Emmanuella Guenova

Melanoma Cell-Intrinsic PD-1 Receptor Functions Promote Tumor Growth

Therapeutic antibodies targeting programmed cell death 1 (PD-1) activate tumor-specific immunity and have shown remarkable efficacy in the treatment of melanoma. Yet, little is known about tumor cell-intrinsic PD-1 pathway effects. Here, we show that murine and human melanomas contain PD-1-expressing cancer subpopulations and demonstrate that melanoma cell-intrinsic PD-1 promotes tumorigenesis, even in mice lacking adaptive immunity. PD-1 inhibition on melanoma cells by RNAi, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, as does engagement of melanoma-PD-1 by its ligand, PD-L1, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuate growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor modulates downstream effectors of mTOR signaling. Our results identify melanoma cell-intrinsic functions of the PD-1:PD-L1 axis in tumor growth and suggest that blocking melanoma-PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy.

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression

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.

Human TH9 Cells Are Skin-Tropic and Have Autocrine and Paracrine Proinflammatory Capacity

Aberrant activation of human TH9 cells may contribute to inflammatory diseases of the skin. TH9 Cells: Immune Cell Specialists One of the main strengths of the immune system is its diverse strategies for fighting off infection. There are the stalwart innate cells and more flexible adaptive cells. However, even among these groups are many more specialized subsets, adapted to fight particular foes. Schlapbach et al. describe a subset of proinflammatory human helper T cells—TH9 cells—that may contribute to inflammatory diseases of the skin. Although TH9 cells had been described in mouse models, little was known about the role of TH9 cells in humans. The authors found that most of these cells were either skin-tropic or skin-resident. These cells produced interleukin-9 but not cytokines common to other T helper cell subsets, and many were specific for Candida albicans, suggesting a protective role against infection. However, these cells were also increased in psoriasis lesions, suggesting that they may contribute to inflammatory disease as well. T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice, but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells coexpressed tumor necrosis factor–α and granzyme B and lacked coproduction of TH1/TH2/TH17 cytokines, and many were specific for Candida albicans. Interleukin-9 (IL-9) production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of interferon-γ, IL-9, IL-13, and IL-17 by skin-tropic T cells. IL-9–producing T cells were increased in the skin lesions of psoriasis, suggesting that these cells may contribute to human inflammatory skin disease. Our results indicate that human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.

Natural Staphylococcus aureus-derived peptidoglycan fragments activate NOD2 and act as potent costimulators of the innate immune system exclusively in the presence of TLR signals

Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus‐derived pathogen‐associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGNpolymer) preparations activated NF‐κB through human Toll‐like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGNpolymer from lgt‐mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGNpolymer, but not PGN itself, activate TLR2. Thus, HPLC‐purified monomeric PGN (PGNmonomer) structures were investigated. Strikingly, PGNmonomer completely lacked NF‐κB activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGNmonomer in concert with various TLR ligands most effectively stimulated DCs to up‐regulate IL‐12p70 and IL‐23 by ≥3‐ to 5‐fold. Consequently, DCs coactivated by PGNmonomer markedly up‐regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGNmonomer failed to coactivate NOD2−/− DCs. This demonstrates that PGNmonomer is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus‐derived PAMPs, including PGNmonomer, providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.—Volz, T., Nega, M., Buschmann, J., Kaesler, S., Guenova, E., Peschel, A., Röcken, M., Götz, F., Biedermann, T. Natural Staphylococcus aureus‐derived peptidoglycan fragments activate NOD2 and act as potent costimulator of the innate immune system exclusively in the presence of TLR signals. FASEB J. 24, 4089–4102 (2010). www.fasebj.org

Nonpathogenic Bacteria Alleviating Atopic Dermatitis Inflammation Induce IL-10-Producing Dendritic Cells and Regulatory Tr1 Cells

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-γ(low) IL-10(high) Tr1 (type 1 regulatory T) cells. These IL-10(high) 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.

Interleukin 23 Expression in Pyoderma Gangrenosum and Targeted Therapy With Ustekinumab

BACKGROUND Interleukin (IL)-23 is involved in the pathogenesis of the chronic inflammatory Crohn disease. Pyoderma gangrenosum (PG) is often associated with and can even be the first manifestation of this disease and has abundant neutrophilic infiltration. Because IL-23 plays a critical role in driving inflammation associated with IL-17 production and especially neutrophil recruitment, we suspect that PG might be driven by a pathogenetic mechanism similar to that of inflammatory bowel diseases or psoriasis. OBSERVATIONS Tissue sample analysis showed highly elevated expression of IL-23 on both transcriptional and protein level in a recalcitrant PG lesion. On the basis on these data, a treatment targeting the p40 subunit of the heterodimeric IL-23 with the monoclonal antibody ustekinumab was started. Therapy with ustekinumab resulted in a significant decrease of IL-23 expression in PG and healing after 14 weeks of treatment. No relapse occurred in a 6-month follow-up period. CONCLUSIONS Our data provide evidence of an IL-23-dominated inflammatory infiltrate in PG. This might specify a new concept for PG pathophysiology and suggests a possibility for using ustekinumab as a therapeutic agent in this disease.

IL-4 abrogates T(H)17 cell-mediated inflammation by selective silencing of IL-23 in antigen-presenting cells.

Significance Interleukin 4 (IL-4) has been shown to be highly protective against delayed type hypersensitivity and organ-specific autoimmune and autoinflammatory reactions in mice and humans, but its mode of action has remained controversial and has failed to be explained solely by redirection of TH1/TH17 toward a TH2-type immune response. Here we uncovered that IL-4 selectively suppresses IL-23 transcription and secretion by cells of the innate immune system. We further describe a previously unidentified therapeutic mode of action of IL-4 in TH17-mediated inflammation, and a physiologically highly relevant approach to selectively target IL-23/TH17-dependent inflammation while sparing IL-12 and TH1 immune responses. 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 TH1 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/TH17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/TH17 responses without blocking IL-12/TH1, selective IL-4–mediated IL-23/TH17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12–dependent TH1 responses.

Toll-like receptor 2 ligands promote chronic atopic dermatitis through IL-4–mediated suppression of IL-10

BACKGROUND Atopic dermatitis (AD) is a T cell-mediated inflammatory skin disease, with TH2 cells initiating acute flares. This inflamed skin is immediately colonized with Staphylococcus aureus, which provides potent Toll-like receptor (TLR) 2 ligands. However, the effect of TLR2 ligands on the development of TH2-mediated AD inflammation remains unclear. OBJECTIVE We investigated the progression of TH2 cell-mediated dermatitis after TLR2 activation. METHODS Using models for acute AD with TH2 cells initiating cutaneous inflammation, we investigated the consequences of TLR2 activation. Dermatitis, as assessed by changes in ear skin thickness and histology, was analyzed in different BALB/c and C57BL/6 wild-type and knockout mouse strains, and immune profiling was carried out by using in vitro and ex vivo cytokine analyses. RESULTS We show that TH2 cell-mediated dermatitis is self-limiting and depends on IL-4. Activation of TLR2 converted the limited TH2 dermatitis to chronic cutaneous inflammation. We demonstrate that the concerted activation of TLR2 and IL-4 receptor on dendritic cells is sufficient for this conversion. As an underlying mechanism, we found that the combinatorial sensing of the innate TLR2 ligands and the adaptive TH2 cytokine IL-4 suppressed anti-inflammatory IL-10 and consequently led to the exacerbation and persistence of dermatitis. CONCLUSION Our data demonstrate that innate TLR2 signals convert transient TH2 cell-mediated dermatitis into persistent inflammation, as seen in chronic human AD, through IL-4-mediated suppression of IL-10. For the first time, these data show how initial AD lesions convert to chronic inflammation and provide another rationale for targeting IL-4 in patients with AD, a therapeutic approach that is currently under development.

TH2 Cytokines from Malignant Cells Suppress TH1 Responses and Enforce a Global TH2 Bias in Leukemic Cutaneous T-cell Lymphoma

Purpose: In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. Experimental Design: We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. Results: Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. Conclusions: A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses. Clin Cancer Res; 19(14); 3755–63. ©2013 AACR.

IL-4-mediated fine tuning of IL-12p70 production by human DC.

IL‐4 is expressed at high levels in allergic diseases and dominates the early phases of multiple acquired immune responses. However, the precise role of IL‐4 during early inflammation and its impact on the differentiation of newly recruited DC precursors remains elusive. In order to characterize the impact of IL‐4 on the differentiation of human DC, we investigated the role of IL‐4 on the differentiation of monocytes into DC. Human DC were differentiated from peripheral blood precursors under either low or high concentrations of IL–4. We analyzed their cytokine profile and capacity to polarize T‐cell differentiation. Concentrations of 5 (low) and 50 (high) ng/mL IL‐4 induced two distinct types of DC. DC differentiated under low‐dose IL‐4 (5 ng/mL) produced almost no IL–12p70, and primed naïve CD4+ T cells allowing IL‐4 secretion and Th2 induction. In contrast, DC generated under high concentrations of IL‐4 (50 ng/mL) produced large amounts of IL‐12p70, low IL‐10 and primed naïve CD4+ T cells to become Th1 cells. Thus, we demonstrate that the Th2 cell cytokine IL‐4 decisively determines the phenotype of ongoing immune responses by orchestrating the functional phenotype of newly immigrating DC precursors.