Murine Inducible Nitric Oxide Synthase Expression Is Essential for Antifungal Defenses in Kidneys during Disseminated Cryptococcus deneoformans Infection.

Disseminated cryptococcosis has a nearly 70% mortality, mostly attributed to CNS infection, with lesser-known effects on other organs. Immune protection against Cryptococcus relies on Th1 immunity with M1 polarization, rendering macrophages fungicidal. The importance of M1-upregulated inducible NO synthase (iNOS) has been documented in pulmonary anticryptococcal defenses, whereas its role in disseminated cryptococcosis remains controversial. Here we examined the effect of iNOS deletion in disseminated (i.v.) C. deneoformans 52D infection, comparing wild-type (C57BL/6J) and iNOS-/- mice. iNOS-/- mice had significantly reduced survival and nearly 100-fold increase of the kidney fungal burden, without increases in the lungs, spleen, or brain. Histology revealed extensive lesions and almost complete destruction of the kidney cortical area with a loss of kidney function. The lack of fungal control was not due to a failure to recruit immune cells because iNOS-/- mice had increased kidney leukocytes. iNOS-/- mice also showed no defect in T cell polarization. We conclude that iNOS is critically required for local anticryptococcal defenses in the kidneys, whereas it appears to be dispensable in other organs during disseminated infection. This study exemplifies a unique phenotype of local immune defenses in the kidneys and the organ-specific importance of a single fungicidal pathway.

Longitudinal Evaluation of Biomarkers in Wound Fluids from Venous Leg Ulcers and Split-thickness Skin Graft Donor Site Wounds Treated with a Protease-modulating Wound Dressing.

Venous leg ulcers represent a clinical challenge and impair the quality of life of patients. This study examines impaired wound healing in venous leg ulcers at the molecular level. Protein expression patterns for biomarkers were analysed in venous leg ulcer wound fluids from 57 patients treated with a protease-modulating polyacrylate wound dressing for 12 weeks, and compared with exudates from 10 acute split-thickness wounds. Wound healing improved in the venous leg ulcer wounds: 61.4% of the 57 patients with venous leg ulcer achieved a relative wound area reduction of ≥ 40%, and 50.9% of the total 57 patients achieved a relative wound area reduction of ≥ 60%. Within the first 14 days, abundances of S100A8, S100A9, neutrophil elastase, matrix metalloproteinase-2, and fibronectin in venous leg ulcer exudates decreased significantly and remained stable, yet higher than in acute wounds. Interleukin-1ß, tumour necrosis factor alpha, and matrix metalloproteinase-9 abundance ranges were similar in venous leg ulcers and acute wound fluids. Collagen (I) α1 abundance was higher in venous leg ulcer wound fluids and was not significantly regulated. Overall, significant biomarker changes occurred in the first 14 days before a clinically robust healing response in the venous leg ulcer cohort.

Victors: a web-based knowledge base of virulence factors in human and animal pathogens.

Virulence factors (VFs) are molecules that allow microbial pathogens to overcome host defense mechanisms and cause disease in a host. It is critical to study VFs for better understanding microbial pathogenesis and host defense mechanisms. Victors (http://www.phidias.us/victors) is a novel, manually curated, web-based integrative knowledge base and analysis resource for VFs of pathogens that cause infectious diseases in human and animals. Currently, Victors contains 5296 VFs obtained via manual annotation from peer-reviewed publications, with 4648, 179, 105 and 364 VFs originating from 51 bacterial, 54 viral, 13 parasitic and 8 fungal species, respectively. Our data analysis identified many VF-specific patterns. Within the global VF pool, cytoplasmic proteins were more common, while adhesins were less common compared to findings on protective vaccine antigens. Many VFs showed homology with host proteins and the human proteins interacting with VFs represented the hubs of human-pathogen interactions. All Victors data are queriable with a user-friendly web interface. The VFs can also be searched by a customized BLAST sequence similarity searching program. These VFs and their interactions with the host are represented in a machine-readable Ontology of Host-Pathogen Interactions. Victors supports the 'One Health' research as a vital source of VFs in human and animal pathogens.

Autocrine IL-10 Signaling Promotes Dendritic Cell Type-2 Activation and Persistence of Murine Cryptococcal Lung Infection.

