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Nickel (Ni) is the most frequent metal allergen and induces Th1-dependent type-IV allergies. In local skin, epidermal Langerhans cells (LCs) and/or dermal dendritic cells (DCs) uptake antigens and migrate to draining lymph nodes (LNs). However, the subsets of antigen-presenting cells that contribute to Ni presentation have not yet been identified. In this study, we analyzed the Ni-binding capabilities of murine DCs using fluorescent metal indicator Newport Green. Elicitation of Ni allergy was assessed after intradermal (i.d.) injection of Ni-treated DCs into ear pinnae of Ni-sensitized mice. The Ni-binding capabilities of MHC class IIhi CD11cint migratory DCs were significantly stronger than those of MHC class IIint CD11chi resident DCs and CD11cint PDCA1+ MHC class IIint B220+ plasmacytoid DCs. Migratory DCs in skin-draining and mandibular LNs showed significantly stronger Ni-binding capabilities than those in mesenteric and medial iliac LNs. An i.d. injection of IL-1β induced the activation of LCs and dermal DCs with strong Ni-binding capabilities. Ni-binding LCs were detected in draining LNs after i.d. challenge with IL-1β and Ni. Moreover, an i.d. injection of Ni-treated DCs purified from skin-draining LNs elicited Ni-allergic inflammation. These results demonstrated that migratory DCs in skin-draining LNs have strong Ni-binding capabilities and elicit Ni allergy.

Conventional dendritic cells (cDCs) scan and integrate environmental cues in almost every tissue, including exogenous metabolic signals. While cDCs are critical in maintaining immune balance, their role in preserving energy homeostasis is unclear. Here, we showed that Batf3-deficient mice lacking conventional type 1 DCs (cDC1s) had increased body weight and adiposity during aging. This led to impaired energy expenditure and glucose tolerance, insulin resistance, dyslipidemia, and liver steatosis. cDC1 deficiency caused adipose tissue inflammation that was preceded by a paucity of NK1.1+ invariant NKT (iNKT) cells. Accordingly, among antigen-presenting cells, cDC1s exhibited notable induction of IFN-γ production by iNKT cells, which plays a metabolically protective role in lean adipose tissue. Flt3L treatment, which expands the dendritic cell (DC) compartment, mitigated diet-induced obesity and hyperlipidemia in a Batf3-dependent manner. This effect was partially mediated by NK1.1+ cells. These results reveal a new critical role for the cDC1-iNKT cell axis in the regulation of adipose tissue homeostasis. We are grateful to the Immunology, Ophthalmology, and ENT Department at the UCM for providing useful discussion and to Gillian Dunphy and Antonia Tomás for critically reading the manuscript. We thank the CNIC and UCM facilities. Funding: Work in the S.I. laboratory is funded by the Spanish Ministerio de Ciencia, Innovación (MICINN), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), RTI2018-094484-BI00, and RYC-2016-19463. EHG is the recipient of an FPI fellowship (PRE2019-087509) from the Spanish Ministry of Science and Innovation. Work in the DS laboratory is funded by the CNIC; the European Research Council (ERC-2016-Consolidator Grant 725091); the MICINN, AEI and FEDER (PID2019-108157RB); Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); Atresmedia (Constantes y Vitales prize); and Fundació La Marató de TV3 (201723). Work in the G.S. laboratory receives funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° ERC 260464, EFSD/Lilly European Diabetes Research Programme GS, 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (Investigadores-BBVA-2017) IN[17]_BBM_BAS_0066, MINECO-FEDER SAF2016-79126-R, EUIN2017-85875, Comunidad de Madrid IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733 and Fundación AECC. IN receives funding from EFSD/Lilly (2019), EFSD Rising star (2019), and JdC— Incorporation (IJC2018-035390-I). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation. Sí

Type B coxsackieviruses (CVB) are enteroviruses responsible for a common infectious myocarditis and pancreatitis. DCs and regulatory T cells (Tregs) are key players in controlling virus replication and regulating the immune response and tissue damage, respectively. However, the mechanisms underlying cellular migration to target tissues remain unclear. In the present study, we found that CVB5 infection induced CCL17 production and controlled the migration of CCR4+ DCs and CCR4+ Tregs to the pancreatic lymph nodes (pLN). CVB5 infection of CCR4−/− mice reduced the migration of the CD8α+ DC subset and reduced DC activation and production of IFN-β and IL-12. Consequently, CCR4−/− mice presented decreased IFN-γ-producing CD4+ and CD8+ T cells, an increased viral load and more severe pancreatitis. In addition, CCR4−/− mice had impaired Treg accumulation in pLN as well as increased T lymphocyte activation. Adoptive transfer of CCR4+ Tregs but not CCR4− Tregs was able to regulate T lymphocyte activation upon CVB5 infection. The present data reveal a previously unknown role for CCR4 in coordinating immune cell migration to CVB-infected tissues and in controlling subsequent pancreatitis. These new insights may contribute to the design of future therapies for acute and chronic infection of non-polio enteroviruses.

Conventional dendritic cells (cDCs) bridge antigen detection to the induction of adaptive immunity, playing crucial roles in host defense. Vanderkerken, Baptista, et al. show that LTα1β2-expressing ILC3s, together with B cells, control the size of the splenic cDC compartment and cDC2 differentiation., The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1β2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTβR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1β2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis.

The present study delineates the interaction of a typical PRRSV1.1 isolate 3267 (moderate virulence) with in vitro derived pig conventional dendritic cells, cDC1, cDC2, and a CD14+ population (designated as CD14+ DCs). cDC1 and cDC2 were not susceptible to 3267 infection, but a fraction of CD14+ DCs were infected. After exposure to the virus, all three DC types remained immature as determined by no increase of maturation molecules (MHC-I, MHC-II, CD80/86, CCR7), no release of cytokines, no modification of antigen presentation abilities, and no alteration of endocytic/phagocytic capabilities. However, when infected MARC-145 cells were used as a source of viral antigens, cDC2 and CD14+ DCs showed a significant increase in the expression of maturation molecules and substantial release of cytokines, notably IL-12/IL-23p40 (by both DC types) and IL-10 (by CD14+ DCs). To address the impact of PRRSV1 3267 on TLR3- and TLR7-mediated activation, cDC1, cDC2, and CD14+ DCs were inoculated by the virus (live or UV-inactivated) for 6 h prior to or simultaneously with the addition of poly I:C (TLR3 ligand) or gardiquimod (TLR7 ligand; not used for cDC1). Compared with using TLR ligand alone, combination with the virus did not result in any alteration to the maturation markers on all DC types but changed the cytokine response to either TLR3 or TLR7 ligand. Pre-exposure of cDC2 or CD14+ DCs to the live virus resulted in an increased production of IFN-α upon poly I:C stimulation, while pre-exposure to UV-inactivated virus tended to enhance the release of IL-10 upon gardiquimod stimulation. Simultaneous addition of the live virus and the TLR ligand either had no effect (mainly in cDC2) or impaired most of the cytokine release after gardiquimod stimulation (in CD14+ DCs). When used as antigen presenting cells, cDC2 pre-inoculated by the live virus before addition of gardiquimod impaired the proliferation of CD4–CD8– T cells. In the case of CD14+ DCs, pre-exposure to the live virus or simultaneously added with TLR3 or TLR7 ligand largely decreased the proliferation of CD4–CD8+ and CD4–CD8+ T-cell subsets. For cDC1, no significant changes were observed in cytokine responses or T-cell proliferation after poly I:C stimulation. Of note, cDC1 had a short life during in vitro culturing, for which the results obtained might be biased. Overall, exposure to PRRSV1 did not induce maturation of cDC1, cDC2, or CD14+ DCs, but modified TLR3 and TLR7-associated responses (except for cDC1), which may affect the development of adaptive immunity during PRRSV1 infection. Moreover, the sensing of infected cells was different from that of the free virus.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3−/− mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b−CD103− double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses., CD40L-expressing chimeric antigen receptor (CAR) T cells show enhanced anti-tumor immunity, but the cellular mechanisms are still unclear. Here we show, by analyzing mice deficient of conventional dendritic cell type 1 (cDC1) that cDC1s are induced by CD40L+ CAR T cells to prime endogenous CD8 T cells for a stronger anti-tumor immune response.

