Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis. We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.Chlamydia trachomatis is an obligate intracellular bacterial pathogen associated with a variety of human diseases. The species of C. trachomatis can be divided into biovars, such as the trachoma biovar and the lymphogranuloma venereum biovar; these are further divided into serovars. In the developing world, serovars A-C are associated with trachoma, one of the oldest diseases caused by C. trachomatis infection, and the leading cause of preventable blindness in the world (reviewed in Ref. 1). Serovars D-K are also of the trachoma biovar and are all associated with genital tract disease as well as conjunctivitis and infant pneumonia. The World Health Organization reported 89 million new cases of genital Chlamydia infections in 1995, demonstrating the extent of this infection worldwide and hinting at the economic burden it has on healthcare (2). In 2003, 877,478 cases of infection with C. trachomatis were reported to the CDC from 50 states and the District of Columbia (3), making it the most common bacterial sexually transmitted infection. The lymphogranuloma venereum (LGV) 2 serovars L1, L2, and L3 are also sexually transmitted but are associated with a more invasive disease known as lymphogranuloma venereum (4). Although LGV is uncommon in the developed world, there have been recent outbreaks of LGV proctitis in the Netherlands and Western Europe (5, 6).The unique developmental cycle of the chlamydiae sets them apart from other bacterial species. C. trachomatis ...

Increased BH(4) by cardiomyocyte-specific overexpression of GTPCH-1 preserves the ability of IPC to elicit myocardial and mitochondrial protection that is impaired by HG, and this action appears to be dependent on NO.

Background-There remains no reliable noninvasive method to detect cardiac transplant rejection. Recently, speckle-tracking 2-dimensional strain echocardiography was shown to be sensitive in early detection of myocardial dysfunction in various models of cardiomyopathy. We aim to determine if 2DSE derived functional indices can detect cardiac transplant rejection.

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

Tetrahydrobiopterin (BH 4 ), a cofactor of inducible nitric-oxide synthase (iNOS), is an important post-translational regulator of NO bioactivity. We examined whether treatment of cardiac allograft recipients with sepiapterin [S-(Ϫ)-2-amino-7,8-dihydro-6-(2-hydroxy-1-oxopropyl)-4-(1H)-pteridinone], a precursor of BH 4 , inhibited acute rejection and apoptosis in cardiac transplants. Heterotopic cardiac transplantation was performed in Wistar-Furth donor to Lewis recipient strain rats. Recipients were treated daily after transplantation with 10 mg/kg sepiapterin. Grafts were harvested on post-transplant day 6 for analysis of BH 4 (high-performance liquid chromatography), expression of inflammatory cytokines (reverse transcription-and real-time polymerase chain reaction), iNOS (Western blots), and NO (Griess reaction and NO analyzer). Histological rejection grade was scored, and graft function was determined by echocardiography. Apoptosis, protein nitration, and oxidative stress were determined by immunohistochemistry. Treatment of allografts with sepiapterin increased cardiac BH 4 levels by 3-fold without changing protein levels of GTP cyclohydrolase, the enzyme that regulates de novo BH 4 synthesis. Sepiapterin decreased inflammatory cell infiltrate and significantly inhibited histological rejection scores and apoptosis similar in magnitude to cyclosporine. Sepiapterin also decreased nitrative and oxidative stress. Sepiapterin caused a smaller increase in left ventricular mass versus untreated allografts but without improving fractional shortening. Sepiapterin did not alter tumor necrosis factor-␣ and interferon-␥ expression, whereas it decreased interleukin (IL)-2 expression. Sepiapterin did not change total iNOS protein or monomer levels, or plasma and tissue NO metabolites levels. It is concluded that the mechanism(s) of antirejection are due in part to decreased apoptosis, protein nitration, and oxidation of cardiomyocytes, which seems to be mediated at the immune level by limiting inflammatory cell infiltration via decreased IL-2-mediated T-lymphocyte expansion.

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.

