A type of immune cell called neutrophils could be responsible for controlling bacterial numbers of an antibiotic-resistant strain of Staphylococcus aureus (MRSA) on human skin before the bacteria get a chance to invade, according to a new study from Karolinska Institutet published in Cell Reports.
The results could provide an explanation for why this superbug is only carried transiently by some people.
The researchers have developed a “humanised” mouse model in which human skin is grafted onto mice, which helps them look at the human tissue response in vivo.
They found that after the skin had been colonised with MRSA, neutrophils were recruited to the skin resulting in the antibiotic-resistant bacteria being killed. This could explain why some people are only transient carriers of MRSA.
The study also highlights how dynamic the human skin is.
The outer layers of the skin were once considered a dry, dead wasteland and a very challenging environment for the microbial inhabitants. However, a body of research is now emerging to show that this is not the case.
“The skin is an incredibly dynamic biological environment where immune cells and microbes stand-off against one another to maintain some kind of equilibrium, a fraught peace,” says Keira Melican, senior researcher at the Department of Neuroscience, Karolinska Institutet, who led the study.
“Breaks in these equilibria typically lead to bad outcomes for humans, and understanding how this process works on the skin could have an impact on how we prevent and treat skin infection in the future.”
Staphylococcus aureus is a common bacterium carried by up to 50 percent of the population.
However, it is also capable of causing some serious infections which are becoming more difficult to treat with the rise of antibiotic resistance.
Understanding the human immune response is a critical part of understanding how to curb the spread of resistant bacteria.
A common problem with using animals as proxies for humans is that parts of their biology is fundamentally different, from physiology to the molecular interactions. By incorporating human elements into our models, it is possible to get a more realistic picture of human biology, in this case, in colonisation by superbugs.
“We hope that our humanised skin model will help make sure that our results are relevant to humans, and not just mice,” says Keira Melican.
The skin is structured into three layers: the epidermis, the dermis, and subcutaneous fat tissue. The epidermis, the outermost layer of the skin, is subdivided into the stratum corneum, stratum lucidum, stratum granulosum, and stratum basale.
The stratum corneum contains corneocytes, which are terminally differentiated keratinocytes. These cells are continuously replenished by keratinocytes localized in the stratum basale . T
he stratum lucidum is a thin, clear layer of dead keratinocytes. Instead of keratin, keratinocytes in the stratum lucidum contain eleidin, a clear intracellular protein, which gives this layer its transparent appearance.
The stratum granulosum is a thin layer between the stratum lucidum and stratum basale. Keratinocytes in the stratum granulosum contain cysteine- and histidine-rich granules, which bind keratin filaments together . The stratum basale contains basal keratinocytes, immune cells such as Langerhans cells and T cells, and melanocytes that provide the skin with pigmentation.
Beneath the epidermis is the dermis, which is further categorized into papillary and reticular sub-layers. In humans, the papillary dermis forms extensions that reach out to the epidermis and contain capillaries that facilitate the transport of nutrients .
Reticular dermis contains skin appendages such as hair follicles, sebaceous glands, and sweat glands.
The reticular dermis is significantly thicker than the papillary dermis due to the dense concentration of collagenous and reticular fibers that are interwoven within this layer. Both dermal layers house fibroblasts, myofibroblasts, and immune cells such as macrophages, lymphocytes, and mast cells. Fibroblasts synthesize an extracellular matrix consisting of collagen, proteoglycans, and elastic fibers that provide the structural integrity of the dermis .
Underlying the dermis is the subcutaneous fat. This layer consists of fibrocytes and adipocytes and is rich in proteoglycans and glycosaminoglycans, which confer mucus-like properties to the layer .
Skin adipose tissue stores energy in the form of fatty acids and functions as an endocrine organ important for glucose homeostasis and lipid metabolism [6,7,8]. This layer also produces a variety of mediators such as growth factors, adipokines, and cytokines, and contains multiple immune cells .
In addition, the subcutaneous fat serves as an insulating layer for the body, as fat is a poor conductor of heat.
Murine models are commonly used in dermatological research. These models are of great value and mimic many pathological aspects observed in human skin disorders. However, it is important to note that there are differences in the skin structure between mice and human.
The thickness of murine epidermis is thinner than that of humans’, which depending on the area of the body, is between six and 32 layers .
The epithelial architecture of human skin contains rete ridges, which are extensions that project into the underlying tissue, and do not exist in murine skin . In contrast to humans, the murine dermis lack sweat glands, excluding the mammary glands in female mice. Furthermore, mice contain a dermal muscular layer, also known as the panniculus carnosus, which is absent in humans .
The skin of other animals such as pigs more closely resembles that of humans , so these animals are increasingly employed in experimental settings. However, murine models are still widely used due to easy access and lower costs and have proved effective for the study of inflammatory diseases of the skin.
The Skin as an Immune Organ
One of the main functions of the skin is to protect the host from invasion, and it does so by employing physical barriers, biomolecules, and an intricate network of resident immune and non-immune cells and skin structures. Furthermore, in the absence of a challenge, resident immune cells promote skin physiological functions. Below we will describe the strategies used by the skin to defend and protect the host, and their contributions to tissue homeostasis.
The barrier function of the epidermis is mainly mediated by corneocytes in the stratum corneum. These cells are organized in a “bricks and mortar” manner, interspersed by lipids such as ceramides, cholesterol, and free fatty acids .
Each corneocyte contains a lipid envelope linked to keratin filament bundles that fill the intracellular compartments of the corneocyte, thus strengthening its rigidity . The stratum corneum is composed of three layers and it is both an outside‒in barrier to prevent the entry of foreign substances and microorganisms, and an inside‒out barrier to prevent water loss .
The formation of the physical barrier of the skin function depends on junction adhesion molecules and tight junction proteins.
Disruptions in the expression or function of these components will result in improper barrier formation or skin disorders. Junctional adhesion molecules (JAMs), claudins, zonula occludins-1 (ZO-1), and occludins are found in epidermal layers. Claudin-1, claudin-7, and JAM-A are found in all layers of the human epidermis , while claudin-1, claudin-12, and JAM-A are ubiquitous in murine epidermal layers [17,18].
al., who showed that deletion of claudin-1 in mice led to death shortly after birth as a result of cutaneous defects ultimately leading to significant loss of fluids .