Dendritic cells (DC) in the lung that induce Th17 differentiation remain incompletely understood, in part because conventional CD11b+ DCs (cDC2) are heterogeneous. Here, we report a population of cDCs that rapidly accumulates in lungs of mice following house dust extract inhalation. These cells are Ly-6C+, are developmentally and phenotypically similar to cDC2, and strongly promote Th17 differentiation ex vivo. Single cell RNA-sequencing (scRNA-Seq) of lung cDC2 indicates 5 distinct clusters. Pseudotime analysis of scRNA-Seq data and adoptive transfer experiments with purified cDC2 subpopulations suggest stepwise developmental progression of immature Ly-6C+Ly-6A/E+ cDC2 to mature Ly-6C–CD301b+ lung resident cDC2 lacking Ccr7 expression, which then further mature into CD200+ migratory cDC2 expressing Ccr7. Partially mature Ly-6C+Ly-6A/E–CD301b– cDC2, which express Il1b, promote Th17 differentiation. By contrast, CD200+ mature cDC2 strongly induce Th2, but not Th17, differentiation. Thus, Th17 and Th2 differentiation are promoted by lung cDC2 at distinct stages of maturation., Dendritic cells in the lung may be specialised to mediate specific types of immune function. Here the authors show that subpopulations of mouse CD11b+ lung DC at different stages of maturation and phenotype can promote Th17 or Th2 CD4+ T cell differentiation.

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis., Dendritic cells initiate and regulate adaptive immunity and differ according to gut anatomical location. Here the authors show that DC residing in the upper and lower intestines show differential PD-L1 and XCR1 expression and drive specific T cell responses to prevent gut inflammation.

Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion (p < 0.01), greater corneal fluorescein staining (p < 0.001), and higher cDC density in the ocular surface (p < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area (p < 0.001) and volume (p < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice (p < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all p < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea (p < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea (p < 0.05). cDCs in contact with nerves demonstrated a larger surface area (p < 0.001) and volume (p < 0.001), however, they exhibited less sphericity (p < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all p < 0.05). Taken together, we present in vivo evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED.

BACKGROUND: The manipulation of dendritic cells (DCs) for cancer vaccination has not reached its full potential, despite the revolution in cancer immunotherapy. DCs are fundamental for CD8+ T cell activation, which relies on cross-presentation of exogenous antigen on MHC-I and can be fostered by immunogenic cancer cell death. Translational and clinical research has focused on in vitro-generated monocyte-derived DCs, while the vaccination efficacy of natural conventional type 1 DCs (cDC1s), which are associated with improved anti-tumor immunity and specialize on antigen cross-presentation, remains unknown. METHODS: We isolated primary spleen mouse cDC1s and established a protocol for fast ex vivo activation and antigen-loading with lysates of tumor cells that underwent immunogenic cell death by UV irradiation. Natural tumor antigen-loaded cDC1s were transferred and their potential for induction of endogenous CD8+ and CD4+ T cell responses in vivo, cancer prevention and therapy were assessed in three grafted cancer models. Further, we tested the efficacy of natural cDC1 vaccination in combination and comparison with anti-PD-1 treatment in two "wildtype" tumor models not expressing exogenous antigens. RESULTS: Herein, we reveal that primary mouse cDC1s ex vivo loaded with dead tumor cell-derived antigen are activated and induce strong CD8+ T cell responses from the endogenous repertoire upon adoptive transfer in vivo through tumor antigen cross-presentation. Notably, cDC1-based vaccines enhance tumor infiltration by cancer-reactive CD8+ and CD4+ T cells and halt progression of engrafted cancer models, including tumors that are refractory to anti-PD-1 treatment. Moreover, combined tumor antigen-loaded primary cDC1 and anti-PD-1 therapy had strong synergistic effects in a PD-1 checkpoint inhibition susceptible cancer model. CONCLUSIONS: This preclinical proof-of-principle study is first to support the therapeutic efficacy of cancer immunotherapy with syngeneic dead tumor cell antigen-loaded mouse cDC1s, the equivalents of the human dendritic cell subset that correlates with beneficial prognosis of cancer patients. Our data pave the way for translation of cDC1-based cancer treatments into the clinic when isolation of natural human cDC1s becomes feasible. Work in the DS laboratory is funded by the CNIC and grant SAF2016–79040-R from Ministerio de Ciencia, Innovación e Universidades (MCIU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER); B2017/BMD-3733 Immunothercan-CM from Comunidad de Madrid; RD16/0015/0018-REEM from FIS-Instituto de Salud Carlos III, MICINN and FEDER; Acteria Foundation; Constantes y Vitales prize (Atresmedia); La Marató de TV3 Foundation (201723); and the European Research Council (ERC-2016-Consolidator Grant 725091). Work at the IM laboratory is funded by grants from MCIU (SAF2014–52361-R and SAF2017–83267-C2–1-R) and by European Commission VII Framework and Horizon 2020 programs (AICR), Fundación de la Asociación Española Contra el Cáncer (AECC), and Fundación BBVA. SKW is supported by a European Molecular Biology Organization Long-term Fellowship (grant ALTF 438–2016) and a CNIC-International Postdoctoral Program Fellowship (grant 17230–2016). SCK is a recipient of a FPU fellowship (FPU16/03142) from the Spanish Ministry of Education, Culture and Sports. IM and DS labs are funded by the European Commission (635122-PROCROP H2020). The CNIC is supported by the MCIU and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505) Sí

