Humanized Mice: 324 (Current Topics in Microbiology and Immunology)

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Immunodeficient mice reconstituted with human immune tissues and cells humanized mice are relevant and robust models for the study of HIV-1 infection, immunopathogenesis, and therapy. The development of combined antiretroviral therapy cART , which can efficiently suppress viral replication, has significantly improved survival and life quality of HIVinfected patients who can both access and tolerate cART 2.

However, cART is not curative and must be continued for life 3 , 4. Thus, there is a great need for the development of novel therapies that can both control the epidemic and cure those individuals who have already been infected with HIV Understanding how HIV-1 infection leads to immunodeficiency is key for the development of new treatments. After more than 30 years of research, the precise mechanism by which HIV-1 infection causes AIDS development is still poorly understood, mainly due to the lack of robust small animal models.

The recent development of humanized mice with functional human immune systems offer a relevant and robust model for the study of HIV-1 infection, replication, pathogenesis, and therapies 6 — 8. In the hu-BLT model, implantation of human thymus tissue under the kidney capsule is combined with HSCs infusion into irradiated adult immunodeficient mice 14 , HIV-1 infection can be treated with the antiretroviral drugs that are used in infected humans 32 — Also like in humans, antiretroviral treatment of HIV-1 infection results in systemic recovery of CD4 T cells in humanized mice.

In addition, both mouse models are used for testing the effectiveness of immunotherapy to inhibit HIV-1 replication, reverse HIV-1 induced immunopathology and control HIV-1 reservoir 25 , 29 , 30 , 34 , 37 — To generate hu-BLT mice, a time consuming and technically difficult surgery procedure is needed to implant the human thymus tissue into the kidney capsule of the mice 14 , Another major difference between these two models is that in NRG-hu HSC mouse, the human T cells are produced in the mouse thymus and presumed to be educated in the context of mouse major histocompatibility complex MHC 10 — Thus, although both models are versatile tools for HIV-1 study, parallelly study to compare the human immune reconstitution, HIV-1 replication, immunopathology, and responses to therapy in both models will help to guide researchers how to balance and decide which system to use.

Human fetal liver and thymus gestational age of 16—20 weeks were obtained from medically or elective indicated termination of pregnancy through a non-profit intermediary working with outpatient clinics Advanced Bioscience Resources, Alameda, CA, USA. Written informed consent of the maternal donors is obtained in all cases, under regulation governing the clinic.

Original Research ARTICLE

All mice were housed and bred in a specific pathogen-free environment. Total lymphocytes were isolated prepared from peripheral blood, spleen, bone marrow BM , and mesenteric lymph nodes mLNs according to standard protocols; red blood cells were lysed with ACK buffer.

Intrahepatic lymphocytes were prepared as described For surface staining, single cell suspension was stained with surface markers and analyzed on a CyAn ADP flow cytometer Dako. Data were analyzed using Summit4. C were all purchased from InvivoGen. The detection limit of the real-time PCR reaction is four copies per reaction. Food formulated with antiretroviral individual drug was prepared as reported with elevated dose modifications Viral outgrowth assay was performed as reported Estimated frequencies of cells with replication-competent HIV-1 were determined by maximum likelihood statistics In all other experiments, significance levels of data were determined by using Prism5 GraphPad Software.

A P value less than 0. The number of animals and replicates is specified in each figure legend. The other difference is that we transplant human HSCs within 3 h after human thymus transplantation. Human immune cell reconstitution in the peripheral blood was detected by flow cytometry 12 weeks after transplantation. The percentage of human B cells was It has been reported that human B cells developed in humanized mice were immature and cannot produce significant level of antigen-specific IgG by vaccination 44 — Plasma was collected at different time points posttreatment.

IL-6 level was detected at 4 h after treatment. Shown D are data from three to four mice each group for each treatment conditions. C and detected cytokine production in the serum. The viral infection data were collected previously by the laboratory. Mice were sacrificed at 10 weeks postinfection. We and others have shown before that as in human patients, cART can efficiently inhibit HIV-1 replication in hu-mice 29 , 33 , 34 , Mice were sacrificed at 12 wpi. The frequency was determined by maximum likelihood statistics. Humanized mice with human immune cells are highly relevant and robust models for HIV-1 study 6 — 8.