Impairment of the components of the skin’s physical barriers can contribute to inflammatory conditions in the skin. For example, the skin of patients with atopic dermatitis exhibits reduced expression levels of ZO-1 and claudin-1 , and knockdown of claudin-1 in mice induces a psoriasis-like condition .
Biomolecules of the Skin
AMPs are amphipathic peptides and are expressed constitutively or induced after cell activation in response to inflammatory or homeostatic stimulation.
The most thoroughly studied AMP families in human skin are the defensins and the cathelicidins, which are produced by a variety of cells in the skin such as keratinocytes, fibroblasts, dendritic cells, monocytes, and macrophages, and sweat and sebaceous glands [22,23].
AMPs are produced as propeptides and become active after proteolytic cleavage. In contrast to defensin family members, which are produced by distinct genes, there is only one gene associated with cathelicidins (cathelicidin antimicrobial peptide—CAMP) . A number of active peptides can be generated via proteolytic cleavage of the inactive CAMP product, but the most studied cathelicidin is LL-37 .
AMPs may act synergistically and have broad activity against microbial species [27,28]. A recent study suggests certain AMPs activity may be pathogen-specific . Recently, due to increased resistance to antibiotics, a focus of active research has been the evaluation of AMPs as therapeutics against infections .
Interestingly, AMPs have roles in modulating host immune responses. Human LL-37 was shown to induce differentiation of monocyte-derived dendritic cells, subsequent cytokine production, and expression of the co-stimulatory molecule CD86 .
LL-37 and β-defensins can also serve as alarmins for keratinocytes by inducing their proliferation and migration . Furthermore, human LL-37 exerts its alarmin effects on immune cells in a synergistic manner with other inflammatory mediators, such as IL-1β .
Human α- and β-defensins serve as chemoattractants for activated neutrophils, memory and naïve T cells and immature dendritic cells , while β-defensins 3 and 4 can recruit monocytes and macrophages . The murine version of β-defensin 2 induces dendritic cell maturation by upregulating the expression of co-stimulatory molecules and the antigen presentation molecule MHCII .
In addition, AMP functions have been associated with the processes of aging and memory [26,32,35]. Interestingly, LL-37 has been shown to exert proangiogenic effects and may also play a role in tissue repair .
Lipids such as sphingomyelin, glucosylceramides, and phospholipids are intermediate molecules, readily converted into sphingosine and dihydrosphingosine, which exert antimicrobial activity against certain bacterial strains such as Staphylococcus aureus, Streptococcus pyogenes, Micrococcus leutus, and Proprionibacterium acnes . These lipids are stored in lamellar bodies found in corneocytes in the stratum corneum .
Sebocytes residing in the sebaceous glands produce sebum, which is rich in lipids such as triacylglycerol, wax esters, non-esterized fatty acids, and squalene. While the functions of sebum are not fully understood, there is a consensus that sebum serves as a “seal” for the hair follicles, thus preventing entry of microbes into the deeper layers of the skin. Intriguingly, a previous study demonstrated that the AMP dermcidin is expressed by sebocytes, suggesting that sebum exerts defensive functions .
Furthermore, sebum can be further processed into free fatty acids by skin commensal bacteria [40,41], and in humans, sebum-derived free fatty acids induce β-defensin 2 expression by sebocytes, further suggesting that sebaceous glands serve an innate defensive role .
pH of the Skin
The pH of human skin is 5.4‒5.9, which makes the skin an inhospitable environment for potential pathogens [43,44]. Furthermore, the dramatic difference in pH levels between the skin and the blood (pH = 7.4) serves as a secondary defensive mechanism in the event that microbes breach the skin tissue and enter the circulation. There are various ways that the skin maintains a low pH.
Filaggrin, a filament-associated protein that binds keratin fibers, is broken down into histidine, which is further processed by histidase, expressed by corneocytes into the acidic metabolite trans-urocanic acid ; this has been implicated in the acidification of the stratum corneum . Fatty acids produced in the stratum corneum also alter the acidity of the skin [41,44]. In addition, sweat glands produce acidic electrolytes and lactic acid, which lowers the pH of the skin  and promotes epidermal turnover .
Furthermore, the physiological pH of the skin is hospitable for commensal bacteria such as Staphylococcus epidermidis, preventing pathogenic strains such as Staphylococcus aureus from establishing infections in the host [49,50].
Immune Cells of the Skin
Skin-resident immune cells promote tissue function in homeostasis and act as sentinels by actively sampling environmental antigens. Both myeloid and lymphoid cell subsets are found in the skin in steady state (Table 1). Some of these resident immune cells migrate to lymph nodes to either induce peripheral tolerance to tissue self-antigens or initiate robust immune responses. In the event of a challenge, such as infections or tissue injury, immune cells resident in the skin and those infiltrating from the periphery interact to create an intricate defense network to resolve the insult and restore the tissue to its original state. In this section we will describe the functions of myeloid and lymphoid cell subsets that are resident to the skin.
Summary of the skin’s immune cells. The location of each immune cell type in the skin tissue and their functions during homeostasis, inflammation, and wound healing are described. N/D: not defined.
|Immune Cell Type||Location in Skin||Functions During Homeostasis||Inflammatory Functions||Functions during Wound Healing|
|Langerhans cells and dDCs||Langerhans cells: Epidermis|
dDCs: Papillary dermis
|Sampling of environmental antigens|
Migrate to lymph nodes to induce tolerance to self-antigens 
Control of commensal-specific T cells in the skin 
|Migration to lymph nodes to induce adaptive immune responses to specific antigens|
Induce T cell responses
Produce pro-inflammatory cytokines and chemokines to recruit peripheral immune cells
|Migrate to draining lymph nodes to prime adaptive responses|
Promote angiogenesis, re-epithelialization, formation of granulation tissue, and growth factor production 
|Macrophages/monocyte-derived macrophages||Papillary and reticular dermis||Hair follicle regeneration/maintenance |
Phagocytosis of cellular debris
|Produce cytokines, chemokines to recruit peripheral immune cells|
Inflammatory macrophages produce inflammatory cytokines (IL-1β, TNFα, IL-6)
Phagocytosis of pathogenic agents and necrotic debris
|Reparative macrophages produce growth factors (VEGF, TGFβ) and regulatory cytokines (IL-10)|
Give rise to de novo fibroblasts and induces their proliferation 
Expression of MMPs during tissue remodeling
Activation of myofibroblasts 
|Mast cells||Papillary and reticular dermis||N/D||Produce inflammatory mediators involved in allergic responses and asthma, and recruitment of immune cells|
Produce inflammatory cytokines and secrete histamine during contact-hypersensitivities
|Induce collagen production (fibrosis) from fibroblasts [57,58,59,60]|
|Eosinophils||Reticular dermis||N/D||Defense against parasites|
Degranulation; release of EPO, MBP, EPX/EDN, and ECP
Infiltrates the skin tissue during eosinophilic dermatoses 
|Neutrophils||Reticular dermis||N/D (not abundant in healthy skin)||Phagocytosis of invading pathogens|
Release of NETs (NETosis) to immobilize pathogens
Production and secretion of coagulation factor XII to induce NETosis 
chemoattractants to recruit other neutrophils to inflamed sites 
|Secretion of laminin 5 β-3 to induce keratinocyte adhesion |
Responsive to VEGF and induce angiogenesis [65,66]
|αβ T lymphocytes||CD8+: Epidermis|
Epidermis and papillary dermis
|Sentinels that can recruit other lymphocytes to the skin|
Found to localize around hair follicles, perhaps to control commensal populations in the proximity 
|Induces antiviral state in the skin through IFNγ mechanism|
T effectors produce cognate cytokines (i.e., IFNγ, IL-4, IL-17
Tregs suppress inflammatory monocytes and other autoreactive immune cells
|Resolution of wound inflammation through Treg-mediated control of inflammatory monocytes |
|γδ T lymphocytes (DETCs and dermal)||DETCs: Epidermis|
Other γδ T cells: Papillary dermis
|Secretion of KGF and IGF-1 to maintain keratinocyte populations|
Migrates to draining lymph nodes after sensing stressed keratinocytes; antitumor immunity 
|Produce IL-17 to induce β-defensin expression from keratinocytes|
Protective against cutaneous S. aureus infection 
Involved in disorders such as psoriasis 
|Secretion of KGFs and IGF-1 to induce expansion of the epidermis |
|Non-γδ CD1-restricted lymphocytes||Papillary and reticular Dermis||N/D||Involved in defense against extracellular pathogens|
Suppress autoreactive cell activity in systemic lupus erythematosus
|iNKT cells may be involved in wound healing by controlling inflammatory neutrophil populations |
|B lymphocytes||Reticular Dermis||N/D||Involved in delayed-type hypersensitivity reactions|
Involved in cutaneous autoimmune diseases via production of autoantibodies specific to components of the skin [74,75]
IL-10-producing Bregs suppress autoreactive lymphocyte activation
|Non-immune cells (i.e., keratinocytes and fibroblasts)||Epidermis and reticular dermis||Contribute to barrier function of the skin|
Produce collagen network to provide structural integrity
|Produce inflammatory cytokines during disease (i.e., psoriasis), osmotic stress, or irradiation [76,77]|
Produce AMPs in response to bacterial detection [78,79,80]
|Migrate to close wound (re-epithelialization) and restore barrier function [7,81]|
Synthesize collagen fibers and other extracellular matrix components in the wound bed
Skin-resident myeloid cells include Langerhans cells, dermal dendritic cells, macrophages, mast cells, and eosinophils. Neutrophils are rarely found in healthy skin and thus are not “skin-resident cells.” However, neutrophils populate the skin in inflammatory conditions and after a wound, and they will be discussed in the wound healing sections of this review.
Skin-resident myeloid cells contribute to skin homeostasis by secreting growth factors needed for the survival of keratinocytes, fibroblasts, and endothelial cells. In addition, they maintain optimal tissue function by phagocytosing debris and apoptotic cells, supporting vasculature integrity, and promoting tolerance.
In inflammatory conditions, myeloid cells respond immediately and produce pro-inflammatory mediators that drive the activation of cells in the local vicinity and infiltration of the affected site by peripheral immune cells. Skin myeloid cells also serve as a liaison between the innate and adaptive immune system.
Langerhans cells (LCs) are the sole myeloid cell type in the epidermis. Phenotypically, LCs are characterized by high expression of MHCII and the presence of langerin+ Birbeck granules [51,82]. LC maintenance depends on keratinocyte-derived IL-34 [82,83,84,85], one of the ligands for the colony stimulating factor-1 receptor (CSF-1R). CSF-1R is constitutively expressed on LCs and global deletion of this receptor results in a total lack of LCs [85,86,87]. CSF1 is dispensable for LC maintenance as CSF1 knockout mice retain their LC populations . LCs are absent in mice deficient in inhibitor of DNA binding 2 (Id2) , or Runt-related transcription factor 3 (Runx3) . Both Id2 and Runx3 are involved in TGFβ signaling [88,89], and TGFβ deficiency also results in the complete loss of LCs.
LCs are derived from two sources, the extra-embryonic yolk sac and fetal liver monocytes; so far, there are no functional attributes unique to each niche . LCs renew from a local progenitor cell , as shown for the first time by Merad et al., via bone marrow chimeric mice in which donor CD45.2+ bone marrow cells were injected into lethally irradiated CD45.1+ recipient mice. After reconstitution and BrdU administration, LCs in the recipient mice were of host origin (CD45.1+), and incorporated BrdU, indicating that LCs are self-replenishing during homeostasis [91,92]. In inflammatory conditions, bone-marrow-derived cells are also able to give rise to short-lived or long-lived LCs in an IL-34-independent manner [85,93]. Long-lived bone-marrow-derived LCs are Id2-dependent [87,94].
