2 human induced pluripotent cell line which was generated under the 646 Human Induced Pluripotent Stem Cell Initiative funded by a grant from the Wellcome Trust Downloaded from http://jvi.asm.org/ on March 2, 2020 at CAMBRIDGE UNIV27 and Medical Research Council, supported 647 by the Wellcome Trust (WT098051) and the 648 NIHR/Wellcome Trust Clinical Research Facility, and Life Science Technologies 649 Corporation provided Cytotune for reprogramming. We thank the Wellcome Trust Sanger Institute Gene editing pipeline for generation of IRF5-/- 650 iPSCs and the Mass spectrometry 651 Facility at the Weatherall Institute of Molecular Medicine for help with CyTOF experiments

Interleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation

Abstract: Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA

Models of <i>KPTN</i>-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.</p

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

EROS/CYBC1 mutations: Decreased NADPH oxidase function and chronic granulomatous disease.

We demonstrate for the first time that EROS (CYBC1/C17ORF62) regulates abundance of the gp91phox-p22phox heterodimer of the phagocyte NADPH oxidase in human cells and that EROS mutations are a novel cause of chronic granulomatous disease.Wellcome Trus

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.status: publishe

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases