Hiroyuki Tezuka

Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells

Immunoglobulin-A has an irreplaceable role in the mucosal defence against infectious microbes. In human and mouse, IgA-producing plasma cells comprise ∼20% of total plasma cells of peripheral lymphoid tissues, whereas more than 80% of plasma cells produce IgA in mucosa-associated lymphoid tissues (MALT). One of the most biologically important and long-standing questions in immunology is why this ‘biased’ IgA synthesis takes place in the MALT but not other lymphoid organs. Here we show that IgA class-switch recombination (CSR) is impaired in inducible-nitric-oxide-synthase-deficient (iNOS-/-; gene also called Nos2) mice. iNOS regulates the T-cell-dependent IgA CSR through expression of transforming growth factor-β receptor, and the T-cell-independent IgA CSR through production of a proliferation-inducing ligand (APRIL, also called Tnfsf13) and a B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF, also called Tnfsf13b). Notably, iNOS is preferentially expressed in MALT dendritic cells in response to the recognition of commensal bacteria by toll-like receptor. Furthermore, adoptive transfer of iNOS+ dendritic cells rescues IgA production in iNOS-/- mice. Further analysis revealed that the MALT dendritic cells are a TNF-α/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNF-α/iNOS-producing dendritic-cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

Interleukin 15–dependent crosstalk between conventional and plasmacytoid dendritic cells is essential for CpG-induced immune activation

The function of interleukin 15 (IL-15) in unmethylated CpG oligodeoxynucleotide (CpG)–induced immune responses remains unknown. Here, in response to CpG, both wild-type and natural killer cell–depleted mice produced IL-12 and became resistant to a lethal dose of Listeria monocytogenes. In contrast, CpG-treated IL-15-deficient mice produced little IL-12 and succumbed to L. monocytogenes. CpG-stimulated conventional dendritic cells (cDCs) were the main producers of both IL-15 and IL-12, but cDCs did not produce IL-12 in the absence of plasmacytoid DCs (pDCs). The cDC-derived IL-15 induced CD40 expression by cDCs. Interaction between CD40 on cDCs and CD40 ligand on pDCs led to IL-12 production by cDCs. Thus, IL-15-dependent crosstalk between cDCs and pDCs is essential for CpG-induced immune activation.

CXCL10-CXCR3 Enhances the Development of Neutrophil-mediated Fulminant Lung Injury of Viral and Nonviral Origin

RATIONALE Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome-coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation. OBJECTIVES We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins. METHODS We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-α/β receptor 1), or TIR domain-containing adaptor inducing IFN-β (TRIF). MEASUREMENTS AND MAIN RESULTS We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a unique CXCR3 receptor via TRIF. CXCL10-CXCR3 acts in an autocrine fashion on the oxidative burst and chemotaxis in the inflamed neutrophils, leading to fulminant pulmonary inflammation. CONCLUSIONS CXCL10-CXCR3 signaling appears to be a critical factor for the exacerbation of the pathology of ARDS. Thus, the CXCL10-CXCR3 axis could represent a prime therapeutic target in the treatment of the acute phase of ARDS of nonviral and viral origins.

Regulation of intestinal homeostasis by dendritic cells.

Summary:  The healthy gut consists of the commensal flora, the epithelial layer, and the gut‐associated lymphoid tissues (GALT). The GALT need to be hyporesponsive to commensal and dietary antigens while possessing the capacity to detect and attack pathogens. Accumulating evidence suggests that dendritic cells (DCs) play integral roles in managing this paradoxical situation and maintaining the complex homeostasis in the gut, which includes the induction of immunoglobulin A (IgA) synthesis. This review outlines the roles of the commensal flora, epithelial layer, and GALT in mucosal homeostasis and inflammatory conditions and highlights recent progress in our understanding of how DCs are involved in IgA synthesis in the gut.

Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages.

Leishmaniases are a major international public health problem, and macrophages are crucial for host resistance to this parasite. To determine if phosphatase and tensin homologue deleted on chromosome ten (Pten), a negative regulator of the PI3K pathway, plays a role in macrophage‐mediated resistance to Leishmania, we generated C57BL/6 mice lacking Pten specifically in macrophages (LysMCrePtenflox/flox mice). Examination of lesions resulting from Leishmania major infection showed that LysMCrePtenflox/flox mice were more susceptible to the parasite than wild‐type (WT) mice in the early phase of the infection, but were eventually able to eliminate the pathogen. In vitro Pten‐deficient macrophages showed a reduced ability to kill parasites in response to IFN‐γ treatment, possibly because the mutant cells exhibited decreased TNF secretion that correlated with reductions in inducible nitric oxide synthase expression and nitric oxide production. In response to various TLR ligands, Pten‐deficient macrophages produced less TNF and IL‐12 but more IL‐10 than WT cells. However, analysis of cells in the lymph nodes draining L. major inoculation sites indicated that both LysMCrePtenflox/flox and WT mice developed normal Th1 responses following L. major infection, in line with the ability of LysMCrePtenflox/flox mice to eventually eliminate the parasite. Our results indicate that the efficient clearance of intracellular parasites requires Pten in macrophages.