Summary Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo., Classical human dendritic cells (cDCs) are rare sentinel cells specialized in regulating adaptive immunity. Here, the authors show that expression of membrane bound FLT3L, along with stem cell factor (SCF) and CXCL12 in stromal cells induces specification of pre/AS-DCs, type 1 and type2 cDC from haematopoietic stem cells.

Dendritic cells (DC) fulfill an essential sentinel function within the immune system, acting at the interface of innate and adaptive immunity. The DC family, both in mouse and man, shows high functional heterogeneity in order to orchestrate immune responses toward the immense variety of pathogens and other immunological threats. In this review, we focus on the Langerin+CD8+ DC subpopulation in the spleen. Langerin+CD8+ DC exhibit a high ability to take up apoptotic/dying cells, and therefore they are essential to prime and shape CD8+ T cell responses. Next to the induction of immunity toward blood-borne pathogens, i.e., viruses, these DC are important for the regulation of tolerance toward cell-associated self-antigens. The ontogeny and differentiation pathways of CD8+CD103+ DC should be further explored to better understand the immunological role of these cells as a prerequisite of their therapeutic application.

Macrophages (m phi) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete m phi populations carry out these distinct functions or if resident m phi change during inflammation. We show here that most resident m phi in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCII hi, but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1(int) cells is present in resting colon and it expands during experimental colitis. Ly6C(hi) CCR2(+) monocytes can give rise to all m phi subsets in both healthy and inflamed colon and we show that the CX3CR1int pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1 hi m phi. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory m phi. Phenotypic analysis of human intestinal m phi indicates that analogous processes occur in the normal and Crohn's disease ileum. These studies show for the first time that resident and inflammatory m phi in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.

