RESUMEN
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
Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.
Asunto(s)
Dermis , Cicatrización de Heridas , Cicatriz/patología , Dermis/patología , Fibroblastos , Folículo Piloso , Humanos , PielRESUMEN
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
It is now evident that NK cells kill bacteria, fungi, and parasites in addition to tumor and virus-infected cells. In addition to a number of recent publications that have identified the receptors and ligands, and mechanisms of cytotoxicity, new insights are reflected in the reports from researchers all over the world at the 17th Meeting of the Society for Natural Immunity held in San Antonio, TX, USA from May 28 through June 1, 2018. We will provide an overview of the field and discuss how the presentations at the meeting might shape our knowledge and future directions in the field.
Asunto(s)
Bacterias/inmunología , Hongos/inmunología , Inmunidad Celular , Células Asesinas Naturales/inmunología , Virus/inmunología , Animales , Congresos como Asunto , Humanos , Inmunidad Innata , Sociedades Científicas , TexasRESUMEN
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
Pulmonary mycoses are among the most feared infections encountered in immunocompromised patients. The problem is amplified by the increasing numbers of chronically immunocompromised patients that have substantially increased both the prevalence and clinical severity of infections caused by fungi. Moreover, fungal infections in this patient population pose challenges in diagnosis and management. Fortunately, recent advances in diagnostics and antifungal therapy, and their direct application to specific diseases, provide important new approaches to this complex and often seriously ill patient population. In this article we review the commonly occurring pulmonary fungal infections in the immunocompromised population with a particular focus on their management.