IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

Identification of a Human Clonogenic Progenitor with Strict Monocyte Differentiation Potential: A Counterpart of Mouse cMoPs.

Monocytes give rise to macrophages and dendritic cells (DCs) under steady-state and inflammatory conditions, thereby contributing to host defense and tissue pathology. A common monocyte progenitor (cMoP) that is strictly committed to the monocyte lineage has been recently identified in mice. Here, we identified human cMoPs as a CLEC12AhiCD64hi subpopulation of conventional granulocyte-monocyte progenitors (cGMPs) in umbilical cord blood and in bone marrow. Human cMoPs gave rise to monocyte subsets without showing any potential for differentiating into myeloid or lymphoid cells. Within the cGMP population, we also identified revised GMPs that completely lacked DC and lymphoid potential. Collectively, our findings expand and revise the current understanding of human myeloid cell differentiation pathways.

Bipotent or Oligopotent? A macrophage and DC progenitor revisited.

Macrophage and dendritic cell (DC) progenitors (MDPs) produce macrophages and DCs but not other hematopoietic lineages. In this issue of Immunity, Sathe et al. (2014) show that isolated MDP populations hardly contain such bipotent progenitors at clonal levels, arguing against the existence of MDPs.

CD86-based analysis enables observation of bona fide hematopoietic responses.

Hematopoiesis is a system that provides red blood cells (RBCs), leukocytes, and platelets, which are essential for oxygen transport, biodefense, and hemostasis; its balance thus affects the outcome of various disorders. Here, we report that stem cell antigen-1 (Sca-1), a cell surface marker commonly used for the identification of multipotent hematopoietic progenitors (Lin-Sca-1+c-Kit+ cells; LSKs), is not suitable for the analysis of hematopoietic responses under biological stresses with interferon production. Lin-Sca-1-c-Kit+ cells (LKs), downstream progenitors of LSKs, acquire Sca-1 expression upon inflammation, which makes it impossible to distinguish between LSKs and LKs. As an alternative and stable marker even under such stresses, we identified CD86 by screening 180 surface markers. The analysis of infection/inflammation-triggered hematopoiesis on the basis of CD86 expression newly revealed urgent erythropoiesis producing stress-resistant RBCs and intact reconstitution capacity of LSKs, which could not be detected by conventional Sca-1-based analysis.

Commitment to dendritic cells and monocytes.

Dendritic cells (DCs) and monocytes are widely conserved immune cells in vertebrates that arise from hematopoietic stem cells via intermediate progenitors. The progenitors that strictly give rise to DCs or monocytes have been recently identified both in humans and in mice, thereby revealing their differentiation pathways. Advances in analysis technologies have further deepened our understanding of the development of DCs and monocytes from progenitor population-based to individual progenitor cell-based commitment. Since DC-committed progenitors, common DC progenitors (CDPs) and precursor conventional DCs (pre-cDCs) do not differentiate into monocytes, DCs are a distinct lineage from monocytes, although monocytes can acquire DC functions upon activation at tissues where they arrive.

A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential.

Macrophage and dendritic cell (DC) progenitors (MDPs) and common DC progenitors (CDPs) are bone marrow (BM) progenitors with DC differentiation potential. However, both MDPs and CDPs give rise to large numbers of conventional DCs (cDCs) and few plasmacytoid DCs (pDCs), implying that more dedicated pDC progenitors remain to be identified. Here we have described DC progenitors with a prominent pDC differentiation potential. Although both MDPs and CDPs express the macrophage colony stimulating factor (M-CSF) receptor (M-CSFR), the progenitors were confined to a M-CSFR(-) fraction, identified as Lin(-)c-Kit(int/lo)Flt3(+)M-CSFR(-), and expressed high amounts of E2-2 (also known as Tcf4) an essential transcription factor for pDC development. Importantly, they appeared to be directly derived from either CDPs or lymphoid-primed multipotent progenitors (LMPPs). Collectively, our findings provide insight into DC differentiation pathways and may lead to progenitor-based therapeutic applications for infection and autoimmune disease.

Monocyte-derived dendritic cells perform hemophagocytosis to fine-tune excessive immune responses.