There are different humanized mouse models available as well as different means to prepare them 6 , 7 , 9. These factors make researchers, especially those who have limited experiences on humanized mouse models difficult to decide which model to choose for their studies. Furthermore, HIV-1 infection induced similar pathology including the depletion of human T cells and activation and exhaustion of T cells.

The rescued human T cells could target the HIV-1 reservoirs with elevated gene expression and clear the reservoir cells as we have reported 29 , Each model has its own advantage and disadvantages. As the time needed for human immune reconstitution is 12—16 weeks in both models, researchers can start their experiments with younger NRG-hu HSC mice.

All animal studies were carried out in accordance with the recommendations of NIH guidelines for housing and care of laboratory animals. LC and LS conceived the study and designed the experiments. LC performed the analyses. LC and LS interpreted the data, wrote the manuscript, and supervised the study. All the authors approved the final version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors thank L. The Supplementary Material for this article can be found online at https: Antiretroviral Therapy Cohort Collaboration. Life expectancy of individuals on combination antiretroviral therapy in high-income countries: Lancet Nat Rev Microbiol 12 Barriers to a cure for HIV: HIV infection, inflammation, immunosenescence, and aging.

Annu Rev Med Curr Opin Virol Humanized mouse models for human immunodeficiency virus infection. Annu Rev Virol 4 1: Zhang L, Su L. Subsequently, the newest types of humanized mice have satisfactory recovery of T cells, B cells, and NK cells despite some variability [ 22 — 24 ]. At the same time, they suffer from diminished monocyte and dendritic cell counts coupled with frequently underperforming function of their humanized leukocytes [ 25 — 27 ].

The immunocompromised host retains a significant part of the native immune system despite all aforementioned interventions. Incomplete eradication of the native immune system results in the development of lymphomas whereby more advanced humanized models have had much longer lag time and a lower propensity allowing for prolonged longitudinal studies [ 30 ].

First, researchers observed that human peripheral blood mononuclear cells PBMCs could engraft successfully [ 14 , 31 ]. While injecting mice with PBMCs resulted in the reconstitution of T cells, the high risk of graft versus host disease limited such models to short-term experiments [ 14 ]. This system preserved the complex interaction between antigen presenting cells APCs and T cells, which is critical in the resolution of acute inflammation [ 8 , 10 , 32 ].

MHC restricted selection of T cells took place in this model. Furthermore, a secondary lymphatic system developed along the human mucosal immune system. This model featured high graft stability and diversity, proportional to the number of cells used for grafting [ 33 — 36 ]. Due to a lack of species-specific cytokines and inefficient ability of host mesenchymal cells to support function of the leukocytes, an emergence of a more complete human immune system was impaired [ 23 , 37 , 38 ].

Lack of interlocking cytokine networks contributed to the poor regulation of leukocyte populations [ 39 ]. Nanoparticles, plasmids, and lentiviruses were tried to boost the supportive environment of mice bone marrow since optimal cytokine environment is pivotal for the emergence of a complete immune system [ 21 , 38 , 39 ]. Despite these efforts and several other modifications, the development of most specialized cells was hampered [ 40 , 41 ]. Even more alarming was the emergence of unintended consequences of genetic manipulation aimed at boosting the production of human cytokines.

For example, Billerbeck et al. Aberration of composition of leukocyte population is one of the critical phenomena of sepsis. Consequently, an introduction of any bias may affect the validity of results and their translation into clinical practice. Currently, engineering of MIRTG mice, which can produce four human cytokines endogenously, is the most sophisticated model even among similar models [ 23 , 40 , 41 ].

Ancillary techniques surrounding grafting procedures have evolved as well. Initially, all animals were subjected to irradiation to remove the native immune system [ 15 — 17 , 19 , 33 , 34 , 36 , 40 ]. However, collateral damage to supportive structures of bone marrow and organs was often present. Humanized mice with Rag2 mutations appeared to be less sensitive to the effects of radiation [ 19 , 41 ].

Some mice strains such as those carrying the c-kit mutation did not need irradiation, but they have not been evaluated in sepsis studies [ 42 ]. Finally, chemical ablation can be used, but it appears to have a detrimental effect on animal survival [ 34 ]. Additionally, reconstitution of the immune system was found to be more efficient and better tolerated in newborn versus adult mice especially if augmented by the injection of human grafting factors [ 12 ]. Reconstitution of the human immune system is a relatively slow process that may take up to 3 months [ 53 ].

