Published papers

Ebola virus (EBOV) initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal EBOV infections. The VP40 matrix protein is a key viral structural protein that is critical for virion egress. Physical and functional interactions between VP40 and host proteins such as Tsg101 and Nedd4 facilitate efficient release of VP40-driven viruslike particles (VLPs) and infectious virus. Here, we show that host suppressor of cytokine signaling 3 (SOCS3) can also bind to EBOV VP40, leading to enhanced ubiquitinylation and egress of VP40. Indeed, titers of infectious EBOV derived from SOCS3 knockout mouse embryonic fibroblasts (MEFs) were significantly reduced compared to those from wild-type (WT) MEFs at 24 and 48 h postinfection. Importantly, this reduced virus yield could be rescued back to WT levels by exogenously expressing SOCS3. Lastly, we show that SOCS3 expression is induced by EBOV glycoprotein (GP) expression and that VLPs containing EBOV VP40 and GP induced production of proinflammatory cytokines, which induced SOCS3 for negative-feedback regulation. These data indicate that host innate immune protein SOCS3 may play an important role in budding and pathogenesis of EBOV.

The transcriptional regulator IκB-ζ is important for the control of apoptosis in keratinocytes. Thus, IκB-ζ-deficient mice develop autoimmune diseases, such as Sjögren’s syndrome. However, T cells also play a pivotal role in Sjögren’s syndrome. To study the role of IκB-ζ in T cells, we generated T cell-specific, IκB-ζ-deficient mice. We observed increased numbers of peripheral effector/memory CD4+ cells and IFN-γ-producing CD4+ cells in 3-week-old mice. We found that IκB-ζ can be up-regulated by TGF-β1 in naïve CD4+ T cells and that it negatively regulates IFN-γ expression. In addition, we generated Treg-specific, IκB-ζ deficient mice and found that IκB-ζ is dispensable for the plasticity and stability of Tregs. However, Tregs from T cell-specific, IκB-ζ-deficient mice have reduced immunoregulatory function. Thus, our data reveal a previously unappreciated role for IκB-ζ in IFN-γ production in T cells and the immunoregulatory function of Tregs.

Objective To elucidate the pathologic role of follicular helper T (Tfh) cells and their subsets in active, untreated IgG4-related disease. Methods Fifteen patients with active, untreated, biopsy-proven IgG4-related disease, 24 patients with primary Sjögren's syndrome (SS), 12 patients with allergic rhinitis, and 23 healthy controls were evaluated. Tfh cells were defined as CD3+CD4+CXCR5+CD45RA- cells. Circulating Tfh cell subsets among CXCR5+CD45RA-CD4+ T cells were defined as Tfh17 cells (CXCR3-CCR6+), Tfh1 cells (CXCR3+CCR6-), or Tfh2 cells (CXCR3-CCR6-). CD19+CD20-CD27+CD38+ cells were defined as plasmablasts. Serum cytokine levels (interleukin-4 [IL-4], IL-10, IL-21, and IL-33) were measured by cytometric bead array or enzyme-linked immunosorbent assay. Results Patients with IgG4-related disease had significantly increased levels of Tfh2 cells compared to healthy controls or patients with primary SS or allergic rhinitis. Increased Tfh2 levels were strongly associated with increased serum IgG4 levels and the IgG4:IgG ratio in IgG4-related disease. A positive correlation was observed between Tfh2 counts, plasmablast counts, and serum IL-4 levels. Interestingly, levels of plasmablasts and serum IL-4 and IgG4 decreased after treatment with glucocorticoids, whereas no obvious change was observed in Tfh2 cell counts. Conclusion The Tfh2 cell count was specifically increased in IgG4-related disease and was correlated with elevated serum levels of IgG4 and IL-4 and plasmablast counts. Tfh2 cells were the only component that was not affected by glucocorticoid treatment, suggesting that Tfh2 cells are the cell type implicated in IgG4-related disease.

