SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

Double-stranded RNA of intestinal commensal but not pathogenic bacteria triggers production of protective interferon-ß.

The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-ß (IFN-ß) production, which protected mice from experimental colitis. The LAB-induced IFN-ß response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-ß than LAB, and dsRNA was not involved in pathogen-induced IFN-ß induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatory and protective immune responses.

Serum-free immunoglobulin E: A useful biomarker of atopy and type 2 asthma in adults with asthma.

BACKGROUND: It has been known that a high serum total immunoglobulin E (IgE) level is a predisposing factor of allergic asthma; however, there are considerable limitations to apply it in clinical practice. OBJECTIVE: To determine the clinical significance of the serum-free IgE level in patients with adult asthma. METHODS: We measured free IgE levels using our homemade enzyme-linked immunosorbent assay by applying a novel IgE TRAP protein (GI innovation, Seoul, Republic of Korea) in sera of adults with asthma (n = 116) compared with healthy controls (n = 32); enzyme-linked immunosorbent assay inhibition test was performed to validate its binding specificity. Associations between asthma-related clinical and laboratory parameters were analyzed. The diagnostic value and cutoff point for detecting atopy and type 2 asthma were determined using receiver operating characteristic curve analysis. RESULTS: The serum-free IgE levels were significantly higher in adults with asthma than in healthy controls and were significantly associated with atopic status and type 2 asthma (all P < .001). In the receiver operating characteristic analysis, serum-free IgE had a significantly greater area under the curve (AUC) than serum total IgE for assessing asthma, especially type 2 asthma (AUC, 0.810 vs 0.743; P = .006 and AUC, 0.729 vs 0.572; P < .001). The optimal cutoff points for predicting atopy and type 2 asthma were 82.8 and 120.8 ng/mL, respectively. CONCLUSION: It is suggested that a higher serum-free IgE level may be a useful biomarker of atopy and type 2 asthma in adults with asthma.

Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5.

The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized. Here we identify a subset of CD11c(hi)CD11b(hi) lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5(+) LPDCs induced the differentiation of naive B cells into immunoglobulin A-producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific interleukin 17-producing T helper cells and type 1 T helper cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of immunoglobulin A-producing cells in the lamina propria and positively regulated the differentiation interleukin 17-producing T helper cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine.

Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice.

BACKGROUND & AIMS: Obesity and metabolic syndrome have been associated with alterations to the intestinal microbiota. However, few studies examined the effects of obesity on the intestinal immune system. We investigated changes in subsets of intestinal CD4+ T-helper (TH) cells with obesity and the effects of gut-tropic TH17 cells in mice on a high-fat diet (HFD). METHODS: We isolated immune cells from small intestine and adipose tissue of C57BL/6 mice fed a normal chow diet or a HFD for 10 weeks and analyzed the cells by flow cytometry. Mice fed a vitamin A-deficient HFD were compared with mice fed a vitamin A-sufficient HFD. Obese RAG1-deficient mice were given injections of only regulatory T cells or a combination of regulatory T cells and TH17 cells (wild type or deficient in integrin ß7 subunit or interleukin 17 [IL17]). Mice were examined for weight gain, fat mass, fatty liver, glucose tolerance, and insulin resistance. Fecal samples were collected before and after T cell transfer and analyzed for microbiota composition by metagenomic DNA sequencing and quantitative polymerase chain reaction. RESULTS: Mice placed on a HFD became obese, which affected the distribution of small intestinal CD4+ TH cells. Intestinal tissues from obese mice had significant reductions in the proportion of TH17 cells but increased proportion of TH1 cells, compared with intestinal tissues from nonobese mice. Depletion of vitamin A in obese mice further reduced the proportion of TH17 cells in small intestine; this reduction correlated with more weight gain and worsening of glucose intolerance and insulin resistance. Adoptive transfer of in vitro-differentiated gut-tropic TH17 cells to obese mice reduced these metabolic defects, which required the integrin ß7 subunit and IL17. Delivery of TH17 cells to intestines of mice led to expansion of commensal microbes associated with leanness. CONCLUSIONS: In mice, intestinal TH17 cells contribute to development of a microbiota that maintains metabolic homeostasis, via IL17. Gut-homing TH17 cells might be used to reduce metabolic disorders in obese individuals.

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5.

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

BACKGROUND: The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. OBJECTIVE: We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. METHODS: B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. RESULTS: B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. CONCLUSION: B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies.

Transcriptional regulator CTR9 inhibits Th17 differentiation via repression of IL-17 expression.

