Self-reactive T cells induce and perpetuate chronic relapsing arthritis.

BACKGROUND: CD4+ T cells play a central role during the early stages of rheumatoid arthritis (RA), but to which extent they are required for the perpetuation of the disease is still not fully understood. The aim of the current study was to obtain conclusive evidence that T cells drive chronic relapsing arthritis. METHODS: We used the rat pristane-induced arthritis model, which accurately portrays the chronic relapsing-remitting disease course of RA, to examine the contribution of T cells to chronic arthritis. RESULTS: Rats subjected to whole-body irradiation and injected with CD4+ T cells from lymph nodes of pristane-injected donors developed chronic arthritis that lasted for more than 4 months, whereas T cells from the spleen only induced acute disease. Thymectomy in combination with irradiation enhanced the severity of arthritis, suggesting that sustained lymphopenia promotes T cell-driven chronic inflammation in this model. The ability of T cells to induce chronic arthritis correlated with their expression of Th17-associated transcripts, and while depletion of T cells in rats with chronic PIA led to transient, albeit significant, reduction in disease, neutralization of IL-17 resulted in almost complete and sustained remission. CONCLUSION: These findings show that, once activated, self-reactive T cells can sustain inflammatory responses for extended periods of time and suggest that such responses are promoted in the presence of IL-17.

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.

Characterization of heme binding to recombinant α1-microglobulin.

BACKGROUND: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. METHODS: To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. RESULTS: The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with KD values in a µM range. CONCLUSIONS: This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.

A1M/α1-microglobulin protects from heme-induced placental and renal damage in a pregnant sheep model of preeclampsia.

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20(th) gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

The mtDNA nt7778 G/T polymorphism affects autoimmune diseases and reproductive performance in the mouse.

Mitochondria are organelles of all nucleated cells, and variations in mtDNA sequence affect a wide spectrum of human diseases. However, animal models for mtDNA-associated diseases are rare, making it challenging to explore mechanisms underlying the contribution of mitochondria. Here, we identify a polymorphism in the mitochondrial genome, G-to-T at position 7778, which results in an aspartic acid-to-tyrosine (D-Y) substitution in the fifth amino acid of the highly conserved N-terminus of ATP synthase 8 (ATP8). Using a series of conplastic strains we show that this polymorphism increases susceptibility to multiple autoimmune diseases, including collagen-induced arthritis, autoimmune diabetes, nephritis and autoimmune pancreatitis. In addition, it impairs reproductive performance in females, but only in the MRL/MpJ strain. We also demonstrate that the mtAtp8 polymorphism alters mitochondrial performance, increasing H(2)O(2) production and affecting mitochondrial structure. Functional analysis reveals that the polymorphism increase the CD4 T cell adaptive potential to an oxidative phosphorylation impaired condition. Our findings provide direct experimental evidence for the role of mitochondria in autoimmunity and reproduction.

Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains.

Previous reports have demonstrated that the mtDNA of mouse common inbred strains (CIS) originated from a single female ancestor and that mtDNA mutations occurred during CIS establishment. This situation provides a unique opportunity to investigate the impact of individual mtDNA variations on complex traits in mammals. In this study, we compiled the complete mtDNA sequences of 52 mouse CIS. Phylogenetic analysis demonstrated that 50 of the 52 CIS descended from a single female Mus musculus domesticus mouse, and mtDNA mutations have accumulated in 26 of the CIS. We then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations. We also generated conplastic strains for mtDNA variants of the four M. musculus subspecies, each of which contains hundreds of mtDNA variations. In total, a panel of conplastic strains was generated for 16 mtDNA variants. Phenotypic analysis of the conplastic strains demonstrated that mtDNA variations affect susceptibility to experimental autoimmune encephalomyelitis and anxiety-related behavior, which confirms that mtDNA variations affect complex traits. Thus, we have developed a unique genetic resource that will facilitate exploration of the biochemical and physiological roles of mitochondria in complex traits.