Platelet phosphatidylserine exposure and microparticle production as health bioindicators in marine mammals.

In human medicine, various pathologies, including decompression sickness, thrombocytopenia, and rheumatoid arthritis, have been linked to changes in cellular microparticles (MP) formation, particularly platelet microparticles (PMP). Similar disorders in marine mammals might be attributed to anthropogenic threats or illnesses, potentially impacting blood PMP levels. Thus, detecting platelet phosphatidylserine (PS) exposure and PMP formation could serve as a crucial diagnostic and monitoring approach for these conditions in marine mammals. Our group has developed a methodology to assess real-time PS exposure and PMP formation specifically tailored for marine mammals. This method, pioneered in species such as bottlenose dolphins, beluga whales, walruses, and California sea lions, represents a novel approach with significant implications for both clinical assessment and further research into platelet function in these animals. The adapted methodology for evaluating PS exposure and PMP formation in marine mammals has yielded promising results. By applying this approach, we have observed significant correlations between alterations in PMP levels and specific pathologies or environmental factors. These findings underscore the potential of platelet function assessment as a diagnostic and monitoring tool in marine mammal health. The successful adaptation and application of this methodology in marine mammals highlight its utility for understanding and managing health concerns in these animals.

Physiological values of phagocytic capacity in marine mammals and alterations during pathological situations.

The study of the immune function in marine mammals is essential to understand their physiology and can help to improve their welfare in the aquariums. Dedicating efforts to studying marine mammal physiology, pathophysiology, and implementing new diagnostic and therapeutic tools promote progress towards preventive medicine in aquariums by facilitating early detection and treatment of diseases. However, biological and clinical research on marine mammals is currently very limited due to difficult access to these species and their biological samples. With this objective, our group has adapted to marine mammals a commercially available assay routinely used to evaluate the phagocytic capacity of monocytes and granulocytes in human whole blood samples. We adapted IngoflowEx kit to bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), walruses (Odobenus rosmarus), Patagonian sea lions (Otaria flavescens), and harbor (Phoca vitulina). In this paper, we report the modifications carried out on the original protocol for their correct functioning in marine mammals. We obtained physiological values of phagocytic capacity in each species after repeated sampling for 4 years in various individuals of each species. Specific results revealed that the % phagocytic cells that ingested E.coli in bottlenose dolphins were 59.6 ± 1.27, in walruses 62.6 ± 2.17, in sea lions 57.5 ± 4.3, and in beluga whales 61.7 ± 1.4. In the case of the % phagocytic cells producing respiratory burst in bottlenose dolphins were 34.2 ± 3.6, in walruses 36.3 ± 4.3, in sea lions 40.8 ± 10.2, and in beluga whales 26.3 ± 3.7. These preliminary results can be used as a reference to detect alterations in phagocytic capacity either by immunosuppression or by exacerbation of the response in infectious inflammatory processes. Clinical applicability of the assay was verified in two clinical cases in which Ingoflow was useful to detect immune alterations in two diseased individuals, before and after the onset of clinical signs.

Antimicrobial Properties of New Polyamines Conjugated with Oxygen-Containing Aromatic Functional Groups.

Antibiotic resistance is now a first-order health problem, which makes the development of new families of antimicrobials imperative. These compounds should ideally be inexpensive, readily available, highly active, and non-toxic. Here, we present the results of our investigation regarding the antimicrobial activity of a series of natural and synthetic polyamines with different architectures (linear, tripodal, and macrocyclic) and their derivatives with the oxygen-containing aromatic functional groups 1,3-benzodioxol, ortho/para phenol, or 2,3-dihydrobenzofuran. The new compounds were prepared through an inexpensive process, and their activity was tested against selected strains of yeast, as well as Gram-positive and Gram-negative bacteria. In all cases, the conjugated derivatives showed antimicrobial activity higher than the unsubstituted polyamines. Several factors, such as the overall charge at physiological pH, lipophilicity, and the topology of the polyamine scaffold were relevant to their activity. The nature of the lipophilic moiety was also a determinant of human cell toxicity. The lead compounds were found to be bactericidal and fungistatic, and they were synergic with the commercial antifungals fluconazole, cycloheximide, and amphotericin B against the yeast strains tested.

Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0.

Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources [...].

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

6-(Methylsulfonyl) Hexyl Isothiocyanate: A Chemopreventive Agent Inducing Autophagy in Leukemia Cell Lines.

