Search

Purpose Reactive oxygen and nitrogen species (ROS/RNS) production and the NF-κB activation are critically involved in inflammatory responses, but knowledge about the temporal dynamics during acute and chronic inflammation is limited. Here, we present a comparative longitudinal in vivo study of both parameters in an experimental model of acute and chronic T cell–driven delayed-type hypersensitivity reaction (DTHR) using noninvasive optical imaging. Procedures Trinitrochlorobenzene (TNCB)-sensitized NF-κB-luciferase-reporter and wild-type mice were TNCB challenged on the right ear to elicit acute DTHR and then repetitively challenged (up to five times) to induce chronic DTHR. Mice were treated with the ROS-scavenging and NF-κB inhibiting molecule N-acetylcysteine (NAC) or underwent sham treatment. ROS/RNS production was noninvasively analyzed in vivo using the ROS-/RNS-sensitive chemiluminescent probe L-012, and NF-κB activation was measured using NF-κB-luciferase-reporter mice. H&E staining, CD3 and myeloperoxidase (MPO) immunohistochemistry (IHC), and quantitative PCR (qPCR) analyses were employed to investigate immune cell infiltration and expression of NF-κB- and ROS-/RNS-driven genes. Results In acute DTHR, we found strongly elevated ROS/RNS production and NF-κB activation 12 h after the 1st TNCB ear challenge, peaking at 24 h after the challenge. In chronic DTHR, ROS production peaked as early as 4 h after the 5th TNCB challenge, whereas NF-κB activity peaked after 12 h. The increase in ROS/RNS production in acute DTHR was higher than the increase in NF-κB activity but the relationship was inverse in chronic DTHR. Treatment with the ROS scavenger NAC had differential effects on ROS/RNS production and NF-κB activation during acute and chronic DTHR. Ex vivo cross-validation by histopathology and qPCR analysis correlated closely with the in vivo imaging results. Conclusions Noninvasive in vivo imaging is capable of assessing the temporal dynamics of ROS/RNS production and NF-κB activation during progression from acute to chronic DTHR and enables monitoring of anti-inflammatory treatment responses. Electronic supplementary material The online version of this article (10.1007/s11307-019-01412-8) contains supplementary material, which is available to authorized users.

To establish strategies for immunotherapy of B-cell lymphoma, it is mandatory to gain deeper insights into the mechanisms of tumor immune escape. In a mouse model of endogenously arising lymphoma, we investigated the impact of IL-10 on the regulation of antitumor responses. Despite progressive functional impairment of NK cells and lack of IFN-γ in the tumor milieu, we found an augmented fraction of T helper type 1 (Th1) cells, which continued to express IFN-γ but also upregulated IL-10 during disease development. Using a lymphoma microenvironment in vitro, we showed that Th1 cells were converted to Foxp3-negative T regulatory type 1 (Tr1) cells, which coexpressed IFN-γ and IL-10 and upregulated PD-1. This differentiation required pre-existing IL-10, which was primarily provided by malignant B cells and dendritic cells. IFN-γ only declined in cells with the uppermost PD-1 levels. Importantly, antibody-mediated IL-10 ablation in vivo improved effector cell functions and significantly suppressed tumor development. While the contribution of IL-10 to cancer immune escape has been controversially discussed in the past, we show that IL-10 suppresses ongoing, potentially protective immune responses in lymphoma and might be a target for immunotherapy.

Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses. Electronic supplementary material The online version of this article (10.1007/s00262-020-02767-6) contains supplementary material, which is available to authorized users.

Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16Ink4a/p19Arf (Cdkn2a) or p21Cip1 (Cdkn1a) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication., The growth of cancer cells can be stably arrested by cytokine-induced senescence. Here, the authors show that cancers with defects in senescence-inducing cell cycle regulator pathways are resistant to immune checkpoint blockade.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.

Dimethyl fumarate (DMF) promotes an IL-17Alow IFN-γlow IL-4+ CD4+ T cell phenotype. Adoptive transfer of in vitro DMF-treated myelin peptide-reactive IL-17Alow IFN-γlow IL-4+ CD4+ T cells prior to immunization for EAE reduces the severity of encephalomyelitis. This beneficial effect of transferred DMF-treated CD4+ T cells requires an early in vivo recall.

Background Chronic inflammatory skin diseases are characterized by controlled proliferation of keratinocytes. Here, activating transcription factor 3 (ATF3) might play a fundamental role. In these inflammatory diseases, proliferation is controlled and only rarely leads to cancer development which can be supported by an inflammatory microenvironment. ATF3 is a dual function protein as it suppresses pro-inflammatory IL-6 and IL-8, but also acts as a pro-oncogenic factor by the suppression of p53. We therefore analyzed ATF3 expression comparing myeloid cells with keratinocytes. Objective To dissect the bi-modal role of ATF3 we pharmacologically induced ATF3 and analyzed its influence on cytokine expression and secretion in a cell type specific manner. Methods Since inflammatory skin diseases can be treated systemically with Cyclosporin A or Dimethylfumarate we stimulated myeloid cells and primary human keratinocytes with these drugs and analyzed gene expression by quantitative real-time PCR. Cytokine secretion was measured by ELISA. Results In the present study, we could show that ATF3 is induced in PBMCs by DMF and weakly by Ebselen, while CsA is the most prominent inducer of ATF3 in keratinocytes without enhancing HO-1 transcription. Further we could show that induction of stress by LPS treatment elevates IL-1β and IL-6 and weakly ATF3 transcription in PBMCs. While transcription of both cytokines is elevated, LPS treatment mediates IL-6 secretion with only little IL-1β secretion. Treatment with DMF dampens LPS-induced transcription. Conclusions Taken together, our results shed light into the different carcinogenic potential of CsA and DMF, which both target ATF3. Collectively our data demonstrate that CsA strongly induces pro-carcinogenic ATF3 in keratinocytes, whereas ATF3 induction by DMF in myeloid cells acts anti-inflammatory.

