Allergin-1 Immunoreceptor Suppresses House Dust Mite-Induced Allergic Airway Inflammation.

House dust mite (HDM) allergens are leading causes of allergic asthma characterized by Th2 responses. The lung-resident CD11b+ dendritic cells (DCs) play a key role in Th2 cell development in HDM-induced allergic asthma. However, the regulatory mechanism of HDM-induced CD11b+ DC activation remains incompletely understood. In this study, we demonstrate that mice deficient in an inhibitory immunoreceptor, Allergin-1, showed exacerbated HDM-induced airway eosinophilia and serum IgE elevation. By using bone marrow-chimeric mice that were sensitized with adoptively transferred HDM-stimulated wild-type or Allergin-1-deficient CD11b+ bone marrow-derived cultured DCs (BMDCs), followed by challenge with HDM, we show that Allergin-1 on the BMDCs suppressed HDM-induced allergic airway inflammation. We also show that Allergin-1 suppressed HDM-induced PGE2 production from CD11b+ BMDCs by inhibiting Syk tyrosine kinase activation through recruitment of SHP-1, subsequently leading to negative regulation of Th2 responses. These results suggest that Allergin-1 plays an important role in regulation of HDM-induced allergic airway inflammation.

Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae.

Invariant natural killer T cells (iNKT cells) are activated by lipid antigens presented by CD1d, but the pathway leading to lipid antigen presentation remains incompletely characterized. Here we show a whole-genome siRNA screen to elucidate the CD1d presentation pathway. A majority of gene knockdowns that diminish antigen presentation reduced formation of glycolipid-CD1d complexes on the cell surface, including members of the HOPS and ESCRT complexes, genes affecting cytoskeletal rearrangement, and ABC family transporters. We validated the role in vivo for the multidrug resistance protein 1 (Mrp1) in CD1d antigen presentation. Mrp1 deficiency reduces surface clustering of CD1d, which decreased iNKT cell activation. Infected Mrp1 knockout mice show decreased iNKT cell responses to antigens from Streptococcus pneumoniae and were associated with increased mortality. Our results highlight the unique cellular events involved in lipid antigen presentation and show how modification of this pathway can lead to lethal infection.

Allergin-1 on mast cells suppresses house dust mite-induced airway hyperresponsiveness in mice.

Although airway hyperresponsiveness (AHR) is a prominent feature of asthma, how it is regulated remains incompletely understood. Allergin-1, an inhibitory immunoglobulin-like receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), is expressed on human and mouse mast cells (MCs) and inhibits high-affinity receptor for IgE (FcεRI)-mediated signaling. Using MC-deficient KitW-sh/W-sh mice and Mas-TRECK mice, which carries a diphtheria toxin (DT)-induced MC deletion system based on il4 enhancer elements, we demonstrate here that MCs are involved in the induction of house dust mite (HDM)-induced AHR. Further, we show that MCs deficient in Allergin-1 exacerbated HDM-induced AHR, but had no effect on airway inflammation. In vitro analysis demonstrated that Allergin-1 inhibited anti-HDM allergen antibody-dependent HDM allergen-mediated degranulation by MCs. Thus, Allergin-1 on MCs plays an important role in the regulation of HDM-induced AHR.

Efficient Transdermal Delivery of Alendronate, a Nitrogen-Containing Bisphosphonate, Using Tip-Loaded Self-Dissolving Microneedle Arrays for the Treatment of Osteoporosis.

To improve the transdermal bioavailability and safety of alendronate (ALN), a nitrogen-containing bisphosphonate, we developed self-dissolving microneedle arrays (MNs), in which ALN is loaded only at the tip portion of micron-scale needles by a dip-coating method (ALN(TIP)-MN). We observed micron-scale pores in rat skin just after application of ALN(TIP)-MN, indicating that transdermal pathways for ALN were created by MN. ALN was rapidly released from the tip of MNs as observed in an in vitro release study. The tip portions of MNs completely dissolved in the rat skin within 5 min after application in vivo. After application of ALN(TIP)-MN in mice, the plasma concentration of ALN rapidly increased, and the bioavailability of ALN was approximately 96%. In addition, the decrease in growth plate was effectively suppressed by this efficient delivery of ALN in a rat model of osteoporosis. Furthermore, no skin irritation was observed after application of ALN(TIP)-MN and subcutaneous injection of ALN, while mild skin irritation was induced by whole-ALN-loaded MN (ALN-MN)-in which ALN is contained in the whole of the micron-scale needles fabricated from hyaluronic acid-and intradermal injection of ALN. These findings indicate that ALN(TIP)-MN is a promising transdermal formulation for the treatment of osteoporosis without skin irritation.

