The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.
The human skin provides the first line of host protection against environmental injury and host defense against pathogens1, 2. Several emerging pathogens, including community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), target the skin for infection and disease1, 3,4,5. Also, vector-borne infectious diseases such as Lyme disease and dengue fever are transmitted via inoculation into the skin by ticks and mosquitos, respectively6. Interactions between keratinocytes, skin fibroblasts, and cutaneous immune cells are involved in initiating the systemic immune response and abrogate pathogen replication and dissemination to other sites of replication7,8,9,10. Thus, the skin provides an ideal vaccination target for inducing immunity against various pathogens, as reflected by the development of several novel vaccine technologies directed at the skin, including skin-patch intradermal vaccines11, 12.
In vivo models for studying environmental insults and pathogens that target the skin and associated cutaneous immune cells primarily involve mice and rats3. These rodent models have improved mechanistic understanding of human diseases; however, significant differences exist between the skin and immune system of humans and rodents3, 13. Rodent skin microanatomy differs from human skin microanatomy due to the rodent skin lacking a multi-layered epidermis, eccrine and apocrine glands, and the papillary, reticular, and hypo-dermal regions of the dermal layer14. Human primary and secondary lymphoid tissues microanatomy differs significantly from that of rodent lymphoid tissues as well, with significant differences in red pulp to white pulp ratio in the spleen and lobulation of the thymus15,16,17. It is well-established that lymphoid tissue microenvironment, including stromal cells, plays a significant role in immune cell development18. Interactions between immune cells and stromal cells in non-lymphoid tissues, such as the skin, play a significant role in modulating tissue-associated immune responses14. Translational gaps may form between clinical studies performed with traditional rodent models, thus highlighting the need for humanized rodent models that can support the engraftment of both human skin and immune system components1.
To address the species gap between rodents and humans, researchers have engrafted the immunodeficient NOD-scid IL2Rγnull (NSG) mouse model, which lacks mature lymphocytes and natural killer (NK) cells and possesses defects in innate immunity, with various human cells and tissues19, 20. Termed humanized-NSG mice, these models exhibit both human immune cell reconstitution and human lymphoid tissue growth and have been used to recapitulate clinical features of human diseases (including skin-associated diseases)21,22,23,24,25. Several separate reports demonstrate that immunodeficient mice support the engraftment of human skin26, 27. Adult human skin-derived from either medical wastes (elective plastic surgery)28, 29 or tissue culture-derived engineered skin (keratinocytes and fibroblasts)24 engrafts successfully in immunodeficient mice. Allogeneic adult human-peripheral blood mononuclear cells (PBMCs) have been introduced into these models to mimic human immune cell-skin interactions with infectious agents24, 29. Although these mouse models demonstrate successful engraftment and development of transplanted human skin and are amenable to the transplantation of allogeneic PBMCs, said platforms are not currently coupled with the engraftment of autologous lymphoid tissues that are critical for a de novo immune response. Humanized mouse models that combine human skin, human immune cells, and human lymphoid structures have yet to be established, despite their potential for developing a functional in vivo system, which could enable studies on human skin-immune cell interactions30. Humanized mouse models with human fetal-derived hematopoietic system and autologous lymphoid tissues are well-established19, 20. Additionally, full-thickness human fetal skin readily engrafts onto immunodeficient mice and develops into adult-like skin due to its high regenerative capability31, 32. Furthermore, human fetal skin exhibits low levels of major histocompatibility complex (MHC) I and II expression, which results in reduced immunogenicity compared to adult skin31, 32. Thus, human fetal-derived tissues and cells provide a feasible means to develop a humanized mouse model with autologous human skin and immune system.
Rats are a major model specie for long-term (greater than one year) studies33; thus, humanized rat models that combine human skin, human immune cells, and human lymphoid structures are being explored for use in long-term in vivo studies of human skin-immune interactions. Reports have demonstrated that nude rats (with moderate immunodeficiency) support adult human skin (split-thickness skin) engraftment and development, albeit host-mediated immune rejection occurs within a few months34,35,36. Evidence also suggests that nude rats support engraftment and development of full-thickness neonatal foreskin37. Recently, an immunodeficient rat, termed Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat, was developed to support engraftment of human cells and tissues into a larger and longer-life span rodent model; this model lacks mature T, B and NK cells38, 39. To date, humanized rat models co-engrafted with human full-thickness skin and autologous lymphoid tissues and immune cells (including cutaneous immune cells) remain to be developed and established28, 40,41,42.
Here, we utilize the immunodeficient NSG mouse and SRG rat models to generate rodent models with human skin, along with autologous lymphoid tissue(s) and autologous immune cells, termed human Skin and Immune System (hSIS)-NSG mice and SRG rats. Adolescent rodents received co-transplants of human full-thickness fetal skin, autologous lymphoid tissues, and autologous hematopoietic stem cells. Additionally, the human skin xenografts were inoculated with CA-MRSA to establish the susceptibility of these hSIS-humanized rodent models to human skin pathogens.
