Search

Highlights from the Science family of journals

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Highlights from the Science family of journals

Highlights from the Science family of journals

Highlights from the Science family of journals

Highlights from the Science family of journals

Highlights from the Science family of journals

The contribution of microglia in neurological disorders is emerging as a leading disease driver rather than a consequence of pathology. RNAseT2‐deficient leukoencephalopathy is a severe childhood white matter disorder affecting patients in their first year of life and mimicking a cytomegalovirus brain infection. The early onset and resemblance of the symptoms to a viral infection suggest an inflammatory and embryonic origin of the pathology. There are no treatments available for this disease as our understanding of the cellular drivers of the pathology are still unknown. In this study, using a zebrafish mutant for the orthologous rnaset2 gene, we have identified an inflammatory signature in early development and an antiviral immune response in mature adult brains. Using the optical transparency and the ex utero development of the zebrafish larvae we studied immune cell behavior during brain development and identified abnormal microglia as an early marker of pathology. Live imaging and electron microscopy identified that mutant microglia displayed an engorged morphology and were filled with undigested apoptotic cells and undigested substrate. Using microglia‐specific depletion and rescue experiments, we identified microglia as drivers of this embryonic phenotype and potential key cellular player in the pathology of RNAseT2‐deficient leukoencephalopathy. Our zebrafish model also presented with reduced survival and locomotor defects, therefore recapitulating many aspects of the human disease. Our study therefore placed our rnaset2 mutant at the forefront of leukodystrophy preclinical models and highlighted tissue‐specific approaches as future therapeutic avenues., Main Points Loss of rnaset2 results in inflammation and lysosomal‐deficient microglia failing to digest dying neurons.Targeting microglia restores embryonic microglial defects to clear neurodevelopmental apoptosis.Mutants have reduced survival and locomotor defects.

Highlights from the Science family of journals

Highlights from the Science family of journals

Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.

Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases. Funding This work has been supported by a European Leukodystrophy Association fellowship (ELA 2016-012F4) to NH, an MRC Programme Grant (MR/M004864/1) to SAR and . JPL is supported by Agence Nationale de la Recherche (grant ANR-16-CE20-0002-03.. Imaging was carried out in the Wolfson Light Microscopy Facility, supported by an MRC grant (G0700091) and a Wellcome Trust grant (GR077544AIA). Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Somatic missense mutations of BTG1 are exclusive to germinal center (GC)-derived B cell lymphomas (~12% of DLBCLs) and are most prevalent in ABC-DLBCL (p=0.0184 vs GCB-DLBCL), particularly in the MCD/cluster 5 subtype, which features extranodal dissemination and unfavorable outcome. However, the relevance, mechanism of action and biological contribution of BTG1 mutations have not been studied. Using a rigorous genomic covariate analysis, we identified BTG1 mutations as a top genetic driver in DLBCL. Furthermore, molecular dynamics simulations indicated that BTG1 recurrent mutations, including the most frequent Q36H, disrupted the protein structure, with likely deleterious functional consequences. To investigate the effect of BTG1 mutation in GC B cells, we generated a conditional Btg1Q36H knock in mouse crossed to the B cell specific Cd19Cre line. Surprisingly, there was no apparent phenotype in GC B cells or other B cell populations. However, placing Btg1 Q36H and WT GC B cells in competition within the same mouse through adoptive transfer revealed a dramatic competitive advantage of Btg1 Q36H cells, virtually taking over the GC reaction. To gain further insight into this striking fitness advantage, we performed RNAseq in Btg1 Q36H GCs, which showed marked enrichment for genes induced in positively selected GC B cells, including MYC targets and biosynthetic pathways. The same genes were also enriched in BTG1 mutant DLBCL patients in 2 independent cohorts. Furthermore, Btg1 Q36H GC B cells displayed greater RNA content and cell size, reflecting increased fitness. Positive selection normally triggers a brief Myc pulse in GC B cells. We therefore crossed our Btg1Q36H mice to the MycGFPprotein fusion reporter and observed higher proportion of Myc GFP+ cells in Btg1 Q36H GCs. For mechanistic studies, we generated isogenic BTG1 Q36H or BTG1 WT human DLBCL cell lines. BTG1 Q36H cells exhibited enrichment for the same positively selected GC B and MYC target genes, as well as greater RNA content and cell size. BTG1 family members were suggested to interact with RNA. Performing RNA-immunoprecipitation, we discovered that ~800 transcripts associated with BTG1 WT, but not BTG1 Q36H. Notably, these corresponded to the same positively selected GC B and MYC target genes, including MYC itself. BTG1 was shown to regulate mRNA stability in other cell types. However, BTG1 Q36H did not alter MYC mRNA stability and instead facilitated MYC protein synthesis, thus disrupting a novel GC context-specific checkpoint mechanism, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. In GC B cells, Myc induction coincides with S phase entry, but G2/M progression requires re-entry into the proliferative dark zone. To characterize GC dynamics in vivo, we performed targeted single cell RNAseq in competing Btg1 Q36H and WT GC B cells and noted earlier and higher proportion of positively selected Btg1 Q36H GC B cells having committed to G2/M and the proliferative program. We confirmed faster S phase completion in competing Btg1 Q36H GC B cells by in vivo EdU/BrdU labelling and greater re-entry into the proliferative dark zone by in vivo antigen delivery to synchronize GC B cells at the time of positive selection. Given that MCD-DLBCLs express high levels of BCL2, we crossed our Btg1Q36H mice to the VavP-Bcl2 model. As compared to VavP-Bcl2, VavP-Bcl2+Btg1 Q36H mice displayed shorter survival (p=0.0005), earlier onset of lymphoma, dysplastic B cell infiltration into non lymphoid organs and they contained highly mutated, selected and clonal tumor B cells. Moribund VavP-Bcl2+Btg1 Q36H mice uniquely featured sheets of large, immunoblastic lymphoma cells, characteristic of ABC-DLBCLs. Most notably, examining ABC-DLBCLs from 5 independent cohorts showed inferior clinical outcome for BTG1 mutant patients (p=0.0011) and independent association of BTG1 mutation with inferior overall survival by multivariable Cox regression (p=0.0190). Collectively, we find that BTG1 mutations mediate lymphomagenesis through an entirely novel mechanism of action that recapitulates the embryonic MYC-dependent "super-competitive" phenotype originally described in Drosophila imaginal disc cells. In the GC, "super-competition" is provided by BTG1 mutation via a subtle acceleration of MYC induction and GC dynamics, conferring dramatic fitness and the potential to transform into aggressive lymphomas. Disclosures Hoehn: Prellis Biologics: Consultancy. Elemento: Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding. Scott: NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; AstraZeneca: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Rich/Genentech: Research Funding. Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees.