Immune checkpoint blockade has revolutionized cancer treatment, Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LP5 signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LP5-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival, These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/− mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/− controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell–cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Melanoma is a malignant tumor in which UVA (320-400 nm) radiation is considered to be an important risk factor. But the role of UVA in melanoma progression toward an invasive phenotype is still not adequately investigated. For most proliferating tumor cells the preference of aerobic glycolysis has been described as the Warburg effect. Here we investigate the effect of UVA irradiation on changes in the Warburg effect and tumor progression toward invasive potential. On UVA irradiation, melanoma cell lines from initial tumors show an induction of the Warburg effect with increased glucose consumption and lactate production, which is at least partially mediated by reactive oxygen species. Associated with UVA treatment and enhanced lactic acid production, tumor-relevant proteases and in situ invasion is upregulated. Simultaneously, UVA increases intracellular concentrations of progression marker transketolase and activated protein kinase Akt, both involved in metabolic changes that increase with proliferation. Using invasion assays we show that lactic acid, resulting from the UVA enhanced and partially reactive oxygen species-mediated Warburg effect, increases the invasive potential of all melanoma cell lines investigated. Therefore, we demonstrate in melanoma cells that production of lactic acid, induced by UVA irradiation, increases invasiveness of melanoma cells via expression of tumor-relevant proteases.

Forty years of research have brought about the development of antibodies that induce effective antitumor immune responses through sustained activation of the immune system. These "immune checkpoint inhibitors" are directed against immune inhibitory molecules, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1). Disruption of the PD-1/PD-L1 interaction improves the intermediate-term prognosis even in patients with advanced stage IV melanoma. One and a half years after treatment initiation, 30-60 % of these patients are still alive. While cancer immunotherapies usually do not eradicate metastases completely, they do cause a regression by 20-80 %. It is well established that the immune system is able to kill tumor cells, and this has also been demonstrated for immunotherapies. Preclinical data, however, has shown that anti-cancer immunity is not limited to killing cancer cells. Thus, through interferon gamma and tumor necrosis factor, the immune system is able to induce stable tumor growth arrest, referred to as senescence. Ensuring patient survival by long-term stabilization of metastatic growth will therefore become a central goal of antitumor immunotherapies. This therapeutic approach is effective in melanoma and non-small-cell lung cancer. Once immunotherapies also have an indication for common cancer types, drug prices will have to drop considerably in order to be able to keep them available to those dependent on such therapies.

Staphylococcusepidermidis colonises human skin without apparent inflammation, but a dominance of S.epidermidis and S.aureus is characteristic of cutaneous microbial dysbiosis in atopic dermatitis (AD). While S.aureus can trigger AD, the role of S.epidermidis is less understood. We characterised consequences of innate immune sensing of lipoteichoic acid (LTA) preparations derived from S.epidermidis (epi-LTA) or S.aureus (aureus-LTA). Therefore, dendritic cell (DC) activation and consecutive priming of antigen-specific T cells following exposure of DC to epi-LTA or aureus-LTA were investigated. Mimicking acute AD, exposure of DC to IL-4 and LTAs was analysed. Exposure to epi-LTA or aureus-LTA activated human immune cells and murine dendritic cells (DCs) via TLR2/MyD88, however, resulting in divergent immune profiles. Differences between LTAs were significant for IL-6, IL-12p40 and IL-12p70 but not for IL-10, which was best reflected by the IL-12p70-to-IL-10 ratio being IL-10-balanced for epi-LTA but pro-inflammatory for aureus-LTA. LTA-exposed DCs activated CD4+ T cells; however, while T-cell-derived IL-10 was equivalent between LTAs, IFN-γ and IL-17 were significantly higher for aureus-LTA. Mimicking acute AD by exposing DCs to IL-4 and LTAs revealed that IL-4 significantly and uniformly suppressed epi-LTA-induced cytokine production, keeping the IL-12p70-to-IL-10 ratio balanced. In contrast, exposure of DCs to aureus-LTA and IL-4 enhanced IL-12p70 but suppressed IL-10 levels, further unbalancing the IL-12p70-to-IL-10 ratio. These data demonstrate opposing immune consequences following exposure to staphylococcal LTAs. Epi-LTA induced IL-10-balanced, aureus-LTA pro-inflammatory immune profiles.

Small interfering RNA (siRNA)–based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10–producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.

Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ-myc-transgenic model of endogenously arising lymphoma and NKG2D-deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN-γ expression, which was effected by MHCI(low) tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D-deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D-deficient λ-myc-transgenic mice was significantly accelerated compared to NKG2D-competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D-L) in late disease stages, this did not occur in NKG2D-deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D-L loss mutants provides a mechanism of tumor escape.

