AbstractType I interferon responses have been considered detrimental to host protection in tuberculosis (TB). We provide novel data to challenge this paradigm, derived from transcriptional profiling of human in vivo immune responses to discover associations with radiographic disease severity in pulmonary TB, combined with mechanistic studies to test causality for observed associations using a zebrafish larval mycobacterial infection model. Type I interferon activity in tissue samples from the site of a standardised mycobacterial challenge, the tuberculin skin test, was associated with less severe human TB disease. Abrogation of type I interferon signalling, by CRISPR-mediated mutagenesis ofstat2, led to increased burden and dissemination ofMycobacterium marinuminfection in zebrafish larvae. The mechanism for increased severity of mycobacterial infection in zebrafish involves reduced recruitment of myeloid cells required to restrict bacterial growth. Our data support a clear host protective role for type I interferon responses in mycobacterial infection, with potential applications for risk-stratification of adverse outcomes and development of a host-directed therapy to mitigate against severe disease.

Age is an important risk factor for infections such as tuberculosis (TB). Telomerase is expressed in immune cells yet leukocyte telomere length declines during ageing suggesting an age-dependent loss of telomerase activity in the immune system. Leukocyte telomere length has been correlated with worse outcomes in TB patients, however the mechanisms linking telomere biology to TB susceptibility and response to therapy are unexplored. Here we use the zebrafish- Mycobacterium marinum model to investigate the role of telomerase in TB resistance. We find depletion and inhibition of Tert, the catalytic subunit of telomerase, increases bacterial burden in zebrafish embryos while small molecule activation of Tert decreases bacterial burden. Depletion of shelterin components did not recapitulate the infection susceptibility phenotype, and infection susceptibility could not be rescued by p53 or STING depletion. Consistent with a previously described role for Tert in developmental hematopoiesis, we find Tert is necessary for demand-driven emergency myelopoiesis to support containment of extended mycobacterial infection. Our findings establish a previously undescribed role for host telomerase in supporting infection demand-driven hematopoiesis to control infection. ### Competing Interest Statement The authors have declared no competing interest. National Medical Research Council, OFIRG22jul-0081 European Research Council, https://ror.org/0472cxd90, 772853 - ENTRAPMENT Wellcome Trust, 226644/Z/22/Z National Natural Science Foundation of China, 82372263, 82402628

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the enterococcal polysaccharide antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called 'decorations', essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.

