RESUMEN

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

Asunto(s)

Acetilcolina , Cloruros , Células Epiteliales , Mucosa Intestinal , Animales , Acetilcolina/metabolismo , Ratones , Cloruros/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Células Epiteliales/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Intestino Delgado/inmunología , Intestino Delgado/parasitología , Intestino Delgado/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Células en Penacho

RESUMEN

Gastrointestinal nematode (GIN) infection has applied significant evolutionary pressure to the mammalian immune system and remains a global economic and human health burden. Upon infection, type 2 immune sentinels activate a common antihelminth response that mobilizes and remodels the intestinal tissue for effector function; however, there is growing appreciation of the impact GIN infection also has on the distal tissue immune state. Indeed, this effect is observed even in tissues through which GINs never transit. This review highlights how GIN infection modulates systemic immunity through (a) induction of host resistance and tolerance responses, (b) secretion of immunomodulatory products, and (c) interaction with the intestinal microbiome. It also discusses the direct consequences that changes to distal tissue immunity can have for concurrent and subsequent infection, chronic noncommunicable diseases, and vaccination efficacy.

Asunto(s)

Microbioma Gastrointestinal , Nematodos , Infecciones por Nematodos , Animales , Humanos , Infecciones por Nematodos/inmunología , Nematodos/inmunología , Nematodos/fisiología , Microbioma Gastrointestinal/inmunología , Inmunomodulación , Interacciones Huésped-Parásitos/inmunología , Parasitosis Intestinales/inmunología , Tolerancia Inmunológica , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/parasitología

RESUMEN

Chemosensory epithelial tuft cells contribute to innate immunity at barrier surfaces, but their differentiation from epithelial progenitors is not well understood. Here, we exploited differences between inbred mouse strains to identify an epithelium-intrinsic mechanism that regulates tuft cell differentiation and tunes innate type 2 immunity in the small intestine. Balb/cJ (Balb) mice had fewer intestinal tuft cells than C57BL/6J (B6) mice and failed to respond to the tuft cell ligand succinate. Most of this differential succinate response was determined by the 50- to 67-Mb interval of chromosome 9 (Chr9), such that congenic Balb mice carrying the B6 Chr9 interval had elevated baseline numbers of tuft cells and responded to succinate. The Chr9 locus includes Pou2af2, which encodes the protein OCA-T1, a transcriptional cofactor essential for tuft cell development. Epithelial crypts expressed a previously unannotated short isoform of Pou2af2 predicted to use a distinct transcriptional start site and encode a nonfunctional protein. Low tuft cell numbers and the resulting lack of succinate response in Balb mice were explained by a preferential expression of the short isoform and could be rescued by expression of full-length Pou2af2. Physiologically, Pou2af2 isoform usage tuned innate type 2 immunity in the small intestine. Balb mice maintained responsiveness to helminth pathogens while ignoring commensal Tritrichomonas protists and reducing norovirus burdens.

Asunto(s)

Mucosa Intestinal , Intestinos , Ratones , Animales , Ratones Endogámicos C57BL , Diferenciación Celular , Succinatos/metabolismo

RESUMEN

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

RESUMEN

Although seemingly unrelated, parasitic worms, venoms, and allergens all induce a type 2 immune response. The effector functions and clinical features of type 2 immunity are well-defined, but fundamental questions about the initiation of type 2 immunity remain unresolved. How are these enormously diverse type 2 stimuli first detected? How are type 2 helper T cells primed and regulated? And how do mechanisms of type 2 initiation vary across tissues? Here, we review the common themes governing type 2 immune sensing and explore aspects of T cell priming and effector reactivation that make type 2 helper T cells a unique T helper lineage. Throughout the review, we emphasize the importance of non-hematopoietic cells and highlight how the unique anatomy and physiology of each barrier tissue shape mechanisms of type 2 immune initiation.

Asunto(s)

Inmunidad , Linfocitos T Colaboradores-Inductores , Células Th2 , Alérgenos

RESUMEN

While the lung bears significant regenerative capacity, severe viral pneumonia can chronically impair lung function by triggering dysplastic remodeling. The connection between these enduring changes and chronic disease remains poorly understood. We recently described the emergence of tuft cells within Krt5+ dysplastic regions after influenza injury. Using bulk and single-cell transcriptomics, we characterized and delineated multiple distinct tuft cell populations that arise following influenza clearance. Distinct from intestinal tuft cells which rely on Type 2 immune signals for their expansion, neither IL-25 nor IL-4ra signaling are required to drive tuft cell development in dysplastic/injured lungs. In addition, tuft cell expansion occurred independently of type I or type III interferon signaling. Furthermore, tuft cells were also observed upon bleomycin injury, suggesting that their development may be a general response to severe lung injury. While intestinal tuft cells promote growth and differentiation of surrounding epithelial cells, in the lungs of tuft cell deficient mice, Krt5+ dysplasia still occurs, goblet cell production is unchanged, and there remains no appreciable contribution of Krt5+ cells into more regionally appropriate alveolar Type 2 cells. Together, these findings highlight unexpected differences in signals necessary for murine lung tuft cell amplification and establish a framework for future elucidation of tuft cell functions in pulmonary health and disease.