Immune checkpoints are accessory molecules that either promote or inhibit T-cell activation. Two inhibitory molecules, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), got high attention, as inhibition of CTLA-4 or PD-1 signaling provides the first immune therapy that significantly improves the survival of patients with metastatic solid cancers. Inhibition of CTLA-4 or PD-1 was first studied in and approved for patients with metastatic melanoma. Blocking immune checkpoints is also efficient in non–small-cell lung cancer, renal cell cancers, hypermutated gastrointestinal cancers, and others. Immune responses, whether directed against infections or against tumors, are divided into 2 phases: an initiation phase and an activation phase, where the immune system recognizes a danger signal and becomes activated by innate signals to fight the danger. This reaction is fundamental for the control of infections and cancer, but needs to be turned off once the danger is controlled, because persistence of this activation ultimately causes severe tissue damage. Therefore, each activation of the immune system is followed by a termination phase, where endogenous immune suppressor molecules arrest immune responses to prevent harmful damage. In the case of cancer immune therapies, therapeutic approaches classically enhanced the initiation and activation of immune responses to increase the emergence and the efficacy of cytotoxic T lymphocytes (CTL) against cancers. In sharp contrast, immune checkpoint blockade focuses on the termination of immune responses by inhibiting immune suppressor molecules. It thus prevents the termination of immune responses or even awakes those CTLs that became exhausted during an immune response. Therefore, blocking negatively regulating immune checkpoints restores the capacity of exhausted CTL to kill the cancer they infiltrate. In addition, they drive surviving cancer cells into a still poorly defined state of dormancy. As the therapy also awakes self-reactive CTL, one downside of the therapy is the induction of organ-specific autoimmune diseases. The second downside is the exorbitant drug price that withdraws patients in need from a therapy that was developed by academic research, which impairs further academic treatment development and financially charges the public health system.

Inherited forms of epidermolysis bullosa are blistering diseases of the skin and mucosa resulting from various gene mutations. Transplantation of bone marrow-derived stem cells might be a promising systemic treatment for severe dystrophic or junctional epidermolysis bullosa, but many key questions remain unresolved. Two open questions of clinical interest are whether systemically transplanted bone marrow-derived stem cells of mesodermal origin might be able to transdifferentiate into keratinocytes with an ectodermal phenotype and whether these cells are also capable of repairing a specific intraepidermal gene defect. To address these questions, we transplanted bone marrow-derived stem cells into mice with a blistering disease exclusively localized to the epidermis resulting from a functional knockout of desmoglein-3 (Dsg3). We found that Dsg3+ donor-derived cells migrate into the recipient epidermis. However, these cells failed to restore the missing Dsg3 mRNA and DSG3 protein expression in the transplanted Dsg3–/– mice. The donor-derived cells found in the epidermis preserved their CD45+ hematopoietic origin, and no transdifferentiation into integrin α6+ keratinocytes or integrin α6+/CD34+ epidermal stem cells occurred. Our results indicate that bone marrow-derived stem cells preserve their mesodermal fate after systemic transplantation and are not capable of treating patients with epidermolysis bullosa with an intraepidermal skin defect.

Background Atopic dermatitis (AD) is a common inflammatory skin disease with dysfunction of the skin barrier, an abnormal immune response and frequent allergies to environmental antigens like food antigens. Clinical observations suggest that certain diets can influence the course of AD. Objective Here we compared the phenotype of food allergen-specific T cells activated through skin or gut allergen exposure to transfer skin inflammation into naive recipients upon epicutaenous allergen challenge. Methods Ovalbumin (OVA) TCR-transgenic mice were treated epicutaneously with OVA or were fed OVA. CD4+ T cells from skin lymph nodes or mesenteric lymph nodes were transferred into naive BALB/c mice, which were challenged with OVA epicutaneously. Skin inflammation was determined by histological parameters. In addition, we analyzed the phenotype of the immune response in lymphoid tissues and in skin tissue. Results TCR-transgenic T cells activated through epicutaneous or oral OVA exposure both migrate to skin lymph nodes after adoptive transfer and epicutaenous OVA exposure. AD-like skin inflammation could only be induced by the transfer of epicutaneously primed OVA T cells. Analysis of the immune phenotype demonstrated an IL-22/IL-17A-dominated immune phenotype of skin-pathogenic T cells. Conclusion IL-22 seems to be the critical cytokine for the development of AD and is induced in this model by epicutaneous sensitization with OVA.

SummarySkin is constantly exposed to bacteria and antigens, and cutaneous innate immune sensing orchestrates adaptive immune responses. In its absence, skin pathogens can expand, entering deeper tissues and leading to life-threatening infectious diseases. To characterize skin-driven immunity better, we applied living bacteria, defined lipopeptides, and antigens cutaneously. We found suppression of immune responses due to cutaneous infection with Gram-positive S. aureus, which was based on bacterial lipopeptides. Skin exposure to Toll-like receptor (TLR)2-6-binding lipopeptides, but not TLR2-1-binding lipopeptides, potently suppressed immune responses through induction of Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs). Investigating human atopic dermatitis, in which Gram-positive bacteria accumulate, we detected high MDSC amounts in blood and skin. TLR2 activation in skin resident cells triggered interleukin-6 (IL-6), which induced suppressive MDSCs, which are then recruited to the skin suppressing T cell-mediated recall responses such as dermatitis. Thus, cutaneous bacteria can negatively regulate skin-driven immune responses by inducing MDSCs via TLR2-6 activation.