The transcriptional regulator IκB-ζ is important for the control of apoptosis in keratinocytes. Thus, IκB-ζ-deficient mice develop autoimmune diseases, such as Sjögren’s syndrome. However, T cells also play a pivotal role in Sjögren’s syndrome. To study the role of IκB-ζ in T cells, we generated T cell-specific, IκB-ζ-deficient mice. We observed increased numbers of peripheral effector/memory CD4+ cells and IFN-γ-producing CD4+ cells in 3-week-old mice. We found that IκB-ζ can be up-regulated by TGF-β1 in naïve CD4+ T cells and that it negatively regulates IFN-γ expression. In addition, we generated Treg-specific, IκB-ζ deficient mice and found that IκB-ζ is dispensable for the plasticity and stability of Tregs. However, Tregs from T cell-specific, IκB-ζ-deficient mice have reduced immunoregulatory function. Thus, our data reveal a previously unappreciated role for IκB-ζ in IFN-γ production in T cells and the immunoregulatory function of Tregs.

Objective To elucidate the pathologic role of follicular helper T (Tfh) cells and their subsets in active, untreated IgG4-related disease. Methods Fifteen patients with active, untreated, biopsy-proven IgG4-related disease, 24 patients with primary Sjögren's syndrome (SS), 12 patients with allergic rhinitis, and 23 healthy controls were evaluated. Tfh cells were defined as CD3+CD4+CXCR5+CD45RA- cells. Circulating Tfh cell subsets among CXCR5+CD45RA-CD4+ T cells were defined as Tfh17 cells (CXCR3-CCR6+), Tfh1 cells (CXCR3+CCR6-), or Tfh2 cells (CXCR3-CCR6-). CD19+CD20-CD27+CD38+ cells were defined as plasmablasts. Serum cytokine levels (interleukin-4 [IL-4], IL-10, IL-21, and IL-33) were measured by cytometric bead array or enzyme-linked immunosorbent assay. Results Patients with IgG4-related disease had significantly increased levels of Tfh2 cells compared to healthy controls or patients with primary SS or allergic rhinitis. Increased Tfh2 levels were strongly associated with increased serum IgG4 levels and the IgG4:IgG ratio in IgG4-related disease. A positive correlation was observed between Tfh2 counts, plasmablast counts, and serum IL-4 levels. Interestingly, levels of plasmablasts and serum IL-4 and IgG4 decreased after treatment with glucocorticoids, whereas no obvious change was observed in Tfh2 cell counts. Conclusion The Tfh2 cell count was specifically increased in IgG4-related disease and was correlated with elevated serum levels of IgG4 and IL-4 and plasmablast counts. Tfh2 cells were the only component that was not affected by glucocorticoid treatment, suggesting that Tfh2 cells are the cell type implicated in IgG4-related disease.

Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1-/- ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

Cytokines from group 2 innate lymphoid cells (ILC2s) have been implicated in acute allergic responses, such as papain-induced lung inflammation. However, the means of homeostatic regulation of ILC2s have not been established. In this study, we demonstrated that Spred1, a negative regulator of the Ras-ERK pathway, plays an important role in the proliferation and apoptosis of ILC2s and in cytokine secretion from ILC2s. Intranasal administration of papain stimulated IL-5 and IL-13 production in the lung, which was enhanced when Spred1 was deleted. In vitro, Spred1-/- ILC2s proliferated faster than wild type ILC2s did and produced higher levels of cytokines in response to IL-33. On the contrary, a MEK inhibitor suppressed ILC2 proliferation and cytokine production. Spred1 deficiency resulted in stabilization of GATA3, which has been shown to play essential roles in the maintenance and cytokine production of ILC2. These data suggest that Spred1 negatively regulates ILC2 development and functions through the suppression of the Ras-ERK pathway.

Colonization with a mixture of Clostridium species has been shown to induce accumulation of induced regulatory T (iTreg) cells in the colon. Transforming growth factor-β (TGF-β) is an essential factor for iTreg cell induction; however, the relationship between Clostridium species and TGF-β remains to be clarified. Here we demonstrated that a gram-positive probiotic bacterial strain, Clostridium butyricum (C. butyricum), promoted iTreg cell generation in the intestine through induction of TGF-β1 from lamina propria dendritic cells (LPDCs). C. butyricum-mediated TGF-β1 induction was mainly Toll-like receptor 2 (TLR2) dependent, and the ERK-AP-1 kinase pathway played an important role. In addition, the autocrine TGF-β-Smad3 transcription factor signal was necessary for robust TGF-β expression in DCs, whereas Smad2 negatively regulated TGF-β expression. Smad2-deficient DCs expressed higher concentrations of TGF-β and were tolerogenic for colitis models. This study reveals a novel mechanism of TGF-β induction by Clostridia through a cooperation between TLR2-AP-1 and TGF-β-Smad signaling pathways.

