Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5-/- Mice

Abstract

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5^-/- mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5^-/- animals tested previously in similar experimental setups.

Keywords: eicosanoids; inflammation; leukotrienes; lipoxygenase; pain; resolvins.

Figure 1

Expression of Alox mRNAs in peritoneal lavage and bone marrow cells of wildtype mice. Cells were prepared from wildtype mice as described in the Materials and Methods section. Total RNA was extracted, reversely transcribed and the different Alox mRNA species were quantified by qRT-PCR using external amplification standards for each target mRNA (mRNAs of different Alox paralogs) and the reference mRNA (Gapdh). (A) qRT-PCR of mouse Alox mRNAs of wildtype peritoneal lavage cells. For this experiment the RNA of the cell preparation obtained from four different animals (n = 4) was pooled and three independent PCR runs were carried out. (B) qRT-PCR of mouse Alox mRNAs of wildtype bone marrow cells. For this experiment, three independent PCR runs were carried out with each RNA pool (n = 4).

Figure 2

Alox5 ex vivo activity assays using whole blood. Alox5-KI mice and corresponding outbred wildtype controls (n = 4 of each genotype) were sacrificed under anesthesia and heparinized blood was drawn. Whole blood was incubated for 30 min at 37 °C with 50 μM calcium ionophoreA23187, cells were spun down and eicosanoid profiles were quantified by LC-MS/MS, as described in the Materials and Methods. For each product, the mean of metabolite formation of wildtype mice was set at 100% and product formation of each individual of either genotype was normalized to this value. Gray columns show wildtype mice, white columns show Alox5-KI mice. genotype was normalized to this value. Gray columns show wildtype mice, white columns show Alox5-KI mice. Dots indicate the individual values for wildtype mice, square indicate the individual values for Alox5-KI mice. Statistical analyses comparing Alox5-KI mice with wildtype animals were carried out with the two-sided Students t-test and the levels of statistical significance are indicated by asterixis (*). * p < 0.05, *** p < 0.001; n.s. indicates no significant differences between the two genotypes. Data are expressed as means ± SEM, n = 4 for each genotype.

Figure 3

Analysis of the Alox products formed during ex vivo activity assays of peritoneal lavage cells and bone marrow cells of Alox5-KI and wildtype mice. Cells were prepared and ex vivo activity assays were carried out (see Materials and Methods). The conjugated dienes formed during the 15 min incubation period were prepared by RP-HPLC and further analyzed by NP/CP-HPLC (see Materials and Methods). Each chromatogram was scaled for the 12S-HETE peak. Four independent individuals (n = 4) were analyzed for each genotype. Representative partial chromatograms are shown, and statistical evaluation of the product patterns is given in Table 1. (A) Wildtype peritoneal lavage cells. (B) Peritoneal lavage cells prepared from Alox5-KI mice. (C) Wildtype bone marrow cells. (D) Bone marrow cells prepared from Alox5-KI mice.

Figure 4

Ex vivo Alox activity assays of bone marrow cells prepared from Alox5-KI mice and corresponding wildtype controls using endogenous substrates. Pooled bone marrow cells of either Alox5-KI mice (n = 5; panels B,D and F) or WT-mice (n = 3, panels A,C and E) were used. For each assay 2 × 10⁷ cells were reconstituted in 0.5 mL PBS and were incubated at room temperature in the presence of 5 μM calcium ionophore A23187 for 5 min. Total lipids were extracted (ethyl acetate), solvent was evaporated, the remaining lipids were reconstituted in 50 μL methanol and analyzed for LTB₄ (panels A,B), 5-HETE (panels C,D) and 15-HETE (panels E,F) by LC-MS/MS. Chromatographic separation of lipid extracts was carried out using a Zorbax Eclipse Plus C18 RP-column (Agilent, Waldbronn, Germany) and for MS/MS detection the QTRAP instrument (Sciex, Darmstadt, Germany) was operated in negative electrospray ionisation mode. Shown are SRM traces of cell extracts for LTB₄ (m/z 335.2 → 195.1), 5-HETE (m/z 319.2 → 115.2) and 15-HETE (m/z 319.2 → 219.2).

Figure 5

Comparison of Alox5-KI and wildtype mice in the EAE inflammation model. Alox5-KI mice and wildtype controls (n = 8 in each group) were taken through the experimental protocol (see Materials and Methods). The clinical EAE scores were determined at the time points indicated. At the end of the experiment the animals were sacrificed at day 22 of the experimental protocol. Data were statistically evaluated using Mann-Whitney U-Test and the mixed ANOVA approach was used to compare the EAE score kinetics of the two genotypes over time. Cumulative incidence was statistically analyzed by Log-Rank test. (A) Onset of disease as indicated by the first appearance of neurological symptoms. (B) EAE score kinetics. Data are means ± SEM. (C) Relative spleen weight vs. brain weight ratios of the two genotypes (n = 8 in each experimental group) at the end of the experimental protocol (day 22).

Figure 6

Comparison of Alox5-KI mice and wildtype controls in the DSS colitis model. Alox5-KI mice (5 males, 5 females) and outbred wildtype controls (5 males, 5 females) were taken through the experimental protocol (see Materials and Methods), and we determined two major clinical readout parameters (body weight, colon length). (A,C) Kinetics of the relative weight loss that were calculated as percentage of the body weight at the time of measurement related to the body weight of the corresponding individual before the onset of DSS treatment. At each time point data were statistically evaluated using the Mann-Whitney U-Test and mixed ANOVA approach was used to compare weight loss kinetics of the two genotypes over time. * means there are no significant differences. Data are expressed as means ± SEM. (B,D) Colon lengths at the end of the experimental protocol. After the animals were sacrificed at the end of the experimental protocol (day 9), the colons were prepared and their lengths were determined.

Figure 7

Comparison of Alox5-KI and wildtype mice in the CCI model of neuropathic pain. Mechanical sensitivity assessed by the von Frey test in paws ipsilateral (A) and contralateral (B) to the CCI. The decrease in the paw withdrawal threshold indicates mechanical hypersensitivity. Heat sensitivity assessed by the Hargreaves test in paws ipsilateral (C) and contralateral (D) to the CCI. The decrease in the paw withdrawal latency indicates heat hypersensitivity. * p < 0.001 vs. values before CCI induction (pre-CCI), one-way repeated measures ANOVA and Bonferroni’s multiple comparison test. Data are expressed as means ± SEM. n = 9 male mice per genotype.