Soyoon Ryoo

The Interleukin-1α Precursor is Biologically Active and is Likely a Key Alarmin in the IL-1 Family of Cytokines

Among the 11 members of the IL-1 family cytokines, the precursors of IL-1α, IL-1β, and IL-33 have relatively long N-terminal pro-sequences of approximately 100 amino acid residues prior to the N-terminus of the mature forms. Compared to the mature forms secreted from the cell, 80–90% of the primary translation product is in the intracellular compartment in the precursor form. However, the precursors are readily released from cells during infections but also with non-infectious conditions such a hypoxia and trauma. In this setting, the precursors act rapidly as “alarmins” in the absence of a processing mechanism to remove the pro-sequence and generate a mature form. In the case of IL-1α, the release of the precursor activates adjacent cells via receptor-mediated signaling. However, there are no data comparing the specific activity of the IL-1α precursor to the mature form. In the present study, we compared the precursor and mature forms of recombinant human IL-1α, IL-1β, and IL-33 proteins on the induction of cytokines from A549 cells as well as from human peripheral blood mononuclear cells (PBMC). Similar to the mature form, the IL-1α precursor was active in inducing IL-6 and TNFα, whereas the precursor forms of IL-1β and IL-33 were not active. On PBMC, precursor and mature IL-1α at 0.04 and 0.2 nM were equally active in inducing IL-6. Given the fact that during necrotic cell death, the IL-1α precursor is released intact and triggers IL-1 receptors on tissue macrophages, these data identify the precursor form of IL-1α as a key player in sterile inflammation.

Paradoxical effects of constitutive human IL-32γ in transgenic mice during experimental colitis

Inflammatory cytokines mediate inflammatory bowel diseases (IBDs) and cytokine blocking therapies often ameliorate the disease severity. IL-32 affects inflammation by increasing the production of IL-1, TNFα, and several chemokines. Here, we investigated the role of IL-32 in intestinal inflammation by generating a transgenic (TG) mouse expressing human IL-32γ (IL-32γ TG). Although IL-32γ TG mice are healthy, constitutive serum and colonic tissue levels of TNFα are elevated. Compared with wild-type (WT) mice, IL-32γ TG mice exhibited a modestly exacerbated acute inflammation early following the initiation of dextran sodium sulfate (DSS)-induced colitis. However, after 6 d, there was less colonic inflammation, reduced tissue loss, and improved survival rate compared with WT mice. Associated with attenuated tissue damage, colonic levels of TNFα and IL-6 were significantly reduced in the IL-32γ TG mice whereas IL-10 was elevated. Cultured colon explants from IL-32γ TG mice secreted higher levels of IL-10 compared with WT mice and lower levels of TNFα and IL-6. Constitutive levels of IL-32γ itself in colonic tissues were significantly lower following DSS colitis. Although the highest level of serum IL-32γ occurred on day 3 of colitis, IL-32 was below constitutive levels on day 9. The ability of IL-32γ to increase constitutive IL-10 likely reduces TNFα, IL-6, and IL-32 itself accounting for less inflammation. In humans with ulcerative colitis (UC), serum IL-32 is elevated and colonic biopsies contain IL-32 in inflamed tissues but not in uninvolved tissues. Thus IL-32γ emerges as an example of how innate inflammation worsens as well as protects intestinal integrity.

Interleukin-32 gamma specific monoclonal antibody and developing IL-32 specific ELISA.

Cytokines are essential coordinators of defensive immune responses for resolving the invasion of pathogens such as bacteria, virus, and fungi. However, dysregulated cytokines are the main cause of various autoinflammatory immune disorders such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Interleukin-32 (IL-32) is a recently described cytokine and characterized as a proinflammatory cytokine. IL-32 stimulates monocytes and macrophages to induce important proinflammatory cytokines (IL-1β, IL-6, and TNFα) and chemokines (IL-8 and MIP-2) by activating the NF-κB and p38 mitogen-activated protein (MAP) kinase pathways. The biological activities of IL-32 are associated with epidemic pathogens, Mycobacterium tuberculosis, influenza A virus, and human immunodeficiency virus (HIV). IL-32 is transcribed as six alternative splice variants (α, β, γ, δ, ɛ, and ζ), with IL-32γ being the most active isoform. However, it is unclear which isoform is related to specific disease activities since there are no high quality antibodies available to measure circulating IL-32 in biological samples of patients. Therefore, we developed specific anti-human IL-32γ monoclonal antibodies from recombinant human IL-32γ, which was expressed in Escherichia coli. The IL-32γ specific monoclonal antibodies recognized IL-32 in cell culture supernatants and serum of IL-32γ transgenic mice. The newly developed IL-32γ monoclonal antibodies will be a useful tool to measure IL-32 level in serum samples of various inflammatory diseases. These monoclonal antibodies will be helpful in investigating the precise function of IL-32 in immune responses and in autoinflammatory diseases.

