Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement.

Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential 'on-off' switch of pDC activity with therapeutic potential.

Serotonin Is Involved in Autoimmune Arthritis through Th17 Immunity and Bone Resorption.

Rheumatoid arthritis is a chronic disease that results in a disabling and painful condition as it progresses to destruction of the articular cartilage and ankylosis of the joints. Although the cause of the disease is still unknown, evidence argues that autoimmunity plays an important part. There are increasing but contradictory views regarding serotonin being associated with activation of immunoinflammatory pathways and the onset of autoimmune reactions. We studied serotonin's involvement during collagen-induced arthritis in wild-type and Tph1(-/-) mice, which have markedly reduced peripheral serotonin levels. In wild-type mice, induction of arthritis triggered a robust increase in serotonin content in the paws combined with less inflammation. In Tph1(-/-) mice with arthritis, a marked increase in the clinical and pathologic arthritis scores was noticed. Specifically, in Tph1(-/-) mice with arthritis, a significant increase in osteoclast differentiation and bone resorption was observed with an increase in IL-17 levels in the paws and in Th17 lymphocytes in the draining lymph nodes, whereas T-regulatory cells were dampened. Ex vivo serotonin and agonists of the 5-HT2A and 5-HT2B receptors restored IL-17 secretion from splenocytes and Th17 cell differentiation in Tph1(-/-) mice. These findings indicate that serotonin plays a fundamental role in arthritis through the regulation of the Th17/T-regulatory cell balance and osteoclastogenesis.

Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice.

Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC(-/-) mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC(-/-) mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC(-/-) mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response.

TLR-induced cytokines promote effective proinflammatory natural Th17 cell responses.

Naive CD4 lymphocytes undergo a polarization process in the periphery to become induced Th17 (iTh17) cells. Using retinoic acid-related orphan receptor γt (RORγt)-gfp mice, we found that RORγt and the transcription factor promyelocytic leukemia zinc finger (PLZF) are valuable new markers to identify the recently described natural Th17 (nTh17) cell population. nTh17 cells are thymically committed to promptly produce large amounts of IL-17 and IL-22. In this study, we show that, in addition to responding to TCR cross-linking, nTh17 cells secrete IL-17 and IL-22 when stimulated with IL-23 plus IL-1ß, either in recombinant form or in supernatants from TLR4-activated dendritic cells. This innate-like ability of RORγt(+) nTh17 cells to respond to TLR4-induced cytokines was not shared by iTh17 cells. The other distinct properties of RORγt(+) nTh17 cells are their high expression of PLZF and their absence from lamina propria; iTh17 cells are found therein. RORγt(+) nTh17 cells are present in the thymus of germ-free RORγt-gfp and IL-6(-/-) RORΓ: t-gfp mice, indicating that these cells do not require symbiotic microbiota or IL-6 for their generation. Finally, we found that PLZF(+)RORγt(+) nTh17 cells represent one of the primary IL-17-producing innate-like T cell populations in a TLR7 imiquimod model of psoriasis-like disorder, indicating their involvement in this kind of lesion. Collectively, our results reveal RORγt and PLZF as characteristic markers for identifying nTh17 cells and demonstrate one of their novel properties: the ability to respond promptly to TLR-dependent proinflammatory stimuli without TCR engagement, placing them as members of the innate-like T cell family.

Serotonin is a key factor for mouse red blood cell survival.

Serotonin (5-HT) is a monoamine originally purified from blood as a vasoactive agent. In nonneuronal tissues, its presence is linked with the expression of tryptophan hydroxylase 1 (TPH1) that catalyzes the rate-limiting step of its synthesis. Targeted disruption in mice of the TPH1 gene results in very low levels of circulating 5-HT. Previous analysis of the TPH1 knockout (TPH1(-/-)) mouse revealed that they develop a phenotype of macrocytic anemia with a reduced half-life of their circulating red blood cells (RBC). In this study, to establish whether the observed reduced half-life of TPH1(-/-) RBC is an intrinsic or an extrinsic characteristic, we compared their survival to RBC isolated from wild-type mice. Both in vivo and in vitro data converge to demonstrate an extrinsic protective effect of 5-HT since presence of 5-HT in the RBC environment protects RBC from senescence. The protective effect played by 5-HT is not mediated through activation of a classical pharmacological pathway as no 5-HT receptors were detected on isolated RBC. Rather, 5-HT acts as an effective antioxidant since reduction of 5-HT circulating levels are associated with a decrease in the plasma antioxidant capacity. We further demonstrate a link between oxidation and the removal of damaged RBC following transfusion, as supplementation with 5-HT improves RBC post-transfusion survival in a mouse model of blood banking.

Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2ß1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

Regulatory CD4+Foxp3+ T cells control the severity of anaphylaxis.

OBJECTIVE: Anaphylaxis is a life-threatening outcome of immediate-type hypersensitivity to allergen, consecutive to mast cell degranulation by allergen-specific IgE. Regulatory T cells (Treg) can control allergic sensitization and mast cell degranulation, yet their clinical benefit on anaphylactic symptoms is poorly documented. Here we investigated whether Treg action during the effector arm of the allergic response alleviates anaphylaxis. METHODS: We used a validated model of IgE-mediated passive systemic anaphylaxis, induced by intravenous challenge with DNP-HSA in mice passively sensitized with DNP-specific IgE. Anaphylaxis was monitored by the drop in body temperature as well as plasma histamine and serum mMCP1 levels. The role of Treg was analyzed using MHC class II-deficient (Aß(°/°)) mice, treatment with anti-CD25 or anti-CD4 mAbs and conditional ablation of Foxp3(+) Treg in DEREG mice. Therapeutic efficacy of Treg was also evaluated by transfer experiments using FoxP3-eGFP knock-in mice. RESULTS: Anaphylaxis did not occur in mast cell-deficient W/W(v) mutant mice and was only moderate and transient in mice deficient for histamine receptor-1. Defects in constitutive Treg, either genetic or induced by antibody or toxin treatment resulted in a more severe and/or sustained hypothermia, associated with a rise in serum mMCP1, but not histamine. Adoptive transfer of Foxp3(+) Treg from either naïve or DNP-sensitized donors similarly alleviated body temperature loss in Treg-deficient DEREG mice. CONCLUSION: Constitutive Foxp3(+) Treg can control the symptomatic phase of mast cell and IgE-dependent anaphylaxis in mice. This might open up new therapeutic avenues using constitutive rather than Ag-specific Treg for inducing tolerance in allergic patients.

CCL22-producing CD8α- myeloid dendritic cells mediate regulatory T cell recruitment in response to G-CSF treatment.

G-CSF prevents type 1 diabetes in the NOD mouse by promoting the local recruitment of T regulatory cells (Tregs). This is an indirect effect because adoptive transfer of G-CSF-induced tolerogenic dendritic cells (DCs) promotes Treg accumulation. However, the identity of the particular DC subset and the molecule(s) mediating this effect remain unknown. We demonstrate in this study that the adoptive transfer of CD11c(high)CD8α(-) DCs isolated from pegylated G-CSF (pegG-CSF) recipients, but not that of other DC subtypes, enhanced the pancreatic recruitment of CD4(+)CD25(+)Foxp3(+) Tregs, which generated increased amounts of TGF-ß. Likewise, only CD11c(high)CD8α(-) DCs from pegG-CSF recipients secreted the chemokine CCL22 at levels that effectively attracted Tregs. PegG-CSF was more efficient at enhancing the synthesis of CCL22 by CD11c(high)CD8α(-) DCs from the pancreatic lymph nodes compared with those from the spleen. Accordingly, CD11c(high)CD8α(-) DCs from the pancreatic lymph nodes of pegG-CSF recipients were more efficient than their splenic counterparts in the recruitment of Tregs upon adoptive transfer. Predictably, CD11c(high)CD8α(-) DCs failed to recruit these Tregs both in vivo and in vitro following intracellular neutralization of CCL22. These data assign a key role to CD8α(-) DCs and CCL22 in Treg recruitment in the protection of NOD mice against type 1 diabetes following the treatment with G-CSF.

Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells.

Diverse hematopoietic progenitors, including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells, mobilized by hematopoietic growth factors or emerging during parasitic infections, display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-γ released by activated effector T cells (Teffs), by up-regulating their Fas ligand (FasL) expression, which enabled them to kill Teffs through apoptosis. In turn, IFN-γ derived from CpG-proBs enhanced IFN-γ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D, adoptively transferred IFN-γ-deficient CpG-proBs did not prevent T1D development. Additionally, CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation.

Early posttransplantation donor-derived invariant natural killer T-cell recovery predicts the occurrence of acute graft-versus-host disease and overall survival.

Invariant natural killer T (iNKT) cells can experimentally dissociate GVL from graft-versus-host-disease (GVHD). Their role in human conventional allogeneic hematopoietic stem cell transplantation (HSCT) is unknown. Here, we analyzed the post-HSCT recovery of iNKT cells in 71 adult allografted patients. Results were compared with conventional T- and NK-cell recovery and correlated to the occurrence of GVHD, relapse, and survival. We observed that posttransplantation iNKT cells, likely of donor origin, recovered independently of T and NK cells in the first 90 days after HSCT and reached greater levels in recipient younger than 45 years (P = .003) and after a reduced-intensity conditioning regimen (P = .03). Low posttransplantation iNKT/T ratios (ie, < 10(-3)) were an independent factor associated with the occurrence of acute GVHD (aGVHD; P = .001). Inversely, reaching iNKT/T ratios > 10(-3) before day 90 was associated with reduced nonrelapse mortality (P = .009) without increased risk of relapse and appeared as an independent predictive factor of an improved overall survival (P = .028). Furthermore, an iNKT/T ratio on day 15 > 0.58 × 10(-3) was associated with a 94% risk reduction of aGVHD. These findings provide a proof of concept that early postallogeneic HSCT iNKT cell recovery can predict the occurrence of aGVHD and an improved overall survival.

Rapamycin combined with TGF-ß converts human invariant NKT cells into suppressive Foxp3+ regulatory cells.

Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-ß. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4(+) iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4(-) counterpart also became positive. All Foxp3(+) iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-ß. Exposure to TGF-ß decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-ß induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3(+) iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4(+) T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-ß plus rapamycin, thus adding a new facet to their complex functional properties.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria.

The role of the IgE-FcεRI complex in malaria severity in Plasmodium falciparum-hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell-deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI(+) neutrophil population, which is not observed in mice hosting a non-ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI(+) neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI(+) neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

Downregulation of basophil-derived IL-4 and in vivo T(H)2 IgE responses by serotonin and other organic cation transporter 3 ligands.

BACKGROUND: Murine basophils can contribute to the T(H)2 polarization of the immune response by providing rapidly large amounts of IL-4, which suggests that pharmacologic downregulation of this cytokine might provide a strategy to attenuate pathologies associated with excessive production. OBJECTIVE: We examined a number of physiological and pharmacologic ligands of the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines, for their inhibitory effect on IL-4 production by basophils, selecting the most efficient compounds for in vivo evaluation in basophil-dependent experimental models. METHODS: IL-4 production by basophils isolated ex vivo or from bone marrow cultures was assessed in response to various stimuli with or without biogenic monoamines or pharmacologic analogs. Selected compounds were administered in vivo to examine their effect on levels of circulating IgE generated during a basophil-dependent T(H)2 response and on basophil activation in mice receiving IL-33. RESULTS: We found a drastic decrease in IL-4 production by stimulated basophils on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by basophils through the specific high-affinity transporters serotonin transporter and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or Slc22a3) but inhibits their function exclusively through the latter. This downregulation is likewise observed in vivo in response to 5-HT and other OCT3 ligands, as well as in human basophils sorted from PMBCs of nonatopic donors. CONCLUSIONS: We provide evidence for a new means of downregulating IL-4 production by basophils, both in vitro and in vivo, through OCT3 targeted by 5-HT and pharmacologic ligands.

Proinflammatory environment dictates the IL-17-producing capacity of human invariant NKT cells.