Development of a drug-coated microneedle array and its application for transdermal delivery of interferon alpha.

Interferon alpha (IFNα) is one of the most famous drugs for the treatment of chronic hepatitis C and various types of human malignancy. Protein drugs, including IFNα, are generally administered by subcutaneous or intramuscular injection due to their poor permeability and low stability in the bloodstream or gastrointestinal tract. Therefore, in the present study, novel IFNα-coated polyvinyl alcohol-based microneedle arrays (IFNα-MNs) were fabricated for the transdermal delivery of IFNα without the painful injection. IFNα was rapidly released from MNs in phosphate buffered solution and these MNs presented piercing ability in the rat skin. Slight erythema and irritation were observed when MNs were applied to the rat skin, but these skin damages completely disappeared within 24 h after removing the IFNα-MNs. Furthermore, the pharmacokinetic parameters of IFNα-MNs were similar to those of IFNα subcutaneous administration. Finally, IFNα-MNs showed a significant antitumor effect in tumor bearing mice similar to that of IFNα subcutaneous administration. These results indicate that IFNα-MNs are a useful biomaterial tool for protein drug therapy and can improve the quality of life in patients by avoidance of painful injections.

Development of a novel self-dissolving microneedle array of alendronate, a nitrogen-containing bisphosphonate: evaluation of transdermal absorption, safety, and pharmacological effects after application in rats.

Alendronate is a nitrogen-containing bisphosphonate that is widely used for the treatment of osteoporosis. In this study, we developed a novel self-dissolving micron-size needle array (microneedle array) containing alendronate, which was fabricated by micromodeling technologies using hyaluronic acid as a basic material. Micron-scale pores in the skin were seen after the application of the alendronate-loaded microneedle array, verifying establishment of transdermal pathways for alendronate. The absorption of alendronate after the application of alendronate-loaded microneedle array was almost equivalent to that after subcutaneous administration, and the bioavailability of alendronate was approximately 90% in rats. Furthermore, delivery of alendronate via this strategy effectively suppressed the decrease in the width of the growth plate in a rat model of osteoporosis. Although mild cutaneous irritation was observed after the application of the alendronate-loaded microneedle array, it resolved by day 15. These findings indicate that this alendronate-loaded microneedle array is a promising transdermal formulation for the treatment of osteoporosis.

An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions.

Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.

Caspase-independent cell death by CD300LF (MAIR-V), an inhibitory immunoglobulin-like receptor on myeloid cells.

The myeloid-associated Ig-like receptor family (CD300) consists of nine activating or inhibitory cell surface receptors preferentially expressed on myeloid cells and are encoded by the genes in a small cluster on mouse chromosome 11. One of the receptors, CD300LF (MAIR-V), has a long cytoplasmic tail containing two consensus ITIMs and an immunoreceptor tyrosine-based switching motif, suggesting that CD300LF regulates the activation of myeloid cells. However, the functional characteristics of this receptor are still incompletely understood. In this study, we demonstrate that cross-linking CD300LF with anti-CD300LF mAb induced cell death in peritoneal macrophages as well as in several transfectants expressing CD300LF. CD300LF-mediated cell death was dependent on the cytoplasmic region but did not require an ITIM or immunoreceptor tyrosine-based switching motif. Scanning electron microscopy revealed a loss of blebs from the surface of the dead cells mediated by CD300LF, a morphological feature similar to that observed in apoptotic cells. However, CD300LF-mediated cell death was not inhibited by a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, or autophagy inhibitors, 3-methyladenine or N-acetyl-L-cystein. Moreover, the splicing isoform of a transcription factor, X-box binding protein-1, which is produced in dead cells as a response to endoplasmic reticulum stress, was not detected. Together, these results indicate that CD300LF mediates caspase and endoplasmic reticulum stress-independent cell death by a novel mechanism.