Because immune responses simultaneously defend and injure the host, the immune system must be finely regulated to ensure the host's survival. Here, we have shown that when injected with high Toll-like receptor ligand doses or infected with lymphocytic choriomeningitis virus (LCMV) clone 13, which has a high viral turnover, inflammatory monocyte-derived dendritic cells (Mo-DCs) engulfed apoptotic erythroid cells. In this process, called hemophagocytosis, phosphatidylserine (PS) served as an "eat-me" signal. Type I interferons were necessary for both PS exposure on erythroid cells and the expression of PS receptors in the Mo-DCs. Importantly, hemophagocytosis was required for interleukin-10 (IL-10) production from Mo-DCs. Blocking hemophagocytosis or Mo-DC-derived IL-10 significantly increased cytotoxic T cell lymphocyte activity, tissue damage, and mortality in virus-infected hosts, suggesting that hemophagocytosis moderates immune responses to ensure the host's survival in vivo. This sheds light on the physiological relevance of hemophagocytosis in severe inflammatory and infectious diseases.

Prolonged high-intensity exercise induces fluctuating immune responses to herpes simplex virus infection via glucocorticoids.

BACKGROUND: Epidemiologic studies have yielded conflicting results regarding the influence of a single bout of prolonged high-intensity exercise on viral infection. OBJECTIVE: We sought to learn whether prolonged high-intensity exercise either exacerbates or ameliorates herpes simplex virus type 2 (HSV-2) infection according to the interval between virus exposure and exercise. METHODS: Mice were intravaginally infected with HSV-2 and exposed to run on the treadmill. RESULTS: Prolonged high-intensity exercise 17 hours after infection impaired the clearance of HSV-2, while exercise 8 hours after infection enhanced the clearance of HSV-2. These impaired or enhanced immune responses were related to a transient decrease or increase in the number of blood-circulating plasmacytoid dendritic cells. Exercise-induced glucocorticoids transiently decreased the number of circulating plasmacytoid dendritic cells by facilitating their homing to the bone marrow via the CXCL12-CXCR4 axis, which led to their subsequent increase in the blood. CONCLUSION: A single bout of prolonged high-intensity exercise can be either deleterious or beneficial to antiviral immunity.

Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction.

Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in T cell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders.

Role of eosinophils in a murine model of inflammatory bowel disease.

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD), which is triggered spontaneously by unknown mechanisms and manifests as chronic and relapsing inflammatory conditions in the colon. Eosinophil infiltration is often observed in the colonic tissue of ulcerative colitis patients. However, the role of eosinophils in the disease has not been well defined. The aim of this study is to investigate the role of eosinophils in colonic inflammation using the murine model of spontaneous colitis. CC chemokine receptor type 3 (CCR3) and interleukin (IL)-10 double knockout mice (CCR3-/-;IL-10-/-) were utilized to evaluate the function of eosinophils in colitis. The levels of colitis were evaluated by colonoscopy, histology, and real-time PCR measurements to determine expression levels of inflammatory cytokines in the colonic tissue. The levels of cytokines produced by T cells in mesenteric lymph nodes were evaluated by ELISA. There was no significant difference in endoscopic and histological scores between the groups of CCR3-/-;IL-10-/- mice and control CCR3+/-;IL-10-/- mice. There was also no significant difference in the expression levels of pro-inflammatory cytokines in the intestinal tissue between the two groups. Similar results were found for IL-17A and interferon gamma (IFN-γ) production from mesenteric lymph node-derived T cells. Our data indicate that eosinophils do not play a significant role in the immunopathology of colitis in IL-10-/- mice.

Monopoiesis in humans and mice.

Monocytes are a widely conserved cell population in vertebrates with important roles in both inflammation and homeostasis. Under both settings, monocytes continuously arise from hematopoietic progenitors in the bone marrow and, on demand, migrate into tissues through the bloodstream. Monocytes are classified into three subsets-classical, intermediate and non-classical-based on their cell surface expression of CD14 and CD16 in humans and Ly6C, CX3CR1 and CCR2 in mice. In tissues, monocytes differentiate further into monocyte-derived macrophages and dendritic cells to mediate innate and adaptive immune responses and maintain tissue homeostasis. Recently, the progenitors that strictly give rise to monocytes were identified in both humans and mice, thereby revealing the monocyte differentiation pathways.

Flexible fate commitment of E2-2high common DC progenitors implies tuning in tissue microenvironments.

