Fungal Clearance Research Articles

CD4 T cell expression of PTIP is necessary for epigenetic maintenance of Th1/Th17 responses during Cryptococcus neoformans infection

Abstract Containment and clearance of opportunistic fungal pathogen Cryptococcus neoformans (CN) requires Th1/Th17 polarized CD4 T cells. Studies show that Th1/Th17 T-cell polarization is linked to enriched chromatin activation at histone 3 lysine 4 (H3K4). DNA repair enzyme PTIP forms a complex with MLL3/4 H3K4 methyl transferases, but its role in regulation of immune-related genes remains unknown. We hypothesized that the absence of PTIP in CD4 T cells would result in a defect in generation and/or maintenance of Th1/Th17 driven immune protection against CN infection. CD4Cre+, PTIP knockout mice (CD4-PTIP-KO) and their control Cre- littermates were infected with CN and their fungal burden and T cell polarization assessed at 3 and 6 weeks post-infection (wpi). There were few statistically significant differences at 3 wpi, but no defects in IFNγ and TNFαproduction in these mice. However, by 6 wpi we observed profound defects in fungal clearance and the expression of hallmarks of Th1 and Th17 responses. CD4-PTIP-KO mice had diminished fungal clearance by 1.5-log and showed diminished frequency of IFNγ, TNFα, and IL-17 producing CD4 T-cells as well as suppressed global production of these cytokines. Furthermore, frequency of IFNγ-IL-17 dual-producing CD4 T cells, previously found to correlate with clearance, was diminished in CD4-PTIP-KO mice. Interestingly no T-cell activation defect (frequency of CD44hi and CD62L low) was altered in CD4-PTIP-KO mice. Collectively, we conclude that PTIP is not necessary for induction of Th1 and Th17 polarized T cells but it is crucial for maintaining robust Th1/Th17 cytokine production by polarized T cells throughout C.neo infection, presumably via epigenetic stabilization of CD4 T cells Th1/Th17 programing.

Early TNFα signaling promotes classical activation of dendritic cells in lung associated lymph nodes to prime development of protective immunity against cryptococcal infection

Abstract Cryptococcus neoformans is a common opportunistic pathogen that requires intact T cell-mediated immunity for protective host defenses. While Th1/Th17 responses contribute to the effective containment and elimination of C. neoformans, Th2 responses are non-protective and can worsen clearance outcomes. Previous studies showed that early tumor necrosis factor alpha (TNFα) signaling is required for optimal clearance, while its absence leads to immune dysregulation and persistent fungal infection. To determine mechanism(s) of early TNFα signaling in the generation of protective anti-cryptococcal immunity, we evaluated the contribution of TNFα to the priming of the innate immune system for optimal T-cell development. In CBA/J mice depleted of TNFα at the time of infection with C. neoformans, we evaluated DC accumulation, activation profile, and interaction frequencies with T cells in lung associated lymph nodes (LALN) in comparison with the infected control mice. We found that early TNFα depletion during pulmonary C. neoformans infection resulted in: 1) lack of pulmonary fungal clearance and enhanced dissemination; 2) decreased Th1/Th17 polarization and CD4+ T cell activation in the LALN; 3) diminished DC accumulation in the LALN; 4) decreased expression of classical activation DC signatures (co-stimulatory molecules CD40 and CD86), pro-Th1 and pro-Th17 cytokines (IL-12b, TNFα, IL-23a and IL-21); and 5) increased expression of the alternative activation DC signatures (Fizz and Gal3) and pro-Th2 cytokines (IL4 and IL-10). Thus, our data indicates early TNFα signaling promotes classical activation of DC in lung associated lymph nodes thereby priming development of protective immunity against cryptococcal infection.