CD1d-reactive invariant NKT (iNKT) cells have been implicated in a number of experimental models of human pathologies. Given the scope of their immunoregulatory activities mediated through distinct cytokine patterns, it has been proposed that this functional diversity originates from distinct iNKT subpopulations. In this study, we report that human CD161(+) iNKT cells are intrinsically endowed with the capacity to generate IL-17, but require TGF-ß, IL-1ß, and IL-23 to carry out this potential. IL-17-producing iNKT cells are already present in cord blood but, in contrast to peripheral blood iNKT cells, they cannot generate IFN-γ. These IL-17 producers respond to aryl hydrocarbon receptor stimulation and express IL-23 receptor and retinoic acid-related orphan receptor C, similar to conventional T helper 17 cells, from which they differ by their restricted ability to coproduce IL-22. In conclusion, IL-17 production by human iNKT cells depends on two critical parameters, namely an intrinsic program and a proinflammatory environment.

Treatment with the TLR7 agonist R848 induces regulatory T-cell-mediated suppression of established asthma symptoms.

The evolution of allergic asthma is tightly controlled by effector and regulatory cells, as well as cytokines such as IL-10 and/or TGF-ß, and it is widely acknowledged that environmental exposure to allergens and infectious agents can influence these processes. In this context, the recognition of pathogen-associated motifs, which trigger TLR activation pathways, plays a critical role with important consequences for disease progression and outcome. We addressed the question whether the TLR7 ligand resiquimod (R848), which has been shown to be protective in several experimental allergic asthma protocols, can also suppress typical asthma symptoms once the disease is established. To this end, we used an OVA-induced experimental model of murine allergic asthma in which R848 was injected after a series of challenges with aerosolized OVA. We found that the treatment attenuated allergic symptoms through a mechanism that required Tregs, as assessed by the expansion of this population in the lungs of mice having received R848, and the loss of R848-mediated suppression of allergic responses after in vivo Treg depletion. IL-10 provided only a minor contribution to this suppressive effect that was largely mediated through a TGF-ß-dependent pathway, a finding that opens new therapeutic opportunities for the pharmacological targeting of Tregs.

Modulation of hematopoiesis through histamine receptor signaling.

Histamine is one of the most versatile biogenic amines targeting a variety of cells through extra- and intracellular binding sites and specific receptors, which trigger different signal transduction pathways. It has been associated with cell growth ever since G. Kahlson demonstrated that its synthesis was increased in rapidly growing tissues of plants and animals. He proposed that the newly formed amine, as opposed to its stored counterpart, might play a major role in growth processes. Later on, a number of investigators provided evidence for the contribution of histamine to the expansion of normal and malignant cells, whether of hematopoietic origin or not. These studies have generated conflicting results, revealing growth-promoting as well as inhibitory effects, most likely because the final outcome of exposure to histamine depends on the signaling pathways triggered by distinct receptors and their differential distribution among the target population. The purpose of the present review is to outline our current understanding of the regulatory functions of histamine during growth and differentiation of hematopoietic progenitors, focusing on those mediated through its H4 receptor.

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma.

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.

Basophils: new players in the cytokine network.

Basophils belong to a myeloid cell population that has been ignored for more than a century, mainly because of its paucity, its lack of specific markers, and the absence of experimental models. Given that in mice, even the mere existence of basophils was contested, they were alluded to as "histamine-producing cells" or "non-T non-B cells" in initial studies. It is now widely acknowledged that basophils respond to various IgE-dependent or -independent stimuli, and are engaged in a complex cross talk with a number of immunocompetent cells (T or B lymphocytes, macrophages, dendritic cells, endothelial cells…). Indeed, on the one hand they are critically involved during the onset, the effector phase and exacerbation of T(H)2 responses through their capacity to generate large amounts of cytokines with pro-T(H)2 functions (IL-4, IL-13 TSLP, IL-25), on the other hand, they contribute to immunoglobulin synthesis and class switching, angiogenesis, autoimmunity, tumor immunity and hematopoiesis by producing cytokines such as IL-6, VEGF, GM-CSF and IL-3. Finally, it has been established that they can present antigens to CD4+ or CD8+ T cells in an MHC class II- or class I-dependent manner, respectively. Taken together, these activities confer important immunoregulatory functions upon basophils, both in innate and adaptive immunity.