Autophagy is a fundamental catabolic process of cellular survival. The role of autophagy in cancer is highly complex: in the early stages of neoplastic transformation, it can act as a tumor suppressor avoiding the accumulation of proteins, damaged organelles, and reactive oxygen species (ROS), while during the advanced stages of cancer, autophagy is exploited by cancer cells to survive under starvation. 6-(Methylsulfonyl) hexyl isothiocyanate (6-MITC) is the most interesting compound in the Wasabia Japonica rizhome. Recently, we proved its ability to induce cytotoxic, cytostatic, and cell differentiation effects on leukemic cell lines and its antimutagenic activity on TK6 cells. In the current study, to further define its chemopreventive profile, Jurkat and HL-60 cells were treated with 6-MITC for 24 h. The modulation of the autophagic process and the involvement of ROS levels as a possible trigger mechanisms were analyzed by flow cytometry. We found that 6-MITC induced autophagy in Jurkat and HL-60 cells at the highest concentration tested and increased ROS intracellular levels in a dose-dependent manner. Our results implement available data to support 6-MITC as an attractive potential chemopreventive agent.

Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1's specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.

Characterization of Early Peripheral Immune Responses in Patients with Sepsis and Septic Shock.

(1) Background: Sepsis is a life-threatening condition caused by an abnormal host response to infection that produces altered physiological responses causing tissue damage and can result in organ dysfunction and, in some cases, death. Although sepsis is characterized by a malfunction of the immune system leading to an altered immune response and immunosuppression, the high complexity of the pathophysiology of sepsis requires further investigation to characterize the immune response in sepsis and septic shock. (2) Methods: This study analyzes the immune-related responses occurring during the early stages of sepsis by comparing the amounts of cytokines, immune modulators and other endothelial mediators of a control group and three types of severe patients: critically ill non-septic patients, septic and septic shock patients. (3) Results: We showed that in the early stages of sepsis the innate immune system attempts to counteract infection, probably via neutrophils. Conversely, the adaptive immune system is not yet fully activated, either in septic or in septic shock patients. In addition, immunosuppressive responses and pro-coagulation signals are active in patients with septic shock. (4) Conclusions: The highest levels of IL-6 and pyroptosis-related cytokines (IL-18 and IL-1α) were found in septic shock patients, which correlated with D-dimer. Moreover, endothelial function may be affected as shown by the overexpression of adhesion molecules such as s-ICAM1 and E-Selectin during septic shock.

New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry.

Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.

Highlighting Curcumin-Induced Crosstalk between Autophagy and Apoptosis as Supported by Its Specific Subcellular Localization.

Curcumin, a major active component of turmeric (Curcuma longa, L.), is known to have various effects on both healthy and cancerous tissues. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying the anticancer effect of curcumin is still unclear. Since there is a recent consensus about endoplasmic reticulum (ER) stress being involved in the cytotoxicity of natural compounds, we have investigated using Image flow cytometry the mechanistic aspects of curcumin's destabilization of the ER, but also the status of the lysosomal compartment. Curcumin induces ER stress, thereby causing an unfolded protein response and calcium release, which destabilizes the mitochondrial compartment and induce apoptosis. These events are also associated with secondary lysosomal membrane permeabilization that occurs later together with an activation of caspase-8, mediated by cathepsins and calpains that ended in the disruption of mitochondrial homeostasis. These two pathways of different intensities and momentum converge towards an amplification of cell death. In the present study, curcumin-induced autophagy failed to rescue all cells that underwent type II cell death following initial autophagic processes. However, a small number of cells were rescued (successful autophagy) to give rise to a novel proliferation phase.

Study of the Apoptotic Effect in Patients with Urological versus Gynaecological Chronic Pelvic Pain.

The objective is to observe if it could be possible to use the apoptosis test to distinguish different aetiologies in chronic pelvic pain syndrome (CPPS). A prospective study was done, 106 patients, 57 had previously been diagnosed with urological chronic pelvic pain (UCPP)/interstitial cystitis (IC) and 49 patients with gynaecological chronic pelvic pain (GCPP). Neoplastic cells cultures were exposed to the urine of patients with UCPP/IC and patients with GCPP. The urine ability to provoque apoptosis on them was analysed. The apoptosis degree was measured by quantifying the percentage of cells in phase subG0, determined by a flow cytometry analysis. It is observed that the cell cultures exposed to urine of patients with UCPP had a significantly higher sub-G1 peak and G2 phase than those of the cells exposed to urine from patient's GCPP. The average values of apoptosis in patients with UCPP were significantly higher to that obtained in -patients having GCPP. With the apoptosis tests having a value >10%, it is considered as positive as well. This means that when we are faced with a patient who has UCPP or non-bladder chronic pelvic pain, the probability of having an UCPP increases by 45% when the apoptosis test is positive for a value >10%. Urine from patients with UCPP has significantly higher apoptotic effect over than the effect produced by urine from patients with GCPP. The apoptosis test could be useful as an illness biomarker.

Oxidative imbalance in low/intermediate-1-risk myelodysplastic syndrome patients: The influence of iron overload.

