Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Effect of topical application of lipopolysaccharide on contact hypersensitivity.

Lipopolysaccharide (LPS) is the principal component of the outer membrane of gram-negative bacteria. The prior oral administration of LPS attenuates inflammatory responses, such as intestinal injury and atopic dermatitis, in mouse models; however, the underlying mechanism remains unclear. Here, we examined the effect of topical LPS application on allergic contact dermatitis and its mechanism of action using a murine contact hypersensitivity (CHS) model. Prolonged LPS application to the skin significantly suppressed 2,4-dinitrofluorobenzene (DNFB)-induced CHS. LPS application to the skin also reduced the phagocytosis of fluorescein isothiocyanate (FITC)-dextran by Langerhans and dendritic cells. Cutaneous cell migration into the skin-draining lymph nodes (LNs) induced by FITC painting was reduced by LPS application. During the CHS response, DNFB application induced T-cell proliferation and inflammatory cytokine production in skin-draining LNs, whereas prolonged LPS application inhibited DNFB-induced T-cell growth and interferon gamma production, indicating suppression of DNFB-induced sensitization. These results suggest that prolonged LPS application suppressed DNFB-induced sensitization and subsequently CHS response. Our findings imply that topical application of LPS may prevent allergic dermatitis such as CHS.

Differential Involvement of Programmed Cell Death Ligands in Skin Immune Responses.

PD-1 is an immunoregulatory receptor that can bind PD-L1 or PD-L2 expressed on stimulated antigen-presenting cells. In this study, isolated antigen-presenting cells (macrophages and dendritic cells) were cultured with IFN-γ, IL-4, or IL-17A, and the expression of PD-L1 and PD-L2 was compared by flow cytometry. Strong upregulation of PD-L1 expression was observed on IFN-γ stimulation of both antigen-presenting cells as well as in response to IL-17A stimulation of macrophages compared with the expression in unstimulated controls. In contrast, only stimulation with IL-4 could upregulate PD-L2 expression on both antigen-presenting cells. Therefore, experiments were performed in murine models, including DNFB-induced contact hypersensitivity, calcipotriol-induced atopic dermatitis-like skin inflammation, and imiquimod-induced psoriasis-like dermatitis models, to trigger IFN-γ‒mediated T helper type (Th)1-, IL-4‒mediated Th2-, and IL-17A‒mediated Th17-type responses, respectively. In both Th1- and Th17-type immunity models, changes in ear thickness were more severe in Pd-l1‒deficient mice than in wild-type or Pd-l2‒deficient mice. In the Th2-type immunity model, changes in thickness in Pd-l2‒deficient mice were more severe than that in wild-type or Pd-l1‒deficient mice. Collectively, PD-L1 has predominant roles in Th1 and Th17 type immunity, whereas PD-L2 is involved in Th2-type immunity.

Entinostat, a histone deacetylase inhibitor, increases the population of IL-10+ regulatory B cells to suppress contact hypersensitivity.

IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].

The Loss of H3K27 Histone Demethylase Utx in T Cells Aggravates Allergic Contact Dermatitis.

The pathogenesis of allergic contact dermatitis (ACD) requires the activation of Ag-specific T cells, including effector and regulatory T cells. The differentiation and function of these T cells is epigenetically regulated through DNA methylation and histone modifications. However, the roles of altered histone H3K27 methylation in T cells in the development of ACD remain unknown. Two types of histone H3K27 demethylases, Utx and Jmjd3, have been reported in mammals. To determine the role of the histone H3K27 demethylase expression of T cells in the development of ACD, we generated T cell-specific, Utx-deficient (Utx KO) mice or Jmjd3-deficient (Jmjd3 KO) mice. Unlike control mice, Utx KO mice had severer symptoms of ACD, whereas Jmjd3 KO mice showed symptoms identical to those in control mice. In Utx KO mice with ACD, the massive infiltration of myeloid cells, including neutrophils and dendritic cells, has been observed. In addition, the expression of proinflammatory cytokines in CD4+ T cells of the draining lymph nodes (LNs) and in CD8+ T cells of the skin was increased in Utx KO mice, whereas the ratio of Foxp3+ regulatory CD4+ T cells to Foxp3- conventional CD4+ T cells was decreased in both the draining LNs and the skin of Utx KO mice with ACD. Furthermore, Foxp3+ regulatory CD4+ T cells of Utx KO mice with ACD expressed a decreased level of CCR4 (a skin-tropic chemokine receptor) in comparison with control. Thus, in CD4+ T cells, Utx could potentially be involved in the regulation of the pathogenesis of ACD.

Putative Immunological Functions of Inducible Skin-Associated Lymphoid Tissue in the Context of Mucosa-Associated Lymphoid Tissue.

