Th2-associated responses and their control in parasitic helminth infections and related disorders

National Institute Of Allergy And Infectious Diseases

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Abstract

To investigate the heterogeneity of Th2 effectors subsets known to be expanded helminth-infected/HDM-sensitized patients (and various control subject cohorts), we characterized their phenotype and function using multiparameter 28-color panel flow cytometry. We identified 3 novel subsets driven by helminth infection and/or allergic sensitization based on a self-organizing map tool. We then sorted these 3 novel subsets and performed single cell RNAseq. Analyses indicated that helminth infection (irrespective of HDM-sensitization status) drives the expansion of a unique effector T cell subsets. To explore the mechanisms underlying the hyperreactive CD4+ response seen in filarial infected/HDM sensitized patients, we developed a murine model for asthmatic inflammation using HDM intranasal sensitization followed by infection with the roundworm Ascaris. We were able to show that environmental aeroallergens drive a lung-specific eosinophil-rich type-2-immune response. This effect is dependent on eosinophils triggered by innate signaling of IL-13Ra1 in the lung., as allergen sensitized-IL-13Ra1 deficient mice were unable to limit parasite development or numbers. To explore further the role played by Th2 cells (and in particular the IL-13/IL-13R signaling axis) in mediating this eosinophil-dependent phenomenon. Using single-cell techniques we showed further that HDM-driven pulmonary inflammation displays a profile characterized by TH2 effector cell induced IL-13 dominated eosinophilic inflammation. Using HDM-sensitized IL-13R1/ mice, we found a marked reduction in the influx of eosinophils into the lungs along with a significant downregulation of both CCL-11 and CCL-24. We further found that eosinophil trafficking to the lung relies on production of IL-13 driven CCL-11 and CCL-24 by fibroblasts and Ly6C+ (so-called classical) monocytes. Moreover, this IL-13 mediated eotaxin-dependent eosinophil influx to the lung tissue required IL-5induced eosinophilia. Finally, we demonstrated that this IL-13driven eosinophil-dominated pulmonary inflammation was critical for limiting bystander lung transiting Ascaris parasites in a model of allergy and helminth interaction. Moreover, preliminary analyses of bulk and single-cell sequencing of HDM-allergic mouse lungs revealed distinct transcriptional profiles for other important inflammatory mediators, including co-stimulatory molecules on T cells. One of the co-stimulatory molecules highly upregulated in effector memory CD4+ T cells in the lungs of HDM-sensitized mice was CD153 or CD30L. Therefore, in collaboration with Daniel Barber, LPD/NIAID, we examined the role of CD30L in the differentiation of allergen-specific Th2 cells in the lung tissue of HDM-sensitized mice. Our preliminary data demonstrated that the HDM-sensitization in the CD30L deficient mice failed to induce an allergen-specific memory CD4+ Th2 cell response in the lungs when compared with WT mice, indicated by the marked reduction in the frequency of allergen-specific IL-13 producing memory CD4 T cells, after in vitro stimulation with HDM extract. Most interestingly, the impairment in the development of an effector Th2 response in the CD30L deficient mice led to diminished HDM-specific IgE levels as well as lower eosinophils numbers in the lung tissue, a finding that suggests a role for CD30L in the pathogenesis of allergic responses in the tissue. Our next step will be to investigate whether anti-CD30L neutralizing antibodies during the effector phase of the HDM-sensitization model in mice could prevent the establishment of an IL-13 driven eotaxin-dependent eosinophil-mediated -type 2 allergic response in the lungs. In an unrelated but equally important set of studies (and in collaboration with Scott Smith at Vanderbilt) we used human hybridoma technology to immortalize IgE encoding B-cells from peripheral blood of subjects with filarial infections and elevated IgE thereby generating ,naturally occurring human IgE mAbs. B-cell cultures were screened in an unbiased manner for IgE production without regard to specificity. Isolated IgE mAbs were then tested for binding to Brugia malayi somatic extracts using ImmunoCAP, immunoblot, and ELISA. Immunoprecipitation followed by mass spectrometry proteomics was used to identify helminth antigens that were then expressed in Escherichia coli for IgE binding characterization. We isolated 56 discrete IgE mAbs from 7 individuals with filarial infections. From these mAbs, we were able to definitively identify 19 filarial antigens. All IgE mAbs targeted filarial excreted/secretory proteins, including a family of previously uncharacterized proteins. Interestingly, the transthyretin-related antigens acted as the dominant inducer of the filaria-specific IgE antibody response. These filaria-specific IgE mAbs were potent inducers of anaphylaxis when passively administered to human FcRIexpressing mice.