Inflammation and immune cell recruitment in the CNS following disseminated Cryptococcus neoformans infection

Abstract Mortality in Cryptococcus neoformans (Cn) infection is chiefly associated with CNS infection but little is known about immune responses against Cn within the CNS. We established an i.v. model of disseminated cryptococcosis that leads to a rapid expansion of Cn in the CNS in order to compare inflammatory readouts in resistant CBA/J with susceptible C57BL/6 mice. Fungal burdens peaked (>107CFU) in the brain by d7 post infection in both models, but in CBA/J mice fungal burden significantly declined between d14 to d21. In contrast, C57BL/6 mice developed persistently high fungal burdens through d21. Consistent with these findings CBA/J mice showed gradual increases in Ly6C+ myeloid, CD4+ and CD8+ T cell infiltration along with production of CCL2, IFNγ and CXCL10, reaching maximum or near maximum levels by day 14. In C57BL6, the development of inflammatory responses was significantly delayed. Production of cytokines and influx of Ly6C+, CD4+ and CD8+ cells was almost absent until d21, but prominently exceeded all readouts from CBA/J at day 21 by several fold. Significant increases in iNOShi CD11bhi MHCIIhi CD11c+ cells and an overabundance of highly activated CD11c+ MHCII+ microglia, hallmarks of rapidly developing CNS pathologies, were also more evident in the brains of C57BL/6 mice at d21. In summary, our findings are consistent with the hypothesis that early recruitment and activation of inflammatory cells in the CNS post-Cn infection, presumably in response to early CCL2-induction, along with subsequent early induction of IFNγ and CXCL10 promotes fungal clearance. In contrast, delayed responses found in C57BL6 mice were ineffective at removing fungal organisms while leading to over exuberant inflammation and detrimental CNS pathology.

Distinctive signalling in NK cells during cytotoxicity to the yeast Cryptococcus neoformans

Abstract Introduction Cryptococcus is a fungal pathogen that subverts the adaptive immune response. Fortunately, NK cells are capable of directly recognizing, triggering cytotoxicity and killing Cryptococcus. Although, NK cytotoxicity against tumor and virally infected targets are well studied, less is known about anti-fungal signaling. In this study, we investigated a novel NK cytotoxicity pathway involving Rac, Src family kinases (SFK), and integrins. Methods The involvement of Rac, SFK, integrin-linked kinase (ILK), and integrins were explored using small molecule inhibitors, siRNA knockdown, activated Rac pull down assays, and immunoblots. Results Our studies found that Cryptococcus activated both SFK and Rac signaling pathways. Small molecule inhibitors of SFK and Rac blocked Erk and PI3K activity. siRNA knockdown of Rac also inhibited PI3K activity, suggesting that Rac was an upstream non-canonical activator of PI3K. Moreover, individually Rac and SFK were not sufficient for activation of PI3K. Inhibition of Rac also resulted in a 90% loss of anti-cryptococcal cytotoxicity. Additionally, inhibitors of ILK prevented Rac activation and caused an 80% reduction in cryptococcal killing. Lastly, siRNA knockdown of beta-1 integrins caused an inhibition of ILK activation and 50% reduction in cryptococcal killing. Conclusion We found beta-1 integrins to be a fungal receptor and Rac an activator of PI3K. These findings build a signaling model of anti-fungal killing that is different from the canonical tumor killing model that involves beta-2 integrins and Rac downstream of PI3K. Our model provides various potential therapeutic targets that could enhance fungal clearance mediated by the innate immune system.

Dual roles of scavenger receptor MARCO in innate versus adaptive immune responses during Cryptococcus neoformans infection

Abstract Resistance to the opportunistic fungal pathogen Cryptococcus neoformans (Cn) is associated with development of Th1/Th17 immune bias while a Th2 immune response results in increased susceptibility. Macrophage Receptor with Collagenous Structure (MARCO) is important for recognition of some pathogens and subsequent activation and polarization of the immune responses by DC and macrophages, however, its role during Cn infection remains unknown. Here we found that in spite of diminished early (innate) control of Cn in the infected lungs, Cn-infected MARCO−/− mice showed improved fungal clearance during the adaptive phase (wk 5) compared to MARCO+/+ mice. MARCO−/− mice showed: 1) improved accumulation of exudate macrophages and CD11b+ DC and their improved activation phenotype (CD80+, CD86+ and MHCIIhigh); 2) increased intracellular IFN-γ and IL-17 production by pulmonary CD4+ and CD8+ T cells; 3) decreased IL-4, IL-5, IL-10 and increased IFN-γ and IL-17 production by splenocytes treated with Cn antigen; 5) diminished systemic level of IgE. Interestingly, in spite of improved clearance, MARCO−/− mice showed decreased frequency of lung resident CD44highCD62LlowCD69+ CD103+CD11a+CCR7− effector memory T cells. We conclude that while important for the innate control of Cn in the lungs, MARCO can be exploited by Cn to interfere with immune polarization leading to an altered, non-protective efferent immune bias, which outweighs potentially beneficial effects of MARCO.

