Vincent Lombardi

Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β-mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells

BACKGROUND A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. OBJECTIVE To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. METHODS In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. RESULTS Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. CONCLUSION The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

Induction of Airway Hyperreactivity by IL-25 Is Dependent on a Subset of Invariant NKT Cells Expressing IL-17RB

IL-25 has been shown to induce Th2 responses and airway hyperreactivity (AHR) in mice, but the mechanism of action is not understood and it is unclear which cells mediate this disease. In this study we show that the receptor for IL-25, IL-17RB, is highly expressed on a subset of naive and activated CD4+ invariant NKT (iNKT) cells, but not on activated T cells. IL-17RB+ iNKT cells produced large amounts of Th2 cytokines that were substantially increased by IL-25 stimulation. Furthermore, IL-17RB+ iNKT cells were capable of restoring AHR in iNKT cell-deficient mice, whereas IL-17RB− iNKT cells failed to reconstitute AHR and lung inflammation. Finally, IL-17RB+ iNKT cells were detected in the lungs of wild-type mice, and induction of AHR by intranasal administration of IL-25 was significantly impaired in iNKT cell-deficient mice. Overall, our data suggest a critical role for iNKT cells in IL-25-mediated AHR. These results may lead to novel therapeutic approaches to target IL-17RB+ iNKT cells for the treatment of allergic asthma.

PD-L1 and PD-L2 modulate airway inflammation and iNKT-cell-dependent airway hyperreactivity in opposing directions

Interactions of the inhibitory receptor programmed death-1 (PD-1) with its ligands, programmed death ligand (PD-L)1 and PD-L2, regulate T-cell activation and tolerance. In this study, we investigated the role of PD-L1 and PD-L2 in regulating invariant natural killer T (iNKT)-cell-mediated airway hyperreactivity (AHR) in a murine model of asthma. We found that the severity of AHR and airway inflammation is significantly greater in PD-L2−/− mice compared with wild-type mice after either ovalbumin (OVA) sensitization and challenge or administration of α-galactosylceramide (α-GalCer). iNKT cells from PD-L2−/− mice produced significantly more interleukin (IL)-4 than iNKT cells from control mice. Moreover, blockade of PD-L2 interactions of wild-type iNKT cells in vitro with monoclonal antibodies (mAbs) resulted in significantly enhanced levels of IL-4 production. In contrast, PD-L1−/− mice showed significantly reduced AHR and enhanced production of interferon-γ (IFN-γ) by iNKT cells. iNKT-deficient Jα18−/− mice reconstituted with iNKT cells from PD-L2−/− mice developed high levels of AHR, whereas mice reconstituted with iNKT cells from PD-L1−/− mice developed lower levels of AHR compared with control. As PD-L2 is not expressed on iNKT cells but rather is expressed on lung dendritic cells (DCs), in which its expression is upregulated by allergen challenge or IL-4, these findings suggest an important role of PD-L2 on lung DCs in modulating asthma pathogenesis. These studies also indicate that PD-L1 and PD-L2 have important but opposing roles in the regulation of AHR and iNKT-cell-mediated activation.

Human Dendritic Cells Stimulated via TLR7 and/or TLR8 Induce the Sequential Production of Il-10, IFN-γ, and IL-17A by Naive CD4+ T Cells

Depending upon which TLRs are triggered, dendritic cells (DCs) may orient the differentiation of naive CD4+ T cells toward either Th1, Th2, regulatory T cells, or the recently defined Th17 lineage. In this study, we report that a dual stimulation of TLR4 and TLR7/8 with LPS plus R848 leads human monocyte-derived DCs (MoDCs) to produce multiple pro- and anti-inflammatory cytokines, including IL-10, IL-12, and IL-23. Surprisingly, a significant variability in the up-regulation of these cytokines is observed in DCs obtained from various healthy donors, with approximately one of three being “high responders.” High responding MoDCs stimulated via TLR4 and TLR7/8 induce naive allogeneic CD4+ T cell to secrete sequentially IL-10 and IFN-γ, and eventually IL-17A, whereas low responding MoDCs only stimulate IFN-γ production. Both TLR7 and TLR8 play a central role in this phenomenon: TLR4 triggering with LPS up-regulates TLR7 expression on human MoDCs from high responders, silencing of either TLR7 or TLR8 mRNAs inhibits cytokine production in LPS plus R848-treated MoDCs, and plasmacytoid DCs constitutively expressing high levels of TLR7 induce the production of IL-10, IFN-γ, and IL-17A by naive T cells when stimulated with R848 alone. Collectively, our results illustrate the synergy between TLR4 and TLR7/8 in controlling the sequential production of regulatory and proinflammatory cytokines by naive CD4+ T cells. The observed polymorphism in DC responses to such TLR-mediated stimuli could explain differences in the susceptibility to infectious pathogens or autoimmune diseases within the human population.