The hSIS-humanized NSG mouse model supports the development of full-thickness human skin, autologous lymphoid tissues (thymus and spleen), and human immune cells
We previously demonstrated that NSG mice support the development of human lymphoid tissues (thymus and spleen) along with autologous immune cells following engraftment of fetal tissues and autologous hematopoietic stem cells17. Several reports demonstrated that immunodeficient mice support the development of human skin following engraftment of human fetal skin43, 44. Here, we hypothesize that NSG mice will support co-engraftment of human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous hematopoietic stem cells. Furthermore, we hypothesize that NSG mice will facilitate human skin and lymphoid tissue development, as well as enable systemic human immune cell reconstitution in transplanted human tissues and the blood. We processed human fetal spleen, thymus, and liver organs into ~ 1 mm3 pieces and isolated autologous human CD34+ hematopoietic stem cells from the fetal liver, then transplanted the tissues and hematopoietic stem cells into irradiated NSG mice. Human skin tissues were obtained from the scalp and dorsum of donors and were used in developing human skin engraftments with and without hair in the mouse model, respectively. Full-thickness human fetal skin was processed via removal of excess fat tissues attached to the subcutaneous layer of the skin, then engrafted over the rib cage, where the mouse skin was previously excised. In three cohorts, the overall success of the human immune system and autologous full-thickness skin development and maintenance for ten weeks or greater was over 75% (Supplementary Fig. 1). Gross analysis of the human skin xenograft in the hSIS-NSG mouse model beginning at two weeks post-transplantation demonstrates wound healing and maturation into adult-like human skin, which is evident at ten weeks post-transplantation (Fig. 1A, Supplementary Fig. 2). A limitation of the human skin in the hSIS-NSG mouse model is the development of dry skin (resulting in hardening) and early signs of murine hair loss (suggestive of graft-versus-host disease) at 20 weeks post-transplantation (Supplementary Fig. 2). The human skin in hSIS-NSG mice also develops human skin appendages, with human hair evident at 12 weeks post-transplantation (Fig. 1B). Gross analysis of human spleen and thymus xenografts in hSIS-humanized NSG mice at ten weeks post-transplantation demonstrated the growth of those lymphoid tissues under the kidney capsule (Fig. 1C)17. In addition to supporting the growth of human spleen and thymus tissues, the hSIS-humanized NSG mouse model supports the reconstitution of the immunodeficient-murine lymph nodes and spleen (Fig. 1C)17. Histochemical analysis of the human skin in hSIS-humanized NSG mice demonstrates the development of the human skin xenograft; the microanatomy of the human skin at ten weeks post-transplantation is comparable to adult human skin, with multiple layers of cells present in the epidermis (Fig. 2A, Supplementary Fig. 3). The human skin xenograft exhibited multiple layers of human keratinocytes (AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis and dermal fibroblasts (Anti-Fibroblasts Antibody+ cells) in the dermis (Fig. 2B). Additionally, the human skin exhibited human immune cell repopulation (human CD45+ cells), including Langerhans cells (hCD207+ cells), macrophages (hCD68+), and T cells (hCD3+) (Fig. 2B, Supplementary Figs. 4 and 5). The human skin in hSIS-humanized NSG mice also exhibited upregulation of alpha-smooth muscle actin-positive (α-SMA+) cells (i.e., blood vessel cells45) during revascularization and wound healing (~ 2 weeks post-transplantation), followed by a reduction in α-SMA+ cells in the healed skin (~ 10 weeks post-transplantation) (Fig. 2B). Histochemical analysis demonstrates the development of the co-transplanted human lymphoid tissues (spleen and thymus) in the renal capsule (~ 10 weeks post-transplantation) (Fig. 2C)17. Human thymus tissue in hSIS-humanized NSG mice exhibits T-cell (human CD3+ cells) reconstitution, with few B cells in the tissue (Fig. 2C)17, 46, 47. Macrophage reconstitution (human CD68+ cells) in the human thymus tissue is restricted to the medulla (Fig. 2C)17, 46, 47. Human spleen tissue in hSIS-humanized NSG mice exhibits macrophage reconstitution, with macrophages predominately in the red-pulp (Fig. 2C)15, 16. The human spleen tissue in hSIS-humanized NSG mice exhibits T and B cell repopulation (human CD3+ and CD20+ cells), with lymphocytes predominately in the white-pulp (Fig. 2C)15. Analysis of the PBMCs in hSIS-humanized NSG mice showed human immune cell reconstitution (hCD45+ cells) (Fig. 3A,B). Various human immune cell (hCD45+) subtypes, namely, αβ T cells (hαβ T cells with a CD4:CD8 ratio average of 1.34:1), γδ T cells (γδ T), NK cells (hNK), B cells (hB), monocytes (hMo) and granulocytes/polymorphonuclear neutrophils (hPMN) were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model (Fig. 3C,D), with analysis performed using appropriate flow cytometry assay controls (Supplementary Fig. 6).