Takahiro Nagatake

A Tecpr1-Dependent Selective Autophagy Pathway Targets Bacterial Pathogens

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy.

The Airway Antigen Sampling System: Respiratory M Cells as an Alternative Gateway for Inhaled Antigens

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2+/− mice. The titers of Ag-specific Abs were as high in Id2−/− mice as in Id2+/− mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Id2-, RORγt-, and LTβR-independent initiation of lymphoid organogenesis in ocular immunity

The eye is protected by the ocular immunosurveillance system. We show that tear duct–associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid–related orphan receptor γt, lymphotoxin (LT) α1β2–LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.

Cutting edge: Uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis.

CD3−CD4+CD45+ inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer’s patches (PP) in the aerodigestive tract. CXCL13−/− mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3−CD4+CD45+ cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13−/− and plt/plt mice, the formation of germinal centers in CXCL13−/− mice was affected, and their population of B cells was much lower than in the NALT of CXCL13+/− mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

Dietary ω3 fatty acid exerts anti-allergic effect through the conversion to 17,18-epoxyeicosatetraenoic acid in the gut

ω3 polyunsaturated fatty acids (PUFAs) have anti-allergic and anti-inflammatory properties, but the immune-metabolic progression from dietary oil remains to be investigated. Here we identified 17,18-epoxyeicostetraenoic acid (17,18-EpETE) as an anti-allergic metabolite generated in the gut from dietary ω3 α-linolenic acid (ALA). Biochemical and imaging mass spectrometry analyses revealed increased ALA and its metabolites, especially eicosapentaenoic acid (EPA), in the intestines of mice receiving ALA-rich linseed oil (Lin-mice). In murine food allergy model, the decreased incidence of allergic diarrhea in Lin-mice was due to impairment of mast cell degranulation without affecting allergen-specific serum IgE. Liquid chromatography–tandem mass spectrometry-based mediator lipidomics identified 17,18-EpETE as a major ω3 EPA-derived metabolite generated from dietary ALA in the gut, and 17,18-EpETE exhibits anti-allergic function when administered in vivo. These findings suggest that metabolizing dietary ω3 PUFAs generates 17,18-EpETE, which is an endogenous anti-allergic metabolite and potentially is a therapeutic target to control intestinal allergies.

Regulatory Role of Lymphoid Chemokine CCL19 and CCL21 in the Control of Allergic Rhinitis

The lymphoid chemokines CCL19 and CCL21 are known to be crucial both for lymphoid cell trafficking and for the structural organization of lymphoid tissues such as nasopharynx-associated lymphoid tissue (NALT). However, their role in allergic responses remains unclear, and so our current study aims to shed light on the role of CCL19/CCL21 in the development of allergic rhinitis. After nasal challenge with OVA, OVA-sensitized plt (paucity of lymph node T cells) mice, which are deficient in CCL19/CCL21, showed more severe allergic symptoms than did identically treated wild-type mice. OVA-specific IgE production, eosinophil infiltration, and Th2 responses were enhanced in the upper airway of plt mice. Moreover, in plt mice, the number of CD4+CD25+ regulatory T cells declined in the secondary lymphoid tissues, whereas the number of Th2-inducer-type CD8α−CD11b+ myeloid dendritic cells (m-DCs) increased in cervical lymph nodes and NALT. Nasal administration of the plasmid-encoding DNA of CCL19 resulted in the reduction of m-DCs in the secondary lymphoid tissues and the suppression of allergic responses in plt mice. These results suggest that CCL19/CCL21 act as regulatory chemokines for the control of airway allergic disease and so may offer a new strategy for the control of allergic disease.

Mode of Bioenergetic Metabolism during B Cell Differentiation in the Intestine Determines the Distinct Requirement for Vitamin B1

Bioenergetic metabolism varies during cell differentiation, but details of B cell metabolism remain unclear. Here, we show the metabolic changes during B cell differentiation in the intestine, where B cells differentiate into IgA(+) plasma cells (PCs). Naive B cells in the Peyers patches (PPs) and IgA(+) PCs in the intestinal lamina propria (iLP) both used the tricarboxylic acid (TCA) cycle, but only IgA(+) PCs underwent glycolysis. These metabolic differences reflected their dependencies on vitamin B1, an essential cofactor for the TCA cycle. Indeed, the diminished activity of the TCA cycle after dietary vitamin B1 depletion decreased the number of naive B cells in PPs without affecting IgA(+) PCs in the iLP. The maintenance of naive B cells by dietary vitamin B1 was required to induce-but not maintain-intestinal IgA responses against oral antigens. These findings reveal the diet-mediated maintenance of B cell immunometabolism in organized and diffuse intestinal tissues.

Implications of nasopharynx-associated lymphoid tissue (NALT) in the development of allergic responses in an allergic rhinitis mouse model

Nasopharynx‐associated lymphoid tissue (NALT) serves as an important inductive site for mucosal immunity in the upper respiratory tract. Despite its importance in the mucosal immune system, little is known regarding the role of NALT in airway allergic immune responses. We aimed to elucidate the role of NALT in the induction of upper airway allergic responses in a mouse model.

Salt‐inducible kinase 3 deficiency exacerbates lipopolysaccharide‐induced endotoxin shock accompanied by increased levels of pro‐inflammatory molecules in mice

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll‐like receptor 4 on macrophages, several signalling cascades co‐operatively up‐regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt‐inducible kinase [SIK, a member of the AMP‐activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up‐regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over‐expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin‐6 (IL‐6), nitric oxide (NO) and IL‐12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up‐regulated in SIK3‐deficient thioglycollate‐elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3‐deficient TEPM suppressed the levels of the above‐mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3‐knockout (KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1‐KO (n = 8), SIK2‐KO (n = 9) and wild‐type (n = 8 or 9) groups. In addition, SIK3‐KO bone marrow transplantation increased LPS sensitivity of the recipient wild‐type mice, which was accompanied by an increased level of circulating IL‐6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS‐stimulated macrophages.

Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell–related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1− cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide–like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.