Effect of antimalarial drugs and malaria pigment ( *-haematin) on monocyte phagocytosis and GTP-cyclohydrolase 1 gene expression.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.During the erythrocytic stage, the malaria parasite digests host cell haemoglobin into amino acids. Toxic haeme is released and is incorporated into an insoluble non-toxic crystal called haemozoin. Haemozoin is released into the blood stream along with the merozoites when the erythrocyte bursts and is phagocytosed by circulating monocytes and macrophages resident in tissues. Phagocytosed haemozoin impairs many functions of the monocytes, including antigen presentation and adhesion to T cells, differentiation and maturation to dendritic cells, erythropoiesis and thrombopoiesis, but stimulates the release of proinflammatory cytokines and activation of metalloproteinase 9 expression. In response to interferon-g secretion by T-helper cells subtype 1, monocytes secrete neopterin, which is used as a marker of a cell mediated immune response. Neopterin is an oxidation product of 7,8-dihydroneopterin, produced by the dephosphorylation of 7,8- dihydroneopterin triphosphate which results from the conversion of guanosine triphosphate that is catalysed by GTP-cyclohydrolase 1. Elevated plasma and urine neopterin levels have been detected in malaria infections and are associated with severe anaemia, respiratory distress, peak temperatures as well as fever- and parasite-clearance times. It has also been reported that monocytic U937 cells treated with P. falciparum-infected red blood cell lysate secrete elevated levels of neopterin. Antimalarial drugs are known to modulate the functions of monocytes, including inhibition of cytokine release, changes in phospholipid metabolism, decrease in expression of cytoadherance receptors as well as TNF receptors and MHC Class I and II molecules, changes in the production of reactive oxygen and nitrogen intermediates, and decreased phagocytosis. However, the effects of antimalarial drugs on haemozoin phagocytosis and GTP-cyclohydrolase 1 mRNA expression by monocytes are unknown. This study aimed to determine the effects of seven antimalarial drugs, amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of latex beads and b-haematin, a synthetic equivalent of haemozoin. Phagocytosis of b-haematin and latex beads by two monocytic cell lines, J774A.1 and U937, as well as peripheral blood mononuclear cells were monitored by enumeration and a novel spectrophotometric method. Patterns of inhibition and activation differed with each cell type investigated, due to the differing stages of cell differentiation. In general, artemisinin, primaquine, pyrimethamine and quinine activated the phagocytosis of b-haematin, whereas amodiaquine and chloroquine inhibited b-haematin phagocytosis. Doxycycline had different effects on each cell type investigated. Artemisinin, chloroquine, primaquine and quinine inhibited latex bead phagocytosis. The remaining drugs had minimal effects on latex bead phagocytosis. Thus, the effects of antimalarial drugs on monocyte phagocytosis appear to be dependent on the substance being phagocytosed. The effects of antimalarial drugs, b-haematin, latex beads, non-infected- and P. falciparuminfected cell lysates on interferon-g-induced neopterin secretion by U937 cells was monitored by GTP-cyclohydrolase 1 mRNA expression using quantitative PCR. Artemisinin, primaquine and quinine down-regulated the interferon-g-induced expression of GTPcyclohydrolase 1 mRNA, but by no greater than 1.7-fold. b-haematin up-regulated mRNA expression by 1.2-fold whereas P. falciparum-infected red blood cell lysate down-regulated the mRNA expression of GTP-cyclohydrolase 1 by 1.6-fold. Quinine and artemisinin, currently used to treat malaria, increased b-haematin phagocytosis suggesting that quinine and artemisinin might promote increased phagocytosis of infected red blood cells and enhance clearance of the parasite from circulation. Increased b- haematin phagocytosis also reduces ICAM-1 expression on the monocyte surface, thereby leading to reduced cytoadherance and sequestration, thus increasing the number of circulating monocytes to phagocytose infected red blood cells. Down regulation of GTPcyclohydrolase 1 mRNA expression by quinine and artemisinin suggested that the drugs reduce the responsiveness of the monocyte to interferon-g. Thus, quinine and artemisinin might also decrease the production of interferon-g-induced proinflammatory cytokines by monocytes, and potentially play a role in maintaining the balance between the pro- and antiinflammatory cytokines that determines the progression from acute to severe malaria. Therefore, in addition to the drug’s ability to kill the malaria parasite, the immunomodulatory effects of the antimalarial drugs may play a role in controlling the pathophysiology associated with the malaria infection

S100A8/A9 Proteins Mediate Neutrophilic Inflammation and Lung Pathology during Tuberculosis

