Naomi M. Scott

Vitamin D 3 deficiency enhances allergen-induced lymphocyte responses in a mouse model of allergic airway disease

To cite this article: Gorman S, Tan DHW, Lambert MJM, Scott NM, Judge MA, Hart PH. Vitamin D3 deficiency enhances allergen‐induced lymphocyte responses in a mouse model of allergic airway disease. Pediatr Allergy Immunol 2012: 23: 83–87

Acute Erythemal Ultraviolet Radiation Causes Systemic Immunosuppression in the Absence of Increased 25-Hydroxyvitamin D3 Levels in Male Mice

Vitamin D is synthesised by ultraviolet (UV) irradiation of skin and is hypothesized to be a direct mediator of the immunosuppression that occurs following UV radiation (UVR) exposure. Both UVR and vitamin D drive immune responses towards tolerance by ultimately increasing the suppressive activities of regulatory T cells. To examine a role for UVR-induced vitamin D, vitamin D3-deficient mice were established by dietary vitamin D3 restriction. In comparison to vitamin D3-replete mice, vitamin D3-deficient mice had significantly reduced serum levels of 25-hydroxyvitamin D3 (25(OH)D3, <20 nmol.L−1) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3, <20 pmol.L−1). Following either acute erythemal UVR, or chronic sub-erythemal UVR (8 exposures over 4 weeks) treatment, serum 25(OH)D3 levels significantly increased in vitamin D3-deficient female but not male mice. To determine if UVR-induced vitamin D was a mediator of UVR-induced systemic immunosuppression, responses were measured in mice that were able (female) or unable (male) to increase systemic levels of 25(OH)D3 after UVR. Erythemal UVR (≥4 kJ/m2) suppressed contact hypersensitivity responses (T helper type-1 or -17), aspects of allergic airway disease (T helper type-2) and also the in vivo priming capacity of bone marrow-derived dendritic cells to a similar degree in female and male vitamin D3-deficient mice. Thus, in male mice, UVR-induced 25(OH)D3 is not essential for mediating the immunosuppressive effects of erythemal UVR.

Altered Immunity and Dendritic Cell Activity in the Periphery of Mice after Long-Term Engraftment with Bone Marrow from Ultraviolet-Irradiated Mice

Alterations to dendritic cell (DC) progenitors in the bone marrow (BM) may contribute to long-lasting systemic immunosuppression (>28 d) following exposure of the skin of mice to erythemal UV radiation (UVR). DCs differentiated in vitro from the BM of mice 3 d after UVR (8 kJ/m2) have a reduced capacity to initiate immunity (both skin and airways) when adoptively transferred into naive mice. Studies in IL-10−/− mice suggested that UV-induced IL-10 was not significantly involved. To investigate the immune capabilities of peripheral tissue DCs generated in vivo from the BM of UV-irradiated mice, chimeric mice were established. Sixteen weeks after reconstitution, contact hypersensitivity responses were significantly reduced in mice reconstituted with BM from UV-irradiated mice (UV-chimeric). When the dorsal skin of UV-chimeric mice was challenged with innate inflammatory agents, the hypertrophy induced in the draining lymph nodes was minimal and significantly less than that measured in control-chimeric mice challenged with the same inflammatory agent. When DCs were differentiated from the BM of UV-chimeric mice using FLT3 ligand or GM-CSF + IL-4, the cells maintained a reduced priming ability. The diminished responses in UV-chimeric mice were not due to different numerical or proportional reconstitution of BM or the hematopoietic cells in blood, lymph nodes, and skin. Erythemal UVR may imprint a long-lasting epigenetic effect on DC progenitors in the BM and alter the function of their terminally differentiated progeny.

Reversible control by vitamin D of granulocytes and bacteria in the lungs of mice: an ovalbumin-induced model of allergic airway disease.

Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We have developed a ‘low dose’ model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 µg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.

