Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.

Intravenous immunoglobulins, cyclosporine, and best supportive care in epidermal necrolysis: Diverse effects on systemic inflammation.

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but potentially life-threatening cutaneous adverse reactions. There is still no consensus on adjuvant treatments, and little is known about their effects on systemic inflammation in SJS/TEN. Our aim was to characterize the systemic and cutaneous immune profiles of SJS/TEN patients and to investigate whether/how intravenous immunoglobulins (IVIG), cyclosporine A (CSA), and best supportive care only (BSCO) affected the systemic immune signature and clinical outcome (6 week-mortality, complications, hospitalization stay). METHODS: We included 16 patients with SJS/TEN, treated with high-dose IVIG (n = 8), CSA (n = 4) or BSCO (n = 4). Serial serum samples were obtained prior-, 5-7 days, and 21 days after treatment onset. Serum levels of inflammation-/immune response-associated proteins were measured by high-throughput proteomics assay (OLINK) and cytotoxic molecules by ELISA. RNA extracted from skin biopsies collected prior treatment was analyzed by Nanostring. RESULTS: Serum inflammatory profiles in SJS/TEN patients were notably characterized by massive upregulation of type 1 immune response and proinflammatory markers. Surprisingly, there was limited overlap between cutaneous and serum immune profiles. Serial serological measurements of immune response markers showed very diverse dynamics between the different treatment groups. IVIG-treated patients showed completely different dynamics and most significant proteomic changes in an early phase (Day 5-7). In all treatment groups, type 1-/inflammatory response markers were dampened at day 21. Clinically, there were no outcome differences. CONCLUSION: Our study demonstrates that BSCO, CSA, and IVIG have very diverse biological effects on the systemic inflammatory response in SJS/TEN, which may not correlate with clinical outcome differences.

Phosphodiesterase-4 Inhibition Reduces Cutaneous Inflammation and IL-1ß Expression in a Psoriasiform Mouse Model but Does Not Inhibit Inflammasome Activation.

Apremilast (Otezla®) is an oral small molecule phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis, psoriatic arthritis, and oral ulcers associated with Behçet's disease. While PDE4 inhibition overall is mechanistically understood, the effect of apremilast on the innate immune response, particularly inflammasome activation, remains unknown. Here, we assessed the effect of apremilast in a psoriasis mouse model and primary human cells. Psoriatic lesion development in vivo was studied in K5.Stat3C transgenic mice treated with apremilast for 2 weeks, resulting in a moderate (2 mg/kg/day) to significant (6 mg/kg/day) resolution of inflamed plaques after 2-week treatment. Concomitantly, epidermal thickness dramatically decreased, the cutaneous immune cell infiltrate was reduced, and proinflammatory cytokines were significantly downregulated. Additionally, apremilast significantly inhibited lipopolysaccharide- or anti-CD3-induced expression of proinflammatory cytokines in peripheral mononuclear cells (PBMCs). Notably, inflammasome activation and secretion of IL-1ß were not inhibited by apremilast in PBMCs and in human primary keratinocytes. Collectively, apremilast effectively alleviated the psoriatic phenotype of K5.Stat3 transgenic mice, further substantiating PDE4 inhibitor-efficiency in targeting key clinical, histopathological and inflammatory features of psoriasis. Despite lacking direct effect on inflammasome activation, reduced priming of inflammasome components upon apremilast treatment reflected the indirect benefit of PDE4 inhibition in reducing inflammation.

Microcrystalline Tyrosine and Aluminum as Adjuvants in Allergen-Specific Immunotherapy Protect from IgE-Mediated Reactivity in Mouse Models and Act Independently of Inflammasome and TLR Signaling.