Background The difficulty of antifungal substances to penetrate keratin and slow nail growth limit the efficacy of topical therapy in onychomycosis. One promising alternative is photodynamic antimicrobial chemotherapy, or PACT: an irradiated photosensitizer creates singlet oxygen molecules which destroy pathogens without damaging human cells. Objective As PACT has demonstrated strong antifungal capabilities, we wanted to investigate its efficacy in an in vitro model of onychomycosis. Methods PACT was tested in a microdilution assay, in an in vitro onychomycosis model as well as in a patient. Results PACT inhibited fungal growth in the microdilution assay with no colonies of T. rubrum detectable. Fungal growth was also inhibited in an onychomycosis model, after 30 min of LED irradiation. Subsequently, a patient with distolateral onychomycosis was treated on three consecutive days and showed significant and durable improvement of nail morphology 6 months after. Conclusion PACT appears to be an effective treatment of onychomycosis in vitro. The promising results need to be validated by clinical trials.

Costimulatory surface molecules and instructive cytokines expressed by dendritic cells (DCs) determine the outcome of an immune response. In malignant disease, DCs are often functionally compromised. In most tumors studied so far, the deficient induction of effective T cell responses has been associated with a blockade of DC maturation, but little has been known on DCs infiltrating malignant B cell lymphoma. Here, we investigated for the first time the phenotypic and functional status of DCs in B cell lymphoma, and we analyzed the network of DCs, tumor cells, natural killer (NK) cells and cytokines present in the tumor micromilieu. Therefor, we used an endogenous myc-transgenic mouse lymphoma model, because transplanted tumor cells foster an IFN-γ-driven Th1 antitumor response rather than an immunosuppressive environment, which is observed in autochthonous neoplasias. Lymphoma-infiltrating DCs showed a mature phenotype and a Th2-inducing cytokine pattern. This situation is in contrast to most human malignancies and mouse models described. Cellular contacts between DCs and tumor cells, which involved CD62L on the lymphoma, caused upregulation of costimulatory molecules, whereas IL-10 primarily derived from lymphoma cells induced an IL-12/IL-10 shift in DCs. Thus, alteration of costimulatory molecules and instructive cytokines was mediated by distinct mechanisms. Normal NK cells were able to additionally modulate DC maturation but this effect was absent in the lymphoma environment where IFN-γ production by NK cells was severely impaired. These data are relevant for establishing novel immunotherapeutic approaches against B cell lymphoma.

The beneficial effects of nonpathogenic bacteria are increasingly being recognized. We reported in a placebo-controlled study with atopic dermatitis (AD) patients that cutaneous exposure to lysates of nonpathogenic bacteria alleviates skin inflammation. To now unravel underlying mechanisms, immune consequences of sensing nonpathogenic bacterium Vitreoscilla filiformis lysate (Vf) were characterized analyzing (1) differentiation of dendritic cells (DCs) and, consecutively, (2) effector functions of DCs and T helper (Th) cells in vitro and in a murine model of AD in NC/Nga mice in vivo. Topical treatment with Vf significantly reduced AD-like inflammation in NC/Nga mice. Importantly, cutaneous exposure to Vf in combination with the allergen FITC significantly also reduced subsequent allergen-induced dermatitis indicating active immune modulation. Indeed, innate sensing of Vf predominantly induced IL-10-producing DCs, which was dependent on Toll-like receptor 2 (TLR2) activation. Vf-induced IL-10+ DCs primed naive CD4+ T helper cells to become regulatory IFN-\textgreekglow IL-10high Tr1 (type 1 regulatory T) cells. These IL-10high Tr1 cells were also induced by Vf in vivo and strongly suppressed T effector cells and inflammation. In conclusion, we show that innate sensing of nonpathogenic bacteria by TLR2 induces tolerogenic DCs and regulatory Tr1 cells suppressing T effector cells and cutaneous inflammation. These findings indicate a promising therapeutic strategy for inflammatory skin diseases like AD. \copyright 2014 The Society for Investigative Dermatology.

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

Background The interplay between microbes and surface organs, such as the skin, shapes a complex immune system with several checks and balances. The first-line defense is mediated by innate immune pathways leading to inflammation. In the second phase specific T cells invade the infected organ, amplifying inflammation and defense. Consecutively, termination of inflammation is crucial to avoid chronic inflammation triggered by microbes, such as in patients with atopic dermatitis. Objective We aimed to elucidate how the Staphylococcus aureus –derived cell-wall component lipoteichoic acid (LTA) governs the second phase of immune responses when high concentrations of LTA access T cells directly through disrupted skin. Methods We analyzed the direct exposure of T cells to LTA in vitro . For in vivo analyses, we used fluorescein isothiocyanate contact hypersensitivity and ovalbumin-induced dermatitis as models for T H 2-mediated cutaneous inflammation. Results We observed that LTA potently suppressed T-lymphocyte activation in a Toll-like receptor 2–independent manner. LTA-exposed T cells did not proliferate and did not produce cytokines. Importantly, these T cells remained completely viable and were responsive to consecutive activation signals on subsequent removal of LTA. Thus LTA exposure resulted in temporary functional T-cell paralysis. In vivo experiments revealed that T-cell cytokine production and cutaneous recall responses were significantly suppressed by LTA. Conclusion We identified a new mechanism through which bacterial compounds directly but temporarily modulate adaptive immune responses.