Prostaglandin E2 imprints a long-lasting effect on dendritic cell progenitors in the bone marrow

Dendritic cells (DCs) that differentiate in vitro from the bone marrow (BM) of mice with prostaglandin E2 (PGE2)‐associated inflammation of the skin, airways, or peritoneal cavity poorly initiate immune responses. To remove in vitro differentiation and allow BM‐derived DCs to seed the periphery under steady‐state conditions, as well as study the molecule proposed responsible, chimeric mice were engrafted for >16 wk with BM cells from mice exposed to PGE2. Serial PGE2‐chimeric mice were established with BM cells from the primary chimeric mice. Immune responses in the airways and skin of the PGE2‐chimeric mice and serial PGE2‐chimeric mice were significantly attenuated. After inflammatory challenges by intranasal LPS, topical fluorescein isothiocyanate, and intraperitoneal alum, DCs, macrophages, and neutrophils trafficked poorly in PGE2‐chimeric mice and serial PGE2‐chimeric mice. Injection of BM‐differentiated DCs from nonchimeric mice restored the reduced immune responses of PGE2‐chimeric mice. DCs from BM of 16‐wk‐engrafted PGE2‐chimeric and serial PGE2‐chimeric mice resembled cells differentiated from BM exposed to PGE2 for only 3 d, demonstrating the long‐lasting effect of PGE2 on DC progenitors. PGE2 attenuates systemic immune responses by modulating myeloid cell progenitors in the BM such that BM‐derived, terminally differentiated myeloid cells have poor trafficking ability to sites of need.

Characterization of regulatory dendritic cells differentiated from the bone marrow of UV-irradiated mice.

When antigen‐loaded dendritic cells (DCs) differentiated from the bone marrow (BM) of UV‐irradiated mice (UV‐BMDCs) were adoptively transferred into naive mice or mice pre‐sensitized with that antigen, the recipients exhibited a reduced immune response following antigen challenge. Hence, UV‐BMDCs are poorly immunogenic and can suppress pre‐existing immunity. The UV‐induced effect on BM‐derived DCs was rapid (observed 1 day after UV radiation), long‐lasting (observed 10 days after UV radiation) and UV dose‐dependent. The mechanism by which UV‐BMDCs could regulate immunity was investigated. The CD11c+ cells, differentiated using granulocyte–macrophage colony‐stimulating factor + interleukin‐4, were confirmed to be DCs because they did not express the myeloid‐derived suppressor cell marker, Gr1. UV‐BMDCs did not display altered antigen uptake, processing or ability to activate T cells in vitro. When gene expression in UV‐BMDCs and DCs differentiated from the BM of non‐irradiated mice (control‐BMDCs) was examined, Ccl7, Ccl8 and CSF1R (CD115) mRNA transcripts were up‐regulated in UV‐BMDCs compared with control‐BMDCs. However, neutralizing antibodies for Ccl7 and Ccl8 did not abrogate the reduced immunogenicity of UV‐BMDCs in vivo. Moreover, the up‐regulation of CSF1R transcript did not correspond with increased receptor expression on UV‐BMDCs. The phenotypes of UV‐BMDCs and control‐BMDCs were similar, with no difference in the expression of CD4, CD8α, CD103, B220 or F4/80, or the regulatory molecules CCR7 (CD197), FasL (CD95L), B7H3 (CD276) and B7H4. However, PDL1 (CD274) expression was reduced in UV‐BMDCs compared with control‐BMDCs following lipopolysaccharide stimulation. In summary, UV‐BMDCs do not express the classical phenotypic or gene expression properties of DCs reported by others as ‘regulatory’ or ‘tolerogenic’.