In homeostasis, LCs anchor themselves within the epidermis through interactions between epithelial cell adhesion molecules (EpCAM) or E-cadherin expressed on LCs and E-cadherin expressed by keratinocytes [51,95,96]. Furthermore, autocrine and paracrine TGFβ signaling restricts LCs in the epidermis by regulating the expression of E-cadherin on LCs [97,98], and increases phagocytic behavior in LCs during steady state . While anchored, LCs sample antigens and, upon activation, they can extend their processes from the cell body outward to the stratum corneum or below toward the stratum basale . LCs participate in tight junction formation and thus can sample the microenvironment without damaging the barrier .
LCs are migratory cells and continually travel to the skin draining lymph nodes to promote tolerance in homeostasis  or to initiate adaptive immune responses [100,101,102]. To migrate from the epidermis to local lymphatic vessels, LCs disengage the adhesion interaction between E-cadherin and EpCAM/E-cadherin. The release of E-cadherin is mediated by β-catenin, which is associated with the intracellular tail of E-cadherin and provides a link between the adhesion molecule and the cytoskeleton . LCs express matrix metalloprotease 9 (MMP9), which allows for the restructuring of the dermal collagen network to create a path for LC migration toward the lymphatic vessel . Inhibition of MMP9 activity reduces LC migration from murine and human epidermal sheets . The migration of LCs (and also of dermal dendritic cells) through the dermis is mediated via CXCR4 signaling after binding to its cognate chemokine CXCL12, produced by dermal fibroblasts .
In the presence of inflammatory mediators and other activators such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), LCs upregulate co-stimulatory molecules and migrate from the epidermis en masse to the draining lymph nodes, where they prime adaptive immune responses in a manner equivalent to that of conventional dendritic cells [51,106,107,108,109]. TGFβ signaling is disrupted during inflammation, thus also promoting LC migration from the epidermis . LCs are professional antigen-presenting cells and activate both CD8+ cytotoxic T lymphocytes and CD4+ helper T lymphocytes. In humans, efficient antigen presentation by LCs is dependent upon caveolin-1, a scaffold protein that serves a multitude of functions including transport of lipids, signal transduction, and membrane trafficking [110,111]. LCs can also exert immunoregulatory and tolerogenic functions [51,112,113]. They were shown to promote tolerance to keratinocyte antigens in the skin by promoting the expansion of desmoglein 3-specific T regulatory cells , while LC depletion led to the formation of anti-desmoglein 3 antibodies resulting in dermatitis in mice .
Dermal Dendritic Cells
Dendritic cells that reside in the dermis are known as dermal dendritic cells (dDCs). In a similar manner to LCs, dDCs migrate to the lymph nodes, and are professional antigen-presenting cells efficient at priming adaptive immune responses [117,118]. It was recently shown that dDCs can induce tolerance to topically applied antigens encountered in the hair follicles .
Unlike LCs, all dDCs are derived from progenitors originating from the bone marrow, with replenishment occurring roughly every seven days [120,121]. The two main subsets of dDCs are the conventional dendritic cell type 1 (cDC1) and the conventional dendritic cell type 2 (cDC2) [122,123]. Murine cDC1s express langerin (CD207), low levels of the fractalkine receptor CX3CR1, the chemokine receptor XCR1, and CD103 (ITGAE), but do not express the myeloid marker CD11b. cDC2s are the most abundant subtype of dDCs in murine skin and express CD11b, CD301b, also known as macrophage galactose-type C-type lectin 2 (MGL2), low levels of langerin , and higher levels of CX3CR1 in comparison to cDC1s . Although a CD103+CD11b+ DC population exists in the intestines [125,126], expression of CD11b and CD103 is mutually exclusive in skin dendritic cells. In the mouse, both cDC1s and cDC2s require stimulation via Flt3 ligand (Flt3l) and CSF2  for development. For population maintenance, cDC1s require basic leucine zipper transcriptional factor 3 (Batf3) and IRF8 [128,129], while cDC2s require IRF4 [129,130,131]. A subset of dendritic cells expressing CD8α exists in lymphoid tissues but not in the skin [123,132]. These cells are capable of cross-presentation, a process during which extracellular antigens, normally processed via the MHCII pathway, are also presented via the MHCI pathway. Like CD8+ DCs, murine dermal cDC1s can cross-present antigens [133,134].
In the mouse, cDC1s are associated with Th1 responses [135,136], while cDC2s are associated with Th2 and Th17 [125,137,138,139]. Th1 responses are mainly specific for intracellular pathogens, while Th2 and Th17 are for extracellular pathogens. Furthermore, both Th1 and Th17 cells are associated with autoimmune disorders, while Th2 cells are associated with asthma and allergic disorders. Mice deficient in Irf8, in which cDC1s are depleted, generate reduced numbers of Th1 cells in the skin in response to cutaneous infection with herpes simplex virus (HSV) . Kruppel-like factor 4 (Klf4) has been shown to be required for maintaining conventional DC populations in the spleen . Intriguingly, Tussiwand et al. demonstrated that expression of Klf4 in dDCs is critical for the induction of Th2 responses in vivo .
dDCs are implicated in maintaining homeostatic interactions between the host and skin-resident commensal bacteria. Naik et al. showed that mice deficient in Batf3 (necessary for cDC1) exhibit reduced populations of CD8+ IL-17+ T cells specific for Staphylococcus epidermidis, suggesting that cDC1s control the generation of commensal-specific T cells in the skin .
Plasmacytoid DCs (pDCs) are a DC subset, found in the skin exclusively under inflammatory conditions. pDCs are mass producers of IFNα, which is essential for viral defense . In mice, pDCs can be identified by the marker B220, which is also expressed by B lymphocytes [142,143,144]. In addition to their antiviral functions, pDCs have been implicated in autoimmune skin disorders such as psoriasis . Interestingly, a recent study showed that depletion of pDCs using CLEC4C-DTR mice treated with bleomycin to induce skin fibrosis exhibited a decrease in dermal thickness and collagen content when compared to WT control mice , suggesting that pDCs play a role in fibrosis.