Fumarates suppress Th1 responses by blocking IL-12 and IL-23 production by dendritic cells via distinct pathways., Fumarates improve multiple sclerosis (MS) and psoriasis, two diseases in which both IL-12 and IL-23 promote pathogenic T helper (Th) cell differentiation. However, both diseases show opposing responses to most established therapies. First, we show in humans that fumarate treatment induces IL-4–producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. In mice, fumarates also generate type II DCs that induce IL-4–producing Th2 cells in vitro and in vivo and protect mice from experimental autoimmune encephalomyelitis. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by increased hemoxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. Induced HO-1 is cleaved, whereupon the N-terminal fragment of HO-1 translocates into the nucleus and interacts with AP-1 and NF-κB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19. As a consequence, GSH depletion by small molecules such as fumarates induces type II DCs in mice and in humans that ameliorate inflammatory autoimmune diseases. This therapeutic approach improves Th1- and Th17-mediated autoimmune diseases such as psoriasis and MS by interfering with IL-12 and IL-23 production.

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFκB, by inhibiting NFκB directly (parthenolide) or by interfering with the inactivation of the NFκB inhibitory protein IκB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca(2+) leakage or glutathione depletion. The present study utilized Western blotting to search for NFκB and IκB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca(2+) (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFκB and IκB-α are expressed in erythrocytes. Targeting the NFκB pathway by Bay 11-7082 (IC(50) ≈ 10 μM) and parthenolide (IC(50) ≈ 30 μM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca(2+) and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFκB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression.

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)Il6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

The balance between GSH-levels and oxidative stress is critical for cell survival. The GSH-levels of erythrocytes are dramatically decreased during infection with Plasmodium spp. We therefore investigated the consequences of targeting GSH for erythrocyte and Plasmodium survival in vitro and in vivo using dimethylfumarate (DMF) at therapeutically established dosage. We first show that noninfected red blood cells (RBC) exposed to DMF undergo changes typical of apoptosis or eryptosis, such as cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine (PS) exposure. DMF did not induce appreciable hemolysis. DMF-triggered PS exposure was mediated by intracellular GSH depletion and reversed by the antioxidative N-acetyl-l-cysteine. DMF treatment controlled intraerythrocyte DNA amplification and in vitro parasitemia of Plasmodium falciparum -infected RBC. In vivo, DMF treatment had no effect on RBC count or GSH levels in noninfected mice. Consistent with its effects on infected RBC, DMF treatment abrogated parasitemia and enhanced the survival of mice infected with Plasmodium berghei from 0% to 60%. In conclusion, DMF sensitizes the erythrocytes to the effect of Plasmodium infection on PS exposure, thus accelerating the clearance of infected erythrocytes. Accordingly, DMF treatment favorably influences the clinical course of malaria. As DMF targets mechanisms within the host cell, it is not likely to generate resistance of the pathogen.

Tumor and immune cells express inhibitory immune checkpoints (ICP) that paralyze tumor infiltrating T cells. ICP-specific antibodies (mAbs) can block and restore T cell functions and promote anti-tumoral effects. Also, tumor-antigen specific IFN-y secreting CD4+ T cells (TA-Th1) mediate strong anti-tumoral effects and can induce senescence in cancer cells. Here, we report on the development and immunological characterization of a highly efficient TA-Th1 and checkpoint-inhibitor based combined immunotherapy (CIT) in a progressed endogenous cancer model. Mice bearing progressed pancreatic islet carcinomas (RIP1-Tag2) and symptomatic low blood glucose levels (BGL; ~80 mg/dl) were treated weekly with TA-Th1 and αPD-L1+αLAG-3 mAbs (PDL1/LAG3) after an initial preparative 2 Gy-whole body radiation. Therapy was monitored by BGL measurements. Additionally, we conducted baseline and follow-up 18F-FDG PET/MRI scans to uncover the splenic glucose metabolism as a consequence of immune cell activation. We performed ex vivo flow cytometry (FC), immunohistochemistry (IHC), and fluorescent microscopy (FM) of tumor tissue and lymphatic organs at early (1 wk) and late (3-4 wks; endpoint of SHAM treated mice) time point of treatment focusing on the immune cell composition, activation patterns and senescence induction. Treatment exclusively with TA-Th1 prolonged the median survival of the mice from 14 to 18 wks (n=14) while ICP blockade without TA-Th1 cells was not efficient at all (PDL1/LAG3; median survival=14wks, n=11). The combination of TA-Th1 and PDL1/LAG3 (CIT) was highly efficient and significantly extended the median survival to 20 wks (n=15, p=0.001). Thus, exclusively CIT-treated mice revealed very small tumors and a strong lymphocytic infiltrate. FC analysis demonstrated early and specific homing of highly activated (CD69) TA-Th1 into the draining LN without impairment of the endogenous CD8+ T cell population. After 4 wks of treatment, we determined an increase in endogenous effector CD4+ and CD8+ T cells exclusively in mice receiving TA-Th1, indicating cross-priming as a possible mechanism of T cell activation. Analysis of p16/Ki67 expression in tumors revealed a strongly enhanced p16 and reduced Ki67 expression exclusively in CIT-treated mice indicating tumor senescence which was not observed in control groups. 18F-FDG-PET/MRI of the spleen showed a significantly increased glucose metabolism in CIT treated mice when compared to SHAM-treatment. In conclusion, our newly developed CIT (2 Gy+TA-Th1+PDL1/LAG3) strongly promoted an anti-tumor immune response in mice with progressed solid cancer. Most importantly, only CIT was able to induce TA-Th1 mediated tumor senescence whereas TA-Th1 or PDL1/LAG3 mAbs alone were inefficient. Moreover, 18F-FDG-PET/MRI of the spleen might represent a novel powerful tool to stratify ICP-blocked responders. Thus, ICP-blockade is applicable to reinforce Th1-cell based immunotherapies and to prolong the lifespan of mice with progressed solid carcinomas. Citation Format: Barbara F. Schörg, Dominik Sonanini, Johannes Schwenck, Christoph Griessinger, Birgit Fehrenbacher, Martin Schaller, Ursula Kohlhofer, Leticia Quintanilla-Martinez, Christian La Fourgère, Martin Röcken, Bernd J. Pichler, Manfred Kneilling. Combining CD4+ T cell transfer and immune checkpoint blockade demonstrates the power of combined immunotherapies for treatment of progressed solid carcinomas in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-105.