The substantial morbidity and mortality caused by invasive fungal pathogens, including Cryptococcus neoformans, necessitates increased understanding of protective immune responses against these infections. Our previous work using murine models of cryptococcal lung infection demonstrated that dendritic cells (DCs) orchestrate critical transitions from innate to adaptive immunity and that IL-10 signaling blockade improves fungal clearance. To further understand interrelationships among IL-10 production, fungal clearance, and the effect of IL-10 on lung DCs, we performed a comparative temporal analysis of cryptococcal lung infection in wild type C57BL/6J mice (designated IL-10+/+) and IL-10-/- mice inoculated intratracheally with C. neoformans (strain 52D). Early and sustained IL-10 production by lung leukocytes was associated with persistent infection in IL-10+/+ mice, whereas fungal clearance was improved in IL-10-/- mice during the late adaptive phase of infection. Numbers of monocyte-derived DCs, T cells, and alveolar and exudate macrophages were increased in lungs of IL-10-/- versus IL-10+/+ mice concurrent with evidence of enhanced DC type-1, Th1/Th17 CD4 cell, and classical macrophage activation. Bone marrow-derived DCs stimulated with cryptococcal mannoproteins, a component of the fungal capsule, upregulated expression of IL-10 and IL-10R, which promoted DC type-2 activation in an autocrine manner. Thus, our findings implicate fungus-triggered autocrine IL-10 signaling and DC type-2 activation as important contributors to the development of nonprotective immune effector responses, which characterize persistent cryptococcal lung infection. Collectively, this study informs and strengthens the rationale for IL-10 signaling blockade as a novel treatment for fungal infections.

Anti-PD-1 Antibody Treatment Promotes Clearance of Persistent Cryptococcal Lung Infection in Mice.

Activation of immunomodulatory pathways in response to invasive fungi can impair clearance and promote persistent infections. The programmed cell death protein-1 (PD-1) signaling pathway inhibits immune effector responses against tumors, and immune checkpoint inhibitors that block this pathway are being increasingly used as cancer therapy. The objective of this study was to investigate whether this pathway contributes to persistent fungal infection and to determine whether anti-PD-1 Ab treatment improves fungal clearance. Studies were performed using C57BL/6 mice infected with a moderately virulent strain of Cryptococcus neoformans (52D), which resulted in prolonged elevations in fungal burden and histopathologic evidence of chronic lung inflammation. Persistent infection was associated with increased and sustained expression of PD-1 on lung lymphocytes, including a mixed population of CD4+ T cells. In parallel, expression of the PD-1 ligands, PD-1 ligands 1 and 2, was similarly upregulated on specific subsets of resident and recruited lung dendritic cells and macrophages. Treatment of persistently infected mice for 4 wk by repetitive administration of neutralizing anti-PD-1 Ab significantly improved pulmonary fungal clearance. Treatment was well tolerated without evidence of morbidity. Immunophenotyping revealed that anti-PD-1 Ab treatment did not alter immune effector cell numbers or myeloid cell activation. Treatment did reduce gene expression of IL-5 and IL-10 by lung leukocytes and promoted sustained upregulation of OX40 by Th1 and Th17 cells. Collectively, this study demonstrates that PD-1 signaling promotes persistent cryptococcal lung infection and identifies this pathway as a potential target for novel immune-based treatments of chronic fungal disease.

Scavenger Receptor MARCO Orchestrates Early Defenses and Contributes to Fungal Containment during Cryptococcal Infection.

The scavenger receptor macrophage receptor with collagenous structure (MARCO) promotes protective innate immunity against bacterial and parasitic infections; however, its role in host immunity against fungal pathogens, including the major human opportunistic fungal pathogen Cryptococcus neoformans, remains unknown. Using a mouse model of C. neoformans infection, we demonstrated that MARCO deficiency leads to impaired fungal control during the afferent phase of cryptococcal infection. Diminished fungal containment in MARCO-/- mice was accompanied by impaired recruitment of Ly6Chigh monocytes and monocyte-derived dendritic cells (moDC) and lower moDC costimulatory maturation. The reduced recruitment and activation of mononuclear phagocytes in MARCO-/- mice was linked to diminished early expression of IFN-γ along with profound suppression of CCL2 and CCL7 chemokines, providing evidence for roles of MARCO in activation of the CCR2 axis during C. neoformans infection. Lastly, we found that MARCO was involved in C. neoformans phagocytosis by resident pulmonary macrophages and DC. We conclude that MARCO facilitates early interactions between C. neoformans and lung-resident cells and promotes the production of CCR2 ligands. In turn, this contributes to a more robust recruitment and activation of moDC that opposes rapid fungal expansion during the afferent phase of cryptococcal infection.

