Disease Tolerance as an Inherent Component of Immunity.

Pathogenic organisms exert a negative impact on host health, revealed by the clinical signs of infectious diseases. Immunity limits the severity of infectious diseases through resistance mechanisms that sense and target pathogens for containment, killing, or expulsion. These resistance mechanisms are viewed as the prevailing function of immunity. Under pathophysiologic conditions, however, immunity arises in response to infections that carry health and fitness costs to the host. Therefore, additional defense mechanisms are required to limit these costs, before immunity becomes operational as well as thereafter to avoid immunopathology. These are tissue damage control mechanisms that adjust the metabolic output of host tissues to different forms of stress and damage associated with infection. Disease tolerance is the term used to define this defense strategy, which does not exert a direct impact on pathogens but is essential to limit the health and fitness costs of infection. Under this argument, we propose that disease tolerance is an inherent component of immunity.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Ligation of Notch receptors in human conventional and plasmacytoid dendritic cells differentially regulates cytokine and chemokine secretion and modulates Th cell polarization.

Notch signaling is involved in multiple cellular processes. Recent data also support the prominent role of Notch signaling in the regulation of the immune response. In this study, we analyzed the expression and function of Notch receptors and ligands on both human blood conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs). The expression and modulation upon TLR activation of Notch molecules partially differed between cDCs and pDCs, but functional involvement of the Notch pathway in both cell types was clearly revealed by specific inhibition using DAPT. Beyond the induction of Notch target genes and modulation of maturation markers, Notch pathway was also involved in a differential secretion of some specific cytokines/chemokines by DC subsets. Whereas Notch ligation induced IL-10 and CCL19 secretion in cDCs, Notch inhibition resulted in a diminished production of these proteins. With regard to pDCs, Notch activation induced TNF-α whereas Notch inhibition significantly abrogated the secretion of CCL19, CXCL9, CXCL10, and TNF-α. Additionally, Notch modulation of DC subsets differentially affected Th polarization of allostimulated T cells. Our results suggest that the Notch pathway may function as an additional mechanism controlling human DC responses, with differential activity on cDCs and pDCs. This control mechanism may ultimately contribute to define the local milieu promoted by these cells under the particular conditions of the immune response.

Maternal dietary exposure to mycotoxin aflatoxin B1 promotes intestinal immune alterations and microbiota modifications increasing infection susceptibility in mouse offspring.

Mycotoxins are secondary metabolites produced by fungi occurring in food that are toxic to animals and humans. Early-life mycotoxins exposure has been linked to diverse pathologies. However, how maternal exposure to mycotoxins impacts on the intestinal barrier function of progeny has not been explored. Here, exposure of pregnant and lactating C57Bl/6J female mice to aflatoxin B1 (AFB1; 400 µg/kg body weight/day; 3 times a week) in gelatine pellets, from embryonic day (E)11.5 until weaning (postnatal day 21), led to gut immunological changes in progeny. The results showed an overall increase of lymphocyte number in intestine, a reduction of expression of epithelial genes related to microbial defence, as well as a decrease in cytokine production by intestinal type 2 innate lymphoid cells (ILC2). While susceptibility to chemically induced colitis was not worsened, immune alterations were associated with changes in gut microbiota and with a higher vulnerability to infection by the protozoan Eimeria vermiformis at early-life. Together these results show that maternal dietary exposure to AFB1 can dampen intestinal barrier homeostasis in offspring decreasing their capability to tackle intestinal pathogens. These data provide insights to understand AFB1 potential harmfulness in early-life health in the context of intestinal infections.

Glycan-based shaping of the microbiota during primate evolution.

Genes encoding glycosyltransferases can be under relatively high selection pressure, likely due to the involvement of the glycans synthesized in host-microbe interactions. Here, we used mice as an experimental model system to investigate whether loss of α-1,3-galactosyltransferase gene (GGTA1) function and Galα1-3Galß1-4GlcNAcß1-R (αGal) glycan expression affects host-microbiota interactions, as might have occurred during primate evolution. We found that Ggta1 deletion shaped the composition of the gut microbiota. This occurred via an immunoglobulin (Ig)-dependent mechanism, associated with targeting of αGal-expressing bacteria by IgA. Systemic infection with an Ig-shaped microbiota inoculum elicited a less severe form of sepsis compared to infection with non-Ig-shaped microbiota. This suggests that in the absence of host αGal, antibodies can shape the microbiota towards lower pathogenicity. Given the fitness cost imposed by bacterial sepsis, we infer that the observed reduction in microbiota pathogenicity upon Ggta1 deletion in mice may have contributed to increase the frequency of GGTA1 loss-of-function mutations in ancestral primates that gave rise to humans.

Ferritin regulates organismal energy balance and thermogenesis.

