Innate immune responses to "Aspergillus fumigatus"

AbstractAspergillus fumigatus is a ubiquitous mould that can cause invasive aspergillosis, a potentially lethal infection in onco-hematological patients. With an incidence rate ranging from 5 to 15%, invasive aspergillosis (IA) is one of the most frequent infections in patients undergoing intensive myeloablative chemotherapy for acute leukaemia or allogenic hematopoietic stem cell transplantation (HSCT). Toll-like receptors (TLRs) are transmembrane proteins located in immune cells, such as macrophages sand dendritic cells, that detect molecular motifs from invading pathogens to initiate immune response mechanisms. Studies suggested a role for TLR2 and TLR4 in the detection of A. fumigatus. However, few data are available on the role of TLR1 and TLR6, both known as TLR2 co-receptors, in innate immune responses to this pathogen.In this study, we used an immunogenic mutant strain of A. fumigatus, together with a wild-type strain, to analyse the role of TLRs and their signalling pathways in the innate immune response to this mould. We show for the first time that this response involves both TLR1 and TLR6 in mouse and TLR1, but not TLR6, in human. We show that, despite the high sequence homology between TLR1 and TLR6, the specificity in the sensing of A. fumigatus relies on the human TLR1 and TLR6 ectodomains. Furthermore, we show that two human single nucleotide polymorphisms (SNPs) (G1805T [S6021] and G239C [R80T]) affect the response to this pathogen. Our work also confirms the role of TLR2 and TLR4 in the detection of A. fumigatus, together with their co-receptors CD 14 and MD2, in both mouse and human, and highlights the nature of the intracellular signaling pathway used by these receptors to mediate the immune response against this pathogen.This study provides a comprehensive analysis of the role of TLRs and their signalling pathways in the innate immune recognition of A. fumigatus and may have important consequences for diagnosis, management and treatment of IA in high risk patients.RésuméAspergillus fumigatus est un champignon saprophyte ubiquitaire qui peut causer l'aspergillose invasive (AI), une infection potentiellement mortelle chez les patients onco-hématologiques. Avec un taux d'incidence de 5 à 15%, l'AI est l'une des infections les plus fréquentes chez les patients subissant une chimiothérapie intensive pour une leucémie aiguë ou une allogreffe de cellules souches hématopoïétiques. Les récepteurs Toll-like (Toll-like receptors, TLRs) sont des protéines transmembranaires placés stratégiquement à la surface de certaines cellules immunitaires, comme les macrophages et les cellules dendritiques. Ces protéines sont capables de détecter des motifs moléculaires à la surface des pathogènes et de déclencher la réponse immunitaire innée. Des études ont suggéré l'implication de TLR2 et TLR4 dans la détection dΆ. fumigatus. Cependant, peu de données sont disponibles sur le rôle de TLR1 et TLR6, qui sont les co-récepteurs de TLR2, dans ce mécanisme de défense immunitaire.Dans cette étude, nous avons utilisé une souche particulièrement immunogénique d'A. fumigatus, ainsi qu'une souche sauvage, pour analyser l'implication des récepteurs TLRs dans la réponse immunitaire à ce champignon filamenteux. Nous montrons pour la première fois que cette détection implique TLR1 et TLR6 chez la souris, et TLR1, mais pas TLR6, chez l'homme. Nous montrons également que la spécificité de détection chez l'homme est due à des séquences spécifiques du domaine extra- membranaire de TLR1 et TLR6, et que des polymorphismes mono-nucléotidiques du récepteur (G1805T [S602I] and G239C [R80T]) influencent la réponse à ce pathogène. Nous confirmons également l'implication de TLR2 et TLR4, avec leurs co-récepteurs CD14 et MD2, dans la détection d'A. fumigatus, chez l'homme et la souris, et mettons en évidence les voies de signalisation cellulaires impliquées dans la réponse immunitaire à ce pathogène.Ces nouvelles connaissances sur le rôle des TLRs et de leurs voies de signalisation cellulaire dans la détection immunitaire innée d'A. fumigatus pourraient influencer le diagnostic, la prévention et le traitement de l'AI chez les patients à haut risque de développer cette infection.

Airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses.

We conducted a 12-year retrospective study to determine the effects that the community respiratory-virus species and the localization of respiratory-tract virus infection have on severe airflow decline, a serious and fatal complication occurring after hematopoietic cell transplantation (HCT). Of 132 HCT recipients with respiratory-tract virus infection during the initial 100 days after HCT, 50 (38%) developed airflow decline < or =1 year after HCT. Lower-respiratory-tract infection with parainfluenza (odds ratio [OR], 17.9 [95% confidence interval {CI}, 2.0-160]; P=.01) and respiratory syncytial virus (OR, 3.6 [95% CI, 1.0-13]; P=.05) independently increased the risk of development of airflow decline < or =1 year after HCT. The airflow decline was immediately detectable after infection and was strongest for lower-respiratory-tract infection with parainfluenza virus; it stabilized during the months after the respiratory-tract virus infection, but, at < or =1 year after HCT, the initial lung function was not restored. Thus, community respiratory virus-associated airflow decline seems to be specific to viral species and infection localization.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Combined radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude mice, advantages and limitations.