This study was undertaken considering that, despite the broad use of the unopposed estrogen replacement therapy in elderly women, data on estrogen influence on the functional capacity of dendritic cells (DCs), and consequently immune response are limited. We examined the influence of 17 beta-estradiol on phenotype, cytokine secretory profile, and allostimulatory and polarizing capacity of splenic (OX62+) conventional DCs from 26-month-old (aged) Albino Oxford rats matured in vitro in the presence of LPS, a TLR4 agonist, and R848, a TLR7/8 agonist In the presence of 17 beta-estradiol, DCs from aged rats exhibited an impaired ability to mature upon stimulation with LPS, as shown by the lower surface density of MHC II and costimulatory CD80 and CD86 molecules. 17 beta-Estradiol alone enhanced CD40 expression in OX62+ DCs without affecting the expression of other costimulatory molecules, thereby confirming that the expression of this molecule is regulated independently from the regulation of other costimulatory molecules. However, although R848 upregulated the expression of MHC II and CD80 and CD40 costimulatory molecules on DCs, 17 beta-estradiol diminished the effect of this TLR agonist only on MHC II expression. In conjunction, the previous findings suggest that LPS and R848 elicit changes in the expression of costimulatory molecules via triggering differential intracellular signaling pathways. Furthermore, 17 beta-estradiol diminished the stimulatory influence of both LPS- and R848-matured OX62+ DCs on allogeneic CD4+ T lymphocyte proliferation in a mixed lymphocyte reaction (MLR). Moreover, as shown in MLR, the exposure to 17 beta-estradiol during LPS- and R848-induced maturation diminished Th1- and enhanced Th17-driving capacity and reduced Th1-driving capacity of OX62+ DCs, respectively. This suggests that LPS and R848 affect not only the surface phenotype, but also functional characteristics of OX62+ DCs triggering distinct intracellular signaling pathways. Collectively, the findings indicate that estrogen directly acting on OX62+ DCs, may affect CD4+ lymphocyte-dependent immune response in aged female rats. (C) 2014 Elsevier B.V. All rights reserved.

Neospora caninum is a coccidian parasite causative of clinical infections in a wide range of animal hosts. The maturation and activation of splenic conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) were studied here in BALB/c mice challenged intraperitoneal with N. caninum tachyzoites. The number of cDCs was found to decrease in the spleen of the infected mice 12 h and 2 days after the parasitic challenge, whereas at day 5 after infection it was significantly above that of mock-infected controls. In contrast, the number of splenic pDCs did not change significantly on infection. In the infected mice, both cell subtypes displayed an activated phenotype with upregulation of costimulatory and MHC class II molecules. This stimulatory effect was more marked at the earliest assessed time point after infection, 12 h, when a clear increase in the frequency of cDCs (CD8alpha(+) and CD8alpha(-)) and pDCs producing interleukin-12 (IL-12) was also observed. N. caninum tachyzoites could be observed by confocal microscopy associated with sorted DCs. Overall, these results present the first evidence that both cDCs and pDCs mediate in vivo the innate immune response to N. caninum infection through the production of IL-12, a key cytokine for host resistance to neosporosis., We are indebted to Dr Andreia Lino, Dr Elsa Seixas and Dr Flávia Lima forfruitful discussions. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) and FEDER grant no POCTI/CVT/38791/MGI/2001 andSUDOE-FEDER IMMUNONET SOE1/P1/E014. Luzia Teixeira was financedby FCT fellowship SFRH/BD/12983/2003

Monocytes consist of two well-defined subsets, the Ly6C+ and Ly6C– monocytes. Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C– monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C– monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C– monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C– monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C– monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C– monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C– or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection., Author Summary The liver is not only a central organ for efficient metabolism of nutrients and for toxin clearance, but also for immune surveillance, including elimination of intravascular infections. However, excess of nutrients like fat or of toxins like alcohol and certain medications, as well as infections can trigger overactive immune responses which destroy the liver. Such chronic inflammations are major worldwide human health problem with often lethal consequences. Thus, understanding the particular function of various liver immune cells could provide original concepts to alleviate damages in this vital organ. Here, we dissected the heterogeneity, dynamics and function of the myeloid/monocytic cell compartment in the liver of mice infected with Trypanosoma congolense parasite. We established that infiltration of Ly6C+ monocyte subset initiated liver injury in infected mice. More importantly, we revealed that another myeloid cell subset for which the role in liver injury remained elusive, the Ly6C- monocyte subset, exerted hepatoprotective function in infected mice by secreting the anti-inflammatory cytokine IL-10 and by inducing, through cell-contact, the differentiation of pathogenic Ly6C+ monocytes into macrophages expressing genes coding for anti-inflammatory molecules. Thus, augmenting Ly6C- monocyte accumulation or functionality may represent a useful intervention strategy complementing anti-infective medication in conditions of liver injury due to chronic infections.