An Enzyme That Inactivates the Inflammatory Mediator Leukotriene B4 Restricts Mycobacterial Infection

While tuberculosis susceptibility has historically been ascribed to failed inflammation, it is now known that an excess of leukotriene A4 hydrolase (LTA4H), which catalyzes the final step in leukotriene B4 (LTB4) synthesis, produces a hyperinflammatory state and tuberculosis susceptibility. Here we show that the LTB4-inactivating enzyme leukotriene B4 dehydrogenase/prostaglandin reductase 1 (LTB4DH/PTGR1) restricts inflammation and independently confers resistance to tuberculous infection. LTB4DH overexpression counters the susceptibility resulting from LTA4H excess while ltb4dh-deficient animals can be rescued pharmacologically by LTB4 receptor antagonists. These data place LTB4DH as a key modulator of TB susceptibility and suggest new tuberculosis therapeutic strategies.


Introduction
An effective host immune response balances effective microbial killing mechanisms and damage to the host itself. Recent work has shown dysregulation of these responses in tuberculosis, either by immunodeficiency or by overexuberant immune activation, worsens outcome by increasing bacterial burdens [1,2]. The balance of pro-and anti-inflammatory eicosanoids is particularly critical in the regulation of the host response to infecting mycobacteria [2,3]. The enzyme LTA4H, which catalyzes the synthesis of LTB 4 from its unstable precursor LTA 4 [4] sits at a key crossroads regulating the balance between the anti-inflammatory lipoxins and pro-inflammatory LTB 4 [2,5]. In zebrafish larvae infected with Mycobacterium marinum, LTA4H deficiency and excess both produce hypersusceptibility with increased bacterial burdens [2].
The susceptibility of LTA4H deficiency is mediated by the lipoxin excess that results from blocking the enzymatic pathway to LTB 4 synthesis rather than compromised LTB 4 production per se [2]. In contrast, excess LTB 4 activity resulting from LTA4H excess plays a critical role in a hyperinflammatory route to increased disease severity [2]. A mechanistic dissection revealed that LTA4H overexpression produces TNF excess during infection and can be rescued by genetic knockdown or pharmacological modulation of TNF [2]. Albeit by a distinct mechanism, LTA4H/TNF excess converges on the same pathway to hypersusceptibility as LTA4H/ TNF deficiency: necrosis of infected macrophages that releases the bacteria into the growth-promoting extracellular environment [6]. While LTA4H/TNF deficiency permits uncontrolled intracellular bacterial growth culminating in macrophage lysis, macrophage necrosis occurs in LTA4H/TNF excess despite an initial reduction in intracellular bacterial growth [2]. LTB 4 is induced in human tuberculosis [7], and the zebrafish findings are corroborated in humans [2]. A human promoter variant that increases LTA4H expression is associated with a similar increase in tuberculosis severity as a low-LTA4H expressing promoter variant. As in zebrafish, the two variants correlate clinically with a high and low inflammatory state, respectively. Importantly, high-activity LTA4H genotypes show strong association with a genotype-dependent response to adjunctive anti-inflammatory therapy in TB meningitis. The implication of LTB 4 as a pharmacologically correctible host susceptibility determinant, led us to investigate whether additional modulators of LTB 4 might influence tuberculosis susceptibility and provide therapeutic targets for adjunctive therapies.

Results and Discussion
We targeted LTB4DH/PTGR1 (henceforth referred to as LTB4DH), an LTB 4 -inactivating enzyme [8,9] not previously implicated in mycobacterial susceptibility (Fig. 1a). In isolated polymorphonuclear leukocytes, LTB 4 is inactivated by omega oxidation via P450 while in other tissues via dehydrogenation through LTB4DH; notably the omega oxidation pathway is not predominant in monocytes, suggesting that dehydrogenation may represent the main inactivation pathway in these immune cells most closely associated with tuberculosis [9][10][11]. Consistent with this, LTB4DH/PTGR1 activity is prevalent in human monocytes (as assessed by a second role for the enzyme in lipoxin modification), but this activity is largely absent in human PMNs [12]. The zebrafish orthologue of human LTB4DH shares 61% amino acid identity (Fig. 2). All key residues shown to interact with NADP+ in the guinea pig LTB4DH crystal structure are conserved between zebrafish, humans and guinea pigs (Fig. 2) [13]. We found baseline ltb4dh RNA levels to be lower in lta4h mutant zebrafish than in wildtype siblings (Fig. 1b), suggesting that the ltb4dh promoter or transcript stability responds to feedback signals within this pathway. RNA levels of the myeloid transcription factor pu.1 were not affected by the lta4h mutation, providing an internal control (Fig. 1b). Thus, production of ltb4dh, the downstream inactivating enzyme, may be limited when its LTB 4 substrate is not produced due to lack of the upstream enzyme responsible for its generation. This interplay suggests that the two enzymes can function together to regulate LTB 4 .
To determine if LTB4DH functions to limit the inflammatory effects of LTB 4, we knocked down ltb4dh expression using antisense morpholinos. Even at baseline, ltb4dh morphants exhibited 2.9+/ 20.4 fold increased tnf RNA expression over wildtype siblings (P = 0.04; three biological replicates). In addition to increased tnf expression, LTA4H-overexpressing zebrafish exhibit increased macrophage recruitment to bacteria introduced into the hindbrain ventricle, a compartment normally lacking macrophages [2]. So too did the LTB4DH morphants (Fig. 1c). Thus both the overexpression of the enzyme that produces LTB 4 and inhibition of its inactivating enzyme result in the signature hyperinflammatory phenotypes attributable to excess levels of LTB 4  findings are consistent with the proposed role of LTB4DH as a negative regulator of LTB 4 activity [8].

. Our in vivo
Thus establishing a functional role for LTB4DH in LTB 4 inactivation, we asked if LTB4DH deficiency produced the same hypersusceptibility phenotype as LTA4H overexpression. ltb