Invasive fungal diseases (IFDs) present a growing clinical challenge, underscoring the urgent need for improved diagnostics, therapeutics and mechanistic understanding. This review highlights the key role of innovative imaging techniques across all scales - ranging from whole-body-level diagnostics and therapy monitoring to host-pathogen interactions at cellular resolution in both clinical and preclinical settings. These imaging modalities facilitate translation of preclinical innovations into clinical applications, accelerating research and advancing IFD management.

Candida albicans is a commensal microbe and opportunistic human pathogen. Candida yeast are recognized and taken up by macrophages via phagocytosis. Macrophage surface receptors bind to specific components of the Candida cell wall. Following phagocytosis, Candida can respond to the host's intracellular environment by switching from a yeast to a hyphal morphology facilitating escape from macrophages and allowing subsequent invasion of host tissues. Various disruptions of Candida's ability to form hyphae have been shown to reduce virulence and fitness in the host. Our previous work concluded that Candida albicans cells lacking AP-2 (apm4Δ/Δ), an endocytic adaptor complex, have increased cell wall chitin and morphologically defective hyphae in vitro. Increased chitin has been correlated with decreased recognition by macrophages, possibly due to masking of cell wall β-glucan, the target for the Dectin-1 immune receptor. Here we test the virulence profiles of apm4Δ/Δ mutant, demonstrating a surprising increase in macrophage phagocytosis that does not occur due to the elevated exposure of β-glucan, highlighting the importance of cell wall components beyond chitin and glucan for macrophage engagement and uptake. Furthermore, the apm4 mutant exhibited parasitism of macrophages, surviving and proliferating within the phagosome, a phenotype that was then replicated with a well-characterized yeast locked mutant, demonstrating the further complexity of C. albicans' ability to evade macrophage responses. Finally, the combined phenotype of reduced hyphal formation but continued proliferation resulted in reduced virulence despite an equivalent burden of infection with wild-type Candida infection, as determined using a zebrafish larval model of candidiasis.

Abstract Background: Camizestrant is a next-generation oral selective estrogen receptor (ER) degrader and pure ER antagonist being investigated in early and advanced breast cancer (BC). In postmenopausal women with advanced ER-positive/human epidermal growth factor receptor 2 (HER2)-negative BC and disease recurrence or progression on or after ≤1 endocrine therapy (ET) in the advanced setting, camizestrant significantly prolonged progression-free survival compared with fulvestrant in the Phase 2 SERENA-2 trial. Camizestrant may also provide benefit in the early BC setting. Following locoregional therapy (surgery ± radiotherapy), standard adjuvant treatment of ET with or without chemotherapy and/or a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) has shown significant benefit in decreasing the risk of recurrence of stage I–III ER-positive/HER2-negative BC. However, BC recurrence as incurable metastatic disease is still common. Thus, adjuvant therapeutic options with improved clinical outcomes are needed. The CAMBRIA-2 study (NCT05952557) aims to assess whether upfront camizestrant can improve outcomes compared with standard ET as adjuvant treatment in patients with ER-positive/HER2-negative early BC with an intermediate-high or high risk of recurrence after definitive locoregional therapy. Trial design: This ongoing Phase 3, randomized, open-label study is enrolling women (pre-, peri-, or postmenopausal) and men with ER-positive/HER2-negative (HER2-negative status defined as immunohistochemistry 0, or 1+, or in situ hybridization-negative) early BC who are at intermediate-high or high risk of recurrence (as defined in the protocol) after having completed definitive locoregional therapy (surgery ± radiotherapy) ± (neo)adjuvant systemic chemotherapy, and who have no evidence of disease. Patients may have received up to 12 weeks of (neo)adjuvant ET prior to randomization. Patients are randomized (1:1) to receive camizestrant 75 mg ± abemaciclib ± luteinizing hormone-releasing hormone (LHRH) agonist or standard ET of the investigator’s choice (any aromatase inhibitor or tamoxifen) ± abemaciclib ± LHRH agonist for up to 7 years. The primary endpoint is invasive BC-free survival (IBCFS) (Standardized Definitions for Efficacy End Points [STEEP] 2.0 criteria). Secondary endpoints include invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) (STEEP 2.0 criteria), overall survival, safety, and health-related quality of life. Primary endpoint analysis will use a stratified log-rank test adjusting for stratification factors, assuming a two-sided significance level of 5%. Approximately 5500 patients will be randomized. Clinical trial identification: NCT05952557 Editorial acknowledgment: Writing assistance was provided by Clare Davis of BOLDSCIENCE Inc., funded by AstraZeneca. Legal entity responsible for the study: AstraZeneca Funding: This study was supported by AstraZeneca. Citation Format: Sibylle Loibl, Yeon Park, Sara Tolaney, Ioanna Gioni, Simon Johnston, Teresa Klinowska, Ingrid A. Mayer, Raquel Nunes, Barbara Pistilli, Mary Stuart, Angela Quintana, Andrew Walding, Michael Gnant. A Phase 3, randomized, open-label study of upfront camizestrant vs standard endocrine therapy as adjuvant treatment for ER-positive/HER2-negative early breast cancer with intermediate-high or high risk of recurrence (CAMBRIA-2) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-07.