Asunto(s)

Citocinas , Gripe Humana , Animales , Bleomicina , Células Caliciformes , Humanos , Pulmón , Ratones

RESUMEN

Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the parasite Heligmosomoides polygyrus bakeri (Hpb) reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory-secretory products reveal that Hpb-mediated revSC generation occurs independently of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness, indicating that helminths compete with their host for control of the intestinal stem cell compartment to promote continuation of their life cycle.

Asunto(s)

Nematospiroides dubius , Infecciones por Strongylida , Animales , Citocinas , Mucosa Intestinal , Intestinos , Ratones , Células Madre

RESUMEN

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Asunto(s)

Alarminas , Mucosa Intestinal , Acilación , Alarminas/inmunología , Antihelmínticos/inmunología , Biomarcadores de Tumor , Citocinas , Proteínas de Unión al ADN , Helmintiasis/inmunología , Humanos , Hiperplasia , Inflamación , Interleucina-33 , Mucosa Intestinal/inmunología , Mebendazol , N-Acetilglucosaminiltransferasas/inmunología , Proteínas Citotóxicas Formadoras de Poros , Factor de Transcripción STAT6/inmunología

RESUMEN

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Asunto(s)

Ácidos y Sales Biliares , Sistema Biliar , Animales , Células Epiteliales/fisiología , Inflamación , Ratones , Neutrófilos

RESUMEN

E-protein transcription factors limit group 2 innate lymphoid cell (ILC2) development while promoting T cell differentiation from common lymphoid progenitors. Inhibitors of DNA binding (ID) proteins block E-protein DNA binding in common lymphoid progenitors to allow ILC2 development. However, whether E-proteins influence ILC2 function upon maturity and activation remains unclear. Mice that overexpress ID1 under control of the thymus-restricted proximal Lck promoter (ID1tg/WT) have a large pool of primarily thymus-derived ILC2s in the periphery that develop in the absence of E-protein activity. We used these mice to investigate how the absence of E-protein activity affects ILC2 function and the genomic landscape in response to house dust mite (HDM) allergens. ID1tg/WT mice had increased KLRG1- ILC2s in the lung compared with wild-type (WT; ID1WT/WT) mice in response to HDM, but ID1tg/WT ILC2s had an impaired capacity to produce type 2 cytokines. Analysis of WT ILC2 accessible chromatin suggested that AP-1 and C/EBP transcription factors but not E-proteins were associated with ILC2 inflammatory gene programs. Instead, E-protein binding sites were enriched at functional genes in ILC2s during development that were later dynamically regulated in allergic lung inflammation, including genes that control ILC2 response to cytokines and interactions with T cells. Finally, ILC2s from ID1tg/WT compared with WT mice had fewer regions of open chromatin near functional genes that were enriched for AP-1 factor binding sites following HDM treatment. These data show that E-proteins shape the chromatin landscape during ILC2 development to dictate the functional capacity of mature ILC2s during allergic inflammation in the lung.

Asunto(s)

Antígenos Dermatofagoides/inmunología , Asma/inmunología , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Alérgenos/inmunología , Animales , Asma/patología , Diferenciación Celular/inmunología , Cromatina/metabolismo , Citocinas/inmunología , Proteínas de Unión al ADN/antagonistas & inhibidores , Femenino , Lectinas Tipo C/genética , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Pyroglyphidae/inmunología , Receptores Inmunológicos/genética , Células Madre/citología , Linfocitos T/citología , Factor de Transcripción AP-1/metabolismo

RESUMEN

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Asunto(s)

Linaje de la Célula , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , MicroARNs/genética , Transcriptoma , Animales , Biomarcadores/metabolismo , Separación Celular , Células Cultivadas , Biología Computacional , Perros , Femenino , Citometría de Flujo , Mucosa Intestinal/citología , Intestino Delgado/citología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/metabolismo , Organoides , RNA-Seq , Análisis de la Célula Individual

RESUMEN

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.

