The human skin is a major target for infection or transmission of several infectious agents of global health significance 1, 2 . Several emerging pathogens, namely community-associated methicillinresistant Staphylococcus aureus (CA-MRSA) and Candida auris, respectively, target the skin for infection and disease 1, [3] [4] [5] . Vector-borne infectious diseases such as Lyme disease, malaria, and dengue are transmitted via ticks and mosquito respectively, via inoculation in the skin. Recent evidence suggests that the cutaneous immune system plays a critical role in modulating the establishment of infections in the skin 6 . In concurrence with the skin's role as a major barrier for host defense against infectious agents, the cutaneous immune system plays a major role in initiating systemic immune response and abrogates pathogen replication and trafficking to other sites of replication [7] [8] [9] [10] . Furthermore, the cutaneous immune system provides an ideal target for inducing human immunity against various infectious agents via vaccination with novel vaccine technologies, such as skin-patch intradermal vaccines 11, 12 . The skin is also a major barrier against environmental insults, including ultraviolet radiation from the Sun; and it is well established that long-term overexposure to ultraviolet radiation induces cutaneous immune suppression 13 .
In vivo models for studying pathogens that target the skin for infection or transmission primarily involve mice and rats 3 . Said rodent models have improved mechanistic understanding of the transmission, replication, and pathogenesis of a myriad of human pathogens; however, significant differences exist between the skin and immune system (including the cutaneous immune system) of humans and rodents 3, 14 . Consequently, translational gaps can form between clinical outcomes and studies performed with conventional rodent models 1 . To addressed the above discussed technical gaps, the severely immunodeficient NOD-scid IL2Rg null (NSG) mouse model, which lacks mature T cells, B cells, and natural killer (NK) cells, along with defects in innate immunity, has been engrafted with various human cells and tissues (termed, humanized NSG mice) to enable investigation of human host-pathogen interactions and recapitulate clinical features, [15] [16] [17] . Recently, a severely immunodeficient rat, termed, Sprague-Dawley-Rag2 tm2hera Il2rg tm1hera (SRG) rat 18, 19 , with comparable immunodeficiency as in the NSG mouse model, was developed to support engraftment of human cells and tissues into a larger and longer-life span rodent model. To date, a humanized rodent model co-engrafted with full-thickness human skin and autologous lymphoid tissues and immune cells (including cutaneous immune cells) remains to be developed and established; albeit mouse and rat models engrafted with human skin, with or without allogeneic human peripheral blood mononuclear cells (PBMCs) (with human T cells the predominant immune cell subset that engraft) have been established [20] [21] [22] [23] .
Here, we employ the severely immunodeficient NSG mouse and SRG rat models to generate human Skin and Immune System (hSIS)-NSG mice and SRG rats for investigating human skin-associated infections and diseases. NSG mice and SRG rats were co-engrafted with full-thickness human fetal skin over the rodent skin-excised rib cage, autologous fetal lymphoid tissues under the renal capsule, along with autologous fetal liver-derived hematopoietic stem cells via intravenous injection. We report that the hSIS-humanized NSG mouse model supports the development of full-thickness human skin, autologous human immune cells, and human thymus and spleen tissues. The hSIS-humanized SRG rat model also supports the development of full-thickness human skin, thymus tissue and autologous human immune cells. We demonstrated that hSIS-humanized rodents support CA-MRSA infection and associated skin pathology to recapitulate clinical presentations in humans.
The hSIS-humanized NSG mouse supports the development of full-thickness human skin, autologous lymphoid tissues (thymus and spleen), and human immune cells.
We previously demonstrated that severely immunodeficient NSG mice support robust development of human lymphoid tissues (thymus and spleen) along with autologous immune cells 24 ; separate studies demonstrated that severely immunodeficient mice support the engraftment and development of human skin 22 . The mouse skin microanatomy differs from human skin microanatomy by the absence of multi-layered epidermis, eccrine and apocrine glands, and the papillary, reticular and hypo-dermal regions of the dermal layer 25 . Additionally, the microanatomy of human primary and secondary lymphoid tissues differs significantly from mouse lymphoid tissues, with significant differences in red pulp to white pulp ratio in the spleen and lobulation in the thymus 24, 26, 27 . Here, we hypothesize that severely immunodeficient NSG mice will support co-engraftment of full-thickness fetal human skin and autologous fetal lymphoid tissues along with hematopoietic stem cells.