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.

Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age -related changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration, and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years old) compared with younger (18-30 years old) donors, alongside downregulation of transcription factors MYC and USF1. In MDMs from young donors, knockdown of MYC or USF1 decreases phagocytosis and chemotaxis and alters the expression of associated genes, alongside adhesion and extracellular matrix remodeling. A concordant dysregulation of MYC and USF1 target genes is also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs leads to an increased cell size, altered morphology, and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age -related functional decline and identify downstream targets to improve macrophage function in aging.

This chapter provides guidance for introducing Cryptococcus neoformans into the zebrafish larvae model system to establish a CNS infection phenotype that mimics cryptococcal meningitis as seen in humans. The method outlines techniques for visualizing different stages of pathology development, from initial to severe infection profiles. The chapter provides tips for real time visualization of the interactions between the pathogen and different aspects of the CNS anatomy and immune system.

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.

Summary Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age-related immunity changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years) compared with younger (18-30 years) donors, alongside downregulation of transcription factors MYC and USF1 with age. In MDMs from young donors, knockdown of MYC or USF1 decreased phagocytosis and chemotaxis and altered expression of genes associated with these functions, as well as adhesion and extracellular matrix remodelling. A concordant dysregulation of MYC and USF1 target genes was also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs led to an increased cell size, altered morphology and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age-related functional decline and identify downstream targets to improve macrophage function in ageing.

Tribbles 3 (TRIB3) modulates lipid and glucose metabolism, macrophage lipid uptake, with a gain-of-function variant associated with increased cardiovascular risk. Here we set out to examine the role of this pseudokinase in atherosclerotic plaque development. Human endarterectomy atherosclerotic tissue specimens analysed by immunofluorescence showed upregulated TRIB3 in unstable plaques and an enrichment in unstable regions of stable plaques. Atherosclerosis was induced in full body Trib3KO and Trib3WT littermate mice by injecting mPCSK9 expressing adeno-associated virus and western diet feeding for 12 weeks. Trib3KO mice showed expanded visceral adipose depot while circulatory lipid levels remained unaltered compared to wildtype mice. Trib3KO mice aortae showed a reduced plaque development and improved plaque stability, with increased fibrous cap thickness and collagen content, which was accompanied by increased macrophage content. Analysis of both mouse and human macrophages with reduced TRIB3 expression showed elongated morphology, increased actin expression and altered regulation of genes involved in extracellular matrix remodelling. In summary, TRIB3 controls plaque development and may be atherogenic in vivo. Loss of TRIB3 increases fibrous cap thickness via altered metalloproteinase expression in macrophages, thus inhibiting collagen and elastic fibre degradation, suggesting a role for TRIB3 in the formation of unstable plaques.

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.

Autophagy leads to degradation of cellular components and has an important role in restricting intracellular pathogens. Autophagy receptors, including p62, target invading intracellular pathogens to the autophagy pathway for degradation. Staphylococcus aureus is a significant pathogen of humans and often life-threatening in the immunocompromised. Increasing evidence demonstrates that S. aureus is an intracellular pathogen of immune cells and may use neutrophils as proliferative niche but the intracellular fate of S. aureus following phagocytosis by neutrophils has not previously been analysed in vivo. In vitro , p62 is able to co-localise with intracellular Staphylococcus aureus , but whether p62 is beneficial or detrimental in host defence against S. aureus in vivo had not been determined. Here we use zebrafish to determine the fate and location of S. aureus within neutrophils throughout infection. We show that Lc3 and p62 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil. We also show rapid Lc3 marking of bacterial phagosomes within neutrophils which may be associated with subsequent bacterial degradation. Finally, we find that p62 is important for controlling cytosolic bacteria demonstrating for the first time a key role of p62 in autophagic control of S. aureus in neutrophils.

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.