PAF complex is an evolutionarily conserved transcriptional complex that associates with RNA polymerase II in the coding region of actively transcribing genes. Although its transcriptional activity is closely related to diverse cellular processes, such as cell-cycle progression or development in mammals, its role in immune responses has not been addressed yet. In this study, we show that CTR9, a component of PAF complex, functions as a repressor of Th17 differentiation. Both mRNA and protein levels of CTR9 were significantly decreased during the differentiation processes of naive T into Th17 effector cells. When CTR9 was depleted, IL-17 expression was induced and differentiation into Th17 cells enhanced. In naive T cells, CTR9 occupied the coding region of Il17a, but dissociated under Th17 in vitro-polarizing conditions. In contrast, both CDC73 and PAF1 were recruited to the Il17a locus under Th17-differentiation conditions. In the IL-6-stimulated splenocytes, expression of CTR9 was decreased, and chromatin-bound CTR9 disappeared in the coding region of Il17a. IL-6 also directly repressed expression of CTR9 gene, as promoter activity of CTR9 was similarly repressed by IL-6 treatment. Moreover, in mice with collagen-induced arthritis, lentivirus-mediated CTR9 overexpression in the joints ameliorated arthritis severity, decreasing the frequency of CD4(+)IL-17(+) T cells in lymph nodes. In conclusion, our data propose a novel feed-forward loop of IL-17 transcriptional regulatory circuit, via IL-6-mediated repression of CTR9 which is a transcriptional repressor of IL-17.

Protective effects of Fc-fused PD-L1 on two different animal models of colitis.

OBJECTIVE: Programmed death-ligand 1 (PD-L1) has been shown to negatively regulate immune responses via its interaction with PD-1 receptor. In this study, we investigated the effects of PD-L1-Fc treatment on intestinal inflammation using two murine models of inflammatory colitis induced by dextran sulfate sodium (DSS) and T-cell transfer. DESIGN: The anti-colitis effect of adenovirus expressing Fc-conjugated PD-L1 (Ad/PD-L1-Fc) and recombinant PD-L1-Fc protein was evaluated in DSS-treated wild-type and Rag-1 knockout (KO) mice. We examined differentiation of T-helper cells, frequency of innate immune cells, and cytokine production by dendritic cells (DCs) in the colon from DSS-treated mice after PD-L1-Fc administration. In Rag-1 KO mice reconstituted with CD4 CD45RB(high) T cells, we assessed the treatment effect of PD-L1-Fc protein on the development of colitis. RESULTS: Administration of Ad/PD-L1-Fc significantly ameliorated DSS-induced colitis, which was accompanied by diminished frequency of interleukin (IL)-17A-producing CD4 T cells and increased interferon-γ-producing CD4 T cells in the colon of DSS-fed mice. The anti-colitic effect of PD-L1-Fc treatment was also observed in DSS-treated Rag-1 KO mice, indicating lymphoid cell independency. PD-L1-Fc modulated cytokine production by colonic DCs and the effect was dependent on PD-1 expression. Furthermore, PD-L1-Fc protein could significantly reduce the severity of colitis in CD4 CD45RB(high) T-cell-transferred Rag-1 KO mice. CONCLUSIONS: Based on the protective effect of PD-L1-Fc against DSS-induced and T-cell-induced colitis, our results suggest that PD-1-mediated inhibitory signals have a crucial role in limiting the development of colonic inflammation. This implicates that PD-L1-Fc may provide a novel therapeutic approach to treat inflammatory bowel disease.

Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection.

Pandemics in poultry caused by the highly pathogenic avian influenza (HPAI) A virus occur too frequently globally, and there is growing concern about the HPAI A virus due to the possibility of a pandemic among humans. Thus, it is important to develop a vaccine against HPAI suitable for both humans and animals. Various approaches are underway to develop such vaccines. In particular, an edible vaccine would be a convenient way to vaccinate poultry because of the behaviour of the animals. However, an edible vaccine is still not available. In this study, we developed a strategy of effective vaccination of mice by the oral administration of transgenic Arabidopsis plants (HA-TG) expressing haemagglutinin (HA) in the endoplasmic reticulum (ER). Expression of HA in the ER resulted in its high-level accumulation, N-glycosylation, protection from proteolytic degradation and long-term stability. Oral administration of HA-TG with saponin elicited high levels of HA-specific systemic IgG and mucosal IgA responses in mice, which resulted in protection against a lethal influenza virus infection with attenuated inflammatory symptoms. Based on these results, we propose that oral administration of freeze-dried leaf powders from transgenic plants expressing HA in the ER together with saponin is an attractive strategy for vaccination against influenza A virus.

Intestinal Lin- c-Kit+ NKp46- CD4- population strongly produces IL-22 upon IL-1ß stimulation.