Alp NJ, Pieper GM. Cardiac myocyte-specific overexpression of human GTP cyclohydrolase I protects against acute cardiac allograft rejection. Am J Physiol Heart Circ Physiol 299: H88 -H96, 2010. First published April 23, 2010; doi:10.1152/ajpheart.00203.2010 is the rate-limiting enzyme for tetrahydrobiopterin (BH 4) synthesis. Decreases in GTPCH activity and expression have been shown in late stages of acute cardiac rejection, suggesting a deficit in BH4. We hypothesized that increasing intracellular levels of BH4 by cardiac myocyte-targeted overexpression of GTPCH would diminish acute cardiac allograft rejection. Transgenic mice overexpressing GTPCH in the heart were generated and crossed on C57BL6 background. Wild-type and transgenic mouse donor hearts were transplanted into BALB/c recipient mice. Left ventricular (LV) function, histological rejection, BH4 levels, and inflammatory cytokine gene expression (mRNA) were examined. Expression of human GTPCH was documented by PCR, Western analysis, and function by a significant (P Ͻ 0.001) increase in cardiac BH4 levels. GTPCH transgene decreased histological rejection (46%; P Ͻ 0.003) and cardiac myocyte injury (eosin autofluorescence; 56%; P Ͻ 0.0001) independent of changes in inflammatory cytokine expression or nitric oxide content. GTPCH transgene decreased IL-2 (88%; P Ͻ 0.002), IL-1R2 (42%; P Ͻ 0.0001), and programmed cell death-1 (67%; P Ͻ 0.0001) expression, whereas it increased fms-like tyrosine kinase 3 (156%; P Ͻ 0.0001) and stromal-derived factor-1 (2; 190%; P Ͻ 0.0001) expression. There was no difference in ejection fraction or fractional shortening; however, LV mass was significantly increased (P Ͻ 0.05) only in wild-type grafts. The decreases in LV mass, cardiac injury, and histological rejection support a protective role of cardiac GTPCH overexpression and increased BH4 synthesis in cardiac allografts. The mechanism of the decreased rejection appears related to decreased T cell proliferation and modulation of immune function by higher expression of genes involved in hematopoietic/stromal cell development and recruitment.tetrahydrobiopterin; rejection; stromal-derived factor 1; left ventricular mass; guanosine 5=-triphosphate cyclohydrolase TETRAHYDROBIOPTERIN (BH 4 ) is a cofactor for only a few known enzymes including three isoforms of nitric oxide (NO) synthases (NOS), four aromatic amino acid hydroxylases, and glyceryl ether monoxygenase (25,30). Synthesis of BH 4 is regulated by the expression of the key enzyme GTP cyclohydrolase I (GTPCH). The discovery of nuclear localization of GTPCH and other proteins involved in BH 4 synthesis (5) coupled with earlier findings that the mitogenic action of BH 4 in various cells is independent of changes in catecholamine and NO synthesis (1) suggests potentially new unknown roles of BH 4 . Thus there is new awareness of the potential regulation of GTPCH/BH 4 for a vast array of biological processes far beyond that regulating NO and catecholamine synthesis including cell proliferation, cell division, apoptosis,...

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (<6 months) have lower BH4:BH2 ratios than hearts of older mice while both GTPCH activity and tissue ascorbate levels were higher in hearts of young than older mice. No evident changes in nitric oxide (NO) production assessed by nitrite and endogenous iron-nitrosyl complexes were detected in any of the age groups. Increased BH4 production in cardiomyocytes resulted in a significant loss of mitochondrial function. Diminished oxygen consumption and reserve capacity was verified in mitochondria isolated from hearts of 12-month old compared to 3-month old mice, even though at 12 months an improved BH4:BH2 ratio is established. Accumulation of 4-hydroxynonenal (4-HNE) and decreased glutathione levels were found in the mGCH hearts and isolated mitochondria. Taken together, our results indicate that the ratio of BH4:BH2 does not predict changes in neither NO levels nor cellular redox state in the heart. The BH4 oxidation essentially limits the capacity of cardiomyocytes to reduce oxidant stress. Cardiomyocyte with chronically high levels of BH4 show a significant decline in redox state and mitochondrial function.