Toward Homeostasis: Regulatory Dendritic Cells from the Bone Marrow of Mice with Inflammation of the Airways and Peritoneal Cavity

Inflammatory mediators from peripheral tissues may control dendritic cell (DC) development in the bone marrow. In this study, DCs (CD11c(+) cells) differentiated from the bone marrow of mice with inflammation of the airways, or the peritoneal cavity had poor priming ability resulting in reduced, long-lived responses to that antigen in vivo. This indicates enhancement of regulatory mechanisms of immune responses through a peripheral tissue-bone marrow axis. If CD11c(+) cells, expanded from the bone marrow of mice with tissue inflammation were antigen pre-loaded and injected into mice already sensitized to that antigen, then subsequent contact hypersensitivity responses were significantly reduced. The effects of inflammation were imprinted in vivo and were independent of in vitro culture conditions for DC differentiation. The effect of tissue inflammation on the bone marrow DC precursors was not detected in mice treated subcutaneously with slow-release indomethacin pellets, suggesting a role for prostanoids, including prostaglandin E(2), in differentiation of regulatory CD11c(+) cells from bone marrow. Our study represents an important homeostatic process with potential for therapeutic use in the future.

Differences in control by UV radiation of inflammatory airways disease in naïve and allergen pre-sensitised mice

Exposure of skin to UV radiation (UVR) prior to allergen exposure can inhibit inflammatory airways disease in mice by reducing effector CD4+ T cells in both the trachea and the airway draining lymph nodes. This study analysed the immunomodulatory properties of UVR delivered to naïve versus allergen pre-sensitised mice. In a model of inflammatory airways disease, BALB/c mice were sensitised by peritoneal injection of the allergen, ovalbumin (OVA) (20 μg/mouse), in the adjuvant, alum (4 mg/mouse), on days 0 and 14. On day 21, the mice were exposed to aerosolised OVA and 24 h later, proliferative responses by the cells in the airway draining lymph nodes were examined. UVR (8 kJ m(-2)) was administered 3 days prior to first OVA sensitisation (day -3), or OVA aerosol challenge (day 18). UVR before sensitisation reduced immune responses associated with expression of allergic airways disease; seven days after first OVA sensitisation, regulation of OVA-induced proliferation in vitro but not in vivo by CD4+CD25+ cells from UV-irradiated mice was detected. UVR administered to pre-sensitised mice regulated allergen responsiveness by cells from the airway draining lymph nodes only with a sensitisation protocol involving allergen and adjuvant at 5% strength of the original dose (1 μg OVA in 0.2 mg alum/mouse). These results suggest that UVR may modulate allergic airways disease by two mechanisms. The first, and more potent, is by reducing effector cells in respiratory tissues and requires UV delivery prior to sensitisation. The second, associated with administration to pre-sensitised mice, is weaker and is detected when the mice are sensitised with lower levels of allergen and adjuvant.

Protection against maternal infection-associated fetal growth restriction: proof-of-concept with a microbial-derived immunomodulator

Infection-associated inflammatory stress during pregnancy is the most common cause of fetal growth restriction and/or miscarriage. Treatment strategies for protection of at-risk mothers are limited to a narrow range of vaccines, which do not cover the bulk of the common pathogens most frequently encountered. Using mouse models, we demonstrate that oral treatment during pregnancy with a microbial-derived immunomodulator (OM85), currently used clinically for attenuation of infection-associated airway inflammatory symptoms in infants–adults, markedly reduces risk for fetal loss/growth restriction resulting from maternal challenge with bacterial lipopolysaccharide or influenza. Focusing on LPS exposure, we demonstrate that the key molecular indices of maternal inflammatory stress, notably high levels of RANTES, MIP-1α, CCL2, KC, and G-CSF (granulocyte colony-stimulating factor) in gestational tissues/serum, are abrogated by OM85 pretreatment. Systems-level analyses conducted in parallel using RNASeq revealed that OM85 pretreatment selectively tunes LPS-induced activation in maternal gestational tissues for attenuated expression of TNF, IL1, and IFNG-driven proinflammatory networks, without constraining Type1-IFN-associated networks central to first-line antimicrobial defense. This study suggests that broad-spectrum protection-of-pregnancy against infection-associated inflammatory stress, without compromising capacity for efficient pathogen eradication, represents an achievable therapeutic goal.

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.