Small intestinal innate lymphoid cells (ILCs) regulate intestinal epithelial cell homeostasis and help to prevent pathogenic bacterial infections by producing IL-22. In a global gene-expression analysis comparing small intestinal ILCs (Lin(-)c-Kit(+)Sca-1(-) cells) with non-ILCs (Lin(-)c-Kit(-)Sca-1(-) cells), we found that Lin(-)c-Kit(+)Sca-1(-) cells highly expressed the mRNAs for Il22, antimicrobial peptides, Csf2rb2 (Il3r), mast cell proteases, and Rorc. We then subdivided the Lin(-)c-Kit(+)Sca-1(-) cells into three groups--Lin(-)c-Kit(+)NKp46(-)CD4(-), Lin(-)c-Kit(+)NKp46(-)CD4(+) (CD4(+) LTi-like cells), and Lin(-)c-Kit(+)NKp46(+) (NKp46(+) ILC22 cells)--and showed that the Lin(-)c-Kit(+)NKp46(-)CD4(-) cells produced the highest level of IL-22 protein after IL-1ß, IL-23, or IL-1ß and IL-23 stimulation. In addition, we showed that the majority of the Lin(-)c-Kit(+)NKp46(-)CD4(-) population was IL-7Rα(+)CD34(-)ß7(int) cells, and IL-7Rα(-) cells could be divided into three subsets (CD34(+)ß7(int), CD34(-)ß7(int), and CD34(int)ß7(hi) cells). The IL-7Rα(+)CD34(-)ß7(int) cells strongly expressed the transcripts for Il17f and Il22 after costimulation with IL-1ß and IL-23. The IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) cells predominantly expressed the transcripts for mast cell proteases and differentiated almost entirely into mast cells after 1 wk in culture medium supplemented with a cytokine mixture, whereas the IL-7Rα(-)CD34(-)ß7(int) cells highly expressed α-defensins and showed no differentiation. Taken together, these findings indicate that the IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) populations are mast cell progenitors, and the IL-7Rα(+)CD34(-)ß7(int) (CD4(-) LTi-like cells) and IL-7Rα(-)CD34(-)ß7(int) populations within Lin(-)c-Kit(+)NKp46(-)CD4(-) cells may control intestinal homeostasis and provide intestinal protection by producing high levels of IL-22 and α-defensins, respectively.

Acidic amino acid residues in the juxtamembrane region of the nucleotide-sensing TLRs are important for UNC93B1 binding and signaling.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

CXCL12 secreted from adipose tissue recruits macrophages and induces insulin resistance in mice.

AIMS/HYPOTHESIS: Obesity-induced inflammation is initiated by the recruitment of macrophages into adipose tissue. The recruited macrophages, called adipose tissue macrophages, secrete several proinflammatory cytokines that cause low-grade systemic inflammation and insulin resistance. The aim of this study was to find macrophage-recruiting factors that are thought to provide a crucial connection between obesity and insulin resistance. METHODS: We used chemotaxis assay, reverse phase HPLC and tandem MS analysis to find chemotactic factors from adipocytes. The expression of chemokines and macrophage markers was evaluated by quantitative RT-PCR, immunohistochemistry and FACS analysis. RESULTS: We report our finding that the chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal cell-derived factor 1), identified from 3T3-L1 adipocyte conditioned medium, induces monocyte migration via its receptor chemokine (C-X-C motif) receptor 4 (CXCR4). Diet-induced obese mice demonstrated a robust increase of CXCL12 expression in white adipose tissue (WAT). Treatment of obese mice with a CXCR4 antagonist reduced macrophage accumulation and production of proinflammatory cytokines in WAT, and improved systemic insulin sensitivity. CONCLUSIONS/INTERPRETATION: In this study we found that CXCL12 is an adipocyte-derived chemotactic factor that recruits macrophages, and that it is a required factor for the establishment of obesity-induced adipose tissue inflammation and systemic insulin resistance.

Constitutive plasmacytoid dendritic cell migration to the splenic white pulp is cooperatively regulated by CCR7- and CXCR4-mediated signaling.

Although the spleen plays an important role in host defense against infection, the mechanism underlying the migration of the innate immune cells, plasmacytoid dendritic cells (pDCs), into the spleen remains ill defined. In this article, we report that pDCs constitutively migrate into the splenic white pulp (WP) in a manner dependent on the chemokine receptors CCR7 and CXCR4. In CCR7-deficient mice and CCR7 ligand-deficient mice, compared with wild-type (WT) mice, substantially fewer pDCs were found in the periarteriolar lymphoid sheath of the splenic WP under steady-state conditions. In addition, the migration of adoptively transferred CCR7-deficient pDCs into the WP was significantly worse than that of WT pDCs, supporting the idea that pDC trafficking to the splenic WP requires CCR7 signaling. WT pDCs responded to a CCR7 ligand with modest chemotaxis and ICAM-1 binding in vitro, and priming with the CCR7 ligand enabled the pDCs to migrate efficiently toward low concentrations of CXCL12 in a CXCR4-dependent manner, raising the possibility that CCR7 signaling enhances CXCR4-mediated pDC migration. In agreement with this hypothesis, CCL21 and CXCL12 were colocalized on fibroblastic reticular cells in the T cell zone and in the marginal zone bridging channels, through which pDCs appeared to enter the WP. Furthermore, functional blockage of CCR7 and CXCR4 abrogated pDC trafficking into the WP. Collectively, these results strongly suggest that pDCs employ both CCR7 and CXCR4 as critical chemokine receptors to migrate into the WP under steady-state conditions.

Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvß1 integrin.

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Ms) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mϕs to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1(+)MHC class II(int)F4/80(hi)CD11b(hi) Mϕs as the main producers of IL-10 via αvß1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mϕs during interactions between IECs and Mϕs. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10-blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvß1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mϕs.

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

Safety, Tolerability, Pharmacokinetics, and pharmacodynamics of YH35324, a novel Long-Acting High-Affinity IgETrap-Fc protein in subjects with Atopy: Results from the First-in-Human study.

BACKGROUND: YH35324, a long-acting IgETrap-Fc fusion protein, is a novel therapeutic agent for immunoglobulin E (IgE)-mediated allergic diseases. This randomized, double-blind, placebo/active-controlled, single ascending dose Phase 1 study assessed the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of YH35324 in subjects with atopy. METHODS: Eligible subjects were healthy subjects or atopic adults with mild allergic rhinitis, atopic dermatitis, food allergy, or urticaria, and a serum total IgE level of 30-700 IU/mL (Part A) or > 700 IU/mL (Part B). In Part A, 35 subjects in 5 cohorts received YH35324 (0.3, 1, 3, 6, and 9 mg/kg), 8 received omalizumab (300 mg), and 9 received placebo. In Part B, 8 subjects received YH35324 and 8 received omalizumab. RESULTS: Twenty subjects (38.5 %) in Part A (YH35324: 37.1 %, omalizumab: 50.0 %, placebo: 33.3 %) and 10 subjects (62.5 %) in Part B (YH35324: 100 %; omalizumab: 25.0 %) experienced treatment-emergent adverse events (TEAEs). TEAEs were mostly grade 1/2; no serious AEs, AE-related treatment discontinuation, or anaphylaxis were reported. YH35324 exhibited dose-proportional increase in Cmax and AUClast over the dose range of 0.3-9 mg/kg. YH35324 rapidly suppressed serum-free IgE levels to a significant extent (< 25 and < 82.8 ng/mL, both P < 0.05) and with longer duration than omalizumab. CONCLUSION: This study showed that YH35324 has a favorable safety profile and is effective in reducing serum-free IgE levels in subjects with atopic conditions.

Pulmonary inflammation induced by bacteria-free outer membrane vesicles from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is often involved in lung diseases such as cystic fibrosis. These bacteria can release outer membrane vesicles (OMVs), which are bilayered proteolipids with diameters of approximately 20 to 250 nm. In vitro, these OMVs activate macrophages and airway epithelial cells. The aim of this study was to determine whether OMVs from P. aeruginosa can induce pulmonary inflammation in vivo and to elucidate the mechanisms involved. Bacteria-free OMVs were isolated from P. aeruginosa cultures. Wild-type, Toll-like receptor (TLR)2 and TLR4 knockout mice were exposed to OMVs by the airway, and inflammation in the lung was assessed using differential counts, histology, and quantification of chemokines and cytokines. The involvement of the TLR2 and TLR4 pathways was studied in human cells using transfection. OMVs given to the mouse lung caused dose- and time-dependent pulmonary cellular inflammation. Furthermore, OMVs increased concentrations of several chemokines and cytokines in the mouse lungs and mouse alveolar macrophages. The inflammatory responses to OMVs were comparable to those of live bacteria and were only partly regulated by the TLR2 and TLR4 pathways, according to studies in knockout mice. This study shows that OMVs from P. aeruginosa cause pulmonary inflammation without live bacteria in vivo. This effect is only partly controlled by TLR2 and TLR4. The role of OMVs in clinical disease warrants further studies because targeting of OMVs in addition to live bacteria may add clinical benefit compared with treating with antibiotics alone.

SIRPα/CD172a regulates eosinophil homeostasis.

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto(-/-)). The SIRPα Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.

Degranulation of Mast Cells as a Target for Drug Development.

Mast cells act as key effector cells of inflammatory responses through degranulation. Mast cell degranulation is induced by the activation of cell surface receptors, such as FcεRI, MRGPRX2/B2, and P2RX7. Each receptor, except FcεRI, varies in its expression pattern depending on the tissue, which contributes to their differing involvement in inflammatory responses depending on the site of occurrence. Focusing on the mechanism of allergic inflammatory responses by mast cells, this review will describe newly identified mast cell receptors in terms of their involvement in degranulation induction and patterns of tissue-specific expression. In addition, new drugs targeting mast cell degranulation for the treatment of allergy-related diseases will be introduced.