B cells are the earliest leukocytes to reconstitute, followed by T cells [ 18 ]. B cells in humanized mice produce all classes of immunoglobulins [ 22 , 53 , 58 — 60 ]. These data should be viewed with caution since the maturation of B cells and antibody class switching from IgM to IgG are particularly inefficient as compared to the human system [ 61 , 62 ].

Effective immunoglobulin class switching is critical for recovery from an acute infectious process. The lack of human IL-6 is considered one of the most important reasons for this inefficiency. Only recently, a novel model of humanized mice with active human IL-6 gene was described. Interestingly, while the immunoglobulin switch was more efficient in this model, the maturation of B cells remained impaired [ 63 ].

Evaluation of these IL-6 boosted mice should be noteworthy as IL-6 is one of the critical cytokines in the development of sepsis. Additionally, the introduction of human stem cell factor, granulocyte macrophage stimulating factor, and IL-3 into the BLT model resulted in more efficient maturation and optimized immunoglobulin production at baseline and after viral infection [ 38 ].

T cells obtained from humanized mice after reconstitution are proficient as measured by the delayed hypersensitivity reaction, but their ability to respond to antigen was suboptimal [ 45 ]. Introduction of the BLT model partially resolved this problem as human T cells rely on grafted fetal thymus for clonal selection [ 53 ]. Since humanized mice are xenotransplanted animals, it is unclear how a state of tolerance to different MHC antigens affects their performance. Conversely, an additional transplant of sensitized dendritic cells alleviated the problem and presented an interesting opportunity for future research [ 54 ].

Altering the cytokine environment was another way to improve the presence of APCs like dendritic cells DCs [ 33 , 54 , 65 — 67 ]. In critical care illnesses, like sepsis, dendritic cells emerge from circulating monocytes that stimulate optimal T cell responses [ 10 , 32 ]. Recently, only one study has investigated monocyte function in sepsis using humanized mice [ 55 ]. Other T cell populations may encounter similar difficulties as well. Since this subtype of T cells plays a critical role in the emergence of the tolerance and the modulation of complex T cell responses, it is unclear how the deficit will affect the evolution of the immune response in the setting of critical care illness.

Potential Pitfalls of the Humanized Mice in Modeling Sepsis

T cells with regulatory properties are present in humanized mice, but their role seems to be conflicting in terms of function and number, and it is greatly influenced by the cytokine environment [ 37 , 69 ]. It is also worth mentioning that balance between different T cell populations may be abnormal in humanized mice and was linked to a deficiency of the human cytokine network [ 22 , 29 , 37 , 45 , 48 ]. Myeloid cells are the last to reconstitute after grafting. Slow and incomplete reconstitution of the myeloid line results in the inability of T cells to mount a proficient response to antigen challenges.

Kraken Interactive

MO from humanized animals were shown to generate a robust T cell response and cytokine production after sepsis [ 35 , 55 ]. Supplementation of the humanized mice with in vitro generated allogeneic DCs can restore T cell responsiveness [ 46 , 64 , 65 ]. DCs emerge in some humanized models on their own or after Flt3 supplementation [ 46 , 49 ]. Such in vivo -generated and antigen-sensitive DCs can trigger T cell response to a specific antigen [ 49 ].

In summary, these studies show that the function of several leukocyte populations can be restored during reconstitution of the immune system. However, the complex nature of the process, dependence on numerous interventions, and unclear functional competency of leukocytes undermine the robustness of the humanized model. Humanized mice were used successfully to study HIV [ 19 , 30 ]. Most recently, several other viral infections were successfully modeled in humanized mice including Zika and West Nile virus [ 59 , 71 ].

Introduction of Epstein-Barr virus reproduced several traits of the infection with high fidelity in humanized mice [ 40 ]. The ability to replicate the trajectory of viral hepatitis, longevity, and mimicry of response to subsequent infections made humanized mice especially suitable for finding the optimal drug to cure hepatitis C [ 71 ]. The endothelial inflammation of the highly lethal dengue virus and other pathological agents causing hemorrhagic fever were investigated in humanized mice, but concerns were raised regarding the accuracy of the model [ 60 , 72 , 73 ].

Of primary concern was the ability of the xenotransplant to mimic vasculitis and interactions between mice endothelium and human immune system [ 55 , 74 — 76 ]. This illustrates a typical shortcoming of humanized mice when the interaction between two organs encounters an interspecies difference that can be overcome only through further modification of the models Table 1.