Furthermore, we hypothesize that severely immunodeficient NSG mice will facilitate human skin and lymphoid tissues development, and enable systemic human immune cell reconstitution in transplanted human tissues and the blood (Figure 1) . We processed human fetal spleen, thymus, and liver organs into ~1 mm^2 tissues and isolated autologous human CD34 + hematopoietic stem cells from the fetal liver, and transplanted the tissues and hematopoietic stem cells into irradiated NSG mice (Figure 1) . 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 mouse skin was previously excised (Figure 1 ). Gross analysis of human skin in hSIS-NSG mice beginning at Figure 2B ) 24 . In addition to supporting the development of human spleen and thymus tissues, the hSIS-humanized NSG mouse model supports reconstitution of the immunodeficient-murine lymph nodes and spleen (Figure 2B ) 24 . The human skin in hSIS-NSG mice also develops human skin appendages (hair) (Supplementary Figure 1) . A limitation of the human skin in the hSIS-NSG mouse model is the development of dry skin and early signs of graft-versus-host disease in and around the human skin graft at 20 weeks post-transplantation, which is associated with rapid aging (Figure 3) .
Histochemical analysis of human skin in hSIS-humanized NSG mice reveals development of the human skin graft; the microanatomy of the human skin at 10 weeks post-transplantation is comparable to adult human skin, and multiple layers of cells are present in the epidermis ( Figure 4A ).
Human skin in hSIS-humanized NSG mice exhibited robust upregulation of alpha-smooth muscle actin-positive (-SMA + ) cells (i.e. blood vessel cells) during revascularization and wound healing (~2 weeks post-transplantation), followed by a reduction in -SMA + cells in healed skin (~10 weeks posttransplantation) ( Figure 4B ). The human skin exhibited robust repopulation with human keratinocytes (AE1/AE3-pan cytokeratin antibody+ cells) in epidermis and dermal fibroblasts (Anti-Fibroblasts Antibody+ cells) in the dermis ( Figure 4B) . Additionally, the human skin exhibited robust human immune cell (human CD45 + cells) repopulation, including Langerhans cells (CD207+ cells) ( Figure   4B ). Co-transplanted human lymphoid tissues (spleen and thymus) developed under the renal capsule (~10 weeks post-transplantation) and exhibited microanatomy comparable to adult human lymphoid organs (Figure 4C ) 24 . Human thymus tissue in hSIS-humanized NSG mice exhibits robust T-cell (human CD3 + cells) reconstitution, with T-cell levels highest in the cortex region and relatively lower in the medulla region ( Figure 4C ) 24, 28, 29 . Macrophage reconstitution (human CD68 + cells) in the human thymus tissue is restricted to the medulla ( Figure 4C ) 24, 29, 30 . Human spleen tissue in hSIShumanized NSG mice exhibits robust macrophage reconstitution, with macrophages predominately in the red-pulp ( Figure 4B ) 26, 27 . Additionally, the human spleen tissue in hSIS-humanized NSG mice exhibit robust T and B cell repopulation (human CD3 + and CD20 + cells), with lymphocytes predominately in the white-pulp (Figure 4B ) 26 . The blood plays a major role in the cutaneous immune system, as it facilitates systemic immune cell trafficking to the skin; analysis of the peripheral blood mononuclear cells (PBMCs) in hSIS-humanized NSG mice showed high (average of ~43%) human immune cell (human CD45 + cells of total leukocytes) reconstitution in the peripheral blood ( Figure 5A) . Additionally, various human immune types, namely, T cells (CD4, CD8, ), B cells, monocytes and granulocytes were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model ( Figure 5B ).