OBJECTIVE: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis. METHODS: We developed a mouse strain, referred herein as FthR26 fl/fl, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5'end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult FthR26Δ/Δ mice, testing whether FTH is required for maintenance of organismal homeostasis. RESULTS: Under standard nutritional Fe supply, Fth deletion in adult FthR26Δ/Δ mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue. CONCLUSION: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation.

Human scavenger protein AIM increases foam cell formation and CD36-mediated oxLDL uptake.

AIM is expressed by macrophages in response to agonists of the nuclear receptors LXR/RXR. In mice, it acts as an atherogenic factor by protecting macrophages from the apoptotic effects of oxidized lipids. In humans, it is detected in atherosclerotic lesions, but no role related to atherosclerosis has been reported. This study aimed to investigate whether the role of hAIM extends beyond inhibiting oxidized lipid-induced apoptosis. To accomplish this goal, functional analysis with human monocytic THP1 cells and macrophages differentiated from peripheral blood monocytes were performed. It was found that hAIM reduced oxLDL-induced macrophage apoptosis and increased macrophage adhesion to endothelial ICAM-1 by enhancing LFA-1 expression. Furthermore, hAIM increased foam cell formation, as shown by Oil Red O and Nile Red staining, as well as quantification of cholesterol content. This was not a result of decreased reverse cholesterol transport, as hAIM did not affect the efflux significantly from [(3)H] Cholesterol-laden macrophages driven by plasma, apoA-I, or HDL2 acceptors. Rather, flow cytometry studies indicated that hAIM increased macrophage endocytosis of fluorescent oxLDL, which correlated with an increase in the expression of the oxLDLR CD36. Moreover, hAIM bound to oxLDL in ELISA and enhanced the capacity of HEK-293 cells expressing CD36 to endocytose oxLDL, as studied using immunofluorescence microscopy, suggesting that hAIM serves to facilitate CD36-mediated uptake of oxLDL. Our data represent the first evidence that hAIM is involved in macrophage survival, adhesion, and foam cell formation and suggest a significant contribution to atherosclerosis-related mechanisms in the macrophage.

Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability.

Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.

Capture of cell-derived microvesicles (exosomes and apoptotic bodies) by human plasmacytoid dendritic cells.

cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cDCs, whereas pDCs display poor capacity to capture cell-derived antigens. However, animal models of organ transplantation suggested a role for pDCs in tolerance induction via phagocytosis of donor antigens. In a transplantation setting, microvesicles, such as apoptotic bodies and exosomes secreted by the graft, may be potential sources of alloantigen. Here, we tested the capacity of human pDCs to capture exosomes and apoptotic bodies from Jurkat T cells. Exosomes and apoptotic bodies were indeed captured by pDCs, although required longer times of incubation when compared with the highly endocytic cDCs. In cDCs and pDCs, exosome capture was more efficient than apoptotic bodies. Endocytosis inhibitors clearly impaired exosome capture by cDCs, although this could not be verified in pDCs as a result of cellular toxicity. Functionally, capture of Jurkat-derived exosomes did not induce nor prevent pDC maturation, and exosome-loaded pDCs induced T cell proliferation, suggesting a link between capture and presentation. Thus, exosomes and apoptotic bodies may be sources of antigen for human pDCs.

TLR-activated conventional DCs promote γ-secretase-mediated conditioning of plasmacytoid DCs.

Cooperative events between DC subsets involve cell contact and soluble factors. Upon viral challenge, murine pDCs induce cDC cooperation through CD40-CD40L interactions and IL-15 secretion, whereas in humans, the same effect is mediated by IFN-α. Conversely, during bacterial infections, pDC maturation may be induced by activated cDCs, although no mechanisms had been described so far. Here, we investigate how human pDCs are "conditioned" by cDCs. Blood-borne DC subsets (cDCs and pDCs) were sorted from healthy donors. IL-3-maintained pDCs were cocultured with LPS-activated, poly (I:C)-activated, or control cDCs [cDC(LPS), cDC(P(I:C)), cDC(CTRL)]. Coculture experiments showed that cDC(LPS)-conditioned pDCs up-regulated maturation markers, such as CD25 and CD86, whereas SNs contained higher amounts of IL-6 and CCL19 compared with control conditions. Gene-expression analyses on sorted cDC(LPS) or cDC(P(I:C)) conditioned pDCs confirmed the induction of several genes, including IL-6 and CCL19 and remarkably, several Notch target genes. Further studies using the γ-secretase/Notch inhibitor DAPT and soluble Notch ligands resulted in a significantly reduced expression of canonical Notch target genes in conditioned pDCs. DAPT treatment also hampered the secretion of CCL19 (but not of IL-6) by cDC(LPS) conditioned pDCs. These results reveal the involvement of γ-secretase-mediated mechanisms, including the Notch pathway, in the cell contact-dependent communication between human DC subsets. The resulting partial activation of pDCs after encountering with mature cDCs endows pDCs with an accessory function that may contribute to T cell recruitment and activation.