Various Types of Dirofilaria immitis Polyproteins Selectively Induce a Th2-Type Immune Response

ABSTRACT Dirofilaria immitis polyproteins (DiAgs) are found as 15-kDa monomeric and 30-kDa dimeric forms in exceretory-secretory products of the adult worm. We evaluated the ability of various types of recombinant DiAg (rDiAg; V1 and V2 as monomers and V1V2, V2V1, V1V1, and V2V2 as dimers) to influence Th1/Th2 immune responses. V1-, V1Vx- and V2-, V2Vx-driven nonspecific immunoglobulin E (IgE) production peaked at 21 and 14 days after administration, respectively. Dimer-induced IgE response was an interesting biphasic pattern with the second peaks on days 35 (V2Vx) or 42 (V1Vx). Absolute amounts of nonspecific IgE production induced with monomers were larger than those observed with dimers at the first peak. The magnitude of cell expansion and interleukin-10 (IL-10) production in mesenteric lymph node (MLN) B-cell induced with rDiAgs was linked to the levels of the first IgE peak in vivo and IgE produced by rDiAg plus IL-4-stimulated B cells in vitro. All rDiAgs failed to augment IgG2c production. V2 and V2Vx elicited IL-4 production by MLN cells more rapidly than V1 and V1Vx. The inhibitory effect of rDiAg on gamma interferon (IFN-γ) production was stronger in monomers than in dimers. Neutralization of IL-10 restored IFN-γ production, whereas the expression of IL-4 and IgE was partly prevented by depletion of IL-10. These results indicate that monomer rather than dimer is an efficient form of DiAg and suggest that the difference of IgE-inducing capacity among these DiAgs is closely associated with the pattern of both B-cell activation and IL-4 production.

Recombinant Dirofilaria immitis Polyprotein That Stimulates Murine B Cells To Produce Nonspecific Polyclonal Immunoglobulin E Antibody

ABSTRACT Nonspecific immunoglobulin E (IgE) production is an event characteristically observed in parasitic helminth infections, but its mechanisms are still unclear. To define these mechanisms, we prepared a recombinant Dirofilaria immitis protein (rDiAg) and assessed its effect on nonspecific IgE production. rDiAg preferentially induced nonspecific IgE production, without eliciting specific IgE production, as well as a Th2-type cytokine profile (high interleukin-4 [IL-4] and IL-10 production but low gamma interferon production) in BALB/c mice. rDiAg significantly elicited the proliferative response of naive B cells. This response was not abolished by polymyxin B, an inhibitor of lipopolysaccharide (LPS), and rDiAg normally expanded splenic B cells from LPS nonresponder C3H/HeJ mice. Thus, the mitogenic effect of rDiAg was not due to LPS contamination. rDiAg also enhanced levels of CD23 expression on splenic B cells. Splenic B cells produced marked levels of IgE when cultured with the combination of rDiAg and IL-4 (rDiAg-IL-4), whereas peritoneal B cells produced negligible levels of IgE. rDiAg-IL-4-induced IgE production by splenic B cells was synergistically increased by coculture with peritoneal B cells. rDiAg-driven IL-10 secretion was higher in peritoneal B cells than in splenic B cells. IgE production by splenic B cells cocultured with peritoneal B cells was decreased to a level comparable to that by splenic B cells in the presence of a neutralizing anti-IL-10 monoclonal antibody. Collectively, these results suggest that rDiAg-induced polyclonal expansion and IgE class switching of splenic B cells contribute to nonspecific IgE production and that these responses are enhanced by peritoneal B-cell-derived IL-10.

A Dirofilaria immitis Polyprotein Up-Regulates Nitric Oxide Production

ABSTRACT We investigated the effect of recombinant Dirofilaria immitis polyprotein (rDiAg) on nitric oxide (NO) production by peritoneal macrophages. rDiAg induced NO production by macrophages from wild-type and lipopolysaccharide-hyporesponsive C3H/HeJ, but not CD40−/−, mice. These results suggest that CD40 is involved in rDiAg-driven NO production by murine macrophages.

Role of SIRPα in regulation of mucosal immunity in the intestine.

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c+ CD11b+ LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady‐state homeostasis of mucosal immunity in the intestine, the flagellin‐stimulated production of IL‐17 or interferon (IFN)‐γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild‐type cells. Moreover, the flagellin‐induced production of IL‐6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL‐10 deficiency. Our data thus suggest that SIRPα expressed on CD11c+ LP cells is important for the production of IL‐17 or IFN‐γ in the LP as well as for the development of colitis induced by IL‐10 deficiency.

Recombinant Dirofilaria immitis-Derived Antigen Can Suppress Passive Cutaneous Anaphylaxis Reactions

Background: High levels of antigen-nonspecific IgE are produced in animals infected with helminth parasites. Generally, the increase in IgE is thought to exacerbate allergic reactions. However, high levels of antigen-nonspecific IgE may alter some features of anaphylactic reactions. To investigate the molecular mechanisms of antigen-nonspecific IgE production induced during filarial infections, we previously constructed rDiAg (recombinant Dirofilaria immitis-derived antigen) in Escherichia coli. In the present study, we examined the effect of rDiAg on the production of antigen-nonspecific IgE and on allergic cutaneous reactions in rats. Methods: Osmotic pumps filled with 200 µg of rDiAg or with 200 µl of PBS (control) were subcutaneously implanted in Wistar rats, and plasma samples were collected weekly thereafter. IgE levels were determined by ELISA. Homologous passive cutaneous anaphylaxis (PCA) reactions with anti-DNP-As IgE were examined 21 days after implantation. 125I-IgE binding assays were examined on peritoneal mast cells from rDiAg-infused rats and control rats. Results: Antigen-nonspecific IgE production was induced in rDiAg-infused rats. PCA reactions were suppressed in rDiAg-infused rats in spite of high levels of IgE and a markedly increased expression of FcΕRI. 125I-IgE did not bind to mast cells derived from rDiAg-infused rats, but it bound dose dependently to mast cells derived from control rats. Conclusion: The present data support the hypothesis that antigen-nonspecific IgE might protect against antigen-specific IgE by means of competition for mast cell receptors. rDiAg is an essential factor to induce antigen-nonspecific IgE in helminth infections.