Yasunobu Yoshikai

Resident Vδ1+ γδ T Cells Control Early Infiltration of Neutrophils after Escherichia coli Infection via IL-17 Production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that γδ T cell population was the major source of IL-17. Mice depleted of γδ T cells by mAb treatment or mice genetically lacking Vδ1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vδ1+ γδ T cells as the source of IL-17. It was further revealed that γδ T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although γδ T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of γδ T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.

Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle

Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6′-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.

The Cytokine RANKL Produced by Positively Selected Thymocytes Fosters Medullary Thymic Epithelial Cells that Express Autoimmune Regulator

The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Roles of Toll-Like Receptors in C-C Chemokine Production by Renal Tubular Epithelial Cells

Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors (TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and lipoprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production, consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-κB inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation with a set of bacterial components.

Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

ABSTRACT Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall ≫ polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.

Identification of CD25+ γδ T Cells As Fetal Thymus-Derived Naturally Occurring IL-17 Producers

We previously reported that resident γδ T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing γδ T cells did not produce IFN-γ or IL-4, similar to Th17 cells. IL-17-producing γδ T cells specifically express CD25 but not CD122, whereas CD122+ γδ T cells produced IFN-γ. IL-17-producing γδ T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-γ-producing CD122+ γδ T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing γδ T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing γδ T cells. The number of thymic IL-17-producing γδ T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vγ6+Vδ1+ γδ T cells. The number of IL-17-producing γδ T cells was decreased in fetal thymus of Vδ1-deficient mice, whereas Vγ5+ fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vγ6+Vδ1+ T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.

Predominant activation and expansion of V gamma 9-bearing gamma delta T cells in vivo as well as in vitro in Salmonella infection.

Gamma delta T cell receptor-positive cells (gamma delta T cells) have recently been implicated to play a role in the protection against infectious pathogens. Serial studies on gamma delta T cells in 14 patients with salmonella infection have revealed that the proportions of gamma delta T cells (mean +/- SD: 17.9 +/- 13.2%) in salmonella infection were significantly increased (P less than 0.01) compared with 35 normal controls (5.0 +/- 2.6%) and 13 patients with other bacterial infections (4.0 +/- 1.4%). Expansion of gamma delta T cells was more prominent in the systemic form (28.9 +/- 10.8%) than in the gastroenteritis form (10.5 +/- 7.9%) of salmonella infection (P less than 0.01). Most in vivo-expanded gamma delta T cells expressed V gamma 9 gene product. Increased activated (HLA-DR+) T cells were observed in all the six patients with the systemic form and four of the seven with gastroenteritis form. Especially in the six with systemic form, gamma delta T cell activation was significantly higher than alpha beta T cell activation at the early stage of illness (P less than 0.01). When peripheral blood lymphocytes from normal individuals were cultured with live salmonella, gamma delta T cells were preferentially activated and expanded and most of them expressed V gamma 9. Purified gamma delta T cells also responded to live salmonella in vitro. The present study suggests that human gamma delta T cells play a role in the protection against salmonella infection in vivo.

Overexpression of IL-15 In Vivo Increases Antigen-Driven Memory CD8+ T Cells Following a Microbe Exposure

To elucidate potential roles of IL-15 in the maintenance of memory CD8+ T cells, we followed the fate of Ag-specific CD8+ T cells directly visualized with MHC class I tetramers coupled with listeriolysin O (LLO)91–99 in IL-15 transgenic (Tg) mice after Listeria monocytogenes infection. The numbers of LLO91–99-positive memory CD8+ T cells were significantly higher at 3 and 6 wk after infection than those in non-Tg mice. The LLO91–99-positive CD8+ T cells produced IFN-γ in response to LLO91–99, and an adoptive transfer of CD8+ T cells from IL-15 Tg mice infected with L. monocytogenes conferred a higher level of resistance against L. monocytogenes in normal mice. The CD44+CD8+ T cells from infected IL-15 Tg mice expressed the higher level of Bcl-2. Transferred CD44+CD8+ T cells divided more vigorously in naive IL-15 Tg mice than in non-Tg mice. These results suggest that IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+ T cells following microbial exposure via promotion of cell survival and homeostatic proliferation.

Lipocalin 2-Dependent Inhibition of Mycobacterial Growth in Alveolar Epithelium

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.

IL-15 Regulates CD8+ T Cell Contraction during Primary Infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257–264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L−CD127− effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.