RESUMEN
Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation.
Asunto(s)
Presentación de Antígeno/inmunología , Criptococosis/inmunología , Cryptococcus gattii/inmunología , Células Dendríticas/inmunología , Cápsulas Fúngicas/inmunología , Evasión Inmune/inmunología , Western Blotting , Proliferación Celular , Humanos , Activación de Linfocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac â PI3K â Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/inmunología , Cryptococcus neoformans/fisiología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Proteínas de Unión al GTP rac/inmunología , Proteína de Unión al GTP rac1/inmunología , Familia-src Quinasas/inmunología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/microbiología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Pironas/farmacología , Quinolinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Familia-src Quinasas/genética , Proteína RCA2 de Unión a GTPRESUMEN
Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.
Asunto(s)
Degranulación de la Célula , Microambiente Celular , Cryptococcus gattii/inmunología , Cryptococcus neoformans/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Perforina/metabolismo , Adhesión Celular , Línea Celular , Células Cultivadas , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Corteza Cerebral/microbiología , Corteza Cerebral/patología , Criptococosis/inmunología , Criptococosis/metabolismo , Criptococosis/microbiología , Criptococosis/patología , Cryptococcus gattii/fisiología , Cryptococcus neoformans/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Células Asesinas Naturales/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Sistema de Señalización de MAP Quinasas , Fosforilación , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Replicación Viral , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.
Asunto(s)
Inmunidad Adaptativa/inmunología , Diferenciación Celular/inmunología , Criptococosis/inmunología , Criptococosis/patología , Cryptococcus gattii/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Evasión Inmune/inmunología , Células Cultivadas , Criptococosis/microbiología , Cryptococcus gattii/crecimiento & desarrollo , Cryptococcus gattii/patogenicidad , Células Dendríticas/microbiología , Humanos , Inmunofenotipificación , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.
Asunto(s)
Cryptococcus neoformans/fisiología , Células Asesinas Naturales/metabolismo , Perforina/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Microdominios de Membrana , Perforina/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fyn/genética , Interferencia de ARN , ARN Interferente Pequeño , Tirosina , Familia-src Quinasas/genéticaRESUMEN
NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.
Asunto(s)
Cryptococcus neoformans/inmunología , Células Asesinas Naturales/inmunología , Viabilidad Microbiana , Perforina/biosíntesis , Degranulación de la Célula , Línea Celular , Recuento de Colonia Microbiana , Humanos , ARN Mensajero/biosíntesisRESUMEN
Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing.
Asunto(s)
Cryptococcus/inmunología , Cryptococcus/patogenicidad , Gránulos Citoplasmáticos/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Cinesinas/metabolismo , Línea Celular , Células Cultivadas , Gránulos Citoplasmáticos/genética , Citotoxicidad Inmunológica , Humanos , Cinesinas/genéticaAsunto(s)
Criptococosis/complicaciones , Cryptococcus gattii , Enfermedades Pulmonares Fúngicas/microbiología , Neumonía/microbiología , Adulto , Criptococosis/diagnóstico , Femenino , Humanos , Enfermedades Pulmonares Fúngicas/diagnóstico , Examen Físico , Neumonía/diagnóstico , Tomografía Computarizada por Rayos XRESUMEN
UNLABELLED: Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. IMPORTANCE: The mechanisms by which NK cells bind directly to pathogens and deploy their deadly cytolytic machinery during microbial host defense are only beginning to be elucidated. With the goal of understanding this process, we used NK cells from HIV-infected patients, which were known to have a defect in killing of Cryptococcus neoformans Taking advantage of previous studies that had shown that IL-12 restored killing, we used the cytokine as a gain-of-function approach to define the relevance of multiple steps in the recognition and cytolytic pathway. We demonstrated that NK cells from HIV-infected patients failed to kill Cryptococcus due to defects in perforin expression, granule polarization, and release of perforin. Additionally, IL-12 restored recognition of C. neoformans through binding of the NK-activating receptor NKp30. These observations identify important mechanisms used by NK cells to kill microbes and determine that defects in NK cells from HIV-infected patients are reversible.
