Publications

TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2-/- and TLR2+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2-/- mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.

<div>Abstract<p>Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism <i>Mycobacterium tuberculosis</i> includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs <i>in vitro</i> and <i>in vivo</i>. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4<sup>+</sup> and CD8<sup>+</sup> T cells to secrete IFN-γ, and induced T-cell–mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA<sub>251–264</sub>-pulsed <i>ex vivo</i> greatly strengthened immune priming <i>in vivo</i>, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy. <i>Cancer Res; 71(8); 2858–70. ©2011 AACR</i>.</p></div>

The mechanisms of induction of liver injury during chronic infection with hepatitis C virus (HCV) are not well understood. Gamma interferon (IFN-γ)-inducible protein 10 (IP-10), a member of the CXC chemokine family, is expressed in the liver of chronic hepatitis C (CHC) patients and selectively recruits activated T cells to the sites of inflammation. Recently, it was shown that a low plasma concentration of IP-10 in CHC patients was closely associated with the outcome of antiviral therapy. In this study, we examined the role of the Toll-like receptor (TLR) pathway on IP-10 production in cells replicating HCV. Among the CXC chemokines, the expression of IP-10 was specifically increased in cells replicating HCV upon stimulation with conventional TLR2 ligands. The enhancement of IP-10 production upon stimulation with TLR2 ligands in cells replicating HCV induced CD44 expression. CD44 is a broadly distributed type I transmembrane glycoprotein and a receptor for the glycosaminoglycan hyaluronan (HA). In CHC patients, the expression of HA in serum has been shown to increase in accord with the progression of liver fibrosis, and HA also works as a ligand for TLR2. In the present study, IP-10 production upon HA stimulation was dependent on the expression of TLR2 and CD44, and a direct association between TLR2 and CD44 was observed. These results suggest that endogenous expression of HA in hepatocytes in CHC patients participates in IP-10 production through an engagement of TLR2 and CD44.

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33 −/− mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2 −/− mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

The chemical synthesis of human interleukin‐2 (IL‐2) , having a core 1 sugar, by a ligation method is reported. Although IL‐2 is a globular glycoprotein, its C‐terminal region, in particular (99‐133), is extremely insoluble when synthesized by solid‐phase method. To overcome this problem, the side‐chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28‐65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL‐2 having full biological activity.

No abstract is provided for this article.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We have shown that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. DAPK-mutant mice, generated by deletion of 74 amino acids from the catalytic kinase domain, were used to investigate the role of the DAPK kinase domain in renal fibrosis following COU. Interstitial collagen and alpha-smooth muscle actin (alpha-SMA) expressions in situ were compared between obstructed kidneys in wild-type and mutant mice. As a result, tubulointerstitial fibrosis, as quantified by interstitial collagen expression, was significantly augmented in mutant kidneys compared with wild-type kidneys following COU. Furthermore, deletion of the kinase domain from DAPK significantly increased the appearance of alpha-SMA-positive myofibroblasts in the renal interstitium during COU. Thus, our results suggest that the kinase domain deleted by gene targeting plays a suppressive role for the development of renal fibrosis through inhibition of the tubular epithelial-to-mesenchymal transition in a mouse model of COU.

Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-γ2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-γ2 in murine lymphopoiesis, the PLC-γ2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-γ2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-γ2 is a critical component of BCR signaling pathways and is required to promote B cell development.

