Project description:The use of C57BL/6 Rag2(-/-)?c(-/-) mice as recipients for xenotransplantation with human immune systems (humanization) has been problematic because C57BL/6 SIRP? does not recognize human CD47, and such recognition is required to suppress macrophage-mediated phagocytosis of transplanted human hematopoietic stem cells (HSCs). We show that genetic inactivation of CD47 on the C57BL/6 Rag2(-/-)?c(-/-) background negates the requirement for CD47-signal recognition protein ? (SIRP?) signaling and induces tolerance to transplanted human HSCs. These triple-knockout, bone marrow, liver, thymus (TKO-BLT) humanized mice develop organized lymphoid tissues including mesenteric lymph nodes, splenic follicles and gut-associated lymphoid tissue that demonstrate high levels of multilineage hematopoiesis. Importantly, these mice have an intact complement system and showed no signs of graft-versus-host disease (GVHD) out to 29 weeks after transplantation. Sustained, high-level HIV-1 infection was observed via either intrarectal or intraperitoneal inoculation. TKO-BLT mice exhibited hallmarks of human HIV infection including CD4(+) T-cell depletion, immune activation, and development of HIV-specific B- and T-cell responses. The lack of GVHD makes the TKO-BLT mouse a significantly improved model for long-term studies of pathogenesis, immune responses, therapeutics, and vaccines to human pathogens.
Project description:BACKGROUND:Oxalobacter formigenes are bacteria that colonize the human gut and degrade oxalate, a component of most kidney stones. Findings of clinical and epidemiological studies suggest that O. formigenes colonization reduces the risk for kidney stones. We sought to develop murine models to allow investigating O. formigenes in the context of its native human microbiome. METHODS:For humanization, we transplanted pooled feces from healthy, noncolonized human donors supplemented with a human O. formigenes strain into recipient mice. We transplanted microbiota into mice that were treated with broad-spectrum antibiotics to suppress their native microbiome, were germ free, or received humanization without pretreatment or received sham gavage (controls). RESULTS:All humanized mice were stably colonized with O. formigenes through 8 weeks after gavage, whereas mice receiving sham gavage remained uncolonized (P < .001). Humanization significantly changed the murine intestinal microbial community structure (P < .001), with humanized germ-free and antibiotic-treated groups overlapping in ?-diversity. Both germ-free and antibiotic-treated mice had significantly increased numbers of human species compared with sham-gavaged mice (P < .001). CONCLUSIONS:Transplanting mice with human feces and O. formigenes introduced new microbial populations resembling the human microbiome, with stable O. formigenes colonization; such models can define optimal O. formigenes strains to facilitate clinical trials.
Project description:Diet and nutritional status are among the most important modifiable determinants of human health. The nutritional value of food is influenced in part by a person's gut microbial community (microbiota) and its component genes (microbiome). Unraveling the interrelations among diet, the structure and operations of the gut microbiota, and nutrient and energy harvest is confounded by variations in human environmental exposures, microbial ecology, and genotype. To help overcome these problems, we created a well-defined, representative animal model of the human gut ecosystem by transplanting fresh or frozen adult human fecal microbial communities into germ-free C57BL/6J mice. Culture-independent metagenomic analysis of the temporal, spatial, and intergenerational patterns of bacterial colonization showed that these humanized mice were stably and heritably colonized and reproduced much of the bacterial diversity of the donor's microbiota. Switching from a low-fat, plant polysaccharide-rich diet to a high-fat, high-sugar "Western" diet shifted the structure of the microbiota within a single day, changed the representation of metabolic pathways in the microbiome, and altered microbiome gene expression. Reciprocal transplants involving various combinations of donor and recipient diets revealed that colonization history influences the initial structure of the microbial community but that these effects can be rapidly altered by diet. Humanized mice fed the Western diet have increased adiposity; this trait is transmissible via microbiota transplantation. Humanized gnotobiotic mice will be useful for conducting proof-of-principle "clinical trials" that test the effects of environmental and genetic factors on the gut microbiota and host physiology. Nearly full-length 16S rRNA gene sequences are deposited in GenBank under the accession numbers GQ491120 to GQ493997.
Project description:Many murine and non-human primate animal models have been recently developed to understand Zika viral pathogenesis. However, a major limitation with these models is the inability to directly examine the human-specific immune response. Here, we utilized a BLT humanized mouse model endowed with a transplanted human immune system. Plasma viremia could be detected within 48h after viral challenge and viremia persisted for as long as 220 days in some mice. Neutralizing human antibody was detected in infected mice and mouse sera showed reactivity with the viral envelope and capsid proteins in a radio-immunoprecipitation assay. Human monocytes/macrophages, B cells and hematopoietic stem cells in the bone marrow were found to be virus infected. These data establish that BLT mice are permissive for Zika viral infection and are capable of generating viral-specific human immune responses thus providing a human surrogate model for future testing of vaccine and antiviral therapeutic candidates.
Project description:The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.
Project description:There are many DEGs between SIVcpz and HIV-1 infected humanized BLT-mice. Overall design: To compare the transcriptome profile of human CD4+T cells between SIVcpz and HIV-1 infected humanized BLT-mice.
Project description:Autism spectrum disorder (ASD) manifests as alterations in complex human behaviors including social communication and stereotypies. In addition to genetic risks, the gut microbiome differs between typically developing (TD) and ASD individuals, though it remains unclear whether the microbiome contributes to symptoms. We transplanted gut microbiota from human donors with ASD or TD controls into germ-free mice and reveal that colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors. The brains of mice colonized with ASD microbiota display alternative splicing of ASD-relevant genes. Microbiome and metabolome profiles of mice harboring human microbiota predict that specific bacterial taxa and their metabolites modulate ASD behaviors. Indeed, treatment of an ASD mouse model with candidate microbial metabolites improves behavioral abnormalities and modulates neuronal excitability in the brain. We propose that the gut microbiota regulates behaviors in mice via production of neuroactive metabolites, suggesting that gut-brain connections contribute to the pathophysiology of ASD.
Project description:Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common ?-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common ?-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID ?-chain(-/-) (NSG); and Rag2(-/-) ?-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common ?-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.
Project description:BLT (bone marrow-liver-thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with human immunodeficiency virus type 1 (HIV-1). We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein, utilizing a rapid prime-boost protocol of poly(lactic-co-glycolic) acid microparticles and a replication-defective herpes simplex virus (HSV) recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide gamma interferon-positive (IFN-?+) T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1-infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although prechallenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these postchallenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5-log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including interleukin-12p70 (IL-12p70) and MIP-1?, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization; however, additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy.IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small-animal model for preclinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active, as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable prechallenge responses, it did increase the magnitude and kinetics of postchallenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.
Project description:The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.