Vasculo-toxic and pro-inflammatory action of unbound haemoglobin, haem and iron in transfusion-dependent patients with haemolytic anaemias.

Increasing evidence suggests that free haem and iron exert vasculo-toxic and pro-inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion-dependent patients with ß-thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic 'free' species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem- and iron-related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble endothelial selectin (sE-selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem-related parameters and soluble intercellular adhesion molecule 1 (sICAM-1), sVCAM-1, interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) with iron-related parameters. While hereditary spherocytosis was associated with the highest IL-6 and TNFα levels, ß-thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions.

Atherosclerosis is aggravated by iron overload and ameliorated by dietary and pharmacological iron restriction.

AIMS: Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE-/- FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). METHODS AND RESULTS: Compared with normo-ferremic ApoE-/- mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE-/- FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE-/- FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. CONCLUSION: Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

Background: Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. Methods: A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. Results: A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. Conclusions: The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy.

Matrix metalloproteinase 9 production by monocytes is enhanced by TNF and participates in the pathology of human cutaneous Leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis (CL) due to L.braziliensis infection is characterized by a strong inflammatory response with high levels of TNF and ulcer development. Less attention has been given to the role of mononuclear phagocytes to this process. Monocytes constitute a heterogeneous population subdivided into classical, intermediate and non-classical, and are known to migrate to inflammatory sites and secrete inflammatory mediators. TNF participates in the induction of matrix metalloproteinases (MMPs). MMP-9 is an enzyme that degrades basal membrane and its activity is controlled by the tissue inhibitor of metalloproteinase. METHODS: Mononuclear cells were obtained from ex-vivo labeling sub-populations of monocytes and MMP-9, and the frequency was determined by flow cytometry. Culture was performed during 72 hours, stimulating the cells with SLA, levels of MMP-9 and TIMP-1 in the supernatants were determined by ELISA. RESULTS: We observed that cells from CL lesions secrete high amounts of MMP-9 when compared to healthy subjects. Although MMP-9 was produced by monocytes, non-classical ones were the main source of this enzyme. We also observed that TNF produced in high level during CL contributes to MMP-9 production. CONCLUSIONS: These observations emphasize the role of monocytes, TNF and MMP-9 in the pathogenesis of L. braziliensis infection.

IL-6 promotes NK cell production of IL-17 during toxoplasmosis.

Previous studies have implicated T cell production of IL-17 in resistance to Toxoplasma gondii as well as the development of immune-mediated pathology during this infection. Analysis of C57BL/6 and C57BL/6 RAG(-/-) mice challenged with T. gondii-identified NK cells as a major innate source of IL-17. The ability of soluble Toxoplasma Ag to stimulate NK cells to produce IL-17 was dependent on the presence of accessory cells and the production of IL-6, IL-23, and TGF-beta. In contrast, these events were inhibited by IL-2, IL-15, and IL-27. Given that IL-6 was one of the most potent enhancers of NK cell production of IL-17, further studies revealed that only a subset of NK cells expressed both chains of the IL-6R, IL-6 upregulated expression of the Th17-associated transcription factor RORgammat, and that IL-6(-/-) mice challenged with T. gondii had a major defect in NK cell production of IL-17. Together, these data indicate that many of the same cytokines that regulate Th17 cells are part of a conserved pathway that also control innate production of IL-17 and identify a major role for IL-6 in the regulation of NK cell responses.