Myoung Ho Jang

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

Aims/hypothesisObesity-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.MethodsWe 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.ResultsWe 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/interpretationIn 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.

Intestinal Epithelial Cell-Derived Semaphorin 7A Negatively Regulates Development of Colitis via αvβ1 Integrin

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Mϕs) 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 IIintF4/80hiCD11bhi Mφs as the main producers of IL-10 via αvβ1 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 αvβ1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mφs.

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.

Combined two-photon microscopy and optical coherence tomography using individually optimized sources.

The combination of two-photon microscopy (TPM) and optical coherence tomography (OCT) is useful in conducting in-vivo tissue studies, because they provide complementary information regarding tissues. In the present study, we developed a new combined system using separate light sources and scanners for individually optimal imaging conditions. TPM used a Ti-Sapphire laser and provided molecular and cellular information in microscopic tissue regions. Meanwhile, OCT used a wavelength-swept source centered at 1300 nm and provided structural information in larger tissue regions than TPM. The system was designed to do simultaneous imaging by combining light from both sources. TPM and OCT had the field of view values of 300 μm and 800 μm on one side respectively with a 20x objective. TPM had resolutions of 0.47 μm and 2.5 μm in the lateral and axial directions respectively, and an imaging speed of 40 frames/s. OCT had resolutions of 5 μm and 8 μm in lateral and axial directions respectively, a sensitivity of 97dB, and an imaging speed of 0.8 volumes per second. This combined system was tested with simple microsphere specimens, and was then applied to image small intestine and ear tissues of mouse models ex-vivo. Molecular, cellular, and structural information of the tissues were visualized using the proposed combined system.

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.

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

Significance Toll-like receptors (TLRs) are key innate immune receptors that sense pathogen- or danger-associated molecules. The members of the TLR family can be classified into two groups based on their subcellular localization patterns—cell surface vs. endolysosomes—but how individual TLR is targeted to its destination is largely unknown. UNC93B1 was regarded to specifically bind the endolysosomal TLRs in the endoplasmic reticulum and deliver them to endolysosomes with no role for the surface-localized TLRs. In this study, we demonstrate that TLR5, a cell surface receptor for bacterial protein flagellin, also binds UNC93B1 and requires it for plasma membrane localization and signaling, thus revealing a previously unappreciated role of UNC93B1 in the regulation of the TLR family. 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.

Delivery of IL-12p40 ameliorates DSS-induced colitis by suppressing IL-17A expression and inflammation in the intestinal mucosa

IL-12p40 homodimer is a natural antagonist of IL-12 and IL-23, which are potent pro-inflammatory cytokines required for Th1 and Th17 immune responses, respectively. It has been reported that Th17 response is involved in inflammatory bowel disease (IBD), a chronic disorder of the digestive system with steadily increasing incidence. Here, we investigated the effects of IL-12p40 delivered via recombinant adenovirus (rAd/IL-12p40) or mesenchymal stem cells (MSC/IL-12p40) in a dextran sulfate sodium salt (DSS)-induced colitis model. Injection of rAd/IL-12p40 or MSC/IL-12p40 efficiently attenuated colitis symptoms and tissue damage, leading to an increased survival rate. Moreover, IL-12p40 delivery suppressed IL-17A, but enhanced IFN-γ production from mesenteric lymph node cells, supporting the preferential suppression of IL-23 by IL-12p40 homodimer in vitro and the suppression of Th17 responses in vivo. Our results demonstrate that IL-12p40 delivery ameliorates DSS-induced colitis by suppressing IL-17A production and inflammation in the intestinal mucosa, providing an effective new therapeutic strategy for IBDs.

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.

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.

Comparative analysis of the effects of anti‐IL‐6 receptor mAb and anti‐TNF mAb treatment on CD4+ T‐cell responses in murine colitis

Background: The efficacy of anti‐tumor necrosis factor monoclonal antibody (anti‐TNF mAb) for Crohns disease (CD) is well established, and anti‐interleukin‐6 receptor (anti‐IL‐6R) mAb has also been reported to be effective in CD. It is, however, unclear if the efficacy and mechanisms of both agents are different in CD therapy. Methods: Using an adoptive transfer colitis model, we compared the efficacy of anti‐IL‐6R mAb, anti‐TNF mAb, and TNF receptor‐Fc fusion protein (TNFR‐Fc), and their modes of action on CD4+ T cells. We also investigated the role of Th1 and Th17 cells in colitis using the same model. Results: The histological scores for the anti‐IL‐6R mAb and anti‐TNF mAb groups but not for TNFR‐Fc group were much lower than that for the control group, and the score was the lowest for the anti‐IL‐6R mAb group. The frequency of proliferating CD4+ T cells was reduced in anti‐IL‐6R mAb and anti‐TNF mAb groups, but not in the TNFR‐Fc group, whereas the frequency of apoptotic CD4+ T cells was similar in all groups. Anti‐IL‐6R mAb suppressed the induction of Th17 cells and increased the frequency of lamina propria regulatory T cells, whereas anti‐TNF mAb exerted no influence on CD4+ T‐cell differentiation. A deficiency in interferon‐γ and/or IL‐17 in CD4+ T cells reduced the severity of colitis. Conclusions: Our findings suggest that suppression of the proliferation of pathogenic CD4+ T cells is the major mode of action of biological agents for colitis therapy. Anti‐IL‐6R mAb might have benefits in CD patients with Th17 dominance and impaired Treg frequency. (Inflamm Bowel Dis 2011)