Colonization with a mixture of Clostridium species has been shown to induce accumulation of induced regulatory T (iTreg) cells in the colon. Transforming growth factor-β (TGF-β) is an essential factor for iTreg cell induction; however, the relationship between Clostridium species and TGF-β remains to be clarified. Here we demonstrated that a gram-positive probiotic bacterial strain, Clostridium butyricum (C. butyricum), promoted iTreg cell generation in the intestine through induction of TGF-β1 from lamina propria dendritic cells (LPDCs). C. butyricum-mediated TGF-β1 induction was mainly Toll-like receptor 2 (TLR2) dependent, and the ERK-AP-1 kinase pathway played an important role. In addition, the autocrine TGF-β-Smad3 transcription factor signal was necessary for robust TGF-β expression in DCs, whereas Smad2 negatively regulated TGF-β expression. Smad2-deficient DCs expressed higher concentrations of TGF-β and were tolerogenic for colitis models. This study reveals a novel mechanism of TGF-β induction by Clostridia through a cooperation between TLR2-AP-1 and TGF-β-Smad signaling pathways.

Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.

Melanoma is the most serious type of skin cancer, with a highly metastatic phenotype. In this report, we show that signal transducing adaptor protein 2 (STAP-2) is involved in cell migration, proliferation, and melanogenesis as well as chemokine receptor expression and tumorigenesis in B16F10 melanoma cells. This was evident in mice injected with STAP-2 shRNA (shSTAP-2)-expressing B16F10 cells, which infiltrated organs in a completely different pattern from the original cells, showing massive colonization in the liver, kidney, and neck but not in the lung. The most important finding was that STAP-2 expression determined tyrosinase protein content. STAP-2 colocalized with tyrosinase in lysosomes and protected tyrosinase from protein degradation. It is noteworthy that B16F10 cells with knocked down tyrosinase showed similar cell characteristics as shSTAP-2 cells. These results indicated that tyrosinase contributed to some cellular events beyond melanogenesis. Taken together, one possibility is that STAP-2 positively regulates the protein levels of tyrosinase, which determines tumor invasion via controlling chemokine receptor expression.

T cells are considered to develop through three stages, from naïve T (Tn) into central memory T (Tcm) and finally into effector memory T (Tem). Among the subsets of Tn, stem cell memory T (Tscm) were recently found to be the least developed memory subset. While this subset was revealed to possess self-reproducibility and multipotentiality, little is known about the relationship between development and polarity. We conducted transcriptome analysis of human CD4+ T subsets and found that Tscm was a clearly distinct subset, located between Tn and Tcm. Surface antigen analysis and differentiation assay showed that the flexibility of polarity and the cytokine production progressively changed as the differentiation of CD4+ T cells advanced. Interestingly, we found that most cells of the CD45RO-CCR7+CCR6+ subset, hitherto considered the naïve precursor of Th17, were in fact Tscm. These findings may advance our understanding of the highly heterogeneous human helper T cells.

T cells are considered to develop through three stages, from naïve T (Tn) into central memory T (Tcm) and finally into effector memory T (Tem). Among the subsets of Tn, stem cell memory T (Tscm) were recently found to be the least developed memory subset. While this subset was revealed to possess self-reproducibility and multipotentiality, little is known about the relationship between development and polarity. We conducted transcriptome analysis of human CD4+ T subsets and found that Tscm was a clearly distinct subset, located between Tn and Tcm. Surface antigen analysis and differentiation assay showed that the flexibility of polarity and the cytokine production progressively changed as the differentiation of CD4+ T cells advanced. Interestingly, we found that most cells of the CD45RO-CCR7+CCR6+ subset, hitherto considered the naïve precursor of Th17, were in fact Tscm. These findings may advance our understanding of the highly heterogeneous human helper T cells.

Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.