Acquired immunity is orchestrated in various lymphoid organs, including bone marrow, thymus, spleen, and lymph nodes in humans. However, mucosa-associated lymphoid tissue (MALT) is evolutionally known to be emerged in the oldest vertebrates as an immunological tissue for acquired immunity, much earlier than the advent of lymph nodes which appeared in endotherms. Furthermore, the lymphocytes which developed in MALT are known to circulate within the limited anatomical areas. Thus, MALT is comprehended as not the structure but the immune network dedicated to local immunity. As for the skin, skin-associated lymphoid tissue (SALT) was previously postulated; however, its existence has not been proven. Our group recently showed that aggregations of dendritic cells, M2 macrophages, and high endothelial venules (HEVs) are essential components to activate effector T cells in the murine contact hypersensitivity model and termed it as inducible SALT (iSALT) since it was a transient entity that serves for acquired immunity of the skin. Furthermore, in various human skin diseases, we reported that the ectopic formation of lymphoid follicles that immunohistochemically analogous to MALT and regarded them as human counterparts of iSALT. These data raised the possibility that SALT can exist as an inducible form, namely iSALT, which shares the biological significance of MALT. In this article, we revisit the evolution of immunological organs and the related components among vertebrates to discuss the conserved functions of MALT. Furthermore, we also discuss the putative characteristics and functions of iSALT in the context of the MALT concept.

Drug and Chemical Allergy: A Role for a Specific Naive T-Cell Repertoire?

Allergic reactions to drugs and chemicals are mediated by an adaptive immune response involving specific T cells. During thymic selection, T cells that have not yet encountered their cognate antigen are considered naive T cells. Due to the artificial nature of drug/chemical-T-cell epitopes, it is not clear whether thymic selection of drug/chemical-specific T cells is a common phenomenon or remains limited to few donors or simply does not exist, suggesting T-cell receptor (TCR) cross-reactivity with other antigens. Selection of drug/chemical-specific T cells could be a relatively rare event accounting for the low occurrence of drug allergy. On the other hand, a large T-cell repertoire found in multiple donors would underline the potential of a drug/chemical to be recognized by many donors. Recent observations raise the hypothesis that not only the drug/chemical, but also parts of the haptenated protein or peptides may constitute the important structural determinants for antigen recognition by the TCR. These observations may also suggest that in the case of drug/chemical allergy, the T-cell repertoire results from particular properties of certain TCR to recognize hapten-modified peptides without need for previous thymic selection. The aim of this review is to address the existence and the role of a naive T-cell repertoire in drug and chemical allergy. Understanding this role has the potential to reveal efficient strategies not only for allergy diagnosis but also for prediction of the immunogenic potential of new chemicals.

Keratinocytes Regulate the Threshold of Inflammation by Inhibiting T Cell Effector Functions.

Whilst the importance of keratinocytes as a first-line defense has been widely investigated, little is known about their interactions with non-resident immune cells. In this study, the impact of human keratinocytes on T cell effector functions was analyzed in an antigen-specific in vitro model of allergic contact dermatitis (ACD) to nickel sulfate. Keratinocytes partially inhibited T cell proliferation and cytokine production. This effect was dependent on the keratinocyte/T cell ratio and was partially reversible by increasing the number of autologous dendritic cells. The inhibition of T cell proliferation by keratinocytes was independent of the T cell subtype and antigen presentation by different professional antigen-presenting cells. Autologous and heterologous keratinocytes showed comparable effects, while the fixation of keratinocytes with paraformaldehyde abrogated the immunosuppressive effect. The separation of keratinocytes and T cells by a transwell chamber, as well as a cell-free keratinocyte supernatant, inhibited T cell effector functions to the same amount as directly co-cultured keratinocytes, thus proving that soluble factor/s account for the observed suppressive effects. In conclusion, keratinocytes critically control the threshold of inflammatory processes in the skin by inhibiting T cell proliferation and cytokine production.

Pituitary adenylate cyclase-activating polypeptide promotes cutaneous dendritic cell functions in contact hypersensitivity.

BACKGROUND: Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified. OBJECTIVE: This study aimed to explore the effect of peripheral nerves on cutaneous immune cells in cutaneous acquired immune responses. METHODS: We analyzed contact hypersensitivity (CHS) responses as a murine model of delayed-type hypersensitivity in absence or presence of resiniferatoxin-induced sensory nerve denervation. We conducted ear thickness measurements, flow cytometric analyses, and mRNA expression analyses in CHS. RESULTS: CHS responses were attenuated in mice that were denervated during the sensitization phase of CHS. By screening neuropeptides, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA expression was decreased in the dorsal root ganglia after denervation. Administration of PACAP restored attenuated CHS response in resiniferatoxin-treated mice, and pharmacological inhibition of PACAP suppressed CHS. Flow cytometric analysis of skin-draining lymph nodes showed that cutaneous dendritic cell migration and maturation were reduced in both denervated mice and PACAP antagonist-treated mice. The expression of chemokine receptors CCR7 and CXCR4 of dendritic cell s was enhanced by addition of PACAP in vitro. CONCLUSION: These findings indicate that a neuropeptide PACAP promotes the development of CHS responses by inducing cutaneous dendritic cell functions during the sensitization phase.