Asunto(s)
Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Infecciones por VIH/complicaciones , Interleucina-12/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/microbiología , Adhesión Celular , Células Cultivadas , Gránulos Citoplasmáticos/metabolismo , Humanos , Perforina/metabolismoRESUMEN
Natural killer (NK) cells are a subset of immune effectors that directly bind and kill fungi via a perforin-dependent mechanism. The receptor mediating this activity and its potential role in disease remain unknown. Using an unbiased approach, we determined that NKp30 is responsible for recognition and killing of the fungal pathogens Cryptococcus and Candida. NKp30 was required for NK cell-fungal conjugate formation, phosphatidylinositol 3-kinase (PI3K) signaling, and perforin release. Because fungal infections are a leading cause of death in AIDS patients, we examined NKp30 expression in HIV-infected patients. NK cells from these patients had diminished NKp30 expression, defective perforin release, and blunted microbicidal activity. Surprisingly, interleukin-12 (IL-12) restored NKp30 expression and fungal killing. Thus, the NKp30 receptor plays a critical role in NK cell antifungal cytotoxicity, and diminished expression of NKp30 is responsible for defective antifungal activity of NK cells from HIV-infected patients, which can be corrected with IL-12.
Asunto(s)
Candida/inmunología , Cryptococcus/inmunología , Infecciones por VIH/inmunología , Interacciones Huésped-Patógeno , Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/biosíntesis , Células Cultivadas , Regulación hacia Abajo , Hongos , Humanos , Viabilidad Microbiana/efectos de los fármacos , Perforina/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Transducción de SeñalRESUMEN
Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inflammatory cytokine production in response to pathogenic molecules, such as ExoS, which is a TLR2 and TLR4 agonist produced by the opportunistic pathogen Pseudomonas aeruginosa. We reasoned that responses to ExoS, a protein, might differ from canonical TLR agonists such as LPS. Stimulating the expression of mCD14 with vitamin D3 enhanced the response to ExoS and LPS. Also, blocking anti-CD14 antibody or removing mCD14 using PLC reduced responses to ExoS and LPS. Furthermore, CD14-deficient cells were unable to bind and respond to ExoS, which was restored by stable transfection of mCD14, indicating that mCD14 was required for the response to ExoS. However, addition of sCD14 to culture enhanced responsiveness to LPS but not ExoS. Moreover, the addition of serum did not alter the response to ExoS but enhanced the response to LPS. Despite differences of adaptor molecule use between ExoS and LPS, lipid antagonists that compete for LPS binding to CD14 also inhibited the response to ExoS. These results highlight a fundamental difference between TLR agonists in their requirements for CD14 and serum components. These results suggest that understanding the dissimilarities and targeting overlapping sites of interaction on CD14 may yield a synergistic, clinical benefit during infections where a variety of TLR agonists are present.
Asunto(s)
ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Membrana Celular/inmunología , Citocinas/biosíntesis , Receptores de Lipopolisacáridos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/enzimología , Transducción de Señal/inmunología , ADP Ribosa Transferasas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citocinas/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Pseudomonas aeruginosa/inmunologíaRESUMEN
Cryptococcosis occurs in immunocompromised and, in special cases, immunocompetent individuals. There have been a number of important advances in the field, but, despite current treatment, patients continue to die of the infection. This article reviews cryptococcosis epidemiology, clinical features, and management. Current knowledge is incomplete, however, so this article also discusses some of the gaps in the present understanding of cryptococcosis. The hope is that current research striving to understand the mechanisms of host evasion of Cryptococcus will result in improved treatment regimens that decrease both the mortality and morbidity of cryptococcosis.
Asunto(s)
Criptococosis , Criptococosis/tratamiento farmacológico , Criptococosis/inmunología , Humanos , Huésped Inmunocomprometido , Enfermedades Pulmonares FúngicasRESUMEN
Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4(+) T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4(+) T cells is defective during HIV infection. This is because CD4(+) T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rbeta, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rbeta precedes granulysin, and preventing the increased expression of IL-2Rbeta using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rbeta, blocking subsequent signaling by IL-2 using IL-2Rbeta-specific blocking Abs abrogates expression of granulysin. Finally, CD4(+) T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rbeta and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rbeta, which in turn is required for production of granulysin. These results provide a mechanism to explain the "late" production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4(+) T cells in HIV-infected patients.