Human dDCs are subdivided into cDC1 (CD141+), cDC2 (CD1c+), and CD14+ dDCs . cDC1s co-express CD304 (neuropilin-1), XCR1, and CD370 (CLEC9A). Unlike murine dDCs, human dDCs do not express langerin [123,146]. Human cDC1s can cross-present in a similar manner to murine cDC1s  and are potent in inducing Th1 responses . cDC2s and CD14+ dDCs co-express CD11b and CX3CR1. Interestingly, human cDC1s and cDC2s are both capable in inducing Th2 responses . Human pDCs express CD304, CD303 and CD123 and like their murine counterparts are only found in inflamed skin. Remarkably, a recent study showed that human peripheral blood pDCs upregulate the expression of B cell maturation antigens upon activation via TLR9 signaling, suggesting that pDCs play a role in the maintenance of plasma cells, in addition to their inflammatory functions .
Macrophages are found in the dermal layer of the skin and require IL-34 for survival [86,150]. Two sources of dermal macrophages have been identified so far. The first source is embryo-derived progenitors that seed the skin prenatally and are self-renewing in a similar fashion to LCs . The second and major source of dermal macrophages is circulating monocytes (monocyte-derived macrophages) that mature once they reach the skin. This population replenishes roughly every 10 days [151,152]. Monocytes that give rise to dermal macrophages express lymphocyte antigen 6C (Ly6C), and home to the skin in a CCR2-dependent manner . As monocytes mature into skin-resident macrophages, the expression of CCR2 is downregulated . CD64 expression is prominent on dermal macrophages and is used as a marker to differentiate them from the dDCs [152,153]. CD36, DC-SIGN, and IL-10 are highly expressed by macrophages isolated from healthy skin, suggesting that they adapt an immunoregulatory phenotype [153,154]. In steady state, macrophages remove cellular debris [129,153], and have also been implicated in homeostatic hair regeneration [54,155]. Macrophages can be localized at post-capillary venules in the skin and secrete chemokines that drive the recruitment of neutrophils . However, the depletion of macrophages does not translate into a reduction of neutrophils in the skin in wounds [152,157], indicating that dermal macrophages are dispensable for neutrophil infiltration.
Macrophages are plastic, and one way to categorize their effector functions is as pro-inflammatory “M1” or anti-inflammatory/pro-repair “M2.” It is important to note that bona fide M1 and M2 macrophages are generated only in vitro, and in vivo macrophages may simultaneously express M1 and M2 markers. Although investigators are beginning to shift away from the M1/M2 macrophage paradigm , we will use this nomenclature to describe and categorize the functions of macrophages as pro-inflammatory or anti-inflammatory/pro-repair. M1 macrophages express inducible nitric oxide synthase (iNOS), and secrete inflammatory cytokines such as TNFα, IL-1β, and IL-6 [159,160]. M2, or “alternatively activated” macrophages, adopt an anti-inflammatory and/or pro-repair phenotype. M2 macrophages can be further subdivided into M2a, M2b, M2c, and M2d. M2a are known to be pro-fibrotic due to TGFβ production . M2b express the co-stimulatory molecule CD86 and as such they are the most pro-inflammatory of the M2 subsets. M2c macrophages are induced by IL-10 or TGFβ, express the Mer tyrosine kinase (MerTK) and promote neovascularization , and have high scavenging and debris clearing activity. M2d cells are responsive to IL-6 and exhibit a few of the properties of tumor-associated macrophages such as secretion of IL-12 and IL-10 [162,163,164,165]. M2d cells are also known to express high levels of the adenosine receptor A2AR in the presence of LPS . A2AR signaling has been shown to attenuate pro-inflammatory cytokine production , suggesting that A2AR-expressing M2d cells may be involved in the resolution of the inflammatory response.
Mast cells are commonly found in the dermal layer. Mast cells enter the skin from the bone marrow as progenitors and mature locally in response to environmental cues . While the exact progenitor cell that gives rise to mast cells has been debated, common markers for committed mast cells progenitor cells in the blood are recognized: Lin–c-Kit+ST2+CD34+CD16/32hiintegrinβ7hi . Skin mast cell maturation requires stem cell factor, derived from keratinocytes, while Th2-related cytokines such as IL-3, IL-4, IL-9 and IL-10 induce and promote the proliferation of mast cells [170,171,172]. Mature mast cells are FcεRI+c-Kit+CD16/32intintegrinβ7lo [173,174]. Intriguingly, the skin’s microbiome regulates the population of mast cells in the skin. Germ-free mice contain fewer mature mast cells in the dermis, and intradermal injection of these mice with staphylococcal-derived lipoteichoic acid induced the expression of stem cell factor in keratinocytes, ultimately resulting in the rescue of the dermal mast cell population .
In humans, mast cells are found in all areas of the skin but are most numerous in the arms and the legs . The density of mast cells in the papillary dermis increases with age and they are most often localized in the proximity of PGP9.5+ nerve fibers expressing vasoactive intestinal peptide (VIP), which was shown to suppress mast cell degranulation . This has been associated with the reduction of the amount of extracellular matrix remodeling in the skin observed during the later stages of life.
Mast cells contain granules containing preformed mediators such as histamine, sulfated proteoglycans, serotonin, and tryptase and/or chymase. In both humans and mice, mast cells resident in the skin express both tryptase and chymase, whereas other tissue-resident mast cells express only tryptase .
Mast cells are classically known for their involvement in allergic reactions as they produce and release copious amounts of histamine when their Fcε receptors are crosslinked via IgE-antigen complexes [179,180]. They also make large amounts of prostaglandin D2 (PGD2), a lipid-derived inflammatory mediator. The role of PGD2 is nebulous because it has been shown to exert both pro-inflammatory and anti-inflammatory roles. PGD2 is well known to mediate inflammatory responses in asthma [181,182]. However, PGD2 has also been shown to exert anti-inflammatory functions in a murine acute lung injury model as mice that cannot produce PGD2 (H-PDGS−/−) exhibited heightened vascular permeability and mRNA levels of pro-inflammatory cytokines in comparison to WT control mice .