In order to determine if 5-fluorouracil (5FU) could potentiate the effect of radioimmunotherapy (RIT), nude mice bearing subcutaneous human colon carcinoma xenografts were treated by 1 or 2 intravenous injection(s) of subtherapeutic doses of 131I labeled F(ab')2 from anti-carcinoembryonic antigen monoclonal antibodies combined with 5 daily intraperitoneal injections of 5FU. Control mice received either 131I F(ab')2 alone, 5FU alone or no treatment. RIT alone induced significant tumor regression, while 5FU alone gave only minimal tumor growth inhibition. The combined treatment group also resulted in long-term tumor regression with tumors remaining significantly smaller than in the RIT alone group. There was however, no significant difference in tumor recurrence time between the groups treated with RIT alone or with RIT + 5FU. Myelotoxicity, the major side effect of RIT, detected by the decrease of peripheral white blood cells (WBC), was shown to be almost identical between the groups receiving only RIT or only 5FU. Surprisingly, there was no cumulative bone marrow toxicity in animals which received 5FU before RIT. Furthermore, in the latter group, the WBC levels after RIT were significantly higher than in the control group receiving only RIT. Taken together, the results demonstrate the higher therapeutic efficiency of RIT as compared to 5FU in this model. They do not show, however, that the combination of the two forms of treatment can induce longer tumor remission. Interestingly, the WBC results suggest that 5FU given before RIT can have a radioprotective effect on bone marrow, possibly by selecting radioresistant bone marrow stem cells.

Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells.

Emerging viral infections after hematopoietic cell transplantation.

This overview summarizes recent data on emerging viruses after hematopoietic cell transplantation (HCT), including adenovirus, BK virus, human metapneumovirus (hMPV), and human herpesvirus (HHV) 6. The increased recognition of these infections is due to improved molecular detection methods, increased surveillance and more profound immunosuppression in the host. Adenovirus can cause serious disease especially in T-cell depleted transplant recipients. Adenovirus viremia is an important risk factor for disease in this setting. BK virus has been associated with hemorrhagic cystitis in HCT recipients. BK viremia is significantly associated with hemorrhagic cystitis. hMPV shows a seasonal distribution and can cause fatal pneumonia in HCT recipients. hMPV may be the etiology of some cases previously categorized as idiopathic pneumonia syndrome. HHV-6 commonly leads to viremia in HCT recipients. HHV-6 has been strongly associated with encephalitis and delayed platelet engraftment. Prospective studies are needed to further examine epidemiology, disease associations, and management strategies for these viruses.

PROX1 Promotes Metabolic Adaptation and Fuels Outgrowth of Wnt(high) Metastatic Colon Cancer Cells.

The Wnt pathway is abnormally activated in the majority of colorectal cancers, and significant knowledge has been gained in understanding its role in tumor initiation. However, the mechanisms of metastatic outgrowth in colorectal cancer remain a major challenge. We report that autophagy-dependent metabolic adaptation and survival of metastatic colorectal cancer cells is regulated by the target of oncogenic Wnt signaling, homeobox transcription factor PROX1, expressed by a subpopulation of colon cancer progenitor/stem cells. We identify direct PROX1 target genes and show that repression of a pro-apoptotic member of the BCL2 family, BCL2L15, is important for survival of PROX1(+) cells under metabolic stress. PROX1 inactivation after the establishment of metastases prevented further growth of lesions. Furthermore, autophagy inhibition efficiently targeted metastatic PROX1(+) cells, suggesting a potential therapeutic approach. These data identify PROX1 as a key regulator of the transcriptional network contributing to metastases outgrowth in colorectal cancer.

Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.

The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34(+) stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells.

Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.

Proliferative and Osteogenic Differentiation Potentials of Human Fetal Bone Cells

BACKGROUND. Human primary fetal bone cells (hFBC) are being characterized for use in bone tissue regeneration. Unlike human mesenchymal stem cells (hMSC), hFBC are partially differentiated with high expansion and regeneration potential. To date, proliferative and osteoblastic differentiation capacities of fetal bone cells remain poorly examined. The goal of this study was to define an environmental culture conditions for optimal proliferation and production of extracellular bone matrix leading to efficient bone repair. METHODS. Human primary FBC derived from our dedicated, consistent banks of bone cells comprising several fetal donors. For proliferation study, monolayer cultures of both cell types were expanded in DMEM or α- MEM media. Osteoblastic differentiation potentials of both hFBC and hMSC were evaluated through RT-PCR. Regulation of osteogenic differentiation by protein ligands Wnt3a and Wnt5a was studied by ALP enzymatic activity measurement. RESULTS. Evaluation of the proliferation rate demonstrated that hFBC proliferated more rapidly in α-MEM medium. Regarding growth factors that could stimulate cell proliferation rate, we observed that PDGF, FGF2 and Wnt3a had positive effects on proliferation of hFBC. Gene expression analysis demonstrated a higher expression of runx2 in hFBC cultured in basal conditions, which was was similar than that was observed in hMSC in osteoinductive culture conditions. Expression of sox9 was very low in hBFC and hMSC, compared to expression observed in fetal cartilage cells. Looking at osteogenic differentiation capacity, ALP activity was positively regulated byWnt5awhen hFBCwere cultured inα-MEM, but not in DMEM. Conversely, Wnt3a was shown to block the effect of osteogenic inductors on differentiation of both cell types. CONCLUSION. Data presented in this study indicate that the proliferation and differentiation of fetal and mesenchymal stem cells is optimal in α- MEM. Evidence for a pre-differentiated state of hBFC was given by extracellular matrix spontaneous mineralization as well as by higher ALP activity levels observed for these cells in baseline culture conditions, in comparison with hMSC. As we showed that, in vitro, hFBC express a higher capacity to differentiate in osteoblasts, they represent an attractive and promising prospect for fundamental research, and specifically for a new generation of skeletal tissue engineering.

Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

The fetal hypothalamus has the potential to generate cells with a gonadotropin releasing hormone (GnRH) phenotype.

BACKGROUND: Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. METHODOLOGY/FINDINGS: Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. CONCLUSIONS/SIGNIFICANCE: This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

Isolation and in vitro chondrogenic potential of human foetal spine cells.

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.