Umeå University's logo

Background: Arsenic (ATO) and retinoic acid (ATRA) are successfully used as chemotherapy-free regimens to treat acute APL. Compared to traditional chemotherapy approaches, this therapy evokes fewer haematological side effects, such as severe neutropenia and thrombocytopenia, but little is known about the impact of the treatment on neutrophil function.

Methods: We included three patients undergoing consolidation treatment for APL. To evaluate the functionality of neutrophils, we assessed chemotaxis, ROS production, and neutrophil extracellular trap (NET) release during different time points of the treatment and compared them with neutrophils from healthy donors.

Results: We revealed that the chemotactic ability of neutrophils isolated from APL patients was decreased before starting each cycle of treatment. However, there was an increase in chemotactic ability in the first week of treatment compared to other time points. Additionally, we observed increased ROS production at the start of the treatment cycle. In vitro exposure of isolated neutrophils from healthy donors to ATO led to decreased chemotaxis at high ATO concentrations exceeding those achieved in vivo, while ROS production was not affected. Chemotaxis and ROS production were not altered by exposure to ATRA in vitro and neither ATO nor ATRA had an effect on neutrophils’ ability to release NETs.

Conclusions: Our study suggests that ATO and ATRA therapy alter neutrophil function by increasing chemotaxis and reducing ROS production. The effect on neutrophil function does not, however, seem to impact infection susceptibility in our patients, indicating that the enhanced functionality might compensate for the lowered neutrophil count.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

The peptide toxin candidalysin, secreted by Candida albicans hyphae, promotes stimulation of neutrophil extracellular traps (NETs). However, candidalysin alone triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leucotoxic hypercitrullination, which is governed by calcium-induced protein arginine deaminase 4 activation and initiation of intracellular signalling events in a dose- and time-dependent manner. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Candidalysin-triggered NLS demonstrate anti-Candida activity, which is resistant to nuclease treatment and dependent on the deprivation of Zn²⁺. This study reveals that C. albicans hyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entangles C. albicans hyphae and efficiently inhibits their growth.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Interaction between microorganisms and their surroundings are generally mediated via the cell wall or cell envelope. An understanding of the overall chemical composition of these surface layers may give clues on how these interactions occur and suggest mechanisms to manipulate them. This knowledge is key, for instance, in research aiming to reduce colonization of medical devices and device-related infections from different types of microorganisms. In this context, X-ray photoelectron spectroscopy (XPS) is a powerful technique as its analysis depth below 10 nm enables studies of the outermost surface structures of microorganism. Of specific interest for the study of biological systems is cryogenic XPS (cryo-XPS). This technique allows studies of intact fast-frozen hydrated samples without the need for pre-treatment procedures that may cause the cell structure to collapse or change due to the loss of water. Previously, cryo-XPS has been applied to study bacterial and algal surfaces with respect to their composition of lipids, polysaccharides and peptide (protein and/or peptidoglycan). This contribution focuses onto two other groups of microorganisms with widely different architecture and modes of life, namely fungi and viruses. It evaluates to what extent existing models for data treatment of XPS spectra can be applied to understand the chemical composition of their very different surface layers. XPS data from model organisms as well as reference substances representing specific building blocks of their surface were collected and are presented. These results aims to guide future analysis of the surface chemical composition of biological systems.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.

The Phagocytosis of fungal structures by neutrophils is a well-documented function of these immune cells. However, neutrophil phagocytosis of hyphal structures in the urine sediment is not usually observed during routine sample evaluation. This is a case of hyphal phagocytosis by neutrophils in the urine of a kidney allograft recipient patient.

A fagocitose de estruturas fúngicas por neutrófilos é uma função bem documentada destas células imunes. No entanto, a fagocitose de hifas por neutrófilos no sedimento urinário não é normalmente observada durante avaliação de rotina de amostras. Este é um caso de fagocitose de hifas por neutrófilos na urina de um paciente receptor de aloenxerto renal.

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryosubstituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 mu m for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale. 

Opportunistic, invasive mycoses in immunocompromised patients remain challenging for health care with unacceptably high levels of morbidity and mortality. Neutrophils are essential in host protection against invasive mycoses. Upon development of acute infection, neutrophils are recruited from circulation to the infected tissue, where they exert a considerable variety of effector functions with the ultimate task to eradicate invading microbes. Effector functions include recognition, phagocytosis and intracellular killing of microorganisms via oxidative and non-oxidative mechanisms, excretion of antimicrobial factors from intracellular storages (degranulation), release of neutrophil extracellular traps (NETs) and of extracellular vesicles (EVs), as well as generation of cytokines and chemokines to modulate immune responses. Herein, we describe recent findings which further our understanding of the roles of neutrophils during opportunistic fungal infections which could serve as starting point for the development of immune-targeted interventions to improve clinical management of affected individuals.