The basic helix-loop-helix transcription factor E2-2 is essential for the development of plasmacytoid dendritic cells (pDCs) but not conventional DCs (cDCs). Here, we generated E2-2 reporter mice and demonstrated that an E2-2high fraction among common DC progenitors, which are a major source of pDCs and cDCs in the steady state, strictly gave rise to pDCs in the presence of Flt3 (Fms-like tyrosine kinase receptor-3) ligand ex vivo or in the secondary lymphoid organs when transferred in vivo. However, in the small intestine, some of these E2-2high progenitors differentiated into cDCs that produced retinoic acid. This transdifferentiation was driven by signaling via the common ß receptor, a receptor for the cytokines IL-3, IL-5 and GM-CSF, which are abundant in the gut. In the presence of GM-CSF and Flt3 ligand, E2-2high-progenitor-derived cDCs consistently induced Foxp3+ Treg cells ex vivo. Our findings reveal the commitment and flexibility of E2-2high progenitor differentiation and imply that pertinent tuning machinery is present in the gut microenvironment.

[Identification of a human progenitor strictly committed to monocytic differentiation: a counterpart of mouse cMoPs].

Monocytes give rise to macrophages and dendritic cells (DCs) under steady-state and inflammatory conditions, thereby contributing to host defense and tissue pathology. Inflammation triggers the differentiation of tissue-infiltrating monocytes into monocyte-derived macrophages and DCs, which are associated with homeostatic host defense reactions and inflammatory diseases. In mice, monocytes are divided into classical Ly6chi- and non-classical Ly6clo-expressing subsets. Ly6clo monocytes are present only in the blood; however, Ly6chi monocytes are found in blood and other tissues (wherein they differentiate into macrophages and DCs). In this context, most Ly6clo monocytes are derived from Ly6chi monocytes. In humans, monocytes comprise major CD14+CD16- and other CD14+CD16+ and CD14loCD16+ monocytes. A monocyte lineage-restricted common monocyte progenitor (cMoP) was previously identified in mice; herein, we introduce human cMoP, which was identified as a CLEC12AhiCD64hi subpopulation of conventional granulocyte-monocyte progenitors (cGMPs) in umbilical cord blood and bone marrow. The human cMoP produced monocyte subsets without showing any potential of differentiating into myeloid or lymphoid cells ex vivo. Within the cGMP population, we also identified revised GMPs that completely lacked DC and lymphoid potential, which sequentially produced cMoPs, pre-monocytes, and monocytes. Collectively, our findings enhance the current understanding of human myeloid cell differentiation pathways.

PI3K-Akt pathway enhances the differentiation of interleukin-27-induced type 1 regulatory T cells.

Interleukin 27 (IL-27) has been identified as a potent cytokine in the differentiation of type 1 regulatory T (Tr1) cells through interactions with several key elements, including transcription factors such as aryl hydrocarbon receptor and IL-21. Autocrine production of IL-21 is known to be important for maintaining IL-10 expression by Tr1 cells. Although previous studies have shown that the phosphoinositide 3-kinase (PI3K) -Akt axis contributes to the differentiation of helper T-cell subsets, the role of the PI3K pathway on Tr1 cell differentiation remains to be elucidated. Here, we demonstrate that suppression of the PI3K-Akt pathway results in impairment of IL-27-induced Tr1 (IL-27-Tr1) cell differentiation in vitro and in vivo. Furthermore, this suppression down-regulates IL-21 receptor expression by Tr1 cells, followed by suppression of IL-10 expression by IL-27-Tr1 cells. These results suggest that the PI3K pathway enhances IL-10 expression by IL-27-Tr1 cells through up-regulation of IL-21 receptors.

Cytosine-Phosphorothionate-Guanine Oligodeoxynucleotides Exacerbates Hemophagocytosis by Inducing Tumor Necrosis Factor-Alpha Production in Mice after Bone Marrow Transplantation.