Rationale: A hallmark of pulmonary tuberculosis (TB) is the formation of granulomas. However, the immune factors that drive the formation of a protective granuloma during latent TB, and the factors that drive the formation of inflammatory granulomas during active TB, are not well defined. Objectives: The objective of this study was to identify the underlying immune mechanisms involved in formation of inflammatory granulomas seen during active TB. Methods: The immune mediators involved in inflammatory granuloma formation during TB were assessed using human samples and experimental models of Mycobacterium tuberculosis infection, using molecular and immunologic techniques. Measurements and Main Results: We demonstrate that in human patients with active TB and in nonhuman primate models of M. tuberculosis infection, neutrophils producing S100 proteins are dominant within the inflammatory lung granulomas seen during active TB. Using the mouse model of TB, we demonstrate that the exacerbated lung inflammation seen as a result of neutrophilic accumulation is dependent on S100A8/A9 proteins. S100A8/A9 proteins promote neutrophil accumulation by inducing production of proinflammatory chemokines and cytokines, and influencing leukocyte trafficking. Importantly, serum levels of S100A8/A9 proteins along with neutrophil-associated chemokines, such as keratinocyte chemoattractant, can be used as potential surrogate biomarkers to assess lung inflammation and disease severity in human TB. Conclusions: Our results thus show a major pathologic role for S100A8/A9 proteins in mediating neutrophil accumulation and inflammation associated with TB. Thus, targeting specific molecules, such as S100A8/A9 proteins, has the potential to decrease lung tissue damage without impacting protective immunity against TB

Mycobacterium Tuberculosis Arrests Host Cycle At The G1/S Transition To Establish Long Term Infection

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle

Mycobacterial WhiB6 Differentially Regulates ESX-1 and the Dos Regulon to Modulate Granuloma Formation and Virulence in Zebrafish

During the course of infection, Mycobacterium tuberculosis (Mtb) is exposed to diverse redox stresses that trigger metabolic and physiological changes. How these stressors are sensed and relayed to the Mtb transcriptional apparatus remains unclear. Here, we provide evidence that WhiB6 differentially regulates the ESX-1 and DosR regulons through its Fe-S cluster. When challenged with NO, WhiB6 continually activates expression of the DosR regulons but regulates ESX-1 expression through initial activation followed by gradual inhibition. Comparative transcriptomic analysis of the holo- and reduced apo-WhiB6 complemented strains confirms these results and also reveals that WhiB6 controls aerobic and anaerobic metabolism, cell division, and virulence. Using the Mycobacterium marinum zebrafish infection model, we find that holo- and apo-WhiB6 modulate levels of mycobacterial infection, granuloma formation, and dissemination. These findings provide fresh insight into the role of WhiB6 in mycobacterial infection, dissemination, and disease development

Comprehensive Examination of the Mouse Lung Metabolome Following <i>Mycobacterium tuberculosis</i> Infection Using a Multiplatform Mass Spectrometry Approach

The mechanisms whereby Mycobacterium tuberculosis (Mtb) rewires the host metabolism in vivo are surprisingly unexplored. Here, we used three high-resolution mass spectrometry platforms to track altered lung metabolic changes associated with Mtb infection of mice. The multiplatform data sets were merged using consensus orthogonal partial least squares-discriminant analysis (cOPLS-DA), an algorithm that allows for the joint interpretation of the results from a single multivariate analysis. We show that Mtb infection triggers a temporal and progressive catabolic state to satisfy the continuously changing energy demand to control infection. This causes dysregulation of metabolic and oxido-reductive pathways culminating in Mtb-associated wasting. Notably, high abundances of trimethylamine-N-oxide (TMAO), produced by the host from the bacterial metabolite trimethylamine upon infection, suggest that Mtb could exploit TMAO as an electron acceptor under anaerobic conditions. Overall, these new pathway alterations advance our understanding of the link between Mtb pathogenesis and metabolic dysregulation and could serve as a foundation for new therapeutic intervention strategies. Mass spectrometry data has been deposited in the Metabolomics Workbench repository (data-set identifier: ST001328)

<i>Mycobacterium tuberculosis</i> arrests host cycle at the G₁/S transition to establish long term infection

<div><p>Signals modulating the production of <i>Mycobacterium tuberculosis (Mtb</i>) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of <i>Mtb</i> virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed <i>Mtb in vitro</i>, intraphagosomal and infected host expression analyses. Our <i>Mtb</i> expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found <i>in vivo</i> to establish <i>Mtb</i> infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that <i>Mtb</i> infection inhibited the macrophage G₁/S transition, and polyketides under WhiB3 control arrested the macrophages in the G₀-G₁ phase. Notably, infection with the <i>Mtb whiB3</i> mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by <i>Mtb</i> WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type <i>Mtb</i>. Thus, our findings demonstrate that <i>Mtb</i> WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby <i>Mtb</i> modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.</p></div

Pathway mapping of <i>Mtb</i> infected macrophages reveals that <i>Mtb</i> WhiB3 regulates the host cell cycle.