PD-L1 on mast cells suppresses effector CD8+ T-cell activation in the skin in murine contact hypersensitivity.

BACKGROUND: The programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1) pathway is known to inhibit the activation of effector CD8+ T cells. However, just how this regulatory pathway is involved in the pathophysiology of CD8+ T-cell-mediated inflammatory skin diseases remains unclear. OBJECTIVE: Our aim was to elucidate the mechanisms by which the PD-1/PD-L1 pathway exerts its regulatory roles in CD8+ T-cell-mediated cutaneous immune responses. METHODS: PD-L1-deficient (Pdl1-/-) mice were used for the murine contact hypersensitivity model. Inflammatory responses such as IFN-γ production from CD8+ T cells in the skin was evaluated by flow cytometry. RESULTS: Compared with wild-type mice, Pdl1-/- mice exhibited exacerbated ear swelling and increased numbers of IFN-γ+ CD8+ T cells in the skin. Adoptive T-cell transfer experiments revealed the involvement of the PD-1/PD-L1 pathway in the elicitation phase of contact hypersensitivity. Bone marrow chimera experiments showed that PD-L1 on radioresistant cells was responsible for this regulatory pathway. Flow cytometric analysis revealed that among the radioresistant cells in the skin, PD-L1 was most highly expressed on mast cells (MCs) before and after elicitation. Administration of anti-PD-L1 blocking antibody during the elicitation phase significantly enhanced ear swelling responses and increased the number of IFN-γ+CD8+ T cells in the skin of wild-type mice, whereas no significant effects were observed in MC-deficient (WBB6F1/J-KitW/KitW-v/J and C57BL/6-KitW-sh/W-sh) mice. The high level of expression of PD-L1 on human skin MCs was confirmed by database analysis and immunohistochemical analysis. CONCLUSION: PD-L1 on MCs negatively regulates CD8+ T-cell activation in the skin.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

Skin immune cell characterization in juvenile and adult Göttingen Minipigs.

The skin hosts a sophisticated immune system involving responses from both innate and adaptive immune cell populations. Swine skin is close to human skin by its structure, composition and function. In addition, the minipig is considered the model of choice in toxicology studies for drugs applied by the dermal route and developed for both the adult and paediatric indications. However, knowledge on the skin immune system in minipigs, particularly in Göttingen Minipigs, is still limited. The objective of our work was first to characterize the main skin immune populations (Langerhans cells, dermal dendritic cells, macrophages and T lymphocytes) in Göttingen Minipigs. In parallel, we compared the skin immune populations from healthy and immunocompromised piglets following oral treatment with cyclosporin A (CsA) at 10 mg/kg/day. We also explored other pathological conditions using a contact dermatitis model in minipigs challenged with a sensitizer, 2,4-dinitrochlorobenzene (DNCB). Langerhans cells and dermal MHCIIlowCD163+ cells were increased one month after oral treatment with CsA at 10 mg/kg/day. The contact dermatitis model in Göttingen Minipigs challenged by DNCB suggested migration of Langerhans cells and dermal dendritic cells as well as T cell recruitment into the skin. These data bring new information in skin immunotoxicology in Göttingen Minipigs and could contribute to a better understanding of the effects of new therapeutic drugs on the developing immune system.

Directional mast cell degranulation of tumor necrosis factor into blood vessels primes neutrophil extravasation.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Perinatal treatment of parents with the broad-spectrum antibiotic enrofloxacin aggravates contact sensitivity in adult offspring mice.

BACKGROUND: Antibiotics, while eliminating pathogens, also partially deplete commensal bacteria. Antibiotic-induced dysbiosis may contribute to the observed rise in "immune-mediated" diseases, including autoimmunity and allergy. The aim of this study is to investigate the impact of perinatal antibiotic treatment on T cell-mediated immune response in adult mice. METHODS: Oral treatment with broad-spectrum antibiotic enrofloxacin during gestation and breastfeeding or breastfeeding or gestation alone was used to evaluate whether antibiotic exposure early in life could modulate contact sensitivity (CS) in adult mice. RESULTS: Here, we demonstrated that enrofloxacin treatment during gestation and breastfeeding, but not during pregnancy or breastfeeding alone, aggravated CS reaction in adult mice measured by ear swelling. These data correlate with increased myeloperoxidase (MPO) activity in the ear extracts and elevated production of IL-6 and IL-17A by auricular lymph node cells (ELNC) and was not influenced by food consumption and body weight. In each dosing regimen, enrofloxacin treatment reduced the relative abundance of Enterococcus spp. but did not influence the relative abundances of Lactobacillus, Clostridium cluster XIVa, XIVab, I, Bacteroidetes, and segmented filamentous bacteria (SFB). However, prolonged enrofloxacin-treatment during both gestation and breastfeeding decreased the relative abundance of Clostridium cluster IV. CONCLUSION: These data show that long-term perinatal enrofloxacin treatment induces intestinal dysbiosis, characterized by decreased levels of anti-inflammatory Clostridium cluster IV, and alters T cell-dependent immune responses, enhancing CS reaction in adult mice.

