Concurrent measurement of microbiome and allergens in the air of bedrooms of allergy disease patients in the Chicago area.

The particulate and biological components of indoor air have a substantial impact on human health, especially immune respiratory conditions such as asthma. To better explore the relationship between allergens, the microbial community, and the indoor living environment, we sampled the bedrooms of 65 homes in the Chicago area using 23the patient-friendly Inspirotec electrokinetic air sampling device, which collects airborne particles for characterization of both allergens and microbial DNA. The sampling device captured sufficient microbial material to enable 16S rRNA amplicon sequencing data to be generated for every sample in the study. Neither the presence of HEPA filters nor the height at which the air sampling device was placed had any influence on the microbial community profile. A core microbiota of 31 OTUs was present in more than three quarters of the samples, comprising around 45% of the relative sequence counts in each bedroom. The most abundant single organisms were Staphylococcus, with other core taxa both human and outdoor-associated. Bacterial alpha diversity was significantly increased in bedrooms that reported having open windows, those with flowering plants in the vicinity, and those in homes occupied by dogs. Porphyromonas, Moraxella, Sutterella, and Clostridium, along with family Neisseraceae, were significantly enriched in homes with dogs; interestingly, cats did not show a significant impact on microbial diversity or relative abundance. While dog allergen load was significantly correlated with bacterial alpha diversity, the taxa that significantly correlated with allergen burden did not exclusively overlap with those enriched in homes with dogs. Alternaria allergen load was positively correlated with bacterial alpha diversity, while Aspergillus allergen load was negatively correlated. The Alternaria allergen load was also significantly correlated with open windows. Microbial communities were significantly differentiated between rural, suburban, and urban homes and houses that were physically closer to each other maintained significantly more similar microbiota. We have demonstrated that it is possible to determine significant associations between allergen burden and the microbiota in air from the same sample and that these associations relate to the characteristics of the home and neighborhoods.

Bedroom exposure to airborne allergens in the Chicago area using a patient-operated sampling device.

BACKGROUND: In current practice, allergens in vacuum-collected dust are used as surrogates for inhalable allergens. We developed an air-sampling device that can be used by patients for direct measurement of airborne allergen concentrations in their own homes. OBJECTIVE: To demonstrate the use of this device to establish allergen concentration reference ranges in a target population and to evaluate associations between patient-reported information and measured allergen concentrations. METHODS: Patients from 5 allergist's practices in the Chicagoland region were provided with instructions, questionnaires, informed consent forms, and samplers to run for 5 days in their bedrooms. Samples were collected from cartridges and assayed by multiplex immunoassays for 12 common household allergens and enzyme-linked immunosorbent assay for ragweed. RESULTS: Unique allergen profiles were obtained for 102 patient homes. Samples with allergen concentrations above the limit of detection were as follows: total dust mite, 28%; cat, 61%; dog, 64%; mouse, 12%; rat, 0%; cockroach, 4%; Alternaria, 6%; Aspergillus, 21%; birch pollen 1%; grass, 8%; and ragweed, 5%. Of those, 75 completed questionnaires, providing meta-data for further analysis. Pet allergens correlated significantly with number of pets owned. Humidity correlated with dust mite allergens, open windows with Alternaria and mouse allergens, and high-efficiency particulate air filter use with reduced levels of several allergens. Many other variables showed no significant correlations. CONCLUSION: The combination of ease of use, high air-sampling rate, and sensitive immunoassays permitted the measurement of airborne allergen concentrations in homes and establishment of reference ranges. Patient-reported information permitted identification of factors that could relate to allergen concentrations and suggested remedial measures.

STING-mediated DNA sensing promotes antitumor and autoimmune responses to dying cells.

Adaptive immune responses to Ags released by dying cells play a critical role in the development of autoimmunity, allograft rejection, and spontaneous as well as therapy-induced tumor rejection. Although cell death in these situations is considered sterile, various reports have implicated type I IFNs as drivers of the ensuing adaptive immune response to cell-associated Ags. However, the mechanisms that underpin this type I IFN production are poorly defined. In this article, we show that dendritic cells (DCs) can uptake and sense nuclear DNA-associated entities released by dying cells to induce type I IFN. Remarkably, this molecular pathway requires STING, but not TLR or NLR function, and results in the activation of IRF3 in a TBK1-dependent manner. DCs are shown to depend on STING function in vivo to efficiently prime IFN-dependent CD8(+) T cell responses to tumor Ags. Furthermore, loss of STING activity in DCs impairs the generation of follicular Th cells and plasma cells, as well as anti-nuclear Abs, in an inducible model of systemic lupus erythematosus. These findings suggest that the STING pathway could be manipulated to enable the rational design of immunotherapies that enhance or diminish antitumor and autoimmune responses, respectively.

Natural killer T (NKT)-B-cell interactions promote prolonged antibody responses and long-term memory to pneumococcal capsular polysaccharides.

Innate-like natural killer T (NKT) cells critically enhance cell and humoral immunity against infections through recognition of conserved microbial lipid antigens presented by CD1d-expressing antigen-presenting cells, and provision of CD40L and cytokine signals. Whereas NKT cells efficiently licensed dendritic cells to prime potent effector and memory T cells, studies based on model antigens such as alphagalactosylceramide-nitrophenyl conjugates concluded that help to B cells was associated with NKT follicular helper differentiation, but limited to short-term responses without induction of memory. We revisited this surprising conclusion in the context of the extracellular encapsulated pathogen Streptococcus pneumoniae, where recognition of lipid and capsular polysaccharide antigens by NKT cells and B cells, respectively, provide critical host protection. Using liposomal nanoparticles displaying synthetic lipid and polysaccharide antigens to elicit pure and direct NKT-B-cell interactions in vivo, we observed intense and prolonged antibody responses with isotype switch, affinity maturation, and long-lasting B-cell memory, despite modest or absent NKT follicular helper differentiation. Furthermore, conditional ablation of Cd1d demonstrated a requirement for a two-step process involving first cognate interactions with dendritic cells, for NKT cell activation, and then with B cells, for induction of isotype switch and memory. Thus, NKT help to B cells represents both a major arm of antimicrobial defense and a promising target for B-cell vaccines.