Mast cells are mass producers of leukotrienes (LTs), LTB4, LTC4, LTD4, and LTE4, with the latter three consisting the cysteinyl leukotrienes (cysLTs). LTs are short-lived lipid inflammatory mediators synthesized via the 5-lipoxygenase (5-LO) pathway. While a multitude of immune cell types express the machinery to produce some of the LTs, mast cells and eosinophils produce the complete battery of LTs. LTB4 has been shown to be effective in attracting neutrophils to sites of inflammation and cell death  and is a potent inducer of mast cell degranulation . However, excess LTB4 amounts in the skin can lead to ineffective defense against Staphylococcus aureus infections in diabetic mice . CysLTs are classically involved in allergic reactions such as rhinitis  and have been shown to activate mast cells in an autocrine manner to induce expression of PGD2, mast cell protease-1 (MCP-1), and histamine in mice and humans afflicted with aspirin-exacerbated respiratory disease .
A variety of cytokines and growth factors are produced by mast cells either constitutively or in response to a stimulus . Many of these cytokines and growth factors such as TNFα and vascular endothelial growth factor (VEGF) may be preformed and packaged in mature mast cell granules [189,190,191]. Proper formation of mast cell granules is mediated mostly by proteoglycan serglycin .
Mast cell-derived IL-1β induces production of histamine and IL-8 in human mast cells, suggesting that IL-1β is part of a positive feedback loop for mast cell activation [193,194]. TNFα produced by mast cells is known to drive migration of dermal dendritic cells to draining lymph nodes in a murine model of hapten-induced contact hypersensitivity . Interestingly, mast cell-derived TNFα is also crucial for maintenance of tolerance toward allogeneic skin grafts in mice . Furthermore, recent studies revealed that mast cells are able to form immunological synapses with γδ T lymphocytes when challenged with dengue virus  and can acquire MHCII expression via vesicle transfer from dendritic cells after administration with dinitrofluorobenzene . These studies suggest that dermal mast cells can prime adaptive responses during cutaneous infections.
Eosinophils are skin-resident cells [199,200], but not much is known about their role in tissue homeostasis. Eosinophilic granules are loaded with potent and toxic proteins: major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN), and eosinophil cationic protein (ECP) . Human and murine eosinophilic granules also contain a variety of preformed cytokines and chemokines released in response to appropriate stimuli [201,202,203]. The release of their granular contents is mediated via a process known as piecemeal degranulation [204,205]. This process denotes degranulation in small portions so as to not jeopardize the viability of the cell. Interestingly, cytokines can be chaperoned to secretory vesicles containing their cognate receptors, thus allowing their rapid release from eosinophils . Like mast cells, eosinophils produce all types of LTs and PGD2 , with the latter being crucial for eosinophilic infiltration of the skin in hypersensitivity reactions such as atopic dermatitis . In addition, eosinophils generate extracellular DNA traps (EET) containing eosinophil granules [208,209]. These traps are believed to play a role in antibacterial defense.
Eosinophils are classically known to promote host defense against parasitic infections . Interestingly, eosinophil-lineage deficient mice exhibit no difference in parasitic burden after Schistosoma mansoni infection . This seems to be site-specific and eosinophils seem to not play a role in helminth clearance in intestinal sites . However, eosinophils were shown to be necessary in intestinal clearance of adult Heligmosomoides polygyrus . Thus, there are still gaps in our understanding of eosinophil-specific responses towards parasitic infections.
The roles of eosinophils in dermatoses, or skin diseases associated with eosinophilia such as allergic contact dermatitis and urticaria, are well elucidated [61,214,215,216]. In eosinophilic dermatoses, extensive eosinophilic degranulation in the skin results in local tissue damage.
Eosinophilic pustular folliculitis is an uncommon dermatosis hallmarked by the presence of pruritic follicular papules and pustules associated with folliculotropic infiltration by eosinophils . Skin lesions are most commonly found on the facial area and would typically last for one to two weeks . The recruitment of eosinophils by PGD2  in eosinophilic disorders occurs by two proposed mechanisms. First, by direct binding of PGD2 onto its cognate receptors on eosinophils, thus inducing chemotaxis. Second, by inducing sebocytes to produce eotaxin-3 , which then recruits eosinophils.
Eosinophilic cellulitis, also known as Wells’ Syndrome, is a rare disorder characterized by multiple large, circular, and usually painful or pruritic edematous erythema . While the cause of this disorder is unclear, it has been well documented that the skin of patients with Wells’ Syndrome is infiltrated by eosinophils along with aggregates of complexes comprised of collagen associated with eosinophilic granular contents [220,221].
3.4.2. Lymphoid Immune Cells
The skin harbors different types of lymphoid cells (Table 1), all of which are important in both steady state and inflammatory responses. Both human and murine skin contain γδ T lymphocytes and αβ T lymphocytes, along with natural killer T cells. γδ T cells are the dominant T cell population in murine skin, while αβ T cells are the dominant T cell population in human skin [222,223].
αβ T Lymphocytes
In both mice and humans, αβ T lymphocytes are found in the epidermis and dermis , and traffic to the skin from the periphery via cutaneous lymphocyte antigen (CLA) interactions with E-selectin (expressed on endothelial cells), which is upregulated under inflammatory conditions [225,226]. αβ T lymphocytes in the skin are resident memory T cells (TRM), which are long-lived and distinct from their circulating counterparts .
Most TRM in the skin are derived from antigen-specific effector T cells, which previously infiltrated the tissue as a result of an infection. After resolution, these TRM cells seed all areas of the skin but are denser in areas of antecedent infection . Following a skin infection, TRM cells are also found in distal organs such as the lung and gastrointestinal tract .
TRM express lower levels of CD28 than effector memory T cells, but can mount robust local recall responses  without emigrating from the tissue to do so . Interestingly, TRM also exert sentinel-like functions by promoting recruitment of other memory T cells from the periphery to sites of infection [230,231].