T Cell-Restricted Notch Signaling Contributes to Pulmonary Th1 and Th2 Immunity during Cryptococcus neoformans Infection.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans Inhibition of T cell-restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ-, IL-5-, and IL-13-producing CD4+ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8+ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.

Local GM-CSF-Dependent Differentiation and Activation of Pulmonary Dendritic Cells and Macrophages Protect against Progressive Cryptococcal Lung Infection in Mice.

Patients with acquired deficiency in GM-CSF are susceptible to infections with Cryptococcus neoformans and other opportunistic fungi. We previously showed that GM-CSF protects against progressive fungal disease using a murine model of cryptococcal lung infection. To better understand the cellular and molecular mechanisms through which GM-CSF enhances antifungal host defenses, we investigated temporal and spatial relationships between myeloid and lymphoid immune responses in wild-type C57BL/6 mice capable of producing GM-CSF and GM-CSF-deficient mice infected with a moderately virulent encapsulated strain of C. neoformans (strain 52D). Our data demonstrate that GM-CSF deficiency led to a reduction in: 1) total lung leukocyte recruitment; 2) Th2 and Th17 responses; 3) total numbers of CD11b(+) dendritic cells (DC) and CD11b(-) and CD11b(+) macrophages (Mϕ); 4) DC and Mϕ activation; and 5) localization of DC and Mϕ to the microanatomic sites of alveolar infection. In contrast, GM-CSF deficiency resulted in increased accumulation of DC and Mϕ precursors, namely Ly-6C(high) monocytes, in the blood and lungs of infected mice. Collectively, these results show that GM-CSF promotes the local differentiation, accumulation, activation, and alveolar localization of lung DC and Mϕ in mice with cryptococcal lung infection. These findings identify GM-CSF as central to the protective immune response that prevents progressive fungal disease and thus shed new light on the increased susceptibility to these infections observed in patients with acquired GM-CSF deficiency.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.

STAT1 signaling is essential for protection against Cryptococcus neoformans infection in mice.

Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the fungus. In contrast, experimental studies show that protective immune responses against cryptococcosis are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99γ (C. neoformans strain H99 engineered to produce murine IFN-γ) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in wild-type and STAT1 knockout (KO) mice infected with either strain H99γ or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99γ and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99γ or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90-100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses.

Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection.

The potent immunoregulatory properties of IL-10 can counteract protective immune responses and, thereby, promote persistent infections, as evidenced by studies of cryptococcal lung infection in IL-10-deficient mice. To further investigate how IL-10 impairs fungal clearance, the current study used an established murine model of C57BL/6J mice infected with Cryptococcus neoformans strain 52D. Our results demonstrate that fungal persistence is associated with an early and sustained expression of IL-10 by lung leukocytes. To examine whether IL-10-mediated immune modulation occurs during the early or late phase of infection, assessments of fungal burden and immunophenotyping were performed on mice treated with anti-IL-10R-blocking Ab at 3, 6, and 9 d postinfection (dpi) (early phase) or at 15, 18, and 21 dpi (late phase). We found that both early and late IL-10 blockade significantly improved fungal clearance within the lung compared with isotype control treatment when assessed 35 dpi. Immunophenotyping identified that IL-10 blockade enhanced several critical effector mechanisms, including increased accumulation of CD4(+) T cells and B cells, but not CD8(+) T cells; specific increases in the total numbers of Th1 and Th17 cells; and increased accumulation and activation of CD11b(+) dendritic cells and exudate macrophages. Importantly, IL-10 blockade effectively abrogated dissemination of C. neoformans to the brain. Collectively, this study identifies early and late cellular and molecular mechanisms through which IL-10 impairs fungal clearance and highlights the therapeutic potential of IL-10 blockade in the treatment of fungal lung infections.

STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

Molecules at the interface of Cryptococcus and the host that determine disease susceptibility.

Cryptococcus neoformans and Cryptococcus gattii, the predominant etiological agents of cryptococcosis, are fungal pathogens that cause disease ranging from a mild pneumonia to life-threatening infections of the central nervous system (CNS). Resolution or exacerbation of Cryptococcus infection is determined following complex interactions of several host and pathogen derived factors. Alternatively, interactions between the host and pathogen may end in an impasse resulting in the establishment of a sub-clinical Cryptococcus infection. The current review addresses the delicate interaction between the host and Cryptococcus-derived molecules that determine resistance or susceptibility to infection. An emphasis will be placed on data highlighted at the recent 9th International Conference on Cryptococcus and Cryptococcosis (ICCC).