Distinct APCs explain the cytokine bias of α-galactosylceramide variants in vivo.

α-Galactosylceramide represents a new class of vaccine adjuvants and immunomodulators that stimulate NKT cells to secrete Th1 and Th2 cytokines. Synthetic variants with short or unsaturated acyl chains exhibit a striking Th2 bias in vivo but no evidence of defect in TCR signaling or stimulation of NKT cells in vitro. Using cd1d1(fl/fl) mice, we demonstrated that distinct APC types explained the cytokine bias in vivo. Whereas NKT stimulation by α-Galactosylceramide required CD1d expression by dendritic cells (DCs), presentation of the Th2 variants was promiscuous and unaffected by DC-specific ablation of CD1d. This DC-independent stimulation failed to activate the feedback loop between DC IL-12 and NK cell IFN-γ, explaining the Th2 bias. Conversely, forced presentation of the Th2 variants by DC induced high IL-12. Thus, lipid structural variations that do not alter TCR recognition can activate distinct Th1 or Th2 cellular networks by changing APC targeting in vivo.

Prolonged antigen storage endows merocytic dendritic cells with enhanced capacity to prime anti-tumor responses in tumor-bearing mice.

Tumor cell vaccination with irradiated autologous tumor cells is a promising approach to activate tumor-specific T cell responses without the need for tumor Ag identification. However, uptake of dying cells by dendritic cells (DCs) is generally a noninflammatory or tolerizing event to prevent the development of autoreactive immune responses. In this study, we describe the mechanisms that confer the potent T cell priming capacity of a recently identified a population of DCs (merocytic DCs [mcDCs]) that potently primes both CD8(+) and CD4(+) T cells to cell-associated Ags upon uptake of apoptotic cells. mcDCs acquired cell-associated materials through a process of merocytosis that is defined by the uptake of small particles that are stored in nonacidic compartments for prolonged periods, sustained Ag presentation, and the induction of type I IFN. T cells primed by mcDCs to cell-associated Ags exhibit increased primary expansion, enhanced effector function, and increased memory formation. By using transgenic T cell transfer models and endogenous models, we show that treatment of tumor-bearing mice with mcDCs that have been exposed to dying tumor cells results in tumor suppression and increased host survival through the activation of naive tumor-specific CD8(+) T cells as well as the reinvigoration of tumor-specific T cells that had been rendered nonresponsive by the tumor in vivo. The potent capacity of mcDCs to prime both CD4(+) and CD8(+) T cells to cell-associated Ags under immunosuppressive conditions makes this DC subset an attractive target for tumor therapies as well as interventional strategies for autoimmunity and transplantation.

Distinct roles of cytolytic effector molecules for antigen-restricted killing by CTL in vivo.

Cytotoxic T lymphocytes (CTLs) represent one of the front lines of defense for the immune system, killing virus-infected and tumor-transformed cells. CTL use at least two mechanisms to induce apoptosis in their targets, one mediated by perforin and granzymes, and the other triggered by the death ligand, CD95 ligand (CD95L). Here, we used an in vivo cytotoxicity assay to measure specific clearance of antigen-bearing target cells in mice that had previously been immunized with noninfectious cell-associated antigens. We found that perforin was dispensable for efficient clearance of antigen-bearing cells from immunized mice, but only if CD95/CD95L was functional; however, there was a delay in target cell clearance in the absence of perforin. In addition, we observed ∼35% target cell clearance in the absence of both perforin and CD95L, which was only slightly abrogated in the presence of a neutralizing anti-tumor necrosis factor (TNF) antibody. The presence of a dominant negative Fas-associated death domain (FADD) did not block target cell clearance and therefore cannot be attributed to known death receptors. Taken together, these data suggest that perforin- and CD95L-dependent killing are complementary at early time points, each can compensate for the absence of the other at later time points, and that there is an additional component of antigen-restricted CTL killing independent of perforin, CD95L, and TNFα.

Dependence of reversibility and progression of mouse neuronopathic Gaucher disease on acid beta-glucosidase residual activity levels.

Genetic and chemically induced neuronopathic mouse models of Gaucher disease were developed to facilitate understanding of the reversibility and/or progression of CNS involvement. The lethality of the skin permeability barrier defect of the complete gene knock out [gba, (glucocerebrosidase) GCase] was avoided by conditional reactivation of a low activity allele (D409H) in keratinocytes (kn-9H). In kn-9H mice, progressive CNS disease and massive glucosylceramide storage in tissues led to death from CNS involvement by the age of 14 days. Conduritol B epoxide (CBE, a covalent inhibitor of GCase) treatment (for 8-12 days) of wild type, D409H, D409V or V394L homozygotes recapitulated the CNS phenotype of the kn-9H mice with seizures, tail arching, shaking, tremor, quadriparesis, extensive neuronal degeneration loss and apoptosis, and death by the age of 14 days. Minor CNS abnormalities occurred after daily CBE injections of 100 mg/kg/day for 6 doses, but neuronal degeneration was progressive and glucosylceramide storage persisted in D409V homozygotes in the 2 to 5 months after CBE cessation; wild type and D409H mice had persistent neurological damage without progression. The persistent CNS deterioration, histologic abnormalities, and glucosylceramide storage in the CBE-treated D409V mice revealed a threshold level of GCase activity necessary for the prevention of progression of CNS involvement.