Objective: To determine whether global reduction of CD68 (cluster of differentiation) macrophages impacts the development of experimental pulmonary arterial hypertension (PAH) and whether this reduction affects the balance of pro- and anti-inflammatory macrophages within the lung. Additionally, to determine whether there is evidence of an altered macrophage polarization in patients with PAH. Approach and Results: Macrophage reduction was induced in mice via doxycycline-induced CD68-driven cytotoxic diphtheria toxin A chain expression (macrophage low [MacLow] mice). Chimeric mice were generated using bone marrow transplant. Mice were phenotyped for PAH by echocardiography and closed chest cardiac catheterization. Murine macrophage phenotyping was performed on lungs, bone marrow-derived macrophages, and alveolar macrophages using immunohistochemical and flow cytometry. Monocyte-derived macrophages were isolated from PAH patients and healthy volunteers and polarization capacity assessed morphologically and by flow cytometry. After 6 weeks of macrophage depletion, male but not female MacLow mice developed PAH. Chimeric mice demonstrated a requirement for both MacLow bone marrow and MacLow recipient mice to cause PAH. Immunohistochemical analysis of lung sections demonstrated imbalance in M1/M2 ratio in male MacLow mice only, suggesting that this imbalance may drive the PAH phenotype. M1/M2 imbalance was also seen in male MacLow bone marrow-derived macrophages and PAH patient monocyte-derived macrophages following stimulation with doxycycline and IL (interleukin)-4, respectively. Furthermore, MacLow-derived alveolar macrophages showed characteristic differences in terms of their polarization and expression of diphtheria toxin A chain following stimulation with doxycycline. Conclusions: These data further highlight a sex imbalance in PAH and further implicate immune cells into this paradigm. Targeting imbalance of macrophage population may offer a future therapeutic option.

Invasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancer and is typically ER+ and ERBB2 non-amplified. There is preclinical evidence that somatic ERBB2 mutation may provide an alternative and tractable mechanism for upregulation of HER2 activity in tumors that do not express HER2 by current clinical criteria. Using large public datasets, we previously demonstrated that targetable ERBB2 mutations are enriched in ILC versus invasive ductal carcinoma (IDC) and are an independent prognostic factor in ILC (HR=3.7, 95% CI 1.2-11.0; p=0.021)*. We next hypothesized that a gene expression signature incorporating HER2 activity due to ERBB2 mutation and / or amplification would validate the prognostic signal we found in ILC. To derive a novel gene expression signature of HER2 activity that accounted for the effect of potentially targetable ERBB2 mutations in ERBB2 non-amplified tumors, we applied a weighted average difference method to gene expression data in cases from the METABRIC 2012 (N=1,980) and TCGA 2015 (N=817) cohorts. To compare our novel gene expression signature with an established signature of HER2 activity, we performed multivariate regression modeling of response to neratinib, a small molecule tyrosine kinase inhibitor of HER1, 2 and 4, using pharmacogenomic data accessed via the CellMinerCDB online portal. We show that our novel HER2 pathway signature score uniquely enriches for ERBB2 mutated tumors. Using a Cox regression model and stratifying gene expression scores into upper versus lower quartiles, we were able to validate the prognostic signal of ERBB2 mutations in ILC tumors (HR for 10-year OS in ILC=2.3, 95% CI 1.04-5.05; p=0.040). In contrast, no relationship was found between ERBB2 mutation status or novel HER2 pathway enrichment score and patient outcome in cases of IDC. We conclude that ERBB2 mutations that are enriched in ILC provide a robust biomarker of HER2 pathway activation and can be detected via gene expression signature. Novel clinical trials of HER2-targeted therapy in ERBB2 non-amplified primary ILC are warranted. *Reference: Kurozumi S et al, Cancer Research 2019, 80(4) suppl: SABCS 2019 Abstract P1-18-06 and medRxiv 2020.01.24.20018622 Citation Format: Mansour Alsaleem, Sasagu Kurozumi, Kartikeya Bhardwaj, Cintia Monteiro, Stacey EP Joosten, Andrew R Green, Takaaki Fujii, Ken Shirabe, Ian O Ellis, Emad A Rakha, Nigel P Mongan, David M Heery, Wilbert Zwart, Steffi Oesterreich, Simon J Johnston. Targetable ERBB2 mutation status is an independent marker of adverse prognosis in estrogen receptor positive, ERBB2 non-amplified primary lobular breast carcinoma: Validation using a novel gene signature of HER2 activation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-11.

