Myun Soo Kim

Aminoacyl tRNA Synthetase–Interacting Multifunctional Protein 1 Acts as a Novel B Cell–Activating Factor In Vitro and In Vivo

Endogenous B cell–activating factors play pivotal roles in defense mechanisms by regulating B cell responses. We previously reported that aminoacyl tRNA synthetase–interacting multifunctional protein 1 (AIMP1) functions as a novel proinflammatory cytokine that activates macrophages and dendritic cells. However, roles of AIMP1 in B cell responses have not been studied. In this study, we investigated the effects of AIMP1 on B cell responses and their underlying mechanisms. AIMP1 induced the expression of surface activation markers on murine B cells and the proliferation of B cells. Additionally, AIMP1 increased the expression of activation-induced deaminase and class switch recombination in B cells. AIMP1 also had synergistic effects on B cell activation when combined with CD40 stimulus. Intracellular signaling experiments showed that AIMP1 activated B cells through a protein kinase C/NF-κB signaling pathway. Importantly, i.v. injection of AIMP1 into mice increased the expression of CD69 on splenic B cells and significantly enhanced Ag-specific Ab production. Taken together, our results show that AIMP1 acts as a novel B cell–activating factor. AIMP1-mediated B cell activation and the involvement of AIMP1 in diseases will provide additional information for therapeutic strategies.

IL-33-matured dendritic cells promote Th17 cell responses via IL-1β and IL-6

Graphical abstract Figure. No Caption available. HighlightsIL33 enhances Th17 cell differentiation through DC maturation.IL33 does not directly induce differentiation of naïve CD4+ T cells to Th17 cells.IL33‐matDCs increase surface molecule‐expressions driving T cell activation.IL33‐matDCs promote Th17 cell responses via DC‐derived IL‐1&bgr; and IL‐6. Abstract IL‐33 is associated with a variety of autoimmune diseases, such as sclerosis, inflammatory bowel disease, and rheumatoid arthritis. Although IL‐33 is mainly involved in the induction of Th2 cells, however, the relationship between IL‐33 and Th17 cells is still largely unknown. In this study, we investigated the effects of IL‐33 on DC‐mediated CD4+ T cell activation and Th17 cell differentiation because DCs are essential cells for presenting self‐antigens to CD4+ T cells in autoimmune disease conditions. OT‐II mice were injected with IL‐33‐treated DCs or untreated DCs that were primed by OVA323‐339 peptide, and their Th17 cell responses were compared. Th17 cell population and IL‐17 expression levels were significantly increased in draining lymph nodes of mice injected with IL‐33‐treated DCs, compared with those in mice injected with untreated DCs. IL‐33 treatment maturated DCs to present self‐antigens and to increase production of proinflammatory cytokines such as IL‐1&bgr; and IL‐6, which have a crucial role in Th17 cell differentiation. We found that the IL‐33‐matured DCs enhanced the expression of an early T cell activation marker (CD69) and the Th17 master transcription factor (ROR&ggr;t), but IL‐33 did not directly affect CD4+ T cell differentiation or increase Th17 polarization. Notably, neutralizing IL‐1&bgr; and/or IL‐6 significantly decreased IL‐17 expression levels and Th17 cell population which were increased by the coculture of CD4+ T cells with IL‐33‐matured DCs, indicating that IL‐33 may induce Th17 cell responses via IL‐1&bgr; and IL‐6 derived from IL‐33‐matured DCs.

Aminoacyl tRNA Synthetase–­Interacting Multifunctional Protein 1 Activates NK Cells via Macrophages In Vitro and In Vivo