Although humanized mice models have provided in vivo platforms for investigating the mechanisms of human diseases, the short-life span and small tissue size/volume of mice are a major limitation for pre-clinical studies. We hypothesized that a larger, severely immunodeficient rodent model with longer life span, namely rat, would support development of an in vivo model with longer experimental window, robust longitudinal studies (i.e. biopsy collection) and provide large tissue volume/size. We further hypothesized that co-transplantation of full-thickness fetal human skin, autologous fetal thymus and liver tissues, and fetal-liver derived hematopoietic stem cells in SRG rat would enable development of human skin, autologous thymus, and human immune cells, termed, hSIS-humanized SRG rat (Figure 1) . We processed human thymus and liver tissues into ~1 mm^2 pieces and isolated autologous CD34 + hematopoietic stem cells from the liver, and transplanted the tissues and hematopoietic stem cells into irradiated SRG rat. Full-thickness human fetal skin was processed via (Figure 7A ). Histochemical analysis of human thymus tissues in hSIS-humanized SRG rats at 9 months post-transplantation, exhibits a microanatomy comparable to the adult human thymus (Figure 7B ). Human thymus tissue in the hSIS-humanized SRG rat exhibits robust human immune cell (human CD45 + cells) reconstitution, including T cell (human CD3 + cells) and macrophage (human CD68 + cells) reconstitution (Figure 7B, 7C) .
Additionally, human T cells in the thymus exhibit robust cytokine response to physiological stimulation using CD3/CD28 beads (Supplementary Figure 3) . Human immune cells in the hSIS-humanized SRG rat also reconstitute the immunodeficient-rat spleen (Figure 7D) , albeit human immune cell reconstitution of the blood was not observed (data not shown).
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection represents a significant public health threat 5 ; thus, understanding the host-pathogen interactions in the human skin is critical for the development of therapeutics. In order to demonstrate that human skin engrafted onto immunodeficient rodents provides a novel approach for modeling human host-skin pathogen interactions; we inoculated (intradermal inoculation) the human skin with CA-MRSA USA300. Figure 4) .
The human skin is the primary barrier and host defense-organ against a myriad of pathogenic microbes (i.e. CA-MRSA) and environmental insults (i.e. ultraviolet radiation) 10 . Additionally, the skin is the primary route of infection for various emerging and re-emerging vector-borne diseases, such as tick-borne disease (i.e. Lyme disease, Rocky Mountain spotted fever, etc.) and mosquito-borne disease (i.e. Zika virus disease, Malaria, Dengue virus disease) 7 , and also provides a robust route for vaccine and other therapeutic administration against these infectious agents 12 . The increasing public health threat of infectious agents that target the skin (i.e. CA-MRSA) and geographic expansion of vector-borne diseases have highlighted the need for robust in vivo models for investigating the human skin-pathogen interactions associated with said infectious agents and evaluating therapeutics 4-6 .
Furthermore, the development of novel vaccine and therapeutic delivery platforms (i.e. skin-patch, microneedle, intradermal-vaccines) targeting the skin also illustrate the need for robust in vivo models with human organ systems for investigating the safety and efficacy of those vaccines and therapeutics against human infectious diseases 12 . Rodent models are the primary platforms for investigating skin-associated infectious agents, the mechanisms of disease and host response/defense, and novel therapeutics. Although rodent models provide insights into the mechanisms of disease and host response/defense against skin-associated infectious agents, several limitations exist in rodent models, resulting in knowledge gaps. It is well established that the human skin exhibits significant structural differences compared to rodent skin 10, 25 . Specifically, human skin is composed of multiple layers and cushioned in subcutaneous fat; rodent skin, however, is composed of a single/few layers, with little to no sub-cutaneous fat 14, 25 . Differences in the types and levels of fatty acids, could play a critical role in immune signaling and regulating the skin microbiome, resulting in significant differences in anti-microbial response between human and rodent skin 1,2 . These differences could significantly impact the translation of mechanistic findings from conventional rodent models to humans.
