ABSTRACT
The development of systems that are more accurate and time-efficient in predicting safety and efficacy of target products in humans are critically important in reducing the cost and duration of pharmaceutical development. To circumvent some of the limitations imposed by the use of animal models, ex vivo systems, such as precision-cut lung slices (PCLS), have been proposed as an alternative for evaluating safety, immunogenicity and efficacy of vaccines and pharmaceuticals. In this study, we have established a human PCLS system and methodology for PCLS cultivation that can provide long-term viability and functionality in culture. Using these techniques, we found that cultured PCLS remained viable for at least 14 d in culture and maintained normal metabolic activity, tissue homeostasis and structural integrity. To investigate whether cultured PCLS remained functional, lipopolysaccharide (LPS) was used as a target stimulating compound. We observed that after an 18-hour incubation with LPS, cultured PCLS produced a set of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6 and IL-10 as well as the enzyme COX-2. Furthermore, cultured PCLS were shown to be capable of generating re-call immune responses, characterized by cytokine production, against antigens commonly found in routine vaccinations against influenza virus and tetanus toxoid. Taken together, these results suggest that human PCLS have the potential to be used as an alternative, high-throughput, ex vivo system for evaluating the safety, and potentially immunogenicity, of vaccines and pharmaceuticals.
Subject(s)
Drug Evaluation, Preclinical/methods , Lung/pathology , Organ Culture Techniques/methods , HumansABSTRACT
The cytokine IL-9, derived primarily from T-helper 9 (Th9) lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also has a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch-test sites from non-atopic patients were assayed using quantitative PCR and immunohistochemistry. The cytokines IFN-γ, IL-4, IL-17A, IL-9, and PU.1, a Th9 associated transcription factor, were elevated when compared with paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+ CD3+ and PU.1+ CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. Peripheral blood mononuclear cells from nickel-allergic patients, but not nonallergic controls, show significant IL-9 production in response to nickel. Blocking studies with mAbs to HLA-DR (but not HLA-A, -B, -C) or chloroquine significantly reduced this nickel-specific IL-9 production. In addition, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9(-/-) mice shows enhanced Th1 lymphocyte immune responses, when compared with wild-type mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.
Subject(s)
Dermatitis, Allergic Contact/immunology , Interleukin-9/immunology , Nickel/immunology , Th1 Cells/immunology , Adult , Aged , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Dermatitis, Allergic Contact/genetics , Dermatitis, Allergic Contact/metabolism , Female , Humans , Interleukin-4/immunology , Interleukin-4/metabolism , Interleukin-9/genetics , Interleukin-9/metabolism , Male , Mice , Mice, Inbred BALB C , Middle Aged , Proto-Oncogene Proteins/metabolism , Th1 Cells/metabolism , Trans-Activators/metabolismABSTRACT
Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⺠effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.