The mobilization of hematopoietic stem/progenitor cells (HSC/HPCs) from the bone marrow (BM) to peripheral blood by granulocyte colony-stimulating factor (G-CSF) is an essential method in clinic. We have shown that the suppression of osteoblastic niche by β-adrenergic signal is critical for this phenomenon (Cell 2006; Cell Stem Cell 2013). Because G-CSF administration causes fever and back pain and these symptoms are ameliorated by non-steroidal anti-inflammatory drugs, we investigated the role of prostaglandin E2 (PGE2) in the BM microenvironment during G-CSF treatment. First, HPC (CFU-C) mobilization by G-CSF (125μg/kg/dose, every 12 hours, 8 divided doses) was significantly augmented in microsomal PGE syntase-1-deficient (mPGES-1-/-) mice (42% increase, n=16, p<0.05), and strongly inhibited by exogenous administration of PGE2 (6mg/kg/day for 2 weeks) to wild-type (WT) mice (52%, n=8-9 p<0.05). These data suggest that G-CSF induces mPGES-1-mediated PGE2 production, which suppresses the HPC mobilization. In the early phase (at 4 doses) of G-CSF administration, mPGES-1 mRNA in BM cells was upregulated (34% increase, n=5, p<0.05). Furthermore, WT mice reconstituted with mPGES-1-/- BM showed higher HPC mobilization than control mice reconstituted with mPGES-1+/+ BM (2.2-fold increase, n=4, p<0.05), which indicated that blood cells might be responsible for additional PGE2 synthesis. To address this, we examined the PGE2 production by ELISA from various lineage cell lines, such as neutrophil precursor 32D, macrophage RAW264.7, B cell Ba/F3, and T cell EL4. No increase was observed by G-CSF in any cell lines; whereas, isoproterenol induced PGE2 production significantly only in 32D culture supernatant (2.4-fold increase compared to vehicle treatment, n=4, p<0.05) accompanied with drastic increase of mPGES-1 mRNA in the cells and norepinephrine showed a similar effect. Primary neutrophils sorted from the BM also demonstrated prompt PGE2 production by isoproterenol (3-fold increase compared to vehicle treatment, n=4, p<0.05) but not by G-CSF. These data suggest that G-CSF-triggered high sympathetic tone stimulates the BM neutrophils to lead PGE2 production. We next assessed the exact roles of PGE2 in HPC mobilization. The inhibitory effect of PGE2 on HPC mobilization was completely abrogated in PGE2 receptor EP4-deficient (EP4-/-) mice, and the chimeric model generated by the reciprocal BM transplantation revealed that it was EP4 in microenvironment, but not in hematopoietic cells, that was critical for this effect. Since PGE2 did not change the CXCL12 behavior, we speculated that PGE2 increased another anchor in the niche, osteopontin (OPN). Immunofluorescence staining demonstrated upregulation of OPN by PGE2 and/or G-CSF in the endosteum, which was abolished in EP4-/- mice. Indeed, the inhibitory effect of PGE2 on HPC mobilization was canceled partially in OPN-/- mice and almost completely in anti-OPN antibody-treated WT mice. PGE2 also inhibited AMD3100-induced HPC mobilization, and this effect was canceled by anti-OPN antibody, which confirmed that PGE2-mediated niche modulation was independent of CXCL12 axis. To assess the induction of OPN by PGE2 directly, we fractionated non-hematopoietic (CD45-CD31-Ter119-) cells isolated from adult femur into three populations, i.e. Sca-1+ALCAM- immature mesenchymal cells, Sca-1-ALCAM- preosteoblasts that favorably support HPCs, and Sca-1-ALCAM+ mature osteoblasts that are most potent to maintain quiescent HSCs in vitro. PGE2 upregulated the OPN protein 2-fold in Sca-1+ALCAM- immature mesenchymal cells in cultures and more dramatically (6-fold) in Sca-1-ALCAM- preosteoblasts as assessed by flow cytometry. In sharp contrast, no OPN induction was observed in Sca-1-ALCAM+ mature osteoblasts. PGE2 failed to induce OPN in all three fractions from EP4-/- mice. In contrast to HPCs, the mobilization of long-term (6 months) repopulating HSCs was not altered in mPGES-1-/- and in PGE2-treated WT mice. This was consistent with the OPN induction profile in fractionated osteoblasts. These results suggest that PGE2 selectively regulates the osteoblastic niche for hematopoietic progenitors, but not for stem cells, by the induction of OPN via EP4 receptor. Collectively, we propose the inter-communication between the mature hematopoietic cells and the niche for their immature progenitors governed by the sympathetic nervous system. Disclosures No relevant conflicts of interest to declare.

Legionella pneumophila (Lp), an important cause of morbidity and mortality from pneumonia, infects alveolar macrophages (AMs) and is recognized by several TLRs as well as Birc1e (NAIP5) and IL-1 converting enzyme-protease activating factor. We examined the role of TLR5 during the murine response to aerosolized Lp infection. At 4 h after infection, Tlr5(-/-) mice had lower numbers of polymorphonuclear neutrophils (PMNs) in their broncho-alveolar lavage fluid in comparison to wild-type (WT) mice. At 24 and 72 h, the PMN recruitment was similar. WT mice infected with a flagellin-deficient strain (LpFlaA-) also showed an impaired early PMN response at 4 h compared with those infected with the WT strain. There was no consistent difference in bacterial counts at any of the time points when comparing the Tlr5(-/-) and WT mice. However, at 6 days after infection, the Tlr5(-/-) mice had increased leukocytic infiltrates in the alveolar and peribronchial interstitial spaces that were consistent with organizing pneumonia. We also examined the role of TLR5 during macrophage infection. In contrast to bone marrow-derived macrophages, AMs secreted TNF-alpha after stimulation with purified flagellin. In addition, WT, but not Tlr5(-/-), AMs produced TNF-alpha after stimulation with Lp. Live LpFlaA- did not induce TNF-alpha secretion in AM. These results suggested that AMs recognize Lp flagellin and that a majority of the Lp-induced TNF-alpha response is TLR5-mediated. Thus, TLR5 mediates recognition of Lp in AMs and performs a distinct role during the in vivo pulmonary immune response through regulation of early PMN recruitment and subsequent later development of pneumonia.

Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.

No abstract is provided for this article.