Retinoic acid-loaded liposomes induce human mucosal CD103+ dendritic cells that inhibit Th17 cells and drive regulatory T-cell development in vitro.

The active vitamin A metabolite, all-trans-retinoic acid (RA), primes precursor dendritic cells (DCs) into a mucosal phenotype with tolerogenic properties characterized by the expression of integrin CD103. CD103+ DCs can counteract pathogenic Th1 and Th17 in inflammatory bowel disease (IBD) or celiac disease (CD). Tolerogenic manipulation of DCs using nanoparticles carrying tolerogenic adjuvants and disease-specific antigens is a valuable treatment strategy to induce antigen-specific mucosal tolerance in vivo. Here, we investigated the effects of RA-loaded liposomes on human DC phenotype and function, including DC-driven T-cell development, both during the generation of monocyte-derived DCs (moDCs) as well as by priming immature moDCs. RA liposomes drove CD103+ DC differentiation as well as ALDH1A2 expression in DCs. Neutrophil-dependent Th17 cell development was reduced by RA-liposome-differentiated and RA-liposome-primed DCs. Moreover, RA liposome treatment shifted T-cell development toward a Th2 cell profile. Importantly, RA liposomes induced the development of IL-10-producing and FoxP3+ regulatory T cells (Tregs) of various Treg subsets, including ICOS+ Tregs, that were potent inhibitors of bystander memory T-cell proliferation. Taken together, RA-loaded liposomes could be a novel treatment avenue for IBD or CD patients.

Aberrant neutrophil degranulation in hospitalized patients with COVID-19 partially remains for 6 months.

Neutrophils are important players in COVID-19, contributing to tissue damage by release of inflammatory mediators, including ROS and neutrophil elastase. Longitudinal studies on the effects of COVID-19 on neutrophil phenotype and function are scarce. Here, we longitudinally investigated the phenotype and degranulation of neutrophils in COVID-19 patients (28 nonhospitalized and 35 hospitalized patients) compared with 17 healthy donors (HDs). We assessed phenotype, degranulation, CXCL8 (IL-8) release, and ROS generation within 8 days, at one or 6 month(s) after COVID-19 diagnosis. For degranulation and ROS production, we stimulated neutrophils, either with ssRNA and TNF or granulocyte-macrophage colony-stimulating factor and N-Formylmethionyl-leucyl-phenylalanine. During active COVID-19, neutrophils from hospitalized patients were more immature than from HDs and were impaired in degranulation and ROS generation, while neutrophils from nonhospitalized patients only demonstrated reduced CD66b+ granule release and ROS production. Baseline CD63 expression, indicative of primary granule release, and CXCL8 production by neutrophils from hospitalized patients were elevated for up to 6 months. These findings show that patients hospitalized due to COVID-19, but not nonhospitalized patients, demonstrated an aberrant neutrophil phenotype, degranulation, CXCL8 release, and ROS generation that partially persists up to 6 months after infection.

Liposomes loaded with vitamin D3 induce regulatory circuits in human dendritic cells.

Introduction: Nanomedicine provides a promising platform for manipulating dendritic cells (DCs) and the ensuing adaptive immune response. For the induction of regulatory responses, DCs can be targeted in vivo with nanoparticles incorporating tolerogenic adjuvants and auto-antigens or allergens. Methods: Here, we investigated the tolerogenic effect of different liposome formulations loaded with vitamin D3 (VD3). We extensively phenotyped monocyte-derived DCs (moDCs) and skin DCs and assessed DC-induced regulatory CD4+ T cells in coculture. Results: Liposomal VD3 primed-moDCs induced the development of regulatory CD4+ T cells (Tregs) that inhibited bystander memory T cell proliferation. Induced Tregs were of the FoxP3+ CD127low phenotype, also expressing TIGIT. Additionally, liposome-VD3 primed moDCs inhibited the development of T helper 1 (Th1) and T helper 17 (Th17) cells. Skin injection of VD3 liposomes selectively stimulated the migration of CD14+ skin DCs. Discussion: These results suggest that nanoparticulate VD3 is a tolerogenic tool for DC-mediated induction of regulatory T cell responses.

Vitamin D3 Priming of Dendritic Cells Shifts Human Neutrophil-Dependent Th17 Cell Development to Regulatory T Cells.