Neutrophilic chemotaxis is essential to immune system response to external threats. During this process cells alternate between directed motion towards the higher concentration of external stimuli and correlated random walk. An individual neutrophil migration can thus be characterised as a stochastic dynamical process driven by an external chemotactic environment that is typically not measured. This introduces the problem of estimating spatially-varying chemoattractant concentration field from the observed migration patterns of cell populations. We propose a solution to this estimation problem in a statistical inference framework. The framework has measured cell positions in the field as inputs and employs the expectation-maximisation algorithm for joint estimation of full cell states and parameters of the chemoattractant field decomposed with cubic B-splines. The performance of the developed algorithm is accessed via process in vivo measurements of cell positions in the injured tail fin of zebrafish. Estimation results for different injury types evidence that the proposed estimation algorithm provides a rigorous connection between mathematical modelling and experimental data.

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease., eLife digest Chronic obstructive pulmonary disease (or COPD) is a serious condition that causes the lungs to become inflamed for long periods of time, leading to permanent damage of the airways. Immune cells known as neutrophils promote inflammation after an injury, or during an infection, to aid the healing process. However, if they are active for too long, they may also cause tissue damage and drive inflammatory diseases including COPD. To limit damage to the body, neutrophils usually have a very short lifespan and die by a regulated process known as apoptosis. Finding ways to stimulate apoptosis in neutrophils may be key to developing better treatments for inflammatory diseases. Cells contain many enzymes known as kinases that control apoptosis and other cell processes. Drugs that inhibit specific kinases are effective treatments for some types of cancer and other conditions, and new kinase-inhibiting drugs are currently being developed. However, it remains unclear which kinases regulate apoptosis in neutrophils or which kinase-inhibiting drugs may have the potential to treat COPD and other inflammatory diseases. To address these questions, Rahman et al. tested over 350 kinase-inhibiting drugs to identify ones that promote apoptosis in neutrophils. The experiments showed that human neutrophils treated with drugs that inhibit the ErbB family of kinases died by apoptosis more quickly than untreated neutrophils. Next, Rahman et al. used zebrafish with injured tail fins as models to study inflammation. Zebrafish treated with one of these drugs – known as Tyrphostin AG825 – had lower levels of inflammation and their neutrophils underwent apoptosis more frequently than untreated zebrafish. Since drugs can have off-target effects, Rahman et al. went on to show using gene-editing technology that reducing the activity of two genes that encode ErbB kinases in zebrafish also decreased the levels of inflammation in the fish. Further experiments used mice that develop inflammation in the lungs similar to COPD in humans. As expected, neutrophils in the lungs of mice treated with Tyrphostin AG825 underwent apoptosis more frequently than those in untreated mice. These dead neutrophils were effectively cleared by other immune cells called macrophages, which also helps limit damage caused by neutrophils. Together, these findings show that Tyrphostin AG825 and other drugs that inhibit ErbB kinases help to reduce inflammation by promoting the death of neutrophils. Since several of these drugs are already used to treat human cancers, it may be possible in the future to repurpose them for use in people with COPD and other long-term inflammatory diseases. Determining whether this is possible is an aim for future studies.