Role of CC chemokine receptor 4 in natural killer cell activation during acute cigarette smoke exposure.

Cigarette smoke (CS)-induced lung injury involves innate immune responses. The activation of innate effector cells is thought to require cross talk with dendritic cells (DCs) and macrophages, but the mediators of interaction are unknown. One candidate, CC chemokine receptor 4 (CCR4), is expressed by innate and adaptive effector cells, and its ligands are produced by DCs and macrophages. Using flow cytometry and confocal microscopy, we defined innate responses of lung myeloid DCs, macrophages, and conventional natural killer (NK) cells in mice exposed to CS over 4 days and examined the contribution of CCR4 using CCR4 knockout (CCR4(-/-)) mice. CS affected populations differently, causing an increase in F4/80(+) macrophages, a reduction in parenchymal CD11c(+)CD11b(+)CD103(-) DCs, but no effect on mucosal CD11c(+)CD11b(-)CD103(+) DCs. CS also induced a population of primed/activated CD69(+) NK cells and bronchoepithelial expression of the stress-related NKG2D receptor-activating protein, retinoic acid early transcript 1. CS-exposed CCR4(-/-) mice were similar to controls regarding effects on DCs and macrophages but displayed substantially impaired NK priming/activation and reduced expression of transcripts for interferon gamma, CXCL10, and retinoic acid early transcript 1. Quantitative confocal microscopy revealed that lungs of CS-exposed CCR4(-/-) mice had significantly reduced contacts of NK cells with CD11c(+) cells. These findings demonstrate that acute CS exposure elicits NK cell responses and suggest that CCR4 promotes NK cell priming/activation by mediating contacts with sentinel cells in the lung.

Scavenger receptor A modulates the immune response to pulmonary Cryptococcus neoformans infection.

Scavenger receptors represent an important class of pattern recognition receptors shown to mediate both beneficial and detrimental roles in host defense against microbial pathogens. The role of the major macrophage scavenger receptor, scavenger receptor A (SRA), in the immune response against the pathogenic fungus, Cryptococcus neoformans, is unknown. To evaluate the role of SRA in anticryptococcal host defenses, SRA(+/+) mice and SRA(-/-) mice were infected intratracheally with C. neoformans. Results show that infection of SRA(-/-) mice resulted in a reduction in the pulmonary fungal burden at the efferent phase (3 wk) compared with SRA(+/+) mice. Improved fungal clearance in SRA(-/-) mice was associated with decreased accumulation of eosinophils and greater accumulation of CD4(+) T cells and CD11b(+) dendritic cells. Additional parameters were consistent with enhanced anticryptococcal immunity in the infected SRA(-/-) mice: 1) increased expression of the costimulatory molecules CD80 and CD86 by lung APCs, 2) decreased expression of Th2 cytokines (IL-4 and IL-13) and IL-10 in lung leukocytes and in cryptococcal Ag-pulsed splenocytes, 3) diminished IgE production in sera, and 4) increased hallmarks of classical pulmonary macrophage activation. These effects were preceded by increased expression of early pro-Th1 genes in pulmonary lymph nodes at the afferent phase (1 wk). Collectively, our data show that SRA can be exploited by C. neoformans to interfere with the early events of the afferent responses that support Th1 immune polarization. This results in amplification of Th2 arm of the immune response and subsequently impaired adaptive control of C. neoformans in the infected lungs.

Implicating exudate macrophages and Ly-6C(high) monocytes in CCR2-dependent lung fibrosis following gene-targeted alveolar injury.