CD11c+ dendritic cells mediate antigen-specific suppression in extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) represents one of the most widespread and effective cell therapies for graft-versus-host disease and other T cell-mediated disorders. However, the key factors affecting the therapeutic efficacy of ECP remain unclear. We hypothesized that therapeutic effects are mediated by ECP-treated antigen-presenting dendritic cells (DC). To test this hypothesis, we used the experimental model of contact hypersensitivity (CHS). The ECP's therapeutic activity improved when the total cell dose of the ECP-treated cells was increased. We used different haptens during sensitization to demonstrate that the anti-inflammatory activity of ECP is antigen-specific. This confirmed the hypothesis that professional antigen-presenting cells are involved in the mode of action. Also, the ECP's therapeutic activity was abrogated by the depletion of CD11c+ DC, which represents fewer than 1% of all the ECP-exposed cells. Finally, we confirm the critical importance of CD11c+ DC for ECP activity by showing that only a few purified CD11c+ DC are sufficient to mediate its therapeutic effect. The finding that ECP-treated, physiological antigen-presenting DC alone mediate antigen-specific modulation of a pathological immune response may result in better-targeted interventions when treating patients.

DOCK8 Expression in Regulatory T Cells Maintains their Stability and Limits Contact Hypersensitivity.

Chronic dermatitis is a hallmark of Dedicator of cytokinesis 8 (DOCK8) deficiency. The migration of DOCK8-deficient T cells to the skin and their survival there have been reported to be defective. Surprisingly, we found that Dock8-/- mice demonstrated an exaggerated contact hypersensitivity (CHS) response to oxazolone with increased ear swelling, T-cell infiltration, and expression of Ifng. To understand the mechanisms of persistent skin inflammation in DOCK8 deficiency, we examined mice with selective deficiency of DOCK8 in T cells or T regulatory cells (Tregs) and found that both have exaggerated CHS. Moreover, oral tolerance to oxazolone, mediated by Tregs, was impaired in Dock8-/- mice. Transfer of Tregs from oxazolone-sensitized wild-type mice, but not Dock8-/- mice, reduced the CHS response of Dock8-/- recipients. Lack of DOCK8 in Tregs resulted in their acquisition of a pathogenic FOXP3+T-bet+IFNγ+ phenotype at CHS sites and promoted their conversion into ex-Tregs. The transfer of Tregs from Dock8-/- mice increased the CHS response of wild-type recipients to oxazolone. Thus, DOCK8 expression in Tregs limits CHS by promoting Treg stability and fitness in inflamed skin. Interventions aimed at ameliorating Treg function may be useful in treating skin inflammation in DOCK8 deficiency.

A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses.

Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses and how such functions are regulated at the molecular level. Here, we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that displays increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity in the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared with conventional CD49a-CD16+ NK cells, thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest that adaptive CD49a+CD16- NK cells are able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory toward chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses in the skin. These results define pathways operative in human antigen-specific adaptive NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.

Suprabasin-null mice retain skin barrier function and show high contact hypersensitivity to nickel upon oral nickel loading.

Suprabasin (SBSN) is expressed not only in epidermis but also in epithelial cells of the upper digestive tract where metals such as nickel are absorbed. We have recently shown that SBSN level is decreased in the stratum corneum and serum of atopic dermatitis (AD) patients, especially in intrinsic AD, which is characterized by metal allergy. By using SBSN-null (Sbsn-/-) mice, this study was conducted to investigate the outcome of SBSN deficiency in relation to AD. Sbsn-/- mice exhibited skin barrier dysfunction on embryonic day 16.5, but after birth, their barrier function was not perturbed despite the presence of ultrastructural changes in stratum corneum and keratohyalin granules. Sbsn-/- mice showed a comparable ovalbumin-specific skin immune response to wild type (WT) mice and rather lower contact hypersensitivity (CHS) responses to haptens than did WT mice. The blood nickel level after oral feeding of nickel was significantly higher in Sbsn-/- mice than in WT mice, and CHS to nickel was elevated in Sbsn-/- mice under nickel-loading condition. Our study suggests that the completely SBSN deficient mice retain normal barrier function, but harbor abnormal upper digestive tract epithelium that promotes nickel absorption and high CHS to nickel, sharing the features of intrinsic AD.