Immune Sensing of Aeroallergen-Associated Double-Stranded RNA Triggers an IFN Response and Modulates Type 2 Lung Inflammation.

The innate immune sensing of allergens or allergen-associated components regulate the development of type 2 inflammatory responses. However, the underlying molecular basis by which allergens or allergen-associated components are detected by innate immune receptors remains elusive. In this study, we report that the most common aeroallergen, house dust mite (HDM), harbors a dsRNA species (HDM-dsRNA) that can activate TLR3-mediated IFN responses and counteract the development of an uncontrolled type 2 immune response. We demonstrate that the mouse strains defective in the dsRNA-sensing pathways show aggravated type 2 inflammation defined by severe eosinophilia, elevated level of type 2 cytokines, and mucus overproduction in a model of allergic lung inflammation. The inability to sense HDM-dsRNA resulted in significant increases in airway hyperreactivity. We further show that the administration of the purified HDM-dsRNA at a low dose is sufficient to induce an immune response to prevent the onset of a severe type 2 lung inflammation. Collectively, these results unveil a new role for the HDM-dsRNA/TLR3-signaling axis in the modulation of a type 2 lung inflammation in mice.

Short-term assays for mesenchymal stromal cell immunosuppression of T-lymphocytes.

Introduction: Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less. Methods: Three potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h. Results: Suppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h. Discussion: Takeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

STING activation in alveolar macrophages and group 2 innate lymphoid cells suppresses IL-33-driven type 2 immunopathology.

2'3'-cGAMP is known as a nonclassical second messenger and small immune modulator that possesses potent antitumor and antiviral activities via inducing the stimulator of IFN genes-mediated (STING-mediated) signaling pathway. However, its function in regulating type 2 immune responses remains unknown. Therefore, we sought to determine a role of STING activation by 2'3'-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2'3'-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperreactivity induced by IL-33 and a fungal allergen, Aspergillus flavus. Mechanistically, upon the respiratory delivery, 2'3'-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING/IFN regulatory factor 3/type I IFN signaling axis to induce the production of inhibitory factors containing IFN-α, which blocked the IL-33-mediated activation of group 2 innate lymphoid (ILC2) cells in vivo. We further demonstrated that 2'3'-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 cells in vitro. Taken together, our findings suggest that STING activation by 2'3'-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2'3'-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.