The alveolar epithelium is characteristically abnormal in fibrotic lung disease, and we recently established a direct link between injury to the type II alveolar epithelial cell (AEC) and the accumulation of interstitial collagen. The mechanisms by which damage to the epithelium induces lung scarring remain poorly understood. It is particularly controversial whether an insult to the type II AEC initiates an inflammatory response that is required for the development of fibrosis. To explore whether local inflammation occurs following a targeted epithelial insult and contributes to lung fibrosis, we administered diphtheria toxin to transgenic mice with type II AEC-restricted expression of the diphtheria toxin receptor. We used immunophenotyping techniques and diphtheria toxin receptor-expressing, chemokine receptor-2-deficient (CCR2(-/-)) mice to determine the participation of lung leukocyte subsets in pulmonary fibrogenesis. Our results demonstrate that targeted type II AEC injury induces an inflammatory response that is enriched for CD11b(+) nonresident exudate macrophages (ExM) and their precursors, Ly-6C(high) monocytes. CCR2 deficiency abrogates the accumulation of both cell populations and protects mice from fibrosis, weight loss, and death. Further analyses revealed that the ExM are alternatively activated and that ExM and Ly-6C(high) monocytes express mRNA for IL-13, TGF-ß, and the collagen genes, COL1A1 and COLIIIA1. Furthermore, the accumulated ExM and Ly-6C(high) monocytes contain intracellular collagen, as detected by immunostaining. Together, these results implicate CCR2 and the accumulation of ExM and Ly-6C(high) monocytes as critical determinants of pulmonary fibrosis induced by selective type II AEC injury.

Interleukin-17A enhances host defense against cryptococcal lung infection through effects mediated by leukocyte recruitment, activation, and gamma interferon production.

Infection of C57BL/6 mice with the moderately virulent Cryptococcus neoformans strain 52D models the complex adaptive immune response observed in HIV-negative patients with persistent fungal lung infections. In this model, Th1 and Th2 responses evolve over time, yet the contribution of interleukin-17A (IL-17A) to antifungal host defense is unknown. In this study, we show that fungal lung infection promoted an increase in Th17 T cells that persisted to 8 weeks postinfection. Our comparison of fungal lung infection in wild-type mice and IL-17A-deficient mice (IL-17A(-/-) mice; C57BL/6 genetic background) demonstrated that late fungal clearance was impaired in the absence of IL-17A. This finding was associated with reduced intracellular containment of the organism within lung macrophages and deficits in the accumulation of total lung leukocytes, including specific reductions in CD11c+ CD11b+ myeloid cells (dendritic cells and exudate macrophages), B cells, and CD8+ T cells, and a nonsignificant trend in the reduction of lung neutrophils. Although IL-17A did not alter the total number of CD4 T cells, decreases in the total number of CD4 T cells and CD8 T cells expressing gamma interferon (IFN-γ) were observed in IL-17A(-/-) mice. Lastly, expression of major histocompatibility complex class II (MHC-II) and the costimulatory molecules CD80 and CD86 on CD11c+ CD11b+ myeloid cells was diminished in IL-17A(-/-) mice. Collectively, these data indicate that IL-17A enhances host defenses against a moderately virulent strain of C. neoformans through effects on leukocyte recruitment, IFN-γ production by CD4 and CD8 T cells, and the activation of lung myeloid cells.

Early induction of CCL7 downstream of TLR9 signaling promotes the development of robust immunity to cryptococcal infection.

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Analysis of treatment results in primary germ cell tumours with mediastinal location: own experience.

INTRODUCTION: Primary germ cell tumours with mediastinal location comprise 1-6% of mediastinal tumours and 2-5% of all germ cell tumours occurring in adults. They are identified mostly in the 3rd decade of life, in 90% of cases in men. The most common symptoms are dyspnea, chest pain, cough, fever and weight loss. The aim of the present study was the analysis of our own results of treatment of primary germ cell tumours with mediastinal location, and a review of the literature concerning this subject. MATERIAL AND METHODS: Five patients (4 males, 1 female) median age 27.8 years (range 23-30 years) treated in the period from 1999 to 2009 in Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Department of Lung Cancer and Chest Tumours in Warsaw, due to germinal tumours with primary mediastinal location, entered the study. RESULTS: All patients received chemotherapy according to the BEP regimen. All patients achieved an objective response to treatment. Two patients died due to disease progression in spite of II- and III-line treatment. Three patients are still in follow-up. The median survival time was 55.8 months (range 8.0-120.0 months). CONCLUSIONS: Primary mediastinal germ cell tumours have worse prognosis than do those with gonadal location. Based on our observations and review of the literature, it can be concluded that the results of treatment of non-seminoma type germ cell tumours with primary mediastinal location remain poor. Patients who develop early recurrence or progression during first-line chemotherapy are particularly at risk of unfavourable outcome. Identification of new standards of treatment in tumours resistant to cisplatin require further studies evaluating the effectiveness of new generation cytostatic drugs.