Whether these modifications make the model closer to reality or more artificial remains to be ascertained.

The success of humanized mice in mimicking viral infections established high expectations for using them to study sepsis [ 2 , 32 ]. Sepsis is a highly prevalent and serious condition that has a profound and prolonged impact on morbidity and mortality [ 6 , 40 , 55 , 74 — 77 ]. Bone marrow suppression closely resembling the natural history of sepsis was seen as well [ 77 ]. However, the degree to which these processes replicate the complexity of the septic response is difficult to assess fully. For example, IL, a critical cytokine for the development of sepsis-related apoptosis, can be studied in humanized mice only after modification of the model still resulting in production of age-dependent IL [ 32 , 39 , 78 ].

Introduction of several human cytokines improved cell recovery but introduced artificially skewed populations [ 37 ]. It was demonstrated that extended depression in bone marrow function is mediated by methylation changes in the PU. However, M-CSF production and biological activities were limited to transplanted stem cells since the mice environment did not provide indigenously produced cross-reactive cytokines [ 66 , 70 ]. It becomes evident that these observations may not reflect clinical reality with so many features absent from the model. Other humans disease models successfully mimicked in humanized mice include Toxic Shock Syndrome Toxin TSST mediated shock and staphylococcal infections [ 40 , 76 ].

To date, only a few research investigations have focused on sepsis in humanized mice [ 10 , 27 , 55 , 67 , 76 , 77 ]. The CLP model of sepsis is by far the most popular for studying sepsis despite several shortcomings of the CLP itself and some unique features of humanized mice undergoing sepsis. Further studies with humanized mice tested clinical compounds for the treatment of sepsis. Autologous stem cell transplants showed a potential to reverse some of the postseptic immune system aberrations, but clinical relevance remains to be seen [ 55 ].

In another example, antibodies to human-specific toxins were tested [ 79 ]. Finally, a researcher utilized humanized mice to test indomethacin as the modulator of the immune response in neonatal sepsis under the assumption that the functional immaturity of the grafted immune system was a good model of neonatal immunity [ 79 , 80 ].

Find uddannelse

These results are guarded due to insufficient evidence as to whether immature humanized mice and neonatal immunology are in fact equivalent. Humanized mice seem to be an appealing choice to investigate the pathology and treatment of sepsis [ 2 , 10 , 32 , 75 ]. However, interest remains relatively low. Many of the authors assume that several limitations of humanized mice prevent broader implementation into mainstream septic research Table 1.

Humanized mice earned justifiable praise from several researchers. However, the inherited problems of this model have been acknowledged by a few [ 8 , 10 , 12 , 29 , 42 , 62 , 77 , 81 ]. First, there exist fundamental differences between human and mice physiology [ 11 ]. More specifically, humanized mice exhibit several differences in the natural history of sepsis. Weight loss and mortality are greater in humanized mice than wild-type mice when short-term and long-term data are analyzed [ 35 , 55 , 67 , 81 ].

Only the introduction of extensive measures antibiotics, fluid resuscitation, and diet modification resulted in animal survival exceeding a couple days [ 35 , 55 ]. Prolonged studies were complicated by the emergence of GVHD and lymphomas as well as the suppressive effect of preserved components of the indigenous mice immune system [ 15 , 29 , 82 ]. Additionally, the recovery of the granulocyte compartment required supplementation of human G-CSF [ 50 ]. Considering that granulocytes are a pivotal defense against microbial infections, this need for supplementation is a shortcoming of humanized mice in mimicking their function and heterogeneity significantly limiting that model [ 2 , 32 ].

The deficit in the granulocyte compartment may also underlie early mortality in sepsis and require a more aggressive therapeutic approach [ 55 ]. Furthermore, all leukocyte types demonstrate a sign of functional immaturity unless remedial measures are implemented [ 25 , 31 , 36 , 46 , 66 ]. The corrective modification may create an artificial condition on its own or only be partially effective [ 37 , 38 ].

In the example of expansion in humanized cells, the imbalance may not reflect the natural history of sepsis or postseptic leukocyte population changes [ 37 ]. Other shifts in T cell population composition were reported, but virtually no study investigated population heterogeneity of monocytes, NK, and B cells in the context of the response to infection [ 35 , 45 , 55 ].

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