Vitamin D3 (VD3) is a potential adjuvant for use in tolerogenic vaccine formulations that target dendritic cells (DCs) for the treatment of chronic inflammatory disorders, e.g., autoimmune diseases. These disorders are often associated with enhanced activity of IL-17-producing T helper 17 (Th17) cells which develop in a DC-driven and neutrophil-dependent fashion. Here, we investigated the effect of VD3 on Candida albicans-specific human T-cell differentiation, since C. albicans is a model pathogen for Th17 cell development. VD3 priming of DCs restricted neutrophil-dependent Th17 cell development and neutrophil-independent Th1 cell formation from naive CD4+ T cells. In line with this, the production of Th1/Th17-polarizing cytokines IL-12 and IL-23 by DCs was reduced by VD3 priming. Development of both FoxP3+CD127lowCD25+ Tregs and IL-10-producing T cells was significantly enhanced in VD3-primed conditions, even in the presence of neutrophils. ICOS+ Tregs, major IL-10 producers, CD69+FoxP3+, and TIGIT+FoxP3+ Tregs were significantly induced by VD3 priming as well. Our data support the potential use of VD3 as an adjuvant to induce tolerance in the treatment of autoimmune disorders, including those in which neutrophils are involved in pathogenesis, since we show that Treg development is enhanced by VD3 even in the presence of neutrophils, while Th17 cell development is restricted.

Uptake Kinetics Of Liposomal Formulations of Differing Charge Influences Development of in Vivo Dendritic Cell Immunotherapy.

Dendritic cells (DCs) control adaptive immunity and are therefore attractive for in vivo targeting to either induce immune activation or tolerance, depending on disease. Liposomes, nanoparticles comprised of a lipid bi-layer, provide a nanoplatform for loading disease-relevant antigen, adjuvant and DC-targeting molecules simultaneously. However, it is yet not fully understood how liposomal formulations affect uptake by DCs and DC function. Here, we examined monocyte-derived DC (moDC) and skin DC uptake of six different liposomal formulations, together with their DC-modulating effect. Contrary to literature, we show using imaging flow cytometry that anionic or neutral liposomes are taken up more efficiently than cationic liposomes by moDCs, or by skin DCs after intradermal injection. None of the formulations yielded significant modulation of DC function as determined by the upregulation of maturation markers and cytokine production. These results suggest that anionic liposomes would be more suitable as vaccine carriers for a dermal application.

Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses.

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.

Antigen Uptake After Intradermal Microinjection Depends on Antigen Nature and Formulation, but Not on Injection Depth.

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

Signal Inhibitory Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on mononuclear phagocytes in skin and colon.

Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is expressed on human blood monocytes and granulocytes and inhibits myeloid effector functions. On monocytes, but not granulocytes, SIRL-1 expression is low or absent in individuals with the single nucleotide polymorphism (SNP) rs612529C. The expression of SIRL-1 in tissue and the influence of rs612529 hereon is currently unknown. Here, we used flow cytometry to determine SIRL-1 expression on immune cells in human blood and three barrier tissues; skin, colon and lung. SIRL-1 was expressed by virtually all neutrophils and eosinophils in these tissues. In contrast, SIRL-1 was not expressed by monocyte-derived cells in skin and colon, whereas it was highly expressed by lung classical monocytes. Lung monocytes from individuals with a rs612529C allele had decreased SIRL-1 expression, consistent with the genotype association in blood. Within the different monocyte subsets in blood and lung, SIRL-1 expression was highest in classical monocytes and lowest in nonclassical monocytes. SIRL-1 was not expressed by dendritic cells in blood and barrier tissues. Together, these results indicate that SIRL-1 is differentially expressed on phagocyte subsets in blood and barrier tissues, and that its expression on monocytes is genotype- and tissue-specific. Immune regulation of monocytes by SIRL-1 may be of particular importance in the lung.

Different Interferon Beta Preparations Induce the Same Qualitative Immune Response in Human Skin.

Interferon beta (IFNß) is used as a first-line treatment for multiple sclerosis (MS) and is injected intramuscularly or subcutaneously (s.c.). The subcutaneous route is considered more immunogenic as it is associated with increased antidrug antibody-positive patients. The skin contains dendritic cells (DCs) and it is unclear whether these contribute to immunogenicity. To assess the effect of IFNß on skin-resident cells, IFNß was injected intradermally (i.d.) ex vivo using a human skin explant model or s.c. in vivo in MS patients. Ex vivo, intradermal IFNß injections reduced migration and enhanced surface CD86 expression of dermal DCs, and an increased expression of HLA-DR+ was observed in skin biopsies taken after subcutaneous IFNß injection (in vivo). In both models, IFNß elevated the expression of several inflammatory cytokines when compared to the control biopsies. Our results show that 3 different IFNß preparations, normalized in dose and injection site, induce similar immune responses, suggesting that the differences in immunogenicity are likely due to the route and frequency of administration.