Allergen immunotherapy (AIT) is the only modality that can modify immune responses to allergen exposure, but therapeutic coverage is low. One strategy to improve AIT safety and efficacy is the use of new or improved adjuvants. This study investigates immune responses produced by microcrystalline tyrosine (MCT)-based vaccines as compared with conventional aluminum hydroxide (alum). Wild-type, immune-signaling-deficient, and TCR-transgenic mice were treated with different Ags (e.g., OVA and cat dander Fel d 1), plus MCT or alum as depot adjuvants. Specific Ab responses in serum were measured by ELISA, whereas cytokine secretion was measured both in culture supernatants by ELISA or by flow cytometry of spleen cells. Upon initiation of AIT in allergic mice, body temperature and further clinical signs were used as indicators for anaphylaxis. Overall, MCT and alum induced comparable B and T cell responses, which were independent of TLR signaling. Alum induced stronger IgE and IL-4 secretion than MCT. MCT and alum induced caspase-dependent IL-1ß secretion in human monocytes in vitro, but inflammasome activation had no functional effect on inflammatory and Ab responses measured in vivo. In sensitized mice, AIT with MCT-adjuvanted allergens caused fewer anaphylactic reactions compared with alum-adjuvanted allergens. As depot adjuvants, MCT and alum are comparably effective in strength and mechanism of Ag-specific IgG induction and induction of T cell responses. The biocompatible and biodegradable MCT seems therefore a suitable alternative adjuvant to alum-based vaccines and AIT.

Expression of CD164 on Malignant T cells in Sézary Syndrome.

Sézary syndrome is a primary cutaneous T-cell lymphoma characterized by pruritic erythroderma, peripheral lymphadenopathy and the presence of malignant T cells in the blood. Unequivocal detection of malignant cells in patients with Sézary syndrome is of important diagnostic, prognostic and therapeutic value. However, no single Sézary syndrome specific cell surface marker has been identified. In a cohort of patients with Sézary syndrome, CD164 expression on total CD4+ lymphocytes was significantly upregulated compared with healthy controls. CD164 expression was in most cases limited to CD4+CD26- malignant T lymphocytes, unequivocally identified using flow-cytometry by the expression of a specific Vß clone for each patient. Increased expression of CD164 may be a promising diagnostic parameter and a potential target for a CD164-linked therapeutic approach in Sézary syndrome.

Severe Sweet's Syndrome with Elevated Cutaneous Interleukin-1ß after Azathioprine Exposure: Case Report and Review of the Literature.

Sweet's syndrome (SS) is a dermatosis with systemic symptoms characterized by tender, red nodules or papules, occasionally covered with vesicles, pustules or bullae, usually affecting the upper limbs, face and neck. SS is frequently observed in patients with leukemia or connective tissue diseases, while it is rather seldom in patients with inflammatory bowel disease. The exact pathogenesis of SS is only partially understood. We report the case of a 50-year-old patient with indeterminate colitis, presenting with a febrile diffuse papulopustular and necrotizing skin eruption that healed with significant scarring and appeared 14 days after onset of treatment with azathioprine. Histological examination revealed the presence of features typical of SS, gene expression analysis very high levels of interleukin-1ß (IL-1ß) mRNA in lesional skin, and immunohistochemistry high levels of IL-1ß at the protein level. SS associated with azathioprine is being increasingly reported and is reviewed herein.

Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1ß. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1ß secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1ß when exposed to Malassezia spp. Moreover, we demonstrate that IL-1ß secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.

Rictor in perivascular adipose tissue controls vascular function by regulating inflammatory molecule expression.

OBJECTIVE: Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. APPROACH AND RESULTS: Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictor(ad-/-)). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictor(ad-/-) mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictor(ad-/-) mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1α, and tumor necrosis factor-α in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictor(ad-/-) mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictor(ad-/-) mice with no effect in rictor(fl/fl) mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. CONCLUSIONS: Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Inflammasome activation and IL-1ß target IL-1α for secretion as opposed to surface expression.

Interleukin-1α (IL-1α) and -ß both bind to the same IL-1 receptor (IL-1R) and are potent proinflammatory cytokines. Production of proinflammatory (pro)-IL-1α and pro-IL-1ß is induced by Toll-like receptor (TLR)-mediated NF-κB activation. Additional stimulus involving activation of the inflammasome and caspase-1 is required for proteolytic cleavage and secretion of mature IL-1ß. The regulation of IL-1α maturation and secretion, however, remains elusive. IL-1α exists as a cell surface-associated form and as a mature secreted form. Here we show that both forms of IL-1α, the surface and secreted form, are differentially regulated. Surface IL-1α requires NF-κB activation only, whereas secretion of mature IL-1α requires additional activation of the inflammasome and caspase-1. Surprisingly, secretion of IL-1α also required the presence of IL-1ß, as demonstrated in IL-1ß-deficient mice. We further demonstrate that IL-1ß directly binds IL-1α, thus identifying IL-1ß as a shuttle for another proinflammatory cytokine. These results have direct impact on selective treatment modalities of inflammatory diseases.