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell–mediated autoimmune diseases. By dissecting Th1 cell–mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into KitW/KitW-v, TNF−/−, and TNFR1−/− mice showed that the signaling defect exclusively affects mast cell–EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1−/− mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1−/− mice. As substitution of TNF−/− mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.

Psoriasis pathogenesis is closely associated with disease-inducing Th1 and Th17 cells. Yet, several studies suggest that aberrant keratinocyte or endothelial cell signalling significantly contributes to disease manifestation. Histological hallmarks of psoriatic skin include the infiltration of multiple immune cells, keratinocyte proliferation and increased dermal vascularity. Formation of new blood vessels starts with early psoriatic changes and disappears with disease clearance. Several angiogenic mediators like vascular endothelial growth factor, hypoxia-inducible factors, angiopoietins and pro-angiogenic cytokines, such as tumour necrosis factor (TNF), interleukin (IL)-8 and IL-17, are up-regulated in psoriasis development. Contact- and mediator-dependent factors derived from keratinocytes, mast cells and immune cells may contribute to the strong blood vessel formation of psoriasis. New technologies and experimental models provide new insights into the role of angiogenesis in psoriasis pathogenesis. Interestingly, many therapies target not only immune cells, but also protein structures of endothelial cells. Here we summarize the role of pro-angiogenic factors in psoriasis development and discuss angiogenesis as a potential target of novel therapies.

Experimental tumor vaccination and adoptive T-cell therapies show that interferon-γ (IFN-γ)–producing CD4+ T helper cells (Th1) can be highly effective in tumor prevention and therapy. Unexpectedly, first vaccine trials in humans revealed that tumor immune therapy may not only be protective, but, on the contrary, even promote tumor progression. Here, we analyzed T-cell immune responses to the epithelial cell adhesion molecule (EpCAM), one of the most common tumor-associated antigens (TAA) serving as immune target in colon cancer patients. Th-cell priming against EpCAM inevitably resulted in interleukin-4 (IL-4)–dominated Th2 responses, even under most stringent Th1-inducing conditions. These EpCAM-reactive Th2 cells rather promoted growth of EpCAM-expressing tumors. To analyze the role of IL-4 in tumor immune evasion, we generated EpCAM-reactive Th1 cells from IL-4.ko mice. These Th1 cells provided tumor-specific protection and established highly protective Th1 memory responses, even in naive BALB/c mice. Inhibition of tumor growth by Th1 cells resulted in intra-tumoral expression of cytokines of the IL-12 family and of IFN-γ. Preventing activation-associated death of Th1 cells further increased intratumoral IFN-γ expression and improved therapeutic efficacy. Thus, human TAA may promote tumor immune evasion by strongly favoring Th2 development.

Adoptive transfer of tumor antigen-specific T helper (Th) cells is a surprisingly potent anti-tumor therapy. Even in RIP1-Tag2 mice with a rapidly growing, aggressive endogenous beta cell tumor Th can significantly extend life time and are more efficient than any other therapy studied. The therapeutic effect of Th cells seems to be independent of tumor cell destruction. It critically relies on three principles: (1) inhibition of tumor angiogenesis, (2) inhibition of beta cell proliferation, and (3) induction of tumor dormancy. As tumor cell destruction by cytotoxic CD8(+) T cells (CTL) largely failed in tumor therapy, induction of tumor dormancy by Th cell-mediated immune responses represents a novel therapeutic option that may be combined with other cytotoxic regimens, e.g., radio- and/or chemotherapy, as it is established for bone marrow transplantation. Importantly, Th cell efficacy strictly requires interferon gamma (IFNgamma) signaling, and in the absence of IFNgamma, Th cells may even worsen tumor diseases. Therefore, using the immune system to control tumor dormancy represents a novel approach, especially as therapy of tumors resistant to conventional therapies. Yet, it is important to underline that Th cell-based antitumor effects critically depend on a functional cytokine network, especially appropriate IFNgamma signaling.

In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly potent VEGF-R2/3 inhibitor with IC 50 of 31/37 nM. The novel 3,4-diaryl-2 H-pyrrole-2-ones were designed on the basis of the modeled binding mode of the corresponding 1 H-pyrrole-2,5-dione (maleimide) VEGF-R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2 H-pyrrole-2-ones and structure-activity relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC 50 values) are presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.