BIRB 796 has Distinctive Anti-inflammatory Effects on Different Cell Types

The pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin (IL)-1β are crucial mediators involved in chronic inflammatory diseases. Inflammatory signal pathways regulate inflammatory cytokine expression-mediated by p38 mitogen activated protein kinase (p38MAPK). Therefore, considerable attention has been given to p38MAPK as a target molecule for the development of a novel anti-inflammatory therapeutics. BIRB 796, one of p38MAPK inhibitor, is a candidate of therapeutic drug for chronic inflammatory diseases. In this study, we investigated the effect of BIRB 796 on inflammatory cytokine productions by lipopolysaccharide (LPS) in different immune cell types. BIRB 796 reduced LPS-mediated IL-8 production in THP-1 cells but not in Raw 264.7 cells. Further analysis of signal molecules by western blot revealed that BIRB 796 sufficiently suppressed LPS-mediated phosphorylation of p38MAPK in both cell types whereas it failed to block inhibitor of kappa B (I-κB) degradation in Raw 264.7 cells. Taken together, these results suggest that the anti-inflammatory function of BIRB 796 depends on cell types.

IL-32γ overexpression accelerates streptozotocin (STZ)-induced type 1 diabetes

Interleukin-32 (IL-32) is a cytokine produced by T lymphocytes, natural killer (NK) cells, monocytes and epithelial cells. There are five splicing variants (α, β, γ, δ, and ε) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice, displaying a high level of IL-32γ expression in the pancreas. We investigated the effect of IL-32γ on streptozotocin (STZ)-induced type 1 diabetes model using IL-32γ TG mice. After a suboptimal diabetogenic dose of STZ administration, IL-32γ TG mice showed significantly increased blood glucose level comparing with that of wild type (WT) mice at day 5. Inflammatory cytokines levels such as, IL-6, TNFα, IFNγ and IL-1β, in pancreas and liver lysates were accessed by a specific cytokine ELISA. The proinflammatory cytokines were significantly enhanced in the pancreas of IL-32γ TG mice comparing to that of WT mice whereas those cytokines levels in liver of IL-32γ TG and WT mice were not changed by STZ. These data indicate that the overexpression of IL-32γ contributes to initial islet β-cells injury and inflammation in pancreas and aggravates STZ-induced type 1 diabetes.

Mycobacterium anyangense sp. nov., a rapidly growing species isolated from blood of Korean native cattle, Hanwoo (Bos taurus coreanae).

From the whole blood of Korean native cattle, Hanwoo (Bos taurus coreanae), a previously undescribed, rapidly growing, scotochromogenic isolate of the genus Mycobacterium is reported. Its 16S rRNA gene sequence, and the sequences of three other genes (hsp65, recA and rpoB) were unique and phylogenetic analysis based on 16S rRNA gene sequence (1420 bp) placed the organism into the rapidly growing Mycobacterium group close to Mycobacterium smegmatis (98.5% sequence similarity). However, phylogenetic analyses based on three different gene sequences (hsp65, recA and rpoB) revealed its location to be distinct from the branch of rapidly growing species. Culture and biochemical characteristics were generally similar to those of Mycobacterium fortuitum. Unique matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS profiles of lipids, unique fatty acid profile, unique mycolic acids profiles and a low DNA-DNA relatedness to M. fortuitum (23.6%) and M. smegmatis (39.7%) strongly supported the taxonomic status of this strain as a representative of a novel species of rapidly growing mycobacteria named Mycobacterium anyangense. The type strain is strain QIA-38(T) ( = JCM 30275(T) = KCTC 29443(T)).

Complete Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype O, Isolated from Gochang, Republic of Korea, in 2016

ABSTRACT The complete genome sequence of a foot-and-mouth disease (FMD) serotype O virus isolated from Gochang, Republic of Korea, is reported here.

Isolation of Mycobacterium bovis from Free-Ranging Wildlife in South Korea

Abstract We demonstrate Mycobacterium bovis infection in wild boar (Sus scrofa) in South Korea. During 2012–15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer (Hydropotes inermis argyropus), raccoon dogs (Nyctereutes procyonoides), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units–variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle (Bos taurus coreanae), Formosan sika deer (Cervus nippon taiouanus), and American elk (Cervus canadensis) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Development of isoform-specific monoclonal antibodies against human IL-18 binding protein.

Interleukin-18 binding protein (IL-18BP) is a soluble antagonist of IL-18 originally discovered while attempting to isolate a soluble receptor by using IL-18-ligand affinity column. IL-18BP has four isoforms (a, b, c, and d) in humans and two isoforms (c and d) in mice. The human isoforms IL-18BPa and IL-18BPc neutralize IL-18 activity sufficiently at an equimolar ratio; however IL-18BPb and IL-18BPd isoforms lack a complete Ig domain at C-terminus and lose the ability to neutralize IL-18 activity. Mouse IL-18BPc and IL-18BPd isoforms, possessing a similar complete Ig domain, also neutralize the biological activity of mouse IL-18 at an equimolar ratio. Here we expressed recombinant proteins of the active human IL-18BP isoforms and developed monoclonal antibodies (MAbs) against human IL-18BP a and c isoforms. We obtained two MAbs (78-4 and 38-3) of human IL-18BPa and two MAbs (18-7 and 29-6) of human IL-18BPc. The MAb clones 18-7 and 29-6 specifically recognized recombinant IL-18BPc in Western blot analyses and ELISA, whereas the MAb clone 78-4 recognized both isoforms in Western blot analyses, but only human IL-18BPa isoform in ELISA. We developed a sandwich ELISA by using the monoclonal antibody specific to human IL-18BPa isoform. The isoform-specific anti-human IL-18BP MAb may be a useful tool in categorizing a distinct group of patients from various autoimmune diseases related to IL-18BP.