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1ß as a key mediator, orchestrating an NF-κB-mediated IL-1ß-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1ß. With the central role of IL-1ß underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1ß antagonists in PRP.

Lymph node tumor metastases: more susceptible than primary tumors to CD8(+) T-cell immune destruction.

Hematopoietic cells, and more particularly, dendritic cells are so called "professional" antigen-presenting cells, which prime CD8(+) T-cell responses. They achieve this by taking up antigens and presenting them to CD8(+) T cells in the draining lymph nodes. This process is called cross-presentation (XP). For most developing tumors, XP of tumor antigens results in CD8(+) T-cell tolerance. In addition to XP, direct presentation by any kind of cell can also occur in lymph nodes. We discuss here how a non-hematopoietic cell can efficiently prime CD8(+) T cells by direct presentation in lymph nodes. Such a T-cell activation pathway is likely to be of importance for the control of cancer metastases that use the lymphatic system to spread.

Killing Two Birds with One Stone: TNF Antagonists Downregulate Systemic IL-1ß in Psoriasis.

Verma et al. (2021) demonstrate that TNF antagonists unexpectedly downregulate systemic IL-1ß by inhibiting noncanonical inflammasome activation in patients with psoriasis. Given the known involvement of IL-1ß in the pathogenesis of psoriasis skin manifestations and associated comorbidities, the findings of Verma et al. (2021) highlight a potential added benefit of targeting TNF in psoriasis.

Resistance to FasL and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in Sezary syndrome T-cells associated with impaired death receptor and FLICE-inhibitory protein expression.

Because of the low proliferative potential of tumor cells in patients with Sézary syndrome (SzS), their accumulation has been suggested to be due to defective regulation of apoptosis. We analyzed the sensitivity to soluble Fas-ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), 2 members of the TNF superfamily in peripheral blood leukocytes (PBL) from patients with SzS. Compared with healthy donors, CD4(+) cells from patients with SzS were completely resistant to FasL in 9 of 16 cases. Of these 9 FasL-resistant cases, 4 revealed a loss in Fas (CD95) expression, whereas the remaining 5 exhibited normal or enhanced Fas expression. In the latter 5 cases, the apoptosis inhibitor cFLIP was overexpressed in CD4(+)/CD26(-) tumor cells compared with CD4(+)/CD26(-) cells from Fas-expressing FasL-sensitive patients and healthy donors. Furthermore, resistance to TRAIL and tumor cell-restricted loss of TRAIL-receptor 2 were observed in 16 of 16 SzS PBLs. It is noteworthy that resistance to FasL could be overcome by the use of a hexameric FasL or upon exposure of SzS cells to interferon-alpha (IFN-alpha) or IFN-gamma, the latter by an increase of Fas expression. Our data on primary SzS lymphocytes reveal frequent resistance to apoptosis induced by FasL and TRAIL, which may contribute to their accumulation in patients with SzS and be relevant at a therapeutic level.

IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes.

Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFRi/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFRi/MEKi skin toxicity. We provide molecular and translational evidence that EGFRi/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFRi/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB and KLF4 binding sites in the human IL-36γ gene promoter. EGFRi/MEKi increased KLF4 expression by blockade of the EGFR/MEK/ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFRi/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.

Extralymphatic tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process.