Tumor-specific T cells are crucial for immunologic control of malignant disease. T cells can be induced in vivo by vaccination or adoptively transferred after activation ex vivo. We investigated the requirements for generating T cells with optimal antitumor effector functions in a murine lymphoma model. Using adoptive transfer, we show that in vivo efficacy of T cells cannot be predicted by tumor reactivity in vitro. A restricted T-cell receptor β chain repertoire of T-cell populations stimulated ex vivo against tumor cells was necessary but not sufficient for tumor protectivity. Tumor elimination furthermore required vaccination of donor mice, hence in vivo priming. The in vivo priming step may allow tumor-specific T cells to accumulate in vitro more rapidly and to survive for longer periods after withdrawal of the antigenic stimulus and adoptive transfer. A possible survival benefit of in vivo induced T cells may be ascribed to the responsiveness to homeostatic cytokines and to unique cytokine milieus encountered in vivo. Most importantly, monoclonal T cells cannot inhibit tumor growth. A prerequisite of tumor rejection was the expression of at least 2 T-cell receptor β chains by transferred T-cell populations. This finding has implications for designing adoptive transfer strategies for the clinic. © 2008 Wiley-Liss, Inc.

The vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (VEGF/VEGFR-2) signal transduction system plays a key role during embryonic vascular development and adult neovascularization. In contrast to many endothelial genes, VEGFR-2 is expressed at low levels in most adult vessels but is strongly upregulated during neovascularization, leading to a pro-angiogenic response. Here, we analyzed the activity of regulatory sequences of the murine Vegfr2 gene during neovessel formation in vivo under ischemic and inflammatory conditions. Hindlimb ischemia was induced in transgenic mice, expressing the LacZ reporter gene under the control of Vegfr2 promoter/enhancer elements. Most vessels in the ischemic muscle tissue showed strong endothelium-specific reporter gene expression, whereas nearly no LacZ-expressing capillaries were observed in untreated control tissue. Cutaneous punch wounds were created to induce angiogenesis under inflammatory conditions, leading to robust LacZ expression in the majority of the blood vessels in the wound tissue. Since the cornea is physiologically avascular, the functionality of these promoter/enhancer elements exclusively in newly formed vessels was confirmed using the cornea micropocket assay. Taken together, our results show that these Vegfr2 regulatory elements are active during adult neovessel formation in general. Therefore, these sequences may prove to be valuable targets for novel endothelium-specific anti-angiogenic as well as pro-angiogenic treatment strategies. They may especially allow directing therapeutic gene expression to sites of adult neovascularization. Moreover, the Vegfr2/LacZ reporter mice represent a powerful model to generally analyze the transcriptional control mechanisms involved in the induction of Vegfr2 expression during adult neovascularization.

Objective Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is entirely independent of T cells and B cells but is strictly dependent on the presence of mast cells. Here, we used this disease model to analyze whether exclusive intraarticular mast cell reconstitution is sufficient for disease induction and whether targeted mast cell silencing can prevent neoangiogenesis and joint destruction, 2 hallmarks of rheumatoid arthritis. Methods Ankle swelling and clinical index scores were determined after injection of either K/BxN mouse–derived serum or control serum in wild-type Kit+/Kit+ mice, congenic mast cell–deficient KitW/KitW-v mice, or mast cell–deficient KitW/KitW-v mice reconstituted with mast cells, either by intraperitoneal or selective intraarticular injection. Angiogenesis was quantified in vivo by measuring activated αvβ3 integrin using 18F–galacto-RGD and positron emission tomography. In addition, staining of joint tissue with hematoxylin and eosin, Giemsa, β3, and α-actin was performed. The effect of mast cell stabilization by treatment with cromolyn or salbutamol was investigated in C57BL/6 or BALB/c mice. Results Comparing wild-type mice, mast cell–deficient KitW/KitW-v mice, and mast cell–reconstituted KitW/KitW-v mice, we first showed that intraarticular and intraperitoneal mast cell engraftment fully restores susceptibility to antibody-induced arthritis, angiogenesis, and αvβ3 integrin activation. Importantly, selective mast cell silencing with either salbutamol or cromolyn prevented αvβ3 integrin activation, angiogenesis, and joint destruction. Conclusion Mast cell engraftment fully restores susceptibility to αvβ3 integrin activation, angiogenesis, and joint destruction in GPI antibody–induced arthritis. Importantly, selective mast cell stabilization prevents αvβ3 integrin activation, angiogenesis, and joint destruction.

As one of the largest organs, the skin forms a mechanical and immunological barrier to the environment. The skin immune system harbors cells of the innate immune system and cells of the adaptive immune system. Signals of the innate immune system typically initiate skin immune responses, while cells and cytokines of the adaptive immune system perpetuate the inflammation. Skin immune responses ensure effective host defense against pathogens but can also cause inflammatory skin diseases. An extensive crosstalk between the different cell types of the immune system, tissue cells, and pathogens is responsible for the complexity of skin immune reactions. Here we summarize the major cellular and molecular components of the innate and adaptive skin immune system.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.

Interleukin 4 (IL-4) can suppress delayed-type hypersensitivity reactions (DTHRs), including organ-specific autoimmune diseases in mice and humans. Despite the broadly documented antiinflammatory effect of IL-4, the underlying mode of action remains incompletely understood, as IL-4 also promotes IL-12 production by dendritic cells (DCs) and IFN-γ-producing T(H)1 cells in vivo. Studying the impact of IL-4 on the polarization of human and mouse DCs, we found that IL-4 exerts opposing effects on the production of either IL-12 or IL-23. While promoting IL-12-producing capacity of DCs, IL-4 completely abrogates IL-23. Bone marrow chimeras proved that IL-4-mediated suppression of DTHRs relies on the signal transducer and activator of transcription 6 (STAT6)-dependent abrogation of IL-23 in antigen-presenting cells. Moreover, IL-4 therapy attenuated DTHRs by STAT6- and activating transcription factor 3 (ATF3)-dependent suppression of the IL-23/T(H)17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/T(H)17 responses without blocking IL-12/T(H)1, selective IL-4-mediated IL-23/T(H)17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12-dependent T(H)1 responses.