Toll-like receptor 2 agonist Pam3CSK4 enhances the induction of antigen-specific tolerance via the sublingual route.

Background Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies.

IL-10-Inducing Adjuvants Enhance Sublingual Immunotherapy Efficacy in a Murine Asthma Model

Background: IL-10-inducing adjuvants could enhance the efficacy of allergy vaccines in establishing allergen-specific tolerance.The aim of this study wasto identify such adjuvants using in vitro cultures of human and murine cells and to evaluate them in a therapeutic murine model of sublingual immunotherapy (SLIT). Methods: Adjuvants stimulating IL-10 gene expression by human or murine immune cells were tested sublingually in BALB/c mice sensitized to ovalbumin (OVA), assessing the reduction in airway hyperresponsiveness (AHR) by whole-body plethysmography. The induction of regulatory T cells (Treg) was evaluated using phenotypic and functional assays. T-cell proliferation in cervical lymph nodes (LNs) was assessed following intravenous transfer of CFSE-labelled OVA-specific T cells and FACS analysis. Results: A combination of 1,25-dihydroxyvitamin D3 plus dexamethasone (VitD3/Dex) as well as Lactobacillus plantarum were found to induce IL-10 production by human and murine dendritic cells (DCs). The former inhibits LPS-induced DC maturation, whereas L. plantarum induces DC maturation. Following stimulation with VitD3/Dex-pretreated DCs, CD4+ naïve T cells exhibit a Treg profile.In contrast, a Th1/Treg pattern of differentiation is observed in the presence of DCs treated with L. plantarum. Both adjuvants significantly enhance SLIT efficacy in mice, in association with either induction of Foxp3+ Treg cells (for VitD3/Dex) or proliferation of OVA-specific T cells in cervical LNs (for L. plantarum). Conclusions: Both VitD3/Dex and L. plantarum polarize naïve T cells towards IL-10-expressing T cells, through distinct mechanisms. As adjuvants, they both enhance SLIT efficacy in a murine asthma model.

Lactic acid bacteria as adjuvants for sublingual allergy vaccines.

We compared immunomodulatory properties of 11 strains of lactic acid bacteria as well as their capacity to enhance sublingual immunotherapy efficacy in a murine asthma model. Two types of bacterial strains were identified, including: (i) potent inducers of IL-12p70 and IL-10 in dendritic cells, supporting IFN-gamma and IL-10 production in CD4+ T cells such as Lactobacillus helveticus; (ii) pure Th1 inducers such as L. casei. Sublingual administration in ovalbumin-sensitized mice of L. helveticus, but not L. casei, reduced airways hyperresponsiveness, bronchial inflammation and proliferation of specific T cells in cervical lymph nodes. Thus, probiotics acting as a Th1/possibly Treg, but not Th1 adjuvant, potentiate tolerance induction via the sublingual route.

CD8α⁺β⁻ and CD8α⁺β⁺ plasmacytoid dendritic cells induce Foxp3⁺ regulatory T cells and prevent the induction of airway hyper-reactivity.

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8β as CD8α−β−, CD8α+β−, or CD8α+β+. In a mouse model of lung inflammation, adoptive transfer of CD8α+β− or CD8α+β+ pDCs prevents the development of airway hyper-reactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3+ regulatory T cells compared with CD8α−β− pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.

The Role of Costimulatory Molecules in Allergic Disease and Asthma

The prevalence of allergic diseases has increased rapidly in recent years. It is well established that the deleterious allergic response is initiated by T-cell recognition of major histocompatibility class II-peptide complexes at the surface of antigen-presenting cells. While this first signal gives antigen specificity to the adaptive immune response, a second nonspecific costimulatory signal is required by T cells to become fully activated. This signal is provided by interactions between antigen-presenting cells and T cells through molecules borne at the surfaces of the two cell types. Depending on the type of molecules involved, this secondary signal can promote the development of an inflammatory allergic reaction or may favor immune regulation. Several molecules of the B7 family (CD80, CD86, PD-1, ICOS, CTLA-4) and tumor necrosis factor receptor family (OX40, CD30, 4-1BB, Fas, CD27, CD40) play an important role in delivering costimulatory signals in early and late phases of allergic response. Therefore, costimulatory molecules involved in promotion or prevention of allergic immune responses are potential targets for the development of novel therapeutic approaches. This review aims to recapitulate our current understanding of the relationship between allergic diseases and costimulatory molecules.