Ficolins promote fungal clearance during the early stage of pulmonary infection with Aspergillus fumigatus.

Abstract Aspergillus fumigatus is a fungal pathogen causing severe and usually fatal invasive infections in immunocompromised patients. The Ficolins are pattern recognition molecules of the lectin pathway of complement and in vitro studies suggest a role for ficolins in the defence against A.fumigatus. However, little is known about their significance in fungal infections in vivo. Thus, our aim of the current project was to establish a murine model of pulmonary A. fumigatus infection to study the relevance of ficolins in host protection against A.fumigatus. Wildtype and ficolin knockout mice were infected intranasally with a sublethal dose of A.fumigatus conidia and the lungs were removed at various time points post-infection to assess the fungal load and expression of pro-inflammatory cytokines. In addition, the concentration of pro-inflammatory cytokines was measured in the bronchoalveolar lavage. After 24 hours of A.fumigatus infection, the pulmonary fungal load was significantly higher in ficolin knockout mice compared to wildtype mice. In contrast, no significant difference in fungal load between ficolin knockout mice and wildtype mice was observed after 12, 48 or 72 hours of infection. Interestingly, the increased fungal load in knockout mice 24 post-infection was accompanied by a significantly impaired induction of pro-inflammatory cytokines compared to wildtype mice. These results show that ficolins promote fungal clearance and modulate the pro-inflammatory cytokine response, demonstrating the importance of ficolins during the initial phase of A. fumigatus infections in vivo.

Dectin-1 and Dectin-2 promote control of the fungal pathogen Trichophyton rubrum independently of IL-17 and adaptive immunity in experimental deep dermatophytosis.

Dermatophytoses are chronic fungal infections, the main causative agent of which is Trichophyton rubrum (T. rubrum). Despite their high occurrence worldwide, the immunological mechanisms underlying these diseases remain largely unknown. Here, we uncovered the C-type lectin receptors, Dectin-1 and Dectin-2, as key elements in the immune response to T. rubrum infection in a model of deep dermatophytosis. Invitro, we observed that deficiency in Dectin-1 and Dectin-2 severely compromised cytokine production by dendritic cells. Invivo, mice lacking Dectin-1 and/or Dectin-2 showed an inadequate pro-inflammatory cytokine production in response to T. rubrum infection, impairing its resolution. Strikingly, neither adaptive immunity nor IL-17 response were required for fungal clearance, highlighting innate immunity as the main checkpoint in the pathogenesis of T. rubrum infection.

Eosinophils subvert host resistance to an intracellular pathogen by instigating non-protective IL-4 in CCR2\u2212/\u2212 mice

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however, their influence on intracellular pathogens is less clear. We previously reported that CCR2−/− mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2−/− animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus, our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.

PKC-δ activation in neutrophils promotes fungal clearance.

The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ-deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

Adjunctive Dexamethasone in HIV-Associated Cryptococcal Meningitis

BackgroundCryptococcal meningitis associated with human immunodeficiency virus (HIV) infection causes more than 600,000 deaths each year worldwide. Treatment has changed little in 20 years, and there are no imminent new anticryptococcal agents. The use of adjuvant glucocorticoids reduces mortality among patients with other forms of meningitis in some populations, but their use is untested in patients with cryptococcal meningitis.MethodsIn this double-blind, randomized, placebo-controlled trial, we recruited adult patients with HIV-associated cryptococcal meningitis in Vietnam, Thailand, Indonesia, Laos, Uganda, and Malawi. All the patients received either dexamethasone or placebo for 6 weeks, along with combination antifungal therapy with amphotericin B and fluconazole.ResultsThe trial was stopped for safety reasons after the enrollment of 451 patients. Mortality was 47% in the dexamethasone group and 41% in the placebo group by 10 weeks (hazard ratio in the dexamethasone group, 1.11; 95% confidence interval [CI], 0.84 to 1.47; P=0.45) and 57% and 49%, respectively, by 6 months (hazard ratio, 1.18; 95% CI, 0.91 to 1.53; P=0.20). The percentage of patients with disability at 10 weeks was higher in the dexamethasone group than in the placebo group, with 13% versus 25% having a prespecified good outcome (odds ratio, 0.42; 95% CI, 0.25 to 0.69; P<0.001). Clinical adverse events were more common in the dexamethasone group than in the placebo group (667 vs. 494 events, P=0.01), with more patients in the dexamethasone group having grade 3 or 4 infection (48 vs. 25 patients, P=0.003), renal events (22 vs. 7, P=0.004), and cardiac events (8 vs. 0, P=0.004). Fungal clearance in cerebrospinal fluid was slower in the dexamethasone group. Results were consistent across Asian and African sites.ConclusionsDexamethasone did not reduce mortality among patients with HIV-associated cryptococcal meningitis and was associated with more adverse events and disability than was placebo. (Funded by the United Kingdom Department for International Development and others through the Joint Global Health Trials program; Current Controlled Trials number, ISRCTN59144167.)

Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration.

Card9- and MyD88-Mediated Gamma Interferon and Nitric Oxide Production Is Essential for Resistance to Subcutaneous Coccidioides posadasii Infection.

Coccidioidomycosis is a potentially life-threatening respiratory disease which is endemic to the southwestern United States and arid regions of Central and South America. It is responsible for approximately 150,000 infections annually in the United States alone. Almost every human organ has been reported to harbor parasitic cells of Coccidioides spp. in collective cases of the disseminated form of this mycosis. Current understanding of the mechanisms of protective immunity against lung infection has been largely derived from murine models of pulmonary coccidioidomycosis. However, little is known about the nature of the host response to Coccidioides in extrapulmonary tissue. Primary subcutaneous coccidioidal infection is rare but has been reported to result in disseminated disease. Here, we show that activation of MyD88 and Card9 signal pathways are required for resistance to Coccidioides infection following subcutaneous challenge of C57BL/6 mice, which correlates with earlier findings of the protective response to pulmonary infection. MyD88(-/-) andCard9(-/-) mice recruited reduced numbers of T cells, B cells, and neutrophils to the Coccidioides-infected hypodermis com pared to wild-type mice; however, neutrophils were dispensable for resistance to skin infection. Further studies have shown that gamma interferon (IFN-γ) production and activation of Th1 cells characterize resistance to subcutaneous infection. Furthermore, activation of a phagosomal enzyme, inducible nitric oxide synthase, which is necessary for NO production, is a requisite for fungal clearance in the hypodermis. Collectively, our data demonstrate that MyD88- and Card9-mediated IFN-γ and nitric oxide production is essential for protection against subcutaneous Coccidioides infection.

Inactivation of Aspergillus flavus spores in a sealed package by cold plasma streamers

The main objective of this study is to investigate the inactivation efficacy of cold streamers in a sealed package on pathogenic fungi Aspergillus flavus (A. flavus) spores that artificially contaminated pistachio surface. To produce penetrating cold streamers, electric power supply was adapted to deposit adequate power into the package. The plasma streamers were generated by an alternating high voltage with carrier frequency of 12.5 kHz which was suppressed by a modulated pulsed signal at frequency of 110 Hz. The plasma exposition time was varied from 8 to 18 min to show the effect of the plasma treatment on fungal clearance while the electrode and sample remained at room temperature. This proved a positive effect of the cold streamers treatment on fungal clearance. Benefits of deactivation of fungal spores by streamers inside the package include no heating, short treatment time and adaptability to existing processes. Given its ability to ensure the safety and longevity of food products, this technology has great potential for utilization in food packaging and processing industry. In this study, moisture and pH changes of pistachio samples after plasma streamers treatment were also investigated.

CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival.