The UK Cellular Microbiology Network Meeting started in 2019 with the vision of supporting and enhancing the cellular microbiology community in the UK and Europe. Since its inception, the network meeting has emerged as an important platform for infection and cell biologists to inspire transformative collaborations and novel research agendas. The network meeting is highly interactive and is focused on promoting early career researchers and discovery science. The fifth meeting in this series was held on 19–20 June 2023 at the iconic London School of Hygiene & Tropical Medicine (LSHTM). Similar to the 2022 event held in Warwick (https://onlinelibrary.wiley.com/do/10.1111/mmi.0040011), the meeting was spread over 2 days so that participants could maximise the dynamic science and downtown location. To represent the ethos of our community, our invited speakers were Pascale Cossart (from Institut Pasteur in Paris, France) and Felix Randow (from MRC LMB in Cambridge, UK). We also had a strong cohort of early career researchers from across the UK and Europe who presented their work (Figure 1). Following coffee and welcoming remarks from Serge Mostowy (LSHTM), the meeting opened with a keynote talk by Pascale Cossart entitled ‘How to give birth to a star’, showcasing the rise of Listeria as a paradigm of cellular microbiology (Cossart, 2023) and her highly decorated career which was recently celebrated in the two joined Cellular Microbiology and Molecular Microbiology Special Issues (Buchrieser, 2020; Pizarro-Cerda, 2020). Pascale talked about her career-spanning body of work, including the discovery of ActA for actin-based motility, the generation of the first humanised mouse model to study Listeria invasion, and the uncovering of numerous transformative mechanisms underlying bacterial virulence. All participants were inspired to hear about her enthusiasm for discovery science, and her vision for cellular microbiology moving forward. Although the field is blooming more than ever, she advised that future generations need to think about infection in the context of evolution and the environment—in other words, the context in which it is happening. The first session was chaired by Serge Mostowy (LSHTM) and Dan Humphreys (Sheffield). Giulia Manigrasso (from Andrew Carter's lab, MRC LMB) presented her work on Orientia tsutsugamushi (in collaboration with Jeanne Salje's lab, CIMR) and how this obligate intracellular pathogen hijacks dynein motility for intracellular transport and autophagy evasion. The next talk was from Samkeliso Lisa Blundell (from David Holden's lab, Imperial College London), describing the Salmonella effector SteD as a co-activator of the E3 ubiquitin ligase WWP2. The last talk of this session was Miguel Hernandez-Gonzalez (from Michael Way's lab, Francis Crick), who is imaging poxvirus assembly and exit from infected cells using state-of-the-art microscopy techniques, including cryo-electron tomography (Hernandez-Gonzalez et al., 2023). The next session was chaired by Phil Elks (Sheffield) and began with Margarida Gomes (from Serge Mostowy's lab, LSHTM), who is using zebrafish infection models to decipher mechanisms underlying trained innate immunity and to guide vaccine studies against shigellosis (Gomes et al., 2023). Next was Thomas Burgess (from Phil Elks' lab, Sheffield) who is also using zebrafish infection models, aiming to boost the innate immune response to infection by the fungal pathogen Candida albicans via modulation of hypoxia-inducible factor (HIF). Following this was Guy Pearson (from Jeremy Carlton's lab, Francis Crick and Kings College London), highlighting a wide variety of advanced imaging technologies as he described how the envelope of SARS-CoV-2 (the causative agent of COVID) hijacks an intracellular trafficking pathway to deacidify lysosomes. This session was followed by flash talks selected from submitted abstracts by early career researchers. Gizem Ozbaykal-Guler (from Serge Mostowy's lab, LSHTM) presented interdisciplinary work testing the role of septin hetero-oligomer composition on cage entrapment of Shigella. Keith Egger (from Charlotte Odendall's lab, Kings College London) discussed work on Salmonella Type III Secretion System (T3SS) SPI-2 effectors and their role in inhibition of interferon signalling. Ines Diaz del Olmo (from Teresa Thurston's lab, Imperial College London) described how Salmonella could promote non-canonical reprogramming of transcriptional pathways; Shan Yin (from James Mason's lab, Kings College London) presented studies of symbioses between bacterial vaginosis-associated bacteria and lactobacilli. Daniel Stark (from Jason King's lab, Sheffield) reported on work using zebrafish infection to study the role of bacterial endosymbionts in host-Mucorales interactions. Stevens Robertin (from Serge Mostowy's lab, LSHTM) described his research using