ALOX12 in human toxoplasmosis

Abstract

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.

FIG 1

Analysis of ALOX12 SNPs. (Upper diagram) Positions of genotyped SNPs within the gene. (Lower diagram) LD plots generated in Haploview using ALOX12 gene data from the NCCCTS cohort. The LD (D′ × 100) between any 2 markers is indicated at the intercept of the markers on the matrix. Where no value is given, linkage disequilibrium (D′) is 1 (LD is 100). Red diamonds indicate the greatest linkage disequilibrium and white diamonds the least. Black lines outline SNPs in haplotype blocks, and SNPs in perfect linkage disequilibrium with each other are represented by red blocks.

FIG 2

Quantitation of arachidonic acid levels in wild-type MonoMac6 cells (MM6) and in MonoMac6 cells engineered to express either a lentivirus shRNA that knocks down ALOX12 or a control, off-target lentivirus shRNA that knocks down TetR. The data are from a minimum of five independent experiments. Error bars indicate standard errors. The mean arachidonic acid levels (standard errors of the means) are 1.78 (±1.38) nmol/mg for uninfected cells and 1.27 (±0.37) nmol/mg for infected cells in the ALOX12 knockdown group, 0.97 (±0.43) nmol/mg in uninfected cells and 0.44 (±0.10) nmol/mg in infected cells in the wild-type group, and 0.72 (±0.13) nmol/mg in uninfected cells and 0.41 (±0.09) nmol/mg in infected cells in the TetR siRNA group. When the MM6 and TetR siRNA groups are combined as the ALOX12 wild-type group, the mean arachidonic acid levels (standard errors of the means) are 0.85 (±0.22) nmol/mg in uninfected cells and 0.43 (±0.07) nmol/mg in infected cells. Uninfected cells with ALOX12 shRNA, no shRNA, or TetR shRNA are shown. Cells infected with T. gondii for 24 h that show the same pattern of shRNA for ALOX12 have higher levels of the substrate, arachidonic acid, than wild-type MonoMac6 cells and off-target shRNA controls. The results listed here in the figure legend are with the raw, untransformed data. The graph, on the other hand, is plotted using log-transformed data. For each cell type, the number of uninfected- and infected-cell samples combined was 32, 34, or 32, respectively. Due to evidence of nonnormality, nonparametric tests were used. Since the MM6 and TetR groups were similar, they were combined and compared to the ALOX12 group. In an overall analysis, stratified by infection status, the ALOX12 group had significantly higher values than the MM6-plus-TetR group (P, 0.05 by a stratified Wilcoxon rank-sum test).

FIG 3

(A) Relative viabilities of wild-type MonoMac6 cells and MonoMac6 cells engineered to express ALOX12 or TetR shRNA after infection with T. gondii. Values were obtained by dividing the absorbance of infected cells by the absorbance of uninfected cells. Blue line, MonoMac6 cells; red line, TetR knockdown cells; green line, ALOX12 knockdown cells. Asterisks indicate a significant difference between ALOX12 knockdown cells and wild-type MM6 cells (P, <0.040). (B) Giemsa-stained cytospin preparations of monocytic cells examined by light microscopy to visualize the effect of ALOX12 gene knockdown on the viability of monocytic cells cultured with or without parasites. Wild-type monocytic cells and monocytic cells modified for the stable knockdown of ALOX12 (ALOX12KO) or off-target tetracycline repressor (TetRep KO) gene transcription were cultured with or without parasites. Cytospin smears were prepared and stained with Giemsa stain for each culture at different days postinfection. Representative images for three independent experiments are shown. Arrows point to infected cells.

FIG 4

Measurement of parasite growth based on fluorescence counts of YFP-expressing parasites. Exactly 10⁴ wild-type MonoMac6, TetR knockdown, or ALOX12 knockdown cells were infected with 5 × 10³ YFP-expressing parasites in 96-well plates. Blue line, MonoMac6 cells; red line, TetR knockdown cells; green line, ALOX12 knockdown cells. Asterisks indicate a significant difference between ALOX12 knockdown cells and wild-type MM6 cells (P, <0.0482).

FIG 5

Analysis of the effects of ALOX12 gene knockdown on the expression of cytokines and caspase-1 in a human monocytic cell line (MonoMac6). Quantitative real-time PCR was performed on cDNA synthesized using equal amounts of total RNA from wild-type MonoMac6 cells (MM) and MonoMac6 cells engineered to express either ALOX12 gene shRNA (ALOX12-KD) or tetracycline repressor gene shRNA (TetR) that had been cultured with (shaded bars) or without (open bars) parasites for 36 h. The IL-1β (A), IL-6 (B), TNF-α (C), and caspase-1 (D) transcript levels were divided by the actin transcript level for each respective sample to yield the relative gene transcript levels. Asterisks indicate significant augmentation of gene expression attributable to infection (P, <0.05). Data are means of results from three independent experiments; error bars, standard errors.

FIG 6

Model of human leukocyte 12-LOX (ALOX12). Amino acids shown in red are encoded by functional SNPs. SNPs in ALOX12 (e.g., rs6502997) are associated with susceptibility to congenital toxoplasmosis (P, <0.0005). In humans, some functional allelic variants of ALOX12 (associated with different cell death or hypertension phenotypes) have an Arg substituted for Gln at position 261 and an Asn substitution for Ser at position 322 in LD. This likely would change the surface charge of 12-LOX (ALOX12), which might change the dimerization of 12-LOX (ALOX12) or its association with other molecules. Promoter region polymorphisms also may contribute to differing outcomes.