Dendritic cell type-specific HIV-1 activation in effector T cells: implications for latent HIV-1 reservoir establishment.

BACKGROUND: Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro. METHODS: Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir. RESULTS: Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells' ability to induce viral gene expression. CONCLUSIONS: In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies.Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation.

Fc gamma receptor-TLR cross-talk elicits pro-inflammatory cytokine production by human M2 macrophages.

M2 macrophages suppress inflammation in numerous disorders, including tumour formation, infection and obesity. However, the exact role of M2 macrophages in the context of several other diseases is still largely undefined. We here show that human M2 macrophages promote inflammation instead of suppressing inflammation on simultaneous exposure to complexed IgG (c-IgG) and TLR ligands, as occurs in the context of diseases such as rheumatoid arthritis (RA). c-IgG-TLR ligand co-stimulation of M2 macrophages selectively amplifies production of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and promotes Th17 responses, which all play a critical role in RA pathology. Induction of pro-inflammatory cytokines on c-IgG co-stimulation mainly depends on Fc gamma receptor IIa (FcγRIIa), which selectively amplifies cytokine gene transcription and induces caspase-1 activation. These data indicate that FcγR-TLR cross-talk may be targeted for treatment to attenuate inflammation in RA, by restoring the anti-inflammatory function of M2 macrophages.

Vitamin D3 metabolite calcidiol primes human dendritic cells to promote the development of immunomodulatory IL-10-producing T cells.

Vitamin D is recognized as a potent immunosuppressive drug. The suppressive effects of vitamin D are attributed to its physiologically active metabolite 1,25 dihydroxy vitamin D3 (calcitriol), which was shown, to prime dendritic cells (DCs) to promote the development of regulatory T (Treg) cells. Despite the potential benefit in treating autoimmune diseases, clinical application of calcitriol is hindered by deleterious side effects manifested by hypercalcemia and hypercalciuria. Conversely, the physiological precursors of calcitriol, vitamin D3 (cholecalciferol) and its first metabolite 25-hydroxy vitamin D3 (calcidiol) are widely applied in the clinic due to their low calcimic burden. However, the mechanisms by which cholecalciferol and calcidiol may modulate adaptive immunity remain elusive. This prompted us to unravel the immunosuppressive capacity of these precursors by assessing their influence on DC functions and the subsequent polarization of naïve CD4(+) T cells. In this study we show that, whereas cholecalciferol has insignificant effects on DC maturation and cytokine production, it only weakly primed DCs to induce suppressive T cells. However, like calcitriol, calcidiol not only exerted an inhibitory effect on DC maturation and cytokine production, and primed DCs to promote the development of suppressive IL-10-producing Treg cells. Strikingly, in contrast to the population of IL-10-producing Treg cells induced by calcitriol-primed DCs, the IL-10-producing Treg cells induced by calcidiol-primed DCs exhibited sustained IFN-γ production in face of their suppressive capacity. Experiments with the steroid synthesis inhibitor ketoconazole indicated that the immunomodulatory features of the precursors are dependent on their conversion into calcitriol. Collectively, calcidiol is a potent immune modulator, which may be more adequate than calcitriol for the treatment of chronic inflammatory diseases, since it is less hypercalcimic. This may be of particular interest for the treatment of allergic disease, where concurrent suppression and sustained IFN-γ production by Treg cells effectively counterbalance the Th2-dominated immune responses.

Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study.