Neutrophils are rapidly recruited to inflammatory sites where they coordinate their migration to form clusters, a process termed neutrophil swarming. The factors which modulate neutrophil swarming during its early stages are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around a pioneer neutrophil, in a three-phase sequence of events. By adopting a high-resolution confocal microscopy approach, we observed the release of cell fragments by early swarming neutrophils. We developed a neutrophil specific histone H2A transgenic reporter line TgBAC(mpx:GFP)i114;Tg(lyz:H2A-mCherry)sh530 to study neutrophil extracellular traps (NETs), and found that endogenous neutrophils recruited to sites of tissue damage released NETs at the start of the swarming process. The optical transparency achieved using the zebrafish model has provided some of the highest resolution imaging of NET release in vivo to date. Using a combination of transgenic reporter lines and DNA intercalating agents, we demonstrate that pioneer neutrophils release extracellular traps during the swarming response, suggesting that cell death signalling via NETosis might be important in driving the swarming response.

Background Inflammation is thought to be a major contributor to post-surgical pain, so non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics. However, compared to rats, considerably less is known as to how successfully these prevent pain in mice. Methods A fluorescent COX-2 selective probe was used for the first time to evaluate the post-surgical anti-inflammatory effects of meloxicam, and automated behaviour analyses (HomeCageScan; HCS), the Mouse Grimace Scale (MGS) and body weight changes to assess its pain-preventative properties. Groups of 8–9 BALB/c mice were subcutaneously injected with saline (0.3 mL) or meloxicam at (1, 5 or 20 mg/kg) 1 h before a 1.5-cm midline laparotomy. The probe or a control dye (2 mg/kg) was injected intravenously 3 h later. Imaging was used to quantify inflammation at 7, 24 and 48 h following surgery. HCS data and MGS scores were respectively obtained from video recordings and photographs before surgery and 24 h later. Results Post-surgical inflammation was dose dependently reduced by meloxicam; with 5 or 20 mg/kg being most effective compared to saline. However, all mice lost weight, MGS scores increased and behavioural activity was reduced by surgery for at least 24 h with no perceivable beneficial effect of meloxicam on any of these potentially pain-associated changes. Conclusions Although meloxicam prevented inflammation, even large doses did not prevent post-laparotomy pain possibly arising due to a range of factors, including, but not limited to inflammation. MGS scoring can be applied by very naive assessors and so should be effective for cage-side use.

SummaryThe contribution of microglia in neurological disorders is emerging as a leading driver rather than a consequence of pathology. RNAseT2-deficient leukoencephalopathy is a severe childhood white matter disorder affecting patients in their first year of life and mimics a cytomegalovirus brain infection. The early onset and resemblance of the symptoms to an immune response suggest an inflammatory and embryonic origin of the pathology. In this study, we identify deficient microglia as an early marker of pathology. Using the ex utero development and the optical transparency of an rnaset2-deficient zebrafish model, we found that dysfunctional microglia fail to clear apoptotic neurons during brain development. This was associated with increased number of apoptotic cells and behavioural defects lasting into adulthood. This zebrafish model recapitulates all aspect of the human disease to be used as a robust preclinical model. Using microglia-specific depletion and rescue experiments, we identified microglia as potential drivers of the pathology and highlight tissue-specific approaches as future therapeutic avenues.