<p><b>(A)</b> Top ten host pathways with the most significant differential regulation in <i>MtbΔwhiB3</i> infected RAW264.7 macrophages relative to wt <i>Mtb</i> H37Rv infected macrophages (p values were calculated with <i>t</i> tests). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s003" target="_blank">S2 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s008" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s009" target="_blank">S6</a> Tables. <b>(B)</b> LC-MS/MS identification and quantification of cytoplasmic proteins with more than 2-fold differential expression (p<0.03, ANOVA) in <i>MtbΔwhiB3</i> infected RAW264.7 macrophages relative to wt <i>Mtb</i> H37Rv infected macrophages. Fold changes were calculated from the means of samples in triplicate. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s010" target="_blank">S7 Table</a>. <b>(C)</b> DNA synthesis in RAW264.7 macrophages infected with wt <i>Mtb</i> H37Rv, <i>MtbΔwhiB3</i> and the complemented (comp) strain (MOI 5) for 24 h. Cells were stained with DAPI (blue) to identify the nuclei and BrdU was added to the cells and incorporated into newly synthesized DNA. <b>(D)</b> Representative dot plots of BrdU incorporation versus surface staining with anti-CD3⁺ (lymphocyte marker), anti-CD11b⁺ and anti-CD11c⁺ (myeloid lineage markers) of lung cells isolated from mice infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain for 6 weeks. CD11b⁺CD11c⁺ double positive cells were also gated and evaluated for BrdU incorporation. <b>(E)</b> Percentage of CD3⁺, CD11b⁺, CD11c⁺ or CD11b⁺CD11c⁺ cells isolated from mouse lungs infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain that are positive for BrdU incorporation. (<b>F</b>) Percentage of total CD3⁺, CD11b⁺, CD11c⁺ or CD11b⁺CD11c⁺ cells isolated from mouse lungs infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain. Data are representative of two independent experiments with three mice per group and were analyzed using the unpaired Student’s <i>t</i> test. For the comparison of <i>MtbΔwhiB3</i> infected mice versus any of the other mouse groups: * p<0.01.</p

Infection with <i>Mtb</i> polyketide mutants do not alter the macrophage cell cycle.

<p><b>(A)</b> Representative dot plots of BrdU and PI incorporation into RAW264.7 macrophages infected with wt <i>Mtb</i>, <i>Mtb Tn</i>:<i>pks2</i>, <i>Mtb Tn</i>:<i>mas</i> or <i>Mtb Tn</i>:<i>ppsA</i> at MOI 5 for the indicated time points. <b>(B-D)</b> Percentage of RAW264.7 cells infected with wt <i>Mtb</i> CDC1551, <i>Mtb Tn</i>:<i>pks2</i>, <i>Mtb Tn</i>:<i>mas</i> or <i>Mtb Tn</i>:<i>ppsA</i>, at MOI 5 for the indicated time points in the <b>(B)</b> G₀-G₁ phase, <b>(C)</b> S phase and <b>(D)</b> G₂-M phases of the macrophage cell cycle. Error bars represent SD from the mean of triplicate experiments. Data are representative of two independent experiments. Unpaired Student’s <i>t</i> Test was used to calculate the p value. #, p≤0.0001; **, p≤0.001; *, p≤0.05; symbols above the bars indicates comparison to macrophages infected with wt <i>Mtb</i>.</p

<i>Mtb</i> polyketides and cell wall surface molecules under WhiB3 control modulate the host cell cycle.

<p>RAW264.7 macrophages <b>(M</b>; untreated) were treated in triplicate with <b>(B)</b> MAME, <b>(C)</b> PDIM, <b>(E)</b> PIM 1,2, <b>(F)</b> SL-1, <b>(H)</b> <i>Mtb</i> total lipid extract, or <b>(I)</b> TDM at the concentrations indicated for 24 h prior to cell cycle analysis. RAW264.7 cells treated with the solvents in which the lipids were dissolved <b>(A, D, G)</b> were used as controls. <b>(J)</b> Percentage of RAW264.7 cells in the sub-G₀, G₀-G₁, S and G₂-M phases of the cell cycle following treatment with 1 μg/ml SL-1, PDIM, PIM 1,2, MAME, TDM, total lipids or vehicle (0.1% DMSO) for 24 h, <b>(K, N)</b> combinations of SL-1 and PDIM at the concentrations indicated for 24 h and <b>(O, L)</b> combinations of PIM 1,2 and PDIM at the indicated concentrations for 24 h. Error bars represent SD of the mean of triplicate experiments. Data are representative of two independent experiments. Unpaired Student’s t-Test was used to calculate the p value. #, p<0.0001; **, p≤0.005 and *, p≤0.05.</p

Model whereby <i>Mtb</i> arrests the macrophage cell cycle to establish long term infection.

<p><i>Mtb</i> WhiB3 is a redox sensor that senses NO and low levels of oxygen (hypoxia) along with <i>in vivo</i> carbon sources available and accordingly modulates bioenergetic metabolism in response to the immediate environment. Bioenergetic homeostasis is essential for the transcription and production of polyketides under WhiB3 control. Lipids and polyketides are released from <i>Mtb</i> into the infected macrophage, where they arrest the host’s cell cycle and modulate the immune response to establish a persistent infection.</p