OBJECTIVE: To assess the generation of reactive oxygen species (ROS) and the involvement of the main antioxidant pathways in low/intermediate-1-risk myelodysplastic syndromes (MDS) with iron overload (IOL). METHODS: We examined the levels of superoxide anion (O2-), hydrogen peroxide (H2O2), antioxidants (glutathione, GSH; superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx), mitochondrial membrane potential (ΔΨm), and by-products of oxidative damage (8-isoprostanes and 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-dG) in 42 MDS patients (28 without IOL at diagnosis, and 14 who developed IOL) and 20 healthy subjects. RESULTS: Patients with IOL showed higher O2- levels (39.4 MFI) than normal controls (22.7 MFI, p=0.0356) and patients at diagnosis (19.4 MFI, p=0.0049). Antioxidant systems, except SOD activity, exhibited significant changes in IOL patients with respect to controls (CAT: 7.1 vs 2.7nmol/ml/min, p=0.0023; GPx: 50.9 vs 76.4nmol/ml/min, p=0.0291; GSH: 50.2 vs 24.1 MFI, p=0.0060). Furthermore, mitochondrial dysfunction was only detected in IOL cases compared to controls (ΔΨm: 3.6 vs 6.4 MFI, p=0.0225). Finally, increased levels of 8-oxo-dG were detected in both groups of patients. CONCLUSION: Oxidative stress is an important but non-static phenomenon in MDS disease, whose status is influenced by, among other factors, the presence of injurious iron.

Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.

BACKGROUND: Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629-1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes. METHODS: Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot. RESULTS: The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules. CONCLUSION: This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society.

WITHDRAWN: Systems Biology and Immune Aging.

The Publisher regrets that this article is an accidental duplication of anarticle that has already been published, http://dx.doi.org/10.1016/j.imlet.2014.09.009. The duplicate article has therefore been withdrawn.

Systems Biology and immune aging.

Many alterations of innate and adaptive immunity are common in the aging population, which reflect a deterioration of the immune system, and have lead to the terms "immune aging" or "immunosenescence". Systems Biology aims to the comprehensive knowledge of the structure, dynamics, control and design that define a given biological system. Systems Biology benefits from the continuous advances in the omics sciences, based on high-throughput and high-content technologies, as well as on bioinformatic tools for data mining and integration. The Systems Biology approach is becoming gradually used to propose and to test comprehensive models of aging, both at the level of the immune system and the whole organism. In this way, immune aging may be described by a dynamic view of the states and interactions of every individual cell and molecule of the immune system and their role in the context of aging and longevity. This mini-review presents a panoramics of the current strategies, tools and challenges for applying Systems Biology to immune aging.

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BACKGROUND: Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival. METHODS: In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed. RESULTS: Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface. CONCLUSIONS: Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

U937 variant cells as a model of apoptosis without cell disintegration.

The variant cell line U937V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.

A VIN1 GUS::GFP fusion reveals activated sucrose metabolism programming occurring in interspersed cells during tomato fruit ripening.

The tomato is a model for fleshy fruit development and ripening. Here we report on the identification of a novel unique cell autonomous/cellular pattern of expression that was detected in fruits of transgenic tomato lines carrying a GFP GUS driven by the fruit specific vacuolar invertase promoter VIN1. The VIN1 promoter sequence faithfully reproduced the global endogenous VIN expression by conferring a biphasic pattern of expression with a second phase clearly associated to fruit ripening. A closer view revealed a salt and pepper pattern of expression characterized by individual cells exhibiting a range of expression levels (from high to low) surrounded by cells with no expression. This type of pattern was detected across different fruit tissues and cell types with some preferences for vascular, sub-epidermal layer and the inner part of the fruit. Cell ability to show promoter activity was neither directly associated with overall ripening - as we find VIN+ and - VIN- cells at all stages of ripening, nor with cell size. Nevertheless the number of cells with active VIN-driven expression increased with ripening and the activity of the VIN promoter seems to be inversely correlated with cell size in VIN+ cells. Gene expression analysis of FACS-sorted VIN+ cells revealed a transcriptionally distinct subpopulation of cells defined by increased expression of genes related to sucrose metabolism, and decreased activity in protein synthesis and chromatin remodeling. This finding suggests that local micro heterogeneity may underlie some aspects (i.e. the futile cycles involving sucrose metabolism) of an otherwise more uniform looking ripening program.

Direct Toll-like receptor-mediated stimulation of hematopoietic stem and progenitor cells occurs in vivo and promotes differentiation toward macrophages.

As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin(-) ) as well as HSPCs (identified as Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-) [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin(-) and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2(-/-) , TLR4(-/-) , or MyD88(-/-) mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system.

Candida albicans induces selective development of macrophages and monocyte derived dendritic cells by a TLR2 dependent signalling.

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-)), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2(-/-) mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2(-/-) mice correlating with their higher fungal burden. Accordingly, emigration of Ly6C(high) monocytes and neutrophils to spleen was increased in TLR2(-/-) mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2(-/-) infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin(-) cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.