Staphylococcus aureus to study septin interactions with Gram-positive bacterial pathogens. Finally, Richard Allen (from Meera Unnikrishnan's lab, Warwick) discussed how S. aureus interacts with macrophages via the Type VII Secretion System (T7SS). The late evening ended with drinks, a dynamic poster session and electric discussion. The following morning session was chaired by Charlotte Odendall (Kings College London). The second keynote speaker, Felix Randow, delivered a highly thought-provoking talk on how cells defend their cytosol against invasive bacteria, revealing unexpected roles for the E3 ligase RNF213 in cell-autonomous immunity (Otten et al., 2021). Magdalena Szczesna (from Teresa Thurston's lab, Imperial College London) presented work on how cytosolic bacteria inhibit RNF213-mediated cell-autonomous immunity (Szczesna et al., 2023). Next, Miriam Kutsch, a new group leader at Heinrich Heine University (Germany), described her research on how guanylate binding proteins (GBPs) activate the non-canonical inflammasome. The last two talks of the morning session showcased local excellence. Lucy Thorne (from Greg Towers' lab at University College London and new group leader at Imperial College London) described the convergent evolution of SARS-CoV-2 variants of concern to enhance innate immune suppression (Thorne et al., 2022). Ann-Kathrin Reuschl (from Clare Jolly's lab at University College London) presented work on enhanced innate immune evasion by SARS-CoV-2 Omicron subvariants (Mesner et al., 2023). In a second set of flash talks, Ramon Garcia Maset (from Jennifer Rohn's lab, University College London), presented research on the role of the urinary microenvironment in biofilm and its effect on antibiotic response in uropathogens. Janis Romanopulos (from James Mason's lab, Kings College London) discussed research into the translation of pleurocidin-derived antimicrobial peptide therapeutics. Ioanna Panagi (from Teresa Thurston's lab, Imperial College London) presented work on the molecular dissection of kinase reprogramming by Salmonella effectors. Yizhou Huang (from Teresa Thurston's lab, Imperial College London) described work analysing antibacterial autophagy during infection with Burkholderia. Nagisa Yoshida (from Naomi McGovern's lab, Cambridge) revealed new tools to study the microbicidal capacity of human placental macrophages across gestation. Finally, Zoe Speirs (from Phil Elks' lab, Sheffield) reported on efforts to modulate macrophages as a therapeutic strategy in a zebrafish infection model of tuberculosis. The flash talk session was followed by poster presentations and networking, with lunch and coffee. The final session was chaired by Jennifer Rohn (University College London). Enrica Pellegrino (from Max Gutierrez's lab, Francis Crick) presented innovative work on the role of peroxisomes during M. tuberculosis infection of human macrophages (Pellegrino et al., 2023). Next was Kathryn Wright (from Stefan Oehlers' lab, Centenary Institute in Australia) who discovered, using zebrafish infection, that mycobacterial infection-induced miR-126 protects the host by suppressing permissive macrophages (Wright et al., 2021). J.J. Awodipe (from Meera Unnikrishnan's lab, Warwick) presented work on early transcriptomic responses of osteoblasts and S. aureus during intracellular infection. The final speaker of the afternoon was Daniel Foulkes (from Stephen Kaye's lab, Liverpool) on exotoxin inhibitors as new therapeutics, describing a drug discovery pipeline from in vitro to in vivo analysis. The meeting closed with a robust community discussion and prize giving, over coffee and snacks. Giulia Manigrasso won the best oral presentation for her work on Orientia, and Ines Diaz del Olmo won the best poster prize for her work on Salmonella. Find out more about their work in their respective interviews published alongside this report (Ines, 2023; Manigrasso, 2023). We are very thankful to our flagship sponsor the Company of Biologists and Molecular Microbiology for promoting the work of talented early career researchers that has been showcased at the UK Cellular Microbiology Meeting since its first edition in 2019 (Figure 2). We also are grateful to LSHTM for helping to coordinate this event. Serge Mostowy: Conceptualization; writing – original draft; writing – review and editing. Charlotte Odendall: Conceptualization; writing – original draft; writing – review and editing. Daniel Humphreys: Conceptualization; writing – original draft; writing – review and editing. Philip M. Elks: Conceptualization; writing – original draft; writing – review and editing. Jennifer L. Rohn: Conceptualization; writing – original draft; writing – review and editing. The authors declare no conflict of interest. No human or animal subjects or materials were used in this commentary. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