The most studied skin TRM are CD8+ T cells. Skin CD8+ TRM were shown to be effective against HSV infections by inducing an IFNγ-mediated antiviral state in the tissue . Additionally, CD8+ TRM cells were recently shown by the Gebhardt group to exert antitumor functions in a murine transplantable melanoma model . CD8+ TRM cells are usually found in the epidermis and may dislocate dendritic epidermal T cells . All CD8+ TRM express CD69, and a high proportion also express CD103 . CD103 is required for development of CD8+ TRM cells in the skin  and mediates adhesion interactions with keratinocytes via an E-cadherin-independent manner .
CD4+ TRM cells also make up a significant portion of the skin-resident lymphocyte population and are found in both the epidermis and dermis . Although the function of CD4+ T cells in the skin is not studied to the same extent as CD8+ TRM, they are often the dominant αβ T cell subset. During steady state, clusters of antigen-presenting cells with CD4+ memory T cells are found around the hair follicles in both murine and human skin, and cells in these clusters circulate between the skin and periphery . These clusters are formed because keratinocytes in the hair follicles produce IL-7 and IL-15, which are required for homeostatic maintenance of the T cell populations .
It is also well established that a proportion of skin CD4+ T cells are T regulatory cells (Tregs). Crosstalk between LCs and Tregs has been shown to play a role in dampening immune responses in contact hypersensitivities . Tregs dysregulation leads to disorders such as pemphigus vulgaris, alopecia areata, and systemic sclerosis [237,238].
Skin-infiltrating and -resident T cells play a role in the pathophysiology of various conditions such as psoriasis, alopecia areata, and vitiligo, in which both CD4+ and CD8+ T cells are involved [239,240,241,242]. In psoriasis, infiltrating T lymphocytes produce IL-17, which is a central part of the disease’s pathophysiology. The hair follicle bulbs and melanocytes are attacked by infiltrating cytotoxic CD8+ T cells in alopecia areata and vitiligo, respectively. CD4+ TRM cells can promote the migration of dDCs from the skin to draining lymph nodes in the context of autoimmune reactions .
Non-Conventional T Cells
The mechanisms by which T lymphocytes recognize peptide antigens complexed with the MHC macromolecules are well delineated. However, T cells can also recognize free soluble antigens and non-peptide antigens complexed with non-classical MHC-like molecules, and these T cells are termed non-conventional. Here we will review the types of non-conventional T cells found in the skin:
γδ T Lymphocytes:
Unlike the αβ T lymphocytes, γδ T cells do not undergo the same stringent negative selection process during development and are released from the thymus in waves, with the first wave of γδ T cells seeding the dermis of the skin [244,245,246].
Most γδ T cells in murine skin are found in the epidermis within the junctions between keratinocytes, and are known as dendritic epidermal T cells (DETCs). Lymphocytes with DETC-like characteristics have yet to be discovered in humans [222,223]. It was recently shown that DETCs are derived from yolk sac progenitor cells and are self-renewing in the epidermis in a similar manner to LCs . DETCs express a canonical γδ T cell receptor and sample antigens via their dendrites, which can extend from their mid cell body located in the apical epidermis to the border with the stratum corneum, maintaining contact with local keratinocytes . DETCs require keratinocyte-derived IL-7 for survival , and IL-15 is also important as DETCs were absent in IL-15−/− mice . On the other hand, DETC-derived IGF-1 and keratinocyte growth factor (KGF) promote keratinocyte proliferation in steady state . It is speculated that DETC-derived homeostatic factors are packaged in granules and transported via the DETCs’ dendrites .
Once activated, DETCs retract their dendrites, adopt a round morphology, and secrete a range of cytokines and growth factors. IL-17-producing DETCs are essential for inducing the expression of β-defensins in the epidermis, thus playing a critical role in antimicrobial defense of the skin . Interestingly, DETCs were shown to migrate to cutaneous draining lymph nodes in a CCR7-dependent process after sensing stressed keratinocytes in an inducible Notch1 knockout mouse model, thus likely playing a role in antitumor immunity .
γδ T cells found in the murine dermis express a Vγ4 chain and thus are distinct from their DETC counterparts that express the Vγ5 chain . Additionally, dermal γδ T lymphocytes require IL-7, but not IL-15, for population maintenance , and are highly motile in comparison to DETCs that remain in close contact with keratinocytes during tissue homeostasis . The functions of dermal γδ T cells in inflammatory skin conditions are well documented. This population of dermal T cells is inclined to produce IL-17 , indicating their significance in cutaneous diseases such as psoriasis [257,258].
γδ T cells in the skin can also play a protective role in cutaneous defense, as a previous study showed that γδ T cell-deficient mice exhibited larger lesions and reduced IL-17 production in response to Staphylococcus aureus compared to WT control mice .
γδ T cells including DETCs are not MHC-restricted  and can recognize soluble antigens [259,260], antigens derived from damaged or stressed cells [261,262,263], or antigens complexed with non-classical MHC molecules such as CD1b, CD1c, and CD1d [264,265] or MHCI-related chain A/B (MICA/MICB) . A recent study suggests that γδ T cells also recognize Btnl proteins, which are part of the B7 superfamily .
Other CD1-Restricted Cells
CD1 molecules are essential for presenting lipids antigens to T cells and are categorized under group I (CD1a, CD1b, CD1c) or group II (CD1d and CD1e) [268,269]. In addition to γδ T cells, some αβ T cells and natural killer T cells are CD1 restricted.