To address these technical gaps, larger animal models, namely pig, with skin structure and microanatomy comparable to the human skin have been employed; however, the size, cost and lack of molecular tools, limits employment of said models for mechanistic studies 31 . An attractive alternative is transplantation of human skin and autologous immune system on immunodeficient small animal (rodent) models, namely mice and rats. Published reports demonstrated that immunodeficient mice support engraftment of human skin 32, 33 . Full-thickness human fetal skin readily engrafts onto immunodeficient mice, and develops into adult-like skin, in part due to the high regenerative capability and low MHC I and II expression, reducing the immunogenicity of the fetal skin compared to adult skin 34, 35 . Furthermore, reports demonstrate that nude rats (moderate immunodeficiency) support human skin (split-thickness skin) engraftment and development, albeit host-mediated immune rejection occurs within a few months [36] [37] [38] . Although several rodent models demonstrate human skin engraftment and development, said platforms have not been coupled to autologous human immune cells and lymphoid tissues, such as in the hSIS-humanized SRG rodent models.
In this study, we demonstrate that co-transplanting human fetal skin along with autologous lymphoid tissues and hematopoietic stems cells in severely immunodeficient adult mice results in robust development of full-thickness, adult-like, human skin, as well as autologous human immune system cells in lymphoid tissues and the blood. In parallel, we demonstrate that co-transplanting human fetal skin along with thymus and hematopoietic stems cells in severely immunodeficient adult rat results in human skin and thymus development along with immune cells in the rat lymphoid organ (spleen), although peripheral immune reconstitution is not observed. Furthermore, major skin appendages such as hair developed on the human skin in both immunodeficient mice and rats. To establish that the human skin xenograft in immunodeficient rats is susceptible to human pathogens, we infected engrafted human skin with CA-MRSA via intradermal inoculation. We demonstrated that the human skin in immunodeficient rats supports CA-MRSA-infection and associated skin lesion, which was comparable to patients. Furthermore, the human skin in immunodeficient mice also supports CA-MRSA-infection and associated skin pathology; albeit the experimental window for infection and associated pathogenesis was restricted to less than 4 months post-transplantation, as the human skin aged rapidly (i.e. excessive dryness) and developed early sign of graft versus host disease beginning at 5 months post-transplantation. hSIS-humanized SRG rats do not exhibit rapid aging (i.e. excessive dryness) of the human skin or signs of graft versus host disease; thus providing a large experimental window (>9 months). Future efforts will improve the peripheral human immune reconstitution in immunodeficient rats.
In summary, we report the development of human Skin and Immune System-humanized (hSIS) NSG mouse and SRG rat models that incorporate human lymphoid tissues, human immune cells, and autologous human full-thickness skin. hSIS-humanized NSG mice and SRG rats provide a means of addressing several limitations in conventional rodents for modeling human skin biology/pathobiology and associated cutaneous immune responses, including microanatomy differences, susceptibility to human infectious agents and associated diseases, and cutaneous immune cell signaling. hSIShumanized NSG mouse and SRG rat models provide robust rodent models for in vivo mechanistic studies of a myriad of human diseases associated with the skin, including cancer 39, 40 , viral infections [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] , bacterial infections 51,52 , etc.; thus our hSIS-humanized rodent models represents a novel in vivo platform for biomedical research. Bank at the University of Pittsburgh. Human fetal tissues for constructing humanized rodents were handled and processed under biosafety level 2 conditions. Male and female rodents were myoablated via gamma radiation using cesium-137 irradiator, with mice receiving a dose of (150 rads) and rats receiving a dose of (500 rads). Myoablated male and female mice were transplanted with human fetal-thymus, liver and spleen in the kidney capsule and autologous CD34 + hematopoietic stem cells (via retroorbital injection of 0.2X10^6 cells) 24 (B cells-hCD19+, T cells-hCD3+, CD4+ T cells-hCD4+ T cells, CD8+ T cells-hCD8+ T cells, T cells-hTCR+ T cells, monocytes-hCD14+/hCD16+ human PBMCs, granulocytes-hCD66b+ human PBMCs) in peripheral blood mononuclear cells (PBMCs) of hSIS-humanized mice at 10-12 weeks post-transplantation. 