Since the first description of the subpopulations of TH1 and TH2 cells, insights into the development and control of these cells as two polarized and physiologically balanced subsets have been generated. In particular, implications of the TH1-TH2 concept for TH cell–mediated skin disorders have been discovered. This article will review the basic factors that control the development of TH1 and TH2 cells, such as the cytokines IL-12 and IL-4 and transcription factors, the possible role of costimulatory molecules, and specialized dendritic cell populations. These regulatory mechanisms will be discussed in the context of polarized TH1 or TH2 skin disorders such as psoriasis and atopic dermatitis. Also presented are the principles that govern how chemokines and chemokine receptors recruit TH1 and TH2 cells to inflammatory sites and how they amplify these polarized TH cell responses. All of these concepts, including a novel role for IL-4–inducing TH1 responses, can contribute to the design of better therapeutic strategies to modulate TH cell–mediated immune responses.

Psoriasis is an autoimmune disease affecting 2-4% of the Caucasian population. Inflammatory processes induce the migration of interferon (IFN) gamma producing Th1 lymphocytes into the skin. These play a key role in the pathogenesis of psoriasis. These Th1 lymphocytes are responsible for the pathological reactions in psoriatic skin leading to keratinocyte hyperproliferation, small vessel proliferation and neutrophilic infiltration. Antigen-presenting cells activate dermal CD4+ T lymphocytes, and various signals can support the polarization of Th1 responses. The main signal for Th1 development is interleukin (IL) 12. After binding to their receptors both IL-12 and IFN-gamma promote intracellular IFN-gamma production by activating signal transducer and activator of transcription (STAT) 4 or 1. STAT1 activation by IFN-gamma is followed by T-bet activation, a master transcription factor for Th1 lymphocytes. In experimental models of Th1-mediated autoimmune diseases immune deviation of polarized autoreactive Th1 into anti-inflammatory Th2 responses generally improves the disease. Therefore new therapeutic approaches based on immunomodulating molecules have been developed for psoriasis, a prototypical Th1-mediated autoimmune disorder. Recently IL-4, the most effective Th2-inducing cytokine, has been shown to be safe and efficient for treating psoriasis. Improvement was associated with the induction of a Th2 phenotype of skin infiltrating lymphocytes. This review summarizes the IL-4 inducing potential of various conventional and newer systemic therapies for psoriasis. Many of these were thought to be primarily immunosuppressive. A review of the literature reveals that most of them can induce IL-4 and Th2, and that Th2 induction may be an underestimated mode of action in the therapy of Th1-mediated autoimmune disease. Further studies are needed to determine the central role of IL-4 in the control of Th1-induced autoimmune disease, namely psoriasis.

Psoriasis is a chronic inflammatory disease of the skin and the joints. Multiple factors contribute to the initiation of psoriasis. They include specific genetic characteristics such as major histocompatibility antigens and psoriasis susceptibility genes, as well as trigger factors, namely streptococcal infections. Today, psoriasis is considered as a T-lymphocyte mediated autoimmune disease, even though the putative autoantigen remains unknown. Bacterial proteins with similarity to structural proteins of keratinocytes are potential target antigens. As in other autoimmune diseases, inflammatory cytokines of the innate immune system initiate a cascade that activates inflammation locally in the skin, in the circulation and most likely also in lymph nodes. IFN-gamma-producing CD4+ Th1-lymphocytes seem to be of central importance in the pathogenesis of psoriasis as they critically influence differentiation and functioning of antigen presenting cells, mast cells, neutrophils and endothelial cells. This inflammatory cascade simultaneously provokes neoangiogenesis in the dermis and proliferation of keratinocytes. Based on this hypothesis, cytokines or anticytokine antibodies that either inhibit T-cell mediated inflammation or transform disease-inducing, pro-inflammatory Th1-lymphocytes into a phenotype with anti-inflammatory properties were tested in psoriasis. As both approaches improved psoriasis, they strongly support the current concept that views psoriasis as a Th1-lymphocyte mediated disease.

Selective skewing of autoreactive interferon-gamma (IFN-gamma)-producing T helper cells (Th1) toward an interleukin-4 (IL-4)-producing (Th2) phenotype can in experimental animals alleviate autoimmune disease without inducing general immunosuppression. In a prospective dose escalation study, we assessed treatment with human IL-4 (rhuIL-4) in 20 patients with severe psoriasis. The therapy was well tolerated, and within six weeks all patients showed decreased clinical scores and 15 improved more than 68%. Stable reduction of clinical scores was significantly better at 0.2-0.5 microg rhuIL-4 than at < or =0.1 microg rhuIL-4 (P = 0.009). In psoriatic lesions, treatment with 0.2-0.5 microg/kg rhuIL-4 reduced the concentrations of IL-8 and IL-19, two cytokines directly involved in psoriasis; the number of chemokine receptor CCR5+ Th1 cells; and the IFN-gamma/IL-4 ratio. In the circulation, 0.2-0.5 microg/kg rhuIL-4 increased the number of IL-4+CD4+ T cells two- to three-fold. Thus, IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and has promise as a potential treatment for psoriasis, a prototypic Th1-associated autoimmune disease.

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP κ L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while κ L chains act via another C-independent but mast cell-dependent pathway.