OBJECTIVE: To study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system. DESIGN: Experimental laboratory study. SETTING: Reproductive biology laboratory, academic medical center. PATIENT(S): Acute lymphoblastic leukemia cells from three patients and testicular cells from three other patients. INTERVENTION(S): Acute lymphoblastic leukemia cells were cultured alone or in combination with testicular cells, at various concentrations, in a system that has recently been developed to propagate human spermatogonial stem cells. MAIN OUTCOME MEASURE(S): Viability of ALL and testicular cells during culture was evaluated by flow cytometry using markers for live/dead cells. Furthermore, the presence of ALL cells among testicular cells was determined by highly sensitive (1:10,000 to 1:100,000 cells) patient-specific antigen-receptor minimal residual disease polymerase chain reaction. The presence of spermatogonia at the end of culture was determined by reverse transcription-polymerase chain reaction for ZBTB16, UCHL1, and GPR125. RESULT(S): The ALL cells cultured separately did not survive beyond 14 days of culture. When cultured together with testicular cells, even at extremely high initial concentrations (40% ALL cells), ALL cells were undetectable beyond 26 days of culture. Reverse transcription-polymerase chain reaction confirmed the presence of spermatogonia at the end of the culture period. CONCLUSION(S): Our pilot study shows that the described testicular cell culture system not only allows for efficient propagation of spermatogonial stem cells but also eliminates contaminating ALL cells.

Langerhans cells favor skin flora tolerance through limited presentation of bacterial antigens and induction of regulatory T cells.

The mechanisms preventing detrimental T-cell responses against commensal skin bacteria remain elusive. Using monocyte-derived and skin-derived dendritic cells (DCs), we demonstrate that epidermal Langerhans cells (LCs), the DCs in the most superficial layer of the skin, have a poor capacity to internalize bacteria because of low expression of FcγRIIa. Furthermore, LCs show deficiency in processing and major histocompatibility complex II (MHC-II)-restricted presentation of bacterial antigens, as a result of a decreased expression of molecules involved in these functionalities. The reduced capacity to take up, process, and present bacterial antigens cannot be restored by LC activation by ectopically expressed Toll-like receptors or by cytokines. Consequently, bacteria-primed LCs poorly restimulate antibacterial memory CD4(+) T cells and inefficiently induce bacteria-specific effector CD4(+) T cells from naive T cells; however, they initiate the development of regulatory Foxp3(+)CD4(+) T cells, which are able to suppress the proliferation of autologous bystander T cells specific for the same bacteria. In contrast, dermal DCs that reside in the deeper dermal layer of the skin efficiently present bacterial antigens and provoke robust antibacterial naive and memory CD4(+) T-cell responses. In conclusion, LCs form a unique DC subset that is adapted at multiple levels for the maintenance of tolerance to bacterial skin flora.

Intradermal application of vitamin D3 increases migration of CD14+ dermal dendritic cells and promotes the development of Foxp3+ regulatory T cells.

The active form of vitamin D3 (VitD) is a potent immunosuppressive drug. Its effects are mediated in part through dendritic cells (DCs) that promote the development of regulatory T cells (Tregs). However, it remains elusive how VitD would influence the different human skin DC subsets, e.g., CD1a(+)/langerin(+) Langerhans cells, CD14(+) DDCs and CD1a(+) DDCs upon administration through the skin route in their natural environment. We addressed this issue by intradermal (ID) administration of VitD in a human skin explant system that closely resembles physiological conditions. ID injection of VitD selectively enhanced the migration of CD14(+) DDCs, a subset known for the induction of tolerance. Moreover, ID injection of VitD repressed the LPS-induced T cell stimulatory capacity of migrating DCs. These migrating DCs collectively induced T cells with suppressive activity and abolished IFN-γ productivity. Those induced T cells were characterized by the expression of Foxp3. Thus, we report the novel finding that ID injection of VitD not only modifies skin DC migration, but also programs these DCs in their natural milieu to promote the development of Foxp3(+) Tregs.

Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells.

BACKGROUND: The vitamin D metabolite 1,25(OH)2D3 (VitD3) is a potent immunosuppressive drug and, among others, is used for topical treatment of psoriasis. A proposed mechanism of VitD3-mediated suppression is priming of dendritic cells (DCs) to induce regulatory T (Treg) cells. OBJECTIVE: Currently, there is confusion about the phenotype of VitD3-induced Treg cells and the DC-derived molecules driving their development. We investigated Treg cell induction after VitD3 priming of 2 distinct skin DC subsets: Langerhans cells (LCs) and dermal dendritic cells (DDCs). METHODS: LCs and DDCs primed with VitD3 were cocultured with allogeneic naive T cells. The phenotype and function of the DCs and induced T cells were analyzed. RESULTS: Both VitD3-primed DC subtypes induced T cells with regulatory activity. Unexpectedly, whereas the Treg cell populations generated by VitD3-primed LCs were CD25(hi)CD127(lo) forkhead box protein 3 (Foxp3)-positive cells, which meet the criteria of classical inducible Treg cells, the T cells developing in response to VitD3-primed DDCs were Foxp3(-) T(R)1 cells expressing IL-10. Inhibition experiments revealed that LC-derived TGF-ß is a key factor in the induction of Foxp3(+) Treg cells, whereas DDC-derived IL-10 is important for the induction of IL-10(+) T(R)1 cells. CONCLUSION: Thus we report the novel finding that distinct but closely related DC subsets are differentially programmed by VitD3 to support development of either TGF-ß-dependent Foxp3(+) Treg cells or IL-10-dependent IL-10(+) Treg cells.

Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2.

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to T(H)-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of T(H)-17-polarizing cytokines IL-1ß and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H)-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H)-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H)-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H)-17 immunity to fungi via Malt1-dependent activation of c-Rel.

Aberrant function of peripheral blood myeloid and plasmacytoid dendritic cells in atopic dermatitis patients.

BACKGROUND: Dendritic cells (DCs) can act both as innate cells in host defense and as antigen-presenting cells for naive T cells in adaptive immunity. These functions, among others, are determined by the level of production of particular cytokines. Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by an initial phase predominated by T(H)2 cytokines that switches into a second, more chronic T(H)1-dominated eczematous phase. OBJECTIVE: To assess to what extent the AD phenotype is associated with an aberrant phenotype and function of DCs. METHODS: Classic CD1c(+)/blood DC antigen (BDCA)-1(+) myeloid (m) DCs and CD304(+)/BDCA4(+) plasmacytoid (p) DCs, the natural IFN-producing cells, were isolated from peripheral blood of patients with AD and healthy controls and analyzed for their phenotype and function. RESULTS: Purified CD1c(+)/BDCA1(+) mDCs from patients with AD showed a selective and dramatic reduction of IL-12p70 and TNF-alpha release. IL-12p70 reduction was attributed to a defective expression of both IL-12p35 and IL-12p40 subunits. Accordingly, mature CD1c(+)/BDCA1(+) mDCs from patients with AD induced considerably less IFN-gamma-producing and more IL-4-producing T(H) cells compared with mDCs from healthy controls. In addition, CD304(+)/BDCA4(+) pDCs from patients with AD produced significantly lower levels of IFN-alpha compared with healthy controls. CONCLUSION: Myeloid DCs and pDCs from patients with AD show defective IL-12, TNF-alpha, and IFN-alpha production, which may contribute to increased susceptibility to infection and to the maintenance of the T(H)2 cell-mediated allergic state in patients with AD.

Human keratinocytes express functional Toll-like receptor 3, 4, 5, and 9.

Keratinocytes are continuously in contact with external stimuli and have the capacity to produce several soluble mediators. Pathogen-associated molecular patterns (PAMPs) are recognized, among others, by Toll-like receptors (TLRs). The functional responses of keratinocytes to different PAMPs have not yet been fully established. Here we show that keratinocytes constitutively express TLR1, 2, 3, 4, 5, 6, 9, and 10 mRNA, but not TLR7 and 8. Stimulation of keratinocytes with TLR3, 4, 5, and 9 ligands resulted in differential immune-associated responses. Tumor necrosis factor-alpha, CXC chemokine ligand 8 (CXCL8), CCL2, and C chemokine ligand 20 (CCL20) release was enhanced in response to all PAMPs tested, in a time- and dose-dependent manner. Only TLR9 ligand CpG-oligodeoxynucleotides (ODNs) and TLR3 ligand poly-I:C could additionally induce type I IFNs. CCL27 production was selectively induced by poly-I:C and flagellin, whereas CXCL9 and CXCL10 were exclusively induced by CpG-ODNs and/or poly-I:C. Upregulation of ICAM-1, HLA-DR, HLA-ABC, FasR, and CD40 was mainly observed in response to poly-I:C, flagellin, and lipopolysaccharide. Furthermore, PAMP triggering resulted in the phosphorylation of phosphorylated-IkappaB alpha and in the nucleus translocation of NF-kappaB p65. Altogether, these findings stress an unexpectedly multifaceted role of keratinocytes in innate immunity as evident by their differential, TLR-mediated responses to PAMPs associated with different classes of pathogens.