Systemic Candida albicans infection causes high morbidity and mortality and is now the leading cause of nosocomial bloodstream infection in the United States. Neutropenia is a major risk factor for poor outcome in infected patients; however, the molecular factors that mediate neutrophil trafficking and effector function during infection are poorly defined. Using a mouse model of systemic candidiasis, we found that the neutrophil-selective CXC chemokine receptor Cxcr1 and its ligand, Cxcl5, are highly induced in the Candida-infected kidney, the target organ in the model. To investigate the role of Cxcr1 in antifungal host defense in vivo, we generated Cxcr1(-/-) mice and analyzed their immune response to Candida. Mice lacking Cxcr1 exhibited decreased survival with enhanced Candida growth in the kidney and renal failure. Increased susceptibility of Cxcr1(-/-) mice to systemic candidiasis was not due to impaired neutrophil trafficking from the blood into the infected kidney but was the result of defective killing of the fungus by neutrophils that exhibited a cell-intrinsic decrease in degranulation. In humans, the mutant CXCR1 allele CXCR1-T276 results in impaired neutrophil degranulation and fungal killing and was associated with increased risk of disseminated candidiasis in infected patients. Together, our data demonstrate a biological function for mouse Cxcr1 in vivo and indicate that CXCR1-dependent neutrophil effector function is a critical innate protective mechanism of fungal clearance and host survival in systemic candidiasis.

Reproducibility of CSF quantitative culture methods for estimating rate of clearance in cryptococcal meningitis

Quantitative cerebrospinal fluid (CSF) cultures provide a measure of disease severity in cryptococcal meningitis. The fungal clearance rate by quantitative cultures has become a primary endpoint for phase II clinical trials. This study determined the inter-assay accuracy of three different quantitative culture methodologies. Among 91 participants with meningitis symptoms in Kampala, Uganda, during August-November 2013, 305 CSF samples were prospectively collected from patients at multiple time points during treatment. Samples were simultaneously cultured by three methods: (1) St. George's 100 mcl input volume of CSF with five 1:10 serial dilutions, (2) AIDS Clinical Trials Group (ACTG) method using 1000, 100, 10 mcl input volumes, and two 1:100 dilutions with 100 and 10 mcl input volume per dilution on seven agar plates; and (3) 10 mcl calibrated loop of undiluted and 1:100 diluted CSF (loop). Quantitative culture values did not statistically differ between St. George-ACTG methods (P= .09) but did for St. George-10 mcl loop (P< .001). Repeated measures pairwise correlation between any of the methods was high (r≥0.88). For detecting sterility, the ACTG-method had the highest negative predictive value of 97% (91%St. George, 60% loop), but the ACTG-method had occasional (∼10%) difficulties in quantification due to colony clumping. For CSF clearance rate, St. George-ACTG methods did not differ overall (mean -0.05±0.07 log10CFU/ml/day;P= .14) on a group level; however, individual-level clearance varied. The St. George and ACTG quantitative CSF culture methods produced comparable but not identical results. Quantitative cultures can inform treatment management strategies.

Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

Evaluación de la Patogenicidad Inhalatoria del Micoinsecticida Beauveria bassiana LF14 en Ratones

&lt;p&gt;Se realizaron pruebas de bioseguridad para evaluar los posibles efectos patogénicos del aislamiento autóctono LF14 de &lt;em&gt;Beauveria bassiana&lt;/em&gt; en ratones albinos NMRI; el cual posee una elevada virulencia hacia &lt;em&gt;Rhodnius prolixus&lt;/em&gt; y &lt;em&gt;Triatoma maculata&lt;/em&gt; (Triatominae), vectores de enfermedad de Chagas en Venezuela. Se administró 1x10&lt;sup&gt;9&lt;/sup&gt; conidias/ml del hongo mediante gotas nasales. Se realizaron observaciones clínicas y de comportamiento, así como de peso corporal en forma diaria y por 14 días. Asimismo, se estimó el aclaramiento fúngico mediante examen directo y cultivo de muestras intranasales, y de infectividad con la toma de muestras de tejidos de varios órganos, incluyendo un estudio histopatológico a los 3, 7 y 14 días después de la inoculación. No ocurrieron muertes ni alteraciones clínico-patológicas discernibles, siendo el tipo de comportamiento activo-normal en el 100% de los casos. El peso promedio corporal se incrementó, tanto en los animales expuestos a LF14 &lt;em&gt;B. bassiana&lt;/em&gt; como los controles, habiendo diferencias entre sexos (p&amp;lt;0.0000). No se detectaron anormalidades en la inspección anatomopatológica ni hubo reacciones tisulares de consideración. Solo se obtuvieron conidias viables sin germinar al día tres de la necropsia en pulmón y tráquea, sugiriendo que no existe evidencia de multiplicación fúngica. Los resultados obtenidos permiten sugerir que el aislamiento &lt;em&gt;B. bassiana&lt;/em&gt; LF14 no es patogénico por vía inhalatoria en &lt;em&gt;Mus musculus&lt;/em&gt; (NMRI).&lt;/p&gt;