Asunto(s)

Citocinas/metabolismo , Mucosa Intestinal/parasitología , Prostaglandina D2/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Infecciones por Strongylida/parasitología , Animales , Femenino , Gastroenteritis/parasitología , Gastroenteritis/patología , Células Caliciformes/patología , Interacciones Huésped-Parásitos/fisiología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Nippostrongylus/patogenicidad , Organoides , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Infecciones por Strongylida/patología

RESUMEN

Tuft cells are rare chemosensory epithelial cells that monitor their environment and relay messages to the surrounding tissue via secretion of neuromodulatory and immunomodulatory molecules. In the small intestine tuft cells detect helminth infection, protist colonization, and bacterial dysbiosis, and initiate a type 2 immune response characterized by tissue remodeling. In the airways, tuft cells sense bacteria, allergens, and noxious stimuli and drive evasive behavior, neuroinflammation, and anti-bacterial responses. Here we summarize the most recent tuft cell research and discuss how these findings have provided insight into tuft cell diversity. Built around a core program of chemosensing, tuft cell receptors and effector functions are tuned to the unique environmental exposure and physiology of their surrounding tissue.

Asunto(s)

Células Epiteliales/inmunología , Intestino Delgado/inmunología , Animales , Humanos , Ligandos

RESUMEN

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Asunto(s)

Cisteína/inmunología , Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Leucotrienos/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Animales , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/inmunología , Araquidonato 5-Lipooxigenasa/metabolismo , Cisteína/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Inmunidad Innata/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Leucotrienos/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/parasitología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nippostrongylus/fisiología , Infecciones por Strongylida/parasitología

RESUMEN

The varied list of agonists that activate innate lymphoid cells (ILCs) continues to grow, but whether and how these signals interact is not well defined, especially in vivo. ILC subsets share master transcription factors, chromatin landscapes, and effector cytokines with their corresponding T helper (Th) cell subsets. Here we discuss how studies of these two cell types can inform each other. Specifically, we outline a framework in which ILC agonists are grouped by the transcription factors they activate. Optimal ILC activation requires at least three items from a 'menu' of non-redundant signals that collectively replicate the STAT and TCR signaling that drives effector Th cell function. This conceptual model may also apply to TCR-independent 'bystander' activation of Th cells.

Asunto(s)

Inmunidad Innata/inmunología , Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Humanos

RESUMEN

Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity.

Asunto(s)

Factor 1 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Interferones/inmunología , Animales , Línea Celular , Células Epiteliales/inmunología , Humanos , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT1/inmunología , Interferón lambda

RESUMEN

Tuft cells were first discovered in epithelial barriers decades ago, but their function remained unclear until recently. In the last 2 years, a series of studies has provided important advances that link tuft cells to infectious diseases and the host immune responses. Broadly, a model has emerged in which tuft cells use chemosensing to monitor their surroundings and translate environmental signals into effector functions that regulate immune responses in the underlying tissue. In this article, we review the current understanding of tuft cell immune function in the intestines, airways, and thymus. In particular, we discuss the role of tuft cells in type 2 immunity, norovirus infection, and thymocyte development. Despite recent advances, many fundamental questions about the function of tuft cells in immunity remain to be answered.

Asunto(s)

Inmunidad Mucosa/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Animales , Humanos , Propiedades de Superficie

Asunto(s)

Inmunidad Innata , Interleucina-13 , Linfocitos , ARN , Células Madre

RESUMEN

Respiratory epithelial cells (EpCs) orchestrate airway mucosal inflammation in response to diverse environmental stimuli, but how distinct EpC programs are regulated remains poorly understood. Here, we report that inhalation of aeroallergens leads to expansion of airway brush cells (BrCs), specialized chemosensory EpCs and the dominant epithelial source of interleukin-25 (IL-25). BrC expansion was attenuated in mice lacking either LTC4 synthase, the biosynthetic enzyme required for cysteinyl leukotriene (CysLT) generation, or the EpC receptor for leukotriene E4 (LTE4), CysLT3R. LTE4 inhalation was sufficient to elicit CysLT3R-dependent BrC expansion in the murine airway through an IL-25-dependent but STAT6-independent signaling pathway. Last, blockade of IL-25 attenuated both aeroallergen and LTE4-elicited CysLT3R-dependent type 2 lung inflammation. These results demonstrate that CysLT3R senses the endogenously generated lipid ligand LTE4 and regulates airway BrC number and function.

Asunto(s)

Células Epiteliales/inmunología , Inflamación/inmunología , Interleucinas/biosíntesis , Receptores de Leucotrienos/inmunología , Animales , Interleucinas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados