Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.     

MCPIP1 Deficiency in Mice Results in Severe Anemia Related to Autoimmune Mechanisms

Autoimmune gastritis is an organ-specific autoimmune disease of the stomach associated with pernicious anemia. The previous work from us and other groups identified MCPIP1 as an essential factor controlling inflammation and immune homeostasis. MCPIP1^-/- developed severe anemia. However, the mechanisms underlying this phenotype remain unclear. In the present study, we found that MCPIP1 deficiency in mice resulted in severe anemia related to autoimmune mechanisms. Although MCPIP1 deficiency did not affect erythropoiesis per se, the erythropoiesis in MCPIP1^-/- bone marrow erythroblasts was significantly attenuated due to iron and vitamin B₁₂ (VB₁₂) deficiency, which was mainly resulted from autoimmunity-associated gastritis and parietal cell loss. Consistently, exogenous supplement of iron and VB₁₂ greatly improved the anemia phenotype of MCPIP1^-/- mice. Finally, we have evidence suggesting that autoimmune hemolysis may also contribute to anemia phenotype of MCPIP1^-/- mice. Taken together, our study suggests that MCPIP1 deficiency in mice leads to the development of autoimmune gastritis and pernicious anemia. Thus, MCPIP1^-/- mice may be a good mouse model for investigating the pathogenesis of pernicious anemia and testing the efficacy of some potential drugs for treatment of this disease.     

MIF Contributes to Trypanosoma brucei Associated Immunopathogenicity Development

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C^high inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.     

Hepcidin-Dependent Regulation of Erythropoiesis during Anemia in a Teleost Fish,Dicentrarchus labrax

Anemia is a common disorder, characterized by abnormally low levels of red blood cells or hemoglobin. The mechanisms of anemia development and response have been thoroughly studied in mammals, but little is known in other vertebrates, particularly teleost fish. In this study, different degrees of anemia were induced in healthy European sea bass specimens (Dicentrarchus labrax) and at pre-determined time points hematological parameters, liver iron content and the expression of genes involved in iron homeostasis and hematopoiesis, with particular attention on hepcidins, were evaluated. The experimental anemia prompted a decrease in hamp1 expression in all tested organs, in accordance to an increased need for iron absorption and mobilization, with slight increases in hamp2 in the kidney and intestine. The liver was clearly the major organ involved in iron homeostasis, decreasing its iron content and showing a gene expression profile consistent with an increased iron release and mobilization. Although both the spleen and head kidney are involved in erythropoiesis, the spleen was found to assume a more preponderant role in the recovery of erythrocyte levels. The intestine was also involved in the response to anemia, through the increase of iron transporting genes. Administration of Hamp1 or Hamp2 mature peptides showed that only Hamp1 affects hematological parameters and liver iron content. In conclusion, the molecular mechanisms of response to anemia present in sea bass are similar to the ones described for mammals, with these results indicating that the two hepcidin types from teleosts assume different roles during anemia.     

Experimental African Trypanosome Infection by Needle Passage or Natural Tsetse Fly Challenge Thwarts the Development of Collagen-Induced Arthritis in DBA/1 Prone Mice via an Impairment of Antigen Specific B Cell Autoantibody Titers

Collagen-induced arthritis is a B cell-mediated autoimmune disease. Recently published studies have demonstrated that in some rare cases pathogens can confer protection from autoimmunity. Trypanosoma brucei parasites are tsetse fly transmitted extracellular protozoans causing sleeping sickness disease in humans and Nagana in livestock in sub-Saharan endemic areas. In the past, we demonstrated that trypanosome infections impair B cell homeostasis and abolish vaccine-induced protection against unrelated antigens. Hence, here we hypothesized that trypanosome infection can affect the onset of CIA by specifically dampening specific B-cell responses and type II collagen antibody titers in DBA/1 prone mice. We observed a substantial delay in the onset of collagen-induced arthritis in T. brucei-infected DBA/1 mice that correlates with a drastic decrease of type II collagen titers of the different IgG isotypes in the serum. Treatment of infected mice with Berenil, a trypanocidal drug, restored the development of CIA-associated clinical symptoms. Interestingly, these data were confirmed by the challenge of immunized DBA/1 prone mice with T. brucei-infected tsetse flies. Together, these results demonstrate that T. brucei infection is impairing the maintenance of the antigen specific plasma B cell pool driving the development of CIA in DBA/1 prone mice.     

Helicobacter pylori Infection Induces Anemia,Depletes Serum Iron Storage,and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice

Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world’s population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6–9 weeks of age, and were necropsied at 27–29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.     

The dietary flavonoid myricetin regulates iron homeostasis by suppressing hepcidin expression

Hepcidin, a master regulator of iron homeostasis, is a promising target in treatment of iron disorders such as hemochromatosis, anemia of inflammation and iron-deficiency anemia. We previously reported that black soybean seed coat extract could inhibit hepcidin expression. Based on this finding, we performed a screen in cultured cells in order to identify the compounds in black soybeans that inhibit hepcidin expression. We found that the dietary flavonoid myricetin significantly inhibited the expression of hepcidin both in vitro and in vivo. Treating cultured cells with myricetin decreased both HAMP mRNA levels and promoter activity by reducing SMAD1/5/8 phosphorylation. This effect was observed even in the presence of bone morphogenic protein-6 (BMP6) and interleukin-6 (IL-6), two factors that stimulate hepcidin expression. Furthermore, mice that were treated with myricetin (either orally or systemically) had reduced hepatic hepcidin expression, decreased splenic iron levels and increased serum iron levels. Notably, myricetin-treated mice increased red blood cell counts and hemoglobin levels. In addition, pretreating mice with myricetin prevented LPS-induced hypoferremia. We conclude that myricetin potently inhibits hepcidin expression both in vitro and in vivo, and this effect is mediated by altering BMP/SMAD signaling. These experiments highlight the feasibility of identifying and characterizing bioactive phytochemicals to suppress hepcidin expression. These results also suggest that myricetin may represent a novel therapy for treating iron deficiency-related diseases.     

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.     

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis.     

Development of a pHrodo-Based Assay for the Assessment of In Vitro and In Vivo Erythrophagocytosis during Experimental Trypanosomosis

Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.     

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23^−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.     

A glycosylphosphatidylinositol-based treatment alleviates trypanosomiasis-associated immunopathology

The GPI-anchored trypanosome variant surface glycoprotein (VSG) triggers macrophages to produce TNF, involved in trypanosomiasis-associated inflammation and the clinical manifestation of sleeping sickness. Aiming at inhibiting immunopathology during experimental Trypanosoma brucei infections, a VSG-derived GPI-based treatment approach was developed. To achieve this, mice were exposed to the GPI before an infectious trypanosome challenge. This GPI-based strategy resulted in a significant prolonged survival and a substantial protection against infection-associated weight loss, liver damage, acidosis, and anemia; the latter was shown to be Ab-independent and correlated with reduced macrophage-mediated RBC clearance. In addition, GPI-based treatment resulted in reduced circulating serum levels of the inflammatory cytokines TNF and IL-6, abrogation of infection-induced LPS hypersensitivity, and an increase in circulating IL-10. At the level of trypanosomiasis-associated macrophage activation, the GPI-based treatment resulted in an impaired secretion of TNF by VSG and LPS pulsed macrophages, a reduced expression of the inflammatory cytokine genes TNF, IL-6, and IL-12, and an increased expression of the anti-inflammatory cytokine gene IL-10. In addition, this change in cytokine pattern upon GPI-based treatment was associated with the expression of alternatively activated macrophage markers. Finally, the GPI-based treatment also reduced the infection-associated pathology in Trypanosoma congolense and Trypanosoma evansi model systems as well as in tsetse fly challenge experiments, indicating potential field applicability for this intervention strategy.     

Increased Iron Stores Correlate with Worse Disease Outcomes in a Mouse Model of Schistosomiasis Infection

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the world''s developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.     

Iron regulatory protein 1 is not an early target of cadmium toxicity in mice, but it is sensitive to cadmium stress in a human epithelial cell line

Disruption of iron homeostasis at the levels of intestinal absorption or erythropoiesis contributes to cadmium toxicity. Cellular iron homeostasis in metazoans is maintained by the iron regulatory proteins (IRPs) that regulate the synthesis of proteins involved in the transport, use, and storage of iron. The effect of cadmium intoxication on this regulatory system has been investigated in a cellular model of human epithelium. Cadmium exposure of HeLa cells did not activate the IRPs; rather, the amount of these proteins relative to that of housekeeping proteins decreased. Accordingly, the transferrin receptor mRNA level decreased upon cadmium insult. In a more integrated investigation, separate groups of mice had free access to different doses of cadmium in drinking water for 3 weeks. Cadmium accumulated in all analyzed organs, but its concentration in mouse tissues did not correlate with changes of the activity of the IRPs. The intoxicated mice did not show any sign of anemia, indicating that iron homeostasis was not immediately disrupted after the onset of cadmium accumulation. These data establish that cadmium destabilizes IRPs in mammalian cells, but that iron imbalance is not an early event of cadmium intoxication.     

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.     

Investigation of Iron Metabolism in Mice Expressing a Mutant Menke’s Copper Transporting ATPase (Atp7a) Protein with Diminished Activity (Brindled; MoBr /y)

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [Mo^{Br /y}]). Mutant mice were rescued by perinatal copper injections, and, after a 7–8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult Mo^{Br /y} mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived Mo^{Br /y} mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and Mo^{Br /y}) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in Mo^{Br /y} mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, Mo^{Br /y} mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.     

Trypanosoma brucei: attenuation by cortcosteroids of the anemia of infected mice

BALBc/J mice infected with pleomorphic stabilate of Trypanosoma brucei develop a severe anemia. The anemia is initially normoblastic and normocytic but becomes macrocytic due to brisk and sustained reticulocytosis. Red blood cells from uninfected animals are destroyed when transfused into infected animals. Neither extensive intravascular hemolysis nor red cell agglutinins were detected in infected animals, but spherocytes were common in the peripheral circulation. Treatment of mice with either dexamethasone or hydrocortisone greatly retarded the development of the anemia. Comparison of the course of infection in these treated mice to that in normal mice suggests that these corticosteroids attenuate the anemia through their immunosuppressive action.