Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity

Ischemic stroke and concomitant gastrointestinal complications- a fatal combination for patient recovery

Stroke is primarily a neurodegenerative disease but can also severely impact the functions of other vital organs and deteriorate disease outcomes. A malfunction of the gastrointestinal tract (GIT), commonly observed in stroke patients, is often characterized by severe bowel obstruction, intestinal microbiota changes and inflammation. Over-activated immune cells after stroke are the major contributors to endorse intestinal inflammation and may induce damage to single-layer epithelial cell barriers. The post-stroke leakage of intestinal barriers may allow the translocation and dissemination of resident microflora to systemic organs and cause sepsis. This overshooting systemic immune reaction fuels ongoing inflammation in the degenerating brain and slows recovery. Currently, the therapeutic options to treat these GIT-associated anomalies are very limited and further research is required to develop novel treatments. In this mini-review, we first discuss the current knowledge from clinical studies and experimental stroke models that provide strong evidence of the existence of post-stroke GIT complications. Then, we review the literature regarding novel therapeutic approaches that might help to maintain GIT homeostasis and improve neurological outcomes in stroke patients

Deriving a germinal center lymphocyte migration model from two-photon data

Recently, two-photon imaging has allowed intravital tracking of lymphocyte migration and cellular interactions during germinal center (GC) reactions. The implications of two-photon measurements obtained by several investigators are currently the subject of controversy. With the help of two mathematical approaches, we reanalyze these data. It is shown that the measured lymphocyte migration frequency between the dark and the light zone is quantitatively explained by persistent random walk of lymphocytes. The cell motility data imply a fast intermixture of cells within the whole GC in approximately 3 h, and this does not allow for maintenance of dark and light zones. The model predicts that chemotaxis is active in GCs to maintain GC zoning and demonstrates that chemotaxis is consistent with two-photon lymphocyte motility data. However, the model also predicts that the chemokine sensitivity is quickly down-regulated. On the basis of these findings, we formulate a novel GC lymphocyte migration model and propose its verification by new two-photon experiments that combine the measurement of B cell migration with that of specific chemokine receptor expression levels. In addition, we discuss some statistical limitations for the interpretation of two-photon cell motility measurements in general

Deriving a germinal center lymphocyte migration model from two-photon data

Recently, two-photon imaging has allowed intravital tracking of lymphocyte migration and cellular interactions during germinal center (GC) reactions. The implications of two-photon measurements obtained by several investigators are currently the subject of controversy. With the help of two mathematical approaches, we reanalyze these data. It is shown that the measured lymphocyte migration frequency between the dark and the light zone is quantitatively explained by persistent random walk of lymphocytes. The cell motility data imply a fast intermixture of cells within the whole GC in approximately 3 h, and this does not allow for maintenance of dark and light zones. The model predicts that chemotaxis is active in GCs to maintain GC zoning and demonstrates that chemotaxis is consistent with two-photon lymphocyte motility data. However, the model also predicts that the chemokine sensitivity is quickly down-regulated. On the basis of these findings, we formulate a novel GC lymphocyte migration model and propose its verification by new two-photon experiments that combine the measurement of B cell migration with that of specific chemokine receptor expression levels. In addition, we discuss some statistical limitations for the interpretation of two-photon cell motility measurements in general

Irf4-dependent CD103+CD11b+ dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus

Objective: Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.Design: POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment.Results: We found that Cd11c-Cre+ Irf4flox/flox mice lack CD103+CD11b+ DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C- macrophages and infiltrating chemokine receptor 2-dependent Ly6C+ monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C+ monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI.Conclusions: Our findings reveal that CD103+CD11b+ DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI

The alarmin interleukin-1α triggers secondary degeneration through reactive astrocytes and endothelium after spinal cord injury

Spinal cord injury (SCI) triggers neuroinflammation, and subsequently secondary degeneration and oligodendrocyte (OL) death. We report that the alarmin interleukin (IL)−1α is produced by damaged microglia after SCI. Intra-cisterna magna injection of IL-1α in mice rapidly induces neutrophil infiltration and OL death throughout the spinal cord, mimicking the injury cascade seen in SCI sites. These effects are abolished through co-treatment with the IL-1R1 antagonist anakinra, as well as in IL-1R1-knockout mice which demonstrate enhanced locomotor recovery after SCI. Conditional restoration of IL-1R1 expression in astrocytes or endothelial cells (ECs), but not in OLs or microglia, restores IL-1α-induced effects, while astrocyte- or EC-specific Il1r1 deletion reduces OL loss. Conditioned medium derived from IL-1α-stimulated astrocytes results in toxicity for OLs; further, IL-1α-stimulated astrocytes generate reactive oxygen species (ROS), and blocking ROS production in IL-1α-treated or SCI mice prevented OL loss. Thus, after SCI, microglia release IL-1α, inducing astrocyte- and EC-mediated OL degeneration

Community standards for open cell migration data

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

During early stages of cancer, neutrophils initiate anti-tumor immune responses in tumor-draining lymph nodes

Tumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1-) and stimulate T cells (CD27+Ki67highPD-1-). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+ immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1-3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy