Lucia Filì

Phenotypic and functional features of human Th17 cells

T helper (Th) 17 cells represent a novel subset of CD4+ T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-γ (Th17/Th1), in the gut of patients with Crohns disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor RORγt, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor susceptibility to regulation by autologous regulatory T cells. Interestingly, these subsets also expressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of RORγt and the production of IL-17, but induced IFN-γ. These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17–producing peripheral blood and tonsillar CD4+ T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identification of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1.

Toll-Like Receptors 3 and 4 Are Expressed by Human Bone Marrow-Derived Mesenchymal Stem Cells and Can Inhibit Their T-Cell Modulatory Activity by Impairing Notch Signaling

Bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM‐derived MSCs expressed high levels of Toll‐like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor κB (NF‐κB) activity, as well as the production of interleukin (IL)‐6, IL‐8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged‐1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti‐Jagged‐1 neutralizing antibody and N[N‐(3,5‐difluorophenacetyl‐l‐alanyl)]‐S‐phenylglycine t‐butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T‐cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T‐cell response in the course of dangerous infections, such as those sustained by double‐stranded RNA viruses or Gram‐negative bacteria, respectively.

Human immature myeloid dendritic cells trigger a TH2-polarizing program via Jagged-1/Notch interaction.

BACKGROUND The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear. OBJECTIVE Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). METHODS IL-4 and IFN-gamma production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. RESULTS Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype. CONCLUSION CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components.

Modified Adenine (9-Benzyl-2-Butoxy-8-Hydroxyadenine) Redirects Th2-Mediated Murine Lung Inflammation by Triggering TLR7

Substitute adenine (SA)-2, a synthetic heterocycle chemically related to adenine with substitutions in positions 9-, 2-, and 8- (i.e., 9-benzyl-2-butoxy-8-hydroxyadenine), induces in vitro immunodeviation of Th2 cells to a Th0/Th1 phenotype. In this article, we evaluate the in vivo ability of SA-2 to affect Th2-mediated lung inflammation and its safety. TLR triggering and NF-κB activation by SA-2 were analyzed on TLR-transfected HEK293 cells and on purified bone marrow dendritic cells. The in vivo effect of SA-2 on experimental airway inflammation was evaluated in both prepriming and prechallenge protocols by analyzing lung inflammation, including tissue eosinophilia and goblet cell hyperplasia, bronchoalveolar lavage fluid cell types, and the functional profile of Ag-specific T cells from draining lymph nodes and spleens. SA-2 induced mRNA expression and production of proinflammatory (IL-6, IL-12, and IL-27) and regulatory (IL-10) cytokines and chemokines (CXCL10) in dendritic cells but down-regulated TGF-β. Prepriming administration of SA-2 inhibited OVA-specific Abs and Th2-driven lung inflammation, including tissue eosinophilia and goblet cells, with a prevalent Foxp3-independent regulatory mechanism. Prechallenge treatment with SA-2 reduced the lung inflammation through the induction of a prevalent Th1-related mechanism. In this model the activity of SA-2 was route-independent, but adjuvant- and Ag dose-dependent. SA-2-treated mice did not develop any increase of serum antinuclear autoantibodies. In conclusion, critical substitutions in the adenine backbone creates a novel synthetic TLR7 ligand that shows the ability to ameliorate Th2-mediated airway inflammation by a complex mechanism, involving Th1 redirection and cytokine-mediated regulation, which prevents autoreactivity.

Influence of total serum IgE levels on the in vitro detection of β‐lactams‐specific IgE antibodies

Background Allergic reactions to β‐lactams are a frequent cause of adverse drug reactions; the diagnosis is based on history, clinical examination, skin testing (prick and intradermal) and demonstration of serum‐specific IgE antibodies (Abs).

Perspectives in vaccine adjuvants for allergen-specific immunotherapy

The design of more powerful adjuvants is a tool of crucial interest to ameliorate vaccination strategies to reduce injections and/or dose of antigen, induce local immunity and obtain better protection. Effective anti-infectious vaccines should elicit protective TH1 responses, cytotoxic CD8+ cells and antibody-forming cells. However, cytokine microenvironment is a key point also in targeted therapeutic vaccinations, such as allergen-specific immunotherapy, where the interference with an already-existing but inappropriate immunity is required. In this case, safe, appropriately conditioning and potentially orally available adjuvants together with delivery to appropriate subsets of dendritic cells would be highly appreciated to properly boost innate immune cells. In fact, aluminium hydroxide, although safe, has been classically associated with the induction of a TH2 response to co-formulated antigens. Thus, detoxified lipopolysaccaride (MPL-A), CpG oligonucleotides, imidazoquinolines and adenine derivatives acting via innate sensors may represent improvements in therapeutic vaccinations for allergy as able to interfere with pathogenic TH2 cells with eventual induction of TH1 differentiation.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

The role of etanercept on the expression of markers of T helper 17 cells and their precursors in skin lesions of patients with psoriasis vulgaris.

Very recently, it has been demonstrated that CD161, retinoic acid—related orphan receptor γt (RORγt) and CC-chemokin receptor 6 (CCR6) can be considered good surface markers to detect T helper 17 cells and their precursors, T cell populations that are considered to play an important role in the pathogenesis of psoriasis. In the present study, we evaluate the clinical involvement by calculating the PASI score and the number of CD4+, CD161+, RORγt+ and CCR6+ cells before and after a 12-week course with etanercept or acitretin in patients with moderate-to-severe, plaque-type psoriasis vulgaris. Ten patients were given etanercept 50 mg twice weekly and 10 patients acitretin 0.4 mg/kg per day, both for 12 weeks. At the baseline and at the end of the treatment PASI was calculated, and skin biopsies were taken to evaluate the expression of CD4, CD161, RORyt and CCR6 by immunohistochemistry. As controls, 10 patients with atopic dermatitis (AD) were included in the study. After 12 weeks, PASI was significantly lower than at the baseline for both groups. However, etanercept-treated patients showed lower PASI than acitretin-treated ones. While CD4+ cell numbers were similar in both diseases, all the other markers, that are considered more specific for Th17 cells and their precursors, were more expressed in psoriasis than in AD. Furthermore, only etanercept, but not acitretin, was able to significantly reduce CD161+, RORγt+ and CCR6+ cells in skin lesions of patients with psoriasis. Our study provides further evidence of the role of Th17 pathway in the pathogenesis of psoriasis. Furthermore, our findings suggest that etanercept is able to downregulate the expression of the recently recognized markers of Th17 cells and their precursors CD161, RORγt and CCR6, while acitretin is not. This activity on the Th17 lineage may contribute to the efficacy of etanercept in the treatment of psoriasis.

Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation

A strategy to improve allergen‐specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8‐OH‐modified adenine. Humoral and cellular responses were analysed in allergen‐sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co‐culture system was also investigated. The nDer p 2‐Conj treatment in nDer p 2‐primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen‐driven interleukin‐13 (IL‐13) production in lung mononuclear cells and IgE, in comparison with nDer p 2‐treated mice. The increase of IgG2a paralleled that of interferon‐γ (IFN‐γ) and IL‐10 in allergen‐stimulated spleen cells. Similar effects were elicited by treatment with OVA‐Conj in an OVA‐driven BALB/c model. The nDer p 2‐Conj or OVA‐Conj redirected memory T helper type 2 cells towards the production of IL‐10 and IFN‐γ also in C57BL/6 mice and when subcutaneously administered. Interleukin‐10, IL‐12 and IL‐27 were produced in vitro by Conj‐stimulated bone marrow dendritic cells, whereas IL‐10 and IFN‐γ were up‐regulated in co‐cultures of CD11c+ and CD4+ T cells from Conj‐treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN‐γ‐ and IL‐10‐ producing memory T cells. The Conj effects on IL‐10−/− and IL‐12−/− mice confirmed the role of IL‐10 and IFN‐γ in inducing a protective and balanced redirection the T helper type 2‐mediated airway inflammation.

Drug fever after a single dose of amoxicillin-clavulanic acid

Although amoxicillin-clavulanic acid (AMX/CLV) can be associated with hypersensitivity reactions, ranging from minor and self-limiting to anaphylaxis, this is the first reported case of a reproducible maculopapular exathema (MPE) with drug fever occurring after a single therapeutic dose, not associated with drug reaction with eosinophilia and systemic symptoms (DRESS). This case presented with delayed onset skin hypersensitivity reactions and fever that were finally associated with increased neutrophil counts plus high C-reactive protein (CRP) levels. The patient was a 14-year-old Caucasian girl with a history of AMX/CLV-associated reactions. In 2012, during an Epstein Barr virus infection, she had an MPE during AMX/CLV therapy. In February 2014, she was given AMX/CLV for a febrile bacterial infection and noted another itchy MPE 12 hours after the first dose. She stopped the drug, and in May 2014, she was referred to the Allergy Unit of Anna Meyer Children’s Hospital for evaluation. Skin tests were performed according to the European Network for Drug Allergy recommendations in the following sequence: (a) skin prick testing (SPT) with 20 mg/mL AMX and AMX/ CLV; and (b) an intradermal (ID) testing with 1/100 and 1/10 dilutions of the full-strength (200 mg/mL) AMX and AMX/CLV concentration at 20to 30-minute intervals. Positive and negative controls for SPT and ID were obtained using histamine (ALK-Abello, Milan, Italy: 10 and 1 mg/mL) and normal saline. A positive skin test result was when the SPT wheal was greater than 3 mm and was accompanied by erythema with a negative saline control result or when the ID was greater than 3 mm from the initial wheal or an increase in the diameter of the initial wheal was associated with a flare and a negative saline control result within 15 minutes. ID tests were also read after 24-72 hours for delayed T-cell-mediated reactions. Specific IgE to amoxicillin, ampicillin, and penicillin G and V were measured (Immunocap RAST, Uppsala, Sweden) and were all negative. Because all of the skin and in vitro tests were negative, an open challenge drug provocation test (DPT) to AMX/CLV using 1/ 10, 2/10, and then 7/10 of the therapeutic dose every 30 minutes (50/mg/kg/day in 2 doses) was administered following the current guidelines. She had no acute reactions, and after 2 hours of observation, she was discharged. After 3 hours from the last dose, she was vomiting and had a fever (39.4 C), headache, and generalized erythema. After 8 hours, she had diarrhea and difficulty sleeping. She was transported to the emergency department of Anna Meyer Children’s Hospital where her vital signs, complete blood count, and CRP levels were found to be within the normal range. She was discharged with the suspicion of a concurrent viral infection. In October 2014, the ID tests with AMX/CLAV were again negative. The DPT with AMX/CLV was repeated with the same graded challenge protocol, and after 3 hours, she again noted vomiting and had a fever, headache, and an itchy MPE. Later she had difficulty sleeping and diarrhea. The next day, she was admitted to the Anna Meyer Children’s Hospital Allergy Unit, where her blood was drawn and the following results were observed: 12.3 mg/mL CRP (normal value < 0.50 mg/dL); white blood cells count (WBC) of 14,950 mmc; neutrophils 90%; and liver function and a erythrocyte sedimentation rate within the normal ranges. After 2 days, the MPE was reduced and the girl no longer had a fever (Figure 1). After a month, a lymphocyte transformation test (LTT), induction of hapten-specific short-term T-cell lines, and cytokine production measurements were performed (see this article’s Online Repository at www.jaci-inpractice.org). The LTT was strongly positive to both AMX and AMX/CLV (Figure 2, A), and the T-cell lines that were specific for AMX and AMX/CLV could be induced. The LTT positivity and induction of the hapten-specific T-cell lines were also confirmed 6 months apart from the reaction (data not shown). A flow cytometric analysis showed high percentages of TNF-a producing T-cell blasts (Figure 2, B). No significant production of CXCL8, IL-6, or IL-17 could be found (data not shown). Drug fever is a rare event occurring in only approximately 3%-5% of adverse drug reactions. In 18%-29% of patients who