RESUMEN
Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14-CD1c+ conventional DCs (cDCs) and CD14-CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA-Foxp3hi and CD45RA-Foxp3lo T cells. Whereas CD45RA-Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA-Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Proteínas de la Membrana/genética , Linfocitos T Reguladores/citología , Animales , Factores de Transcripción Forkhead/metabolismo , Técnicas de Transferencia de Gen , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Trasplante de Células Madre Hematopoyéticas , Humanos , Proteínas de la Membrana/administración & dosificación , Ratones , Quimera por TrasplanteRESUMEN
CCR5-tropic (R5) immunodeficiency virus type 1 (HIV-1) strains are highly transmissible during the early stage of infection in humans, whereas CXCR4-tropic (X4) strains are less transmissible. This study aimed to explore the basis for early phase R5 and X4 HIV-1 infection in vivo by using humanized mice dually challenged with R5 HIV-1NLAD8-D harboring DsRed and X4 HIV-1(NL-E) harboring EGFP. Whereas R5 HIV-1 replicated well, X4 HIV-1 caused only transient viremia with variable kinetics; however, this was distinct from the low level but persistent viremia observed in mice challenged with X4 HIV-1 alone. Flow cytometric analysis of HIV-1-infected cells revealed that X4 HIV-1 infection of CCR5(+)CD4(+) T cells was significantly suppressed in the presence of R5 HIV-1. X4 HIV-1 was more cytopathic than R5 HIV-1; however, this was not the cause of restricted X4 HIV-1 infection because there were no significant differences in the mortality rates of CCR5(+) and CCR5(-) cells within the X4 HIV-1-infected cell populations. Taken together, these results suggest that restricted infection of CCR5(+)CD4(+) T cells by X4 HIV-1 (occurring via a still-to-be-identified mechanism) might contribute to the preferential transmission of R5 HIV-1 during the early phase of infection.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/inmunología , Receptores CCR5/inmunología , Receptores CXCR4/inmunología , Viremia/patología , Replicación Viral/inmunología , Animales , Linfocitos T CD4-Positivos/citología , VIH-1/patogenicidad , Ratones , Viremia/mortalidad , Viremia/transmisiónRESUMEN
Live attenuated measles virus (MV) has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT)-PCR that simultaneously measures nucleocapsid (N) and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing enhanced green fluorescent protein both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.
RESUMEN
Humanized mice reconstituted with human hematopoietic cells have been developed as an experimental animal model for human immunodeficiency virus type 1 (HIV-1) infection. Myeloablative irradiation is usually performed to augment the engraftment of donor hematopoietic stem cells (HSCs) in recipient mice; however, some mouse strains are susceptible to irradiation, making longitudinal analysis difficult. We previously attempted to construct humanized NOD/SCID/JAK3(null) (hNOJ) mice, which were not irradiated prior to human HSC transplantation. We found that, over time, many of the reconstituted CD4(+) T cells expanded with an activated effector memory phenotype. Therefore, the present study used hNOJ mice that were irradiated (hNOJ (IR+)) or not (hNOJ (IR-)) prior to human HSC transplantation to examine whether the development and cellularity of the reconstituted CD4(+) T cells were influenced by the degree of chimerism, and whether they affected HIV-1 infectivity. Indeed, hNOJ (IR+) mice showed a greater degree of chimerism than hNOJ (IR-) mice. However, the conversion of CD4(+) T cells to an activated effector memory phenotype, with a high percentage of cells showing Ki-67 expression, occurred in both hNOJ (IR+) and hNOJ (IR-) mice, probably as a result of lymphopenia-induced homeostatic expansion. Furthermore, when hNOJ (IR+) and hNOJ (IR-) mice, which were selected as naïve- and memory CD4(+) T cell subset-rich groups, respectively, were infected with CCR5-tropic HIV-1 in vivo, virus replication (as assessed by the plasma viral load) was delayed; however, the titer subsequently reached a 1-log higher level in memory-rich hNOJ (IR-) mice than in naïve-rich hNOJ (IR+) mice, indicating that virus infectivity in hNOJ mice was affected by the different status of the reconstituted CD4(+) T cells. Therefore, the hNOJ mouse model should be used selectively, i.e., according to the specific experimental objectives, to gain an appropriate understanding of HIV-1 infection/pathogenesis.