The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models.Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB.Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 105 CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2 (control), p<0.005). Along with that, NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 103 PMN/µl +/- 0.2 (NFATc1LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged.Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil Recruitment Is Regulated By Adamts-13 in a Murine Model of Invasive Aspergillosis

▪Introduction: During inflammation von Willebrand factor (VWF) multimers are secreted as an acute phase protein whereupon the size and the prothrombotic activity play an essential role. The size of VWF multimers is regulated by the specific proteolytic activity of ADAMTS-13 (a disintegrin and metalloprotease with ThromboSpondin type 1 repeats-13) which is diminished under several pathological conditions. Employing a murine model of invasive pulmonary aspergillosis (IPA) we aimed to determine the relevance of this regulatory pathway for innate inflammatory responses and polymorphonuclear neutrophil (PMN) recruitment which is crucial for fungal clearance and survival.Methods: IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. 24 h after infection some mice were sacrificed for analysis of fungal load and histology. To assess fungal load as CFU lung homogenates were plated and cultured on Sabourough agar plates. Paraffin sections of the lungs were prepared and stained with mouse complement component C3d antibody for histological analysis. In addition, degranulation, oxidative burst activity and CD62L shedding were assessed in PMN of wildtype and Adamts13-/- mice. Chemotactic properties of A. fumigatus -activated and control serum from wildtype and knock-out mice was evaluated by migration of purified human PMN, isolated by dextran sedimentation and Histopaque® centrifugation, in a transwell assay (Corning® HTS Transwell®-96 well permeable support; 3µm).Results: Adamts13 deficiency in mice was accompanied by a worse outcome of IPA infections compared to wildtype controls. Adamts13-/- mice displayed more severe signs of disease and a lethal course was observed in about 24% of animals. Compared to Adamts13-/- mice all neutropenic mice developed lethal IPA after infection, but all wild-type mice survived the disease. Besides, examination of the lungs revealed a higher fungal burden along with increased signs of acute lung injury, complement deposition and levels of pro-inflammatory cytokines in Adamts13-/- mice. Furthermore a more pronounced perivascular leukocyte infiltration was observed in histological sections indicating a dysregulated inflammatory response in Adamts13-/- mice. Importantly, the activation of neutrophil effector functions in response to TLR agonist or in PMN-mediated fungal killing of conidia or hyphae revealed no general defect in vitro. Despite enhanced complement deposition in the lungs in Adamts13-/- mice, PMN migration towards complement-activated serum revealed unaltered chemotactic properties comparing A. fumigatus -activated serum of wildtype and knock-out mice.Conclusion: In acute inflammatory processes such as fungal pneumonia the proteolytic regulation of VWF by ADAMTS-13 is an important mechanism to control PMN recruitment and outcome of infections. DisclosuresNo relevant conflicts of interest to declare.

Response of Cytokines and Hydrogen Peroxide to Sporothrix schenckii Exoantigen in Systemic Experimental Infection.

The response of hydrogen peroxide (H2O2) and cytokines during an experimental sporotrichosis in male Swiss mice was assessed over a period of 10 weeks by monitoring macrophage activation challenged with exoantigen (ExoAg) from the fungus Sporothrix schenckii. The studied endpoints were: H2O2 production, fungal burden at spleen, apoptosis in peritoneal macrophages, and IL-1β, IL-6, IL-2, IL-10 production. During the two first weeks of infection was observed low burden of yeast in spleen and high response of H2O2, IL-2, and IL-1β. The weeks of highest fungal burden (fourth-sixth) coincided with major apoptosis in peritoneal macrophages, normal production of IL-6 and lower production of H2O2, IL-2, and IL-1β, suggesting a role for these three last in the early control of infection. On the other hand, IL-1β (but not IL-6) was recovered since the sixth week, suggesting a possible role in the late phase of infection, contributing to the fungal clearance in conjunction with the specific mechanisms. The IL-10 was elevated until the sixth, principally in the second week. These results evidences that ExoAg is involved in the host immune modulation, influencing the S. Schenckii virulence, and its role is related with the time of the infection in the model used.