Metastases often develop in lymphoid organs. However, the immunologic mechanism allowing such invasion is not known because these organs are considered to be hostile to tumor cells. Here, we analyzed the interactions between tumor cells and CD8(+) T cells in such lymphoid organs. Tumor cells implanted into lymph nodes were able to induce tumor-specific cytotoxic CD8(+) T-cell responses, conducting to tumor rejection, in contrast to primary extralymphatic tumors rapidly anergizing T cells in draining lymph nodes (DLN) via a cross-presentation process. This abortive CD8(+) T-cell response to extralymphatic tumor is independent of the subcellular localization of antigen in tumor cells and is consistent with a lack of CD4(+) T-cell help. Notably, this anergy was potent enough to allow successful DLN implantation of tumor cells. Such distant cross-tolerization of tumor-specific CD8(+) T cells may be a determinant permissive event leading to invasion of DLN. In this situation, metastatic tumor cells do not need to down-regulate their immunogenicity to spread.

Culprit Drugs Induce Specific IL-36 Overexpression in Acute Generalized Exanthematous Pustulosis.

Acute generalized exanthematous pustulosis (AGEP) is a severe adverse cutaneous drug reaction. Although an involvement of drug-specific T cells has been reported, the physiopathology of AGEP and mechanism of neutrophilic skin inflammation remain incompletely understood. Recently, mutations in IL-36RN, the gene encoding the IL-36 receptor antagonist, have been reported to be more frequent in AGEP patients and pustular psoriasis. Here, we show that IL-36 cytokines, in particular IL-36γ, are highly expressed in lesional skin of AGEP patients, keratinocytes and macrophages being a major source of IL-36γ. Such an IL-36γ overexpression was not observed in patients with drug-induced maculopapular rash. In vitro, the causative drug specifically induced IL-36γ release either directly by the patient's peripheral blood monocytes or indirectly by keratinocytes in the presence of autologous peripheral blood mononuclear cells. Such culprit drug induction of IL-36γ secretion in vitro was specific for AGEP and involved toll-like receptor 4 sensing the drug/albumin complex as a danger signal. Our results suggest that IL-36γ secretion by monocytes/macrophages and keratinocytes in response to culprit drug exposure likely plays a key role in the pathogenesis of AGEP.

Divergent LAG-3 versus BTLA, TIGIT, and FCRL3 expression in Sézary syndrome.

In Sézary syndrome (SS) impaired T-cell function and cytokine profile lead to immune evasion. Immune checkpoints non-redundantly regulate immune responses and targeting them is promising. We evaluated the expression of BTLA, CTLA-4, FCRL3, LAG-3, and TIGIT in tumor and non-tumor SS T-cells.Compared to CD4+ T helper cells from ten healthy individuals, tumor cells of eight SS patients had a significant upregulation of BTLA (1.5-fold; p < .0001), FRCL3 (2.2-fold; p < .0028) and TIGIT (2.2-fold; p < .0003) expression. In contrast, we found a reduced expression of LAG-3+ cells in the blood of tumor patients (0.5-fold; p < .0014). Only weak alternations between tumor, non-tumor cells, and healthy controls were observed regarding CTLA-4 (0.5-fold; p < .2022). Our results show a diverse expression pattern of immune-regulatory molecules in SS patients. As these molecules are essential in the regulation of T-cell mediated tumor surveillance and defense, their specific targeting might be of clinical relevance.

Vaccinating against Acne: Benefits and Potential Pitfalls.

Acne vulgaris is treated with antibiotics and retinoids, but side effects are numerous. Novel safe and efficient therapies are still needed. Wang et al. demonstrate that the secreted virulence factor Christie-Atkins-Munch-Peterson factor 2 from Propionibacterium acnes, a bacterium involved in acne pathogenesis, promotes inflammatory responses. This proinflammatory property could be inhibited by antibodies to Christie-Atkins-Munch-Peterson factor 2, suggesting Christie-Atkins-Munch-Peterson factor 2 as a candidate target in acne vaccination. This work supports the concept of acne immunotherapy, but questions about selection of target antigens remain open.

Propionibacterium acnes Strains Differentially Regulate the Fate of Th17 Responses in the Skin.

Agak et al. demonstrate that different strains of Propionibacterium acnes, a bacterium colonizing pilosebaceous units in healthy skin and acne, have the ability to induce T helper type 17 cells secreting either IFN-γ or IL-10 and exhibiting either pathogenic or protective properties, respectively. This work contributes to growing evidence indicating that the phenotype of T helper type 17 cells is largely dependent on their microbiological environment.