Aminoacyl tRNA synthetase–interacting multifunctional protein 1 (AIMP1) has been reported to have antitumor effects in various tumor models. However, mechanisms by which AIMP1 ameliorates tumorigenesis are not well understood. As NK cells are a major cell type involved in antitumor activities and AIMP1 is known to activate professional APCs, we determined whether AIMP1 induced NK cell activation directly or via these APCs. AIMP1 induced the expression of surface activation markers on murine NK cells in total splenocytes, although AIMP1 did not directly induce these activation markers of NK cells. The inductive effect of AIMP1 on NK cell activation disappeared in macrophage-depleted splenocytes, indicating that macrophages were required for the AIMP1-induced activation of NK cells. Furthermore, coculture experiments showed that AIMP1 activated NK cells in the presence of isolated macrophages, but failed to activate NK cells when cultured alone or with dendritic cells or B cells. Although AIMP1 significantly promoted TNF-α production by macrophages, the secreted TNF-α partially affected the NK cell activation. Transwell cocultivation analysis revealed that direct contact between macrophages and NK cells was required for the AIMP1-induced NK cell activation. In addition, AIMP1 significantly enhanced cytotoxicity of NK cells against Yac-1 cells. Furthermore, the in vivo administration of AIMP1 also induced NK cell activation systemically with a macrophage-dependent manner. Importantly, AIMP1 dramatically reduced the lung metastasis of melanoma cells, which was mediated by NK cells. Taken together, our results show that AIMP1 induces antitumor responses by NK cell activation mainly via macrophages.

IgA+ plasma cells in murine intestinal lamina propria as a positive regulator of Treg differentiation

Continuous exposure to commensal bacteria gives rise to a complex intestinal immune system that maintains local tolerance, which requires Foxp3‐expressing Treg. Recently, the regulation of TFH function by plasma cells has been reported, but effects of intestinal LP‐PCs, one of the richest plasma cells in the body, on T cell differentiation have not been studied. Here, we investigated whether IgA+ LP‐PCs from murine small intestines had effects on T cell differentiation. Surprisingly, when IgA+ LP‐PCs were cocultured with CD4+ T cells, Foxp3 expression was increased significantly in CD4+CD25− T cells. Results using the Transwell coculture system revealed that soluble factors from LP‐PCs, TGF‐β, and RA were involved in the induction of Foxp3 expression. Furthermore, Foxp3+CD25− T cells were decreased in PP after intestinal depletion of plasma cells. In addition, intestinal colony transfer from SPF to germ‐free mice was demonstrated to generate IgA+ LP‐PCs and Foxp3+ T cells with meaningful correlation in LP. We report for the first time that IgA+ LP‐PCs induce Foxp3 expression in T cells through TGF‐β and RA. LP‐PCs generated by commensal bacteria may play a crucial role in intestinal immunity through the induction of Treg, as well as IgA production.

MST1 deficiency promotes B cell responses by CD4+ T cell-derived IL-4, resulting in hypergammaglobulinemia

MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.

R‐phycoerythrin‐conjugated antibodies are inappropriate for intracellular staining of murine plasma cells

Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE‐conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye‐conjugated form of antibody has been rarely reported. In the present study, we discovered that PE‐conjugated antibodies, but not FITC‐ or APC‐antibodies, selectively stained lamina propria plasma cells (LP‐PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP‐PC‐selective staining with PE‐antibodies was not due to interactions of antibody‐epitope or antibody‐Fc receptor. This unexpected staining by PE‐antibody was not dependent on the mouse strain of LP‐PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP‐PCs were also selectively stained by PE‐conjugated antibodies. Taken together, these results show that PE‐conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

l-Asparaginase-mediated downregulation of c-Myc promotes 1,25(OH)2D3-induced myeloid differentiation in acute myeloid leukemia cells

Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long‐term remission. Although differentiation induction therapy utilizing 1,25(OH)2D3 (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l‐asparaginase (ASNase)‐mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3‐driven phenotypic and functional differentiation of three‐different AML cell lines into monocyte/macrophages, along with c‐Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c‐Myc is a critical factor for improving VD3 efficacy. c‐Myc‐dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3‐mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML.

Costunolide, a sesquiterpene lactone, inhibits the differentiation of pro-inflammatory CD4+ T cells through the modulation of mitogen-activated protein kinases

CD4+ T cell activation and adequate differentiation into effector T helper (Th) cells are crucial for mediating adaptive immune responses to cope with foreign pathogens. Despite the significant role of Th cells, excessive increases in their numbers result in inflammatory and autoimmune diseases. In this study, we investigated the effects of costunolide, a plant-derived natural compound with an anti-inflammatory activity, in regulating Th cells and the underlying mechanisms. Costunolide significantly decreased cell populations of differentiated Th1, Th2, and Th17 subsets under Th subset-polarizing conditions, while exerting statistically negligible effects on Treg cell differentiation. Furthermore, costunolide inhibited the expression level of Th subset-polarizing master genes such as T-bet, GATA3, and RORγt, indicating that costunolide inhibits the differentiation of CD4+ T cells into Th subsets. Additionally, costunolide suppressed the proliferative activity of CD4+ T cells and the expression of CD69 activation marker on CD4+ T cells. When the molecular targets of costunolide were investigated, phosphorylation of ERK and p38 was found to be decreased under Th subset-polarizing conditions, whereas activity of JNK remained unchanged. U0126, an ERK inhibitor, and SB203580, a p38 inhibitor, decreased the expression of CD69 upon TCR stimulation and inhibited CD4+ T cell differentiation, indicating that both ERK and p38 are suggested to be critical molecular targets of costunolide. Taken together, these results suggest that costunolide inhibits the differentiation of CD4+ T cells by suppressing ERK and p38 activities and can be an effective therapeutic agent for T cell-mediated immune diseases.

Comparative analysis of proteins in the culture supernatants of human intestinal epithelial cells infected with the wild-type and rtxE mutant of Vibrio vulnificus

Bacterial virulence factors and secreted extracellular proteins from damaged host cells following infection have been recognized as key mediators in the pathophysiological alterations observed in septic shock, and have also been shown to have a synergistic influence on bacterial pathogenicity. We hypothesized that during infections, virulence factors as well as host-shed proteins may synergistically influence aspects of the pathogenicity of V. vulnificus, such as primary septic shock and overproduction of proinflammatory cytokines. However, virulence factors and host-derived proteins have yet to be clearly evaluated during V. vulnificus infection. In this study, we analyzed and compared the proteins in conditioned supernatants generated from co-cultures of host cells and either wild-type or rtxE mutant V. vulnificus using LC-QTOF-MS/MS analysis. In a previous study, we determined that the culture supernatants of the rtxE mutant V. vulnificus-infected INT-407 cells induced significantly lower levels of IL-8 production from human intestinal epithelial cells than did the culture supernatants of wild-type V. vulnificus-infected INT-407 cells. LC-QTOF-MS/MS analysis results demonstrated that levels of proteins such as HSP90 α/β, 14-3-3 γ, PRX II, hnRNP K, β-actin, α-tubulin and V. vulnificus flagellin were significantly lower in the culture supernatants of rtxE mutant V. vulnificus-infected INT-407 cells than in the culture supernatants of wild-type V. vulnificus-infected INT-407 cells. These results demonstrate that V. vulnificus RTX toxins acting via rtxE, a transporter of virulence factors, play a very important role in the pathogenesis of V. vulnificus, as well as in its initial role in inducing pathogenic mediators from host cells.

Vibrio vulnificus RtxA Is a Major Factor Driving Inflammatory T Helper Type 17 Cell Responses in vitro and in vivo

T helper type 17 (Th17) cells are a subset of pro-inflammatory T helper cells that mediate host defense and pathological inflammation. We have previously reported that host dendritic cells (DCs) infected with Vibrio vulnificus induce Th17 responses through the production of several pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6. V. vulnificus produces RTX toxin (RtxA), an important virulence factor that determines successful pathophysiology. In this study, we investigated the involvement of RtxA from V. vulnificus in Th17 cell induction through the activation and maturation of DCs. The increased expression of the DC surface marker CD40 caused by V. vulnificus wild-type infection was reduced by rtxA gene mutation in V. vulnificus. The mRNA and protein levels of Th17 polarization-related cytokines also decreased in V. vulnificus rtxA mutant-infected DCs. In addition, the co-culture of Th cells and DCs infected with rtxA mutant V. vulnificus resulted in reduction in DC-mediated Th17 responses. Th17 cell responses in the small intestinal lamina propria decreased in mice inoculated with V. vulnificus rtxA mutant as compared to those inoculated with the wild-type strain. These decreases in DC maturation, Th17-polarizing cytokine secretion, and Th17 responses attributed to rtxA mutation were restored following infection with the rtxA revertant strain. Furthermore, the mutation in the hlyU gene encoding the activator of rtxA1 gene reproduced the results observed with rtxA mutation. Taken together, V. vulnificus, by means of RtxA, induces inflammatory Th17 responses, which may be associated with adaptive responses of the host against V. vulnificus infection.