Abstract Invasive lobular carcinoma accounts for 10-15% of breast cancer cases and typically expresses hormone receptors. Despite this, lobular tumours respond less well to endocrine therapy, and long-term patient outcomes are inferior to those with other breast cancer subtypes. In primary lobular tumours, HER2 is rarely overexpressed due to relative infrequency of ERBB2 amplification. However, somatic mutation of ERBB2 may provide an alternative and tractable mechanism for upregulation of HER2 activity, which can be effectively targeted using a second generation HER2/EGFR tyrosine kinase inhibitor such as neratinib. In this study we performed comparative in silico analysis of ER/PR(+), HER2(-) cases of invasive lobular (N=352) and ductal carcinoma (N=1,820) from the three largest primary breast cancer clinical datasets with somatic mutations called from next generation sequencing (TCGA, METABRIC and MSK, N=2,172). Using existing functional data from in vitro, cell line and xenograft experiments, ERBB2 mutations were stratified into known ‘oncogenic‘ or ‘uncharacterized‘ and integrated with lymph node status, tumour size and grade. The primary endpoint was overall survival as these data were available across datasets. We find that lobular tumours comprise 11% of all cases but contain 40% of the somatic mutations in ERBB2 in the dataset. The overall rate of mutated ERBB2 in lobular cases is 6% (N=21) versus 1.8% (N=32) in ductal cases (p<0.0001). Using a Cox regression model, we show that oncogenic ERBB2 mutation status independently predicts overall poor survival only in lobular cases (HR 3.6, 95% CI 1.57 - 8.26; p=0.0026). For patients with lobular disease, overall survival was significantly lower if tumours harboured oncogenic ERBB2 mutation (median 64 versus 170 months, p<0.0002). We conclude that ERBB2 mutations are enriched in ERBB2 non-amplified, ER/PR(+) lobular tumours and predict poor outcome despite use of adjuvant endocrine therapy according to standard protocols. Targeted sequencing of ERBB2 should be available for patients with lobular breast cancer and represents an actionable strategy to improve patient outcomes. Citation Format: Sasagu Kurozumi, Andrew R Green, Ian O Ellis, Emad A Rakha, Takaaki Fujii, Ken Shirabe, David Heery, Wilbert Zwart, Steffi Oesterreich, Simon J Johnston. Targetable ERBB2 mutations independently predict an aggressive phenotype in primary, ERBB2 non-amplified, hormone receptor positive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-06.

Cryptococcus neoformans is a facultative intracellular pathogenic yeast which causes cryptococcal meningitis. Infection most commonly occurs via the lungs in humans and is cleared without clinical signs in the immunocompetent, but may cause life-threatening infection in immunocompromised. C. neoformans can be phagocytosed by host phagocytes, but may manipulate the intracellular niche of phagocytes for replication and dissemination. The interaction of macrophages with cryptococcal cells has been studied in detail but little is known about the interaction between human peripheral blood mononuclear cells (PBMC) and C. neoformans . PBMCs are rapidly recruited to the site of initial infection in the lung, peripheral tissues, and also respond to cryptococci that disseminate via the bloodstream. Therefore, deciphering the interactions between PBMCs and cryptococci is an important but neglected aspect of our understanding of the immune response during cryptococcal infection. Here, using time lapse imaging of primary human PBMCs in vitro , we are able to measure the PBMC response to cryptococci. Using this approach we find that naïve, undifferentiated human monocytes phagocytose cryptococcal cells, and that aggregates (swarms) of monocytes and T cells often form in response to engulfment of cryptococci. Interestingly, we find a correlation between the size of the PBMC aggregates and proliferative ability of the cryptococci within. While these aggregates slow intracellular cryptococcal growth, cryptococci are able to replicate within this niche and escape from PBMCs to replicate extracellularly. These results provide evidence for PBMC control of cryptococcal infection and provide a model for the in vitro study of cryptococcal granuloma biology.

Multi-cohort analysis demonstrated that cytoplasmic cyclin E expression in primary breast tumors predicts aggressive disease. However, compared to their younger counterparts, older patients have favorable tumor biology and are less likely to die of breast cancer. Biomarkers therefore require interpretation in this specific context. Here, we assess data on cytoplasmic cyclin E from a UK cohort of older women alongside a panel of >20 biomarkers. Between 1973 and 2010, 813 women >= 70 years of age underwent initial surgery for early breast cancer, from which a tissue microarray was constructed (n = 517). Biomarker expression was assessed by immunohistochemistry. Multivariate analysis of breast cancer-specific survival was performed using Cox's proportional hazards. We found that cytoplasmic cyclin E was the only biological factor independently predictive of breast cancer-specific survival in this cohort of older women (hazard ratio (HR) = 6.23, 95% confidence interval (CI) = 1.93-20.14; p = 0.002). At ten years, 42% of older patients with cytoplasmic cyclin E-positive tumors had died of breast cancer versus 8% of negative cases (p < 0.0005). We conclude that cytoplasmic cyclin E is an exquisite marker of aggressive tumor biology in older women. Patients with cytoplasmic cyclin E-negative tumors are unlikely to die of breast cancer. These data have the potential to influence treatment strategy in older patients.