ABSTRACT Innate immune responses to inflammation and infection are complex and represent major challenges for developing much needed new treatments for chronic inflammatory diseases and drug-resistant infections. To be ultimately successful, the immune response must be balanced to allow pathogen clearance without excess tissue damage, processes controlled by pro- and anti-inflammatory signals. The roles of anti-inflammatory signalling in raising an appropriate immune response are underappreciated, representing overlooked potential drug targets. This is especially true in neutrophils, a difficult cell type to study ex vivo owing to a short lifespan, dogmatically seen as being highly pro-inflammatory. Here, we have generated and describe the first zebrafish transgenic line [TgBAC(arg2:eGFP)sh571] that labels expression of the anti-inflammatory gene arginase 2 (arg2) and show that a subpopulation of neutrophils upregulate arginase soon after immune challenge with injury and infection. At wound-healing stages, arg2:GFP is expressed in subsets of neutrophils and macrophages, potentially representing anti-inflammatory, polarised immune cell populations. Our findings identify nuanced responses to immune challenge in vivo, responses that represent new opportunities for therapeutic interventions during inflammation and infection.

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles 1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo , we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of pro-inflammatory factors il-1 β and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis. ### Competing Interest Statement The authors have declared no competing interest.

Candida spp. cause 750,000 cases per annum of invasive disease worldwide, with up to a 50% mortality rate. Poor efficacy of current antifungals, lack of vaccines and rising antifungal resistance rates point towards an urgent need to develop new therapies. Candida albicans is a human commensal fungus that can cause life-threatening invasive infection in immunocompromised individuals. C. albicans is able to manipulate host macrophages and neutrophils to escape phagosomal killing and has previously been shown to suppress reactive nitrogen species (RNS) production in vitro . However, the effects of C. albicans on RNS in vivo and the molecular and cellular mechanisms involved remain unclear. Using a zebrafish model, we aimed to characterise RNS suppression by C. albicans in vivo . We demonstrate that C. albicans suppressed neutrophil RNS both proximally and distally to the infection site in a partially active process, with heat-killed C. albicans not reducing RNS to the same extent as live fungi. Using a car1 Δ mutant, we show that fungal arginase is partially responsible for the reduction in neutrophil RNS. Stabilisation of Hif-1α, a transcription factor with a key role in immune regulation, rescued neutrophil RNS production during C. albicans infection, leading to improved infection outcomes. The protective effect of Hif-1α stabilisation was neutrophil- and nitric oxide synthase-dependent. Together, these data demonstrate that Hif-1α stabilisation can restore the neutrophil RNS response in C. albicans infection, leading to improved infection outcomes, highlighting the potential of targeting Hif-1α and RNS in host directed therapies against fungal infections.

PDF file - 56K, Table S1, shows relative increase in EGFP fluorescence levels of phd3: EGFP in vhl-/- embryos vs wild-type. Table S2 shows the effect of DMBA treatment on the rates of LOH for the vhl locus in 2.5 month old wild-type and vhl+/- fish. Table S3 shows the increased number of macroscopic phd3: GFP positive areas induced by DMBA vhl-/+ adults as compared to wild-type, they are likely to correspond to vhl-/- tumors. Table S4 shows preferential induction of renal tubule proliferation in vhl heterozygotes.

To date, very little is known regarding the earliest cellular and molecular events within the initial preneoplastic cell (PNC) niche that might determine the outcome of PNC development. Here, we combine in vivo live imaging and single cell transcriptomics to investigate the immediate cellular and molecular changes that occur in PNCs and responding innate immune cells within the first 24 hours following tumour initiation. We do so using an inducible transgenic zebrafish model of tumour initiation, in which oncogenic HRAS is conditionally expressed within the zebrafish epidermis. We found that, when expressed within basal keratinocytes, oncogenic HRAS was sufficient to drive several cancer hallmarks in nascent PNCs and their niche. Within 8 hours of oncogenic HRAS induction, basal keratinocytes began to dedifferentiate, followed by a bifurcation in PNC fate towards either a cellular state characterised by partial Epithelial-Mesenchymal-Transition (pEMT) and cancer stem cell features or a more differentiated cell state resembling suprabasal keratinocytes. By directly integrating data from PNCs and human SCC patients we show that both of these PNC states likely persist with established tumours, whilst the pEMT state corresponded with a more aggressive phenotype. Strikingly, markers of the PNC subpopulation that underwent pEMT correlated with poor prognosis in multiple human cancer types in the TCGA database. In addition, the pEMT PNC subpopulation expressed higher levels of cytokines that modulate neutrophil development and function, suggesting a major role for these cells in governing pro-tumour neutrophil responses. Indeed, we found that granulopoiesis was altered within PNC-bearing larvae, with a large proportion of neutrophils switching to an Arginase 2 positive phenotype that functioned to promote PNC proliferation. Similar to tumour associated neutrophils in the mammalian system, the cxcr1/2 – Il8 axis is involved in mediating the PNC-promoting function of neutrophils in our model. Thus, oncogenic RAS, when activated in the permissive cellular environment is sufficient to drive enabling cancer hallmarks, such as cellular plasticity and inflammation from the inception of the preneoplastic stage of cancer development. Furthermore, these same features may persist to drive malignant progression.

AbstractTuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance.Mycobacterium tuberculosisparasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infectionin vivoare limited. Here, we identify that human Tribbles 1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteriain vivo, we used a zebrafishMycobacterium marinum(Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues.trib1overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely,trib1knockdown exhibited increased infection. Mechanistically,trib1overexpression significantly increased the levels of pro-inflammatory factorsil-1β and nitric oxide. The host-protective effect oftrib1was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infectionin vivoand suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

PDF file - 817K, Figure S1 shows in vivo expression of phd3::EGFP in wild-type and vhl-/- Figure S2 shows that phd3::EGFP mRNA expression recapitulates endogenous phd3 expression. Figure S3 shows that EGFP expression in phd3::EGFP embryos is Hif dependent. Figure S4 shows that EGFP expression in phd3::EGFP embryos can be activated by inhibitors of PHD hydroxylases. Figure S5 shows the half life of the fluorescence of the phd3::EGFP line. Figure S6 shows that the phd3::EGFP transgene acts as an in vivo marker for detecting LOH events at the vhl locus in vhl+/- fish. Figure S7 shows morphology of gonadal tumors after DMBA treatment. Figure S8 shows the sequence analysis of the vhl locus in dissected tumors. Figure S9 shows that vhl mRNA levels are decreased in EGFP+ tumors in vhl+/- fish.

Immunocompromised individuals are at high risk of developing severe fungal infections with high mortality rates, while fungal pathogens pose little risk to most healthy people. Poor therapeutic outcomes and growing antifungal resistance pose further challenges for treatments. Identifying specific immunomodulatory mechanisms exploited by fungal pathogens is critical for our understanding of fungal diseases and development of new therapies. A gap currently exists between the large body of literature concerning the innate immune response to fungal infections and the potential manipulation of host immune responses to aid clearance of infection. This review considers the innate immune mechanisms the host deploys to prevent fungal infection and how these mechanisms fail in immunocompromised hosts. Three clinically relevant fungal pathogens (Candida albicans, Cryptococcus spp. and Aspergillus spp.) will be explored. This review will also examine potential mechanisms of targeting the host therapeutically to improve outcomes of fungal infection.

The innate immune response to inflammatory stimuli must be finely balanced to produce an appropriate pro-inflammatory response while allowing a subsequent return to homeostasis. In recent years, in vivo transgenic zebrafish models have shed light on the temporal regulation of the pro-inflammatory innate response to immune challenges. However, until now, there have been no zebrafish transgenic models of anti-inflammatory signalling. We compared existing expression data of arginase genes in zebrafish neutrophils and macrophages, strong candidates for an anti-inflammatory marker, and identified that arginase 2 is the most highly expressed Arginase in zebrafish immune cells. We developed an arginase 2 (arg2) bacterial artificial chromosome (BAC) transgenic line, TgBAC(arg2:eGFP)sh571, driving GFP expression under the control of the arg2 promoter. We show that, under resting conditions, arg2:GFP is expressed in ionocytes, matching the in situ hybridisation pattern. Upon immune challenge by injury, bacterial and fungal insults, arg2:GFP is predominantly expressed in neutrophils at early timepoints post-insult. Later in infections, arg2:GFP is expressed in cells associated with foci of infection (including neutrophils and macrophages), alongside liver expression. Our data indicate that arginase 2 is predominantly expressed in neutrophils after immune challenge and suggest that anti-inflammatory signals coincide with pro-inflammatory signals during early wound and infection responses.

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.

EDITORIAL article Front. Immunol., 30 November 2021Sec. Molecular Innate Immunity Volume 12 - 2021 | https://doi.org/10.3389/fimmu.2021.810346

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

We describe new open source software called QuantiFish for rapid quantitation of fluorescent foci in zebrafish larvae, to support infection research in this animal model. QuantiFish extends the conventional measurements of bacterial load and number of bacterial foci to include measures for dissemination of infection. These are represented by the proportions of bacteria between foci and their spatial distribution. We showcase these measures by comparison of intravenous and hindbrain routes of Mycobacterium marinum infection, which are indistinguishable by measurement of bacterial load and not consistently differentiated by the number of bacterial foci. The intravenous route showed dose dependent dissemination of infection, reflected by increased spatial dispersion of bacteria and lower proportions of bacteria distributed across many foci. In contrast, hindbrain infection resulted in localised disease, limited to a smaller area and higher proportions of bacteria distributed across fewer foci. The application of QuantiFish may extend beyond models of infection, to study other pathologies such as metastatic cancer.