Humans contain both group I and group II CD1-restricted T cells, while mice only contain group II CD1-restricted T cells. LCs express CD1a, which was recently shown to regulate Th17-mediated inflammatory responses in the skin . Autoreactive T lymphocytes specific to squalene, wax esters, and triglycerides home to the skin, and these lipids are presented by CD1a . CD1b molecules present a diverse array of mycobacterial lipids such as mycolic acid, glucose monomycolate, and phosphatidyl mannosides [272,273,274,275]. CD1c molecules have been shown to present self-lipid antigens such as methyl-lipophosphotidic acid, which is known to aggregate in leukemic cells . A previous study showed that mice containing autoreactive CD1c-restricted T cells were efficient in eliminating human leukemic cells, suggesting that these autoreactive CD1c-restricted T cells play a role in tumor immunity . Unlike the TRM lymphocytes, CD1-restricted T cells have not been documented to be present in healthy skin, but are readily found in the context of inflammatory conditions. Both CD1b and CD1d have been documented in human cutaneous Borrelia burgdorferi infections . Group I CD1-restricted T lymphocytes elicit IgE responses when presented with phospholipid antigens derived from cyprus pollens, suggesting that they contribute to pollen hypersensitivities . CD1a expressed on LCs is central on skin inflammatory conditions such as atopic eczema [270,279] and psoriasis . Furthermore, group I CD1-restricted T cells have been implicated to play a role in controlling the microbiota inhabiting the skin. Group I CD1-restricted T cells have also been well documented in the defense against Mycobacterium tuberculosis [281,282].
Group II CD1d-restricted T lymphocytes have been more extensively studied in mice. CD1d is expressed by keratinocytes and dermal dendritic cells . Most group II CD1d-restricted T cells are also known as invariant natural killer T cells (iNKT). iNKT cells are involved in hypersensitivity reactions as they produce IL-4 in response to haptens in the skin, leading to sensitized iNKT cell accumulation . Similar to the group I CD1-restricted T cells, iNKT cells also exhibit antitumor activity . Interestingly, it has also been suggested that iNKT cells play a role in suppressing autoimmune responses in the skin, as a deficiency of CD1d resulted in increased numbers of skin lesions in a MRL-lpr/lpr murine model of systemic lupus erythematosus . Furthermore, small non-lipid synthetic molecules can activate T cells via CD1d presentation . CD1e molecules are expressed in the Golgi compartments of immature DCs and are later localized in the lysosomes of mature DCs . CD1e is known to be crucial for the antigen processing of bacterial glycolipids to CD1b-restricted T cells . However, not much is known about the roles of CD1e in cutaneous inflammation.
B cells are rather sparse in the skin in steady state and it is unclear whether they are indeed resident to the skin [290,291,292]. However, the roles of B lymphocytes in skin inflammatory conditions are well documented. In humans, B cells are found in elevated levels in cutaneous diseases such as atopic eczema, cutaneous leishmaniasis, and cutaneous sclerosis [293,294,295]. B cells are found in the reticular dermis over the course of these diseases and are associated with increased levels of IgM, IgE, and IgG. In a similar manner to T lymphocytes, B lymphocytes traffic to the skin tissue via CLA . It has been suggested that the CCL20-CCR6 axis is important in addition to CLA for B cell homing from lymph to the skin [291,297]. A population of B-1-like cells is found in inflamed skin in both humans and mice and migrates from the peritoneum via integrin α4β1 . B cells also play a role in delayed-type hypersensitivity reactions in the skin. Peripheral B-1 cells produce allergen-specific IgM antibodies as soon as day one post-induction with either ovalbumin or keyhole limpet hemocyanin. This leads to the formation of immune complexes between the IgM antibodies and allergens and activation of the complement cascade mechanisms, ultimately resulting in recruitment of T cells to the affected skin site [299,300]. Autoimmune bullous diseases are characterized by the presence of autoantibodies that are reactive to structural proteins of the epidermis . Pemphigus vulgaris is such an example in which desmoglein-3 autoantibodies lead to the formation of blisters in the skin . Intriguingly, a population of regulatory B cells (Bregs) have been suggested to play suppressive roles in certain cutaneous inflammatory conditions. Bregs exert their immunosuppressive functions through the production of large amounts of the regulatory cytokine IL-10 . In an imiquimod-induced murine psoriasis model, mice that lacked most B cells (CD19−/−) exhibited more severe symptoms when compared to their WT counterparts. Adoptive transfer of WT B cells to the CD19−/− mice ameliorated psoriasis symptoms, suggesting that a population within these B cells play a therapeutic role in alleviating the disorder .
3.4.3. Non-Immune Cells
Pattern recognition receptors (PRR) are expressed by most cells of the skin and have been characterized on keratinocytes, fibroblasts, adipocytes, melanocytes, and endothelial cells . Activation of these receptors results in the production of cytokines and chemokines by non-immune skin cells, thus participating in the local immune response [306,307,308,309].
Keratinocyte-derived inflammatory responses have been extensively studied. These cells express almost all intracellular and extracellular PRRs and produce a variety of cytokines, chemokine and AMPs to protect the host against infection [78,79,80]. As mentioned earlier, keratinocytes both in the epidermis and skin elements are in constant interaction with local immune cells and produce factors crucial in homeostasis and in tissue repair [85,235,248,249]. Other studies demonstrated that keratinocytes produce IL-33 in response to hypoosmotic stress , and that human and murine keratinocytes produce IL-6 and IL-1β mediated by NFκB signaling in response to UVB irradiation . Evidence has also shown that keratinocytes can produce inflammatory cytokines and chemokines mediated by STAT3 signaling in the presence of IL-25 in a murine psoriasis model, suggesting that keratinocytes play a crucial role in the pathophysiology of the disease . Additionally, keratinocytes have mechanisms in place to control overactivation of inflammatory responses .
Fibroblasts immunomodulatory functions have also been well delineated. They express PRRs, synthesize many cytokines, and were shown to produce AMPs . Dermal fibroblasts and keratinocytes produce serum amyloid A in response to PRR signaling , which is believed to induce the production of pro-inflammatory cytokines from various immune cells . A recent investigation showed that human fibroblasts cultured in vitro produced massive amounts of TNFα, IL-1β, IL-6, IL-8, and IL-25 when subjected to thermal stress .
More information: “Neutrophil recruitment to noninvasive MRSA at the stratum corneum of human skin mediates transient colonization”. Anette Schulz, Long Jiang, Lisanne de Vor, Marcus Ehrström, Fredrik Wermeling, Liv Eidsmo and Keira Melican. Cell Reports, online 29 October 2019, DOI: 10.1016/j.celrep.2019.09.055
Journal information: Cell Reports
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