In this study, we have constructed humice with significant levels of human platelets, hepatocytes, monocytes/macrophages, and other immune cells. Injection of both lab-adapted and clinical strains of DENV2 leads to a systemic infection with characteristic symptoms, such as transient leukopenia, elevation of blood monocyte levels, thrombocytopenia, and liver damage. In particular, we show that the specific depletion of human platelets is not mediated by antibodies or a reduced production of hTPO in the liver but by reduction of human megakaryocytes and progenitor cells in the BM of the infected mice. These findings identify the likely mechanism of thrombocytopenia and suggest the utility of humice in studying DENV infection in a human cell context.
Compared to the previous reports of DENV infection in humanized NSG mice (16
), our humanized mouse model of DENV infection not only shares many common features but also has some unique advantages. Jaiswal et al. used the DENV2 NGC strain (18
), where viremia was detected till 21 dpi in 80% of the mice and the DENV RNA was detected in spleen, BM, and liver, while Mota and Rico-Hesse used different strains of DENV2 (16
), where viremia was detected up to 18 dpi in all the infected mice as well as in the BM and spleen. Similar to the previous studies, i.v. administration of both a lab-adapted and a clinical isolate of DENV2 induced systemic infection in the spleen, LN, BM, and liver in our model. Viremia was detected in a significant proportion of mice even at 28 dpi in NGC-infected humice, where human cells but not mouse cells were infected, in contrast to NGC's original isolation by serial passaging in the brains of suckling mice (24
). This may be due to the decades of lab adaptation of the virus in mosquito cells. In humans, DENV causes an acute infection, and viremia disappears in about a week. The much longer period of viremia in the humice could be due to the slow clearance of the infection associated with the poor immune responses in humice as indicated by the lack of significant levels of dengue-specific antibodies.
Engraftment of NSG mice with human fetal liver CD34+
cells is the key difference between our and previous studies (where engraftment was done with human cord blood CD34+
cells), and this had resulted in the generation of human hepatocytes in the mouse liver (19
). It has been reported that in fetuses, the liver accounts for up to 95% of TPO production (25
). Thus, it is likely that production of TPO by human hepatocytes leads to a higher level of human platelets in mice reconstituted with human fetal liver CD34+
cells as opposed to mice reconstituted with cord blood CD34+
cells. This major advantage in our model leads to the induction of thrombocytopenia in infected mice which is specific to human platelets despite the presence of numerous mouse platelets. This is in contrast to a drop of total platelets in infected humice reported by Mota and Rico-Hesse (16
), while Jaiswal et al. (18
) did not observe any change in platelets even though the same strain of DENV was used. Interestingly, in our model, thrombocytopenia was observed in humice even with viremia levels below the detection limit. This raises the possibility that thrombocytopenia could be used as a more sensitive marker for monitoring infection during testing of potential therapeutics in our model. Also, DENV infection induces additional hematological changes such as transient leukopenia in the blood and elevated levels of blood monocytes in our model, as in humans. We observed liver damage indicated by elevated levels of liver transaminases (ALT and AST) in the circulation of infected humice. Liver damage is believed to be caused by the infiltrating immune cells (26
), which was also observed in our model. Finally, we observed a transient decrease of the CD4-to-CD8 ratio in human T cells in the BM of infected mice, which has also been reported in the blood of patients during the severe phase of infection (27
). Thus, our humanized mouse model of DENV infection captures some key clinical features of DENV infection, including thrombocytopenia.
The depletion of human platelets in our model enabled us to investigate the underlying mechanisms of thrombocytopenia. Two mechanisms, antibody-mediated platelet clearance in the periphery and decreased platelet production in the BM, have been proposed to account for thrombocytopenia in humans. Although the involvement of the BM has been suggested nearly 5 decades ago (11
), until recently, the focus has been primarily on the mechanism of peripheral clearance mediated by dengue virus-specific or cross-reactive antibodies, which mark the platelets for clearance. We excluded antibody-mediated depletion as a significant contributing factor to thrombocytopenia in humice because of poor antibody induction, consistent with previous reports in humice (23
). Our result, however, does not exclude the involvement of antibodies in platelet depletion in humans, as dengue-specific antibodies are induced following infection. We did not detect plasma leakage in the infected mice, and thus the loss of platelets due to plasma leakage can also be excluded.
Our study points to reduction of platelet production as the major cause of thrombocytopenia in humice. Following infection, we detected a significant drop in the level of megakaryocytes and the CD34+ CD41+ progenitors that give rise to megakaryocytes in the BM. In contrast, the level of hTPO transcripts in the liver did not change after DENV infection, suggesting that the reduction of megakaryocytes and their progenitors in the BM was not due to a lack of human TPO in infected mice. We did not detect viral RNA in the purified human megakaryocytes (data not shown), suggesting that either direct infection of megakaryocytes may not be a major cause of their depletion in our model or our qRT-PCR assay was not sufficiently sensitive (100 copies per reaction) to detect the infected megakaryocytes. We observed direct DENV infection and immune responses in the BM as indicated by the change of the CD4-to-CD8 T cell ratio, which might alter the BM microenvironment, resulting in inhibition of human megakaryocytopoiesis and therefore thrombocytopenia. While the precise details still need to be worked out, our findings show that the decreased production of platelets due to inhibition of megakaryocyte development is a key mechanism underlying DENV-induced thrombocytopenia in humice.
In summary, we have established a humanized mouse model of DENV infection that recaptures some of the key clinical symptoms in humans, including thrombocytopenia. Using this model, we have elucidated the mechanism underlying the depletion of human platelets, helping to gain insight into DENV-induced thrombocytopenia in humans. Although absence of a strong antibody response is a drawback of the current model, it has significant advantages over other DENV infection models, including the induction of thrombocytopenia and studying DENV infection in the context of human cells. Improving antibody response in the humanized mice will likely make the model even more useful for studying other aspects of DENV infection, such as antibody-dependent enhancement.