Hemophagocytic syndrome (HPS) is frequently associated with hematopoietic stem cell transplantation and is treated with some benefit derived from TNF-α inhibitors. However, the mechanisms of how HPS occurs and how a TNF-α inhibitor exerts some benefit to HPS management have remained unclear. We evaluated the effect of toll-like receptor (TLR) ligands, especially focusing on cytosine-phosphorothionate-guanine oligodeoxynucleotide (CpG), a TLR9 ligand, on HPS in mice that underwent transplantation with syngeneic or allogeneic bone marrow (BM) cells (Syn-BMT, Allo-BMT), or with allogeneic BM cells plus splenocytes to promote graft-versus-host disease (GVHD mice). Hemophagocytosis was a common feature early after all BMT, but it subsided in Syn-BMT and Allo-BMT mice. In GVHD mice, however, hemophagocytosis persisted and was accompanied by upregulated production of IFN-γ but not TNF-α, and it was suppressed by blockade of IFN-γ but not TNF-α. A single injection of the TLR9 ligand CpG promoted HPS in all BMT mice and was lethal in GVHD mice, accompanied by greatly upregulated production of TNF-α, IL-6, and IFN-γ. Blocking of TNF-α, but not IL-6 or IFN-γ, suppressed CpG-induced HPS in all BMT mice and rescued GVHD mice from CpG-induced mortality. Thus, TLR9 signaling mediates TNF-α-driven HPS in BMT mice and is effectively treated through TNF-α inhibition.

Non-myeloablative preconditioning with ACK2 (anti-c-kit antibody) is efficient in bone marrow transplantation for murine models of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7±4.92% donor chimerism at 16weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.

Novel interferon-based pre-transplantation conditioning in the treatment of a congenital metabolic disorder.

Hematopoietic stem cell (HSC) gene therapy is a potentially curative treatment modality for monogenic hematological diseases and storage disorders. It is necessary, however, to establish pre-bone marrow (BM) transplant conditioning regimens that minimize DNA damage and toxicity. Type I interferon (IFN) signaling activates quiescent HSCs and enables them to be sensitive to 5-fluorouracil (FU)-mediated cytotoxicity, thus implying a molecular basis for improving HSC transplant outcomes. Here we show that type I IFN preconditioning, without irradiation or DNA alkylating agents, significantly enhanced the HSC engraftment efficiency in wild-type (WT) recipient mice. The importance of active type I IFN signaling in HSC recipients was further demonstrated using mice lacking IFN regulatory factor 2 (IRF2), a transcriptional suppressor of type I IFN signaling. In both WT and Irf2(-/-) recipients, active type I IFN signaling greatly enhanced the sensitivity to 5-FU or low-dose irradiation of HSCs. Importantly, IFN-based pre-BM transplant conditioning was also applicable to the treatment of Sly syndrome, a congenital storage disorder with ß-glucuronidase deficiency, in which it restored enzyme expression at the HSC level and reciprocally reduced pathological glycosaminoglycan storage. Our findings suggest type I IFN-based preconditioning, combined with HSC transplantation, as a novel nongenotoxic treatment of some congenital diseases.

Human CD1c⁺ myeloid dendritic cells acquire a high level of retinoic acid-producing capacity in response to vitamin D3.

All-trans-retinoic acid (RA) plays a critical role in maintaining immune homeostasis. Mouse intestinal CD103⁺ dendritic cells (DCs) produce a high level of RA by highly expressing retinal dehydrogenase (RALDH)2, an enzyme that converts retinal to RA, and induce gut-homing T cells. However, it has not been identified which subset of human DCs produce a high level of RA. In this study, we show that CD1c⁺ blood myeloid DCs (mDCs) but not CD141(high) mDCs or plasmacytoid DCs exhibited a high level of RALDH2 mRNA and aldehyde dehydrogenase (ALDH) activity in an RA- and p38-dependent manner when stimulated with 1α,25-dihydroxyvitamin D3 (VD3) in the presence of GM-CSF. The ALDH activity was abrogated by TLR ligands or TNF. CD103⁻ rather than CD103⁺ human mesenteric lymph node mDCs gained ALDH activity in response to VD3. Furthermore, unlike in humans, mouse conventional DCs in the spleen and mesenteric lymph nodes gained ALDH activity in response to GM-CSF alone. RALDH2(high) CD1c⁺ mDCs stimulated naive CD4⁺ T cells to express gut-homing molecules and to produce Th2 cytokines in an RA-dependent manner. This study suggests that CD1c⁺ mDCs are a major human DC subset that produces RA in response to VD3 in the steady state. The "vitamin D-CD1c⁺mDC-RA" axis may constitute an important immune component for maintaining tissue homeostasis in humans.

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/ß receptor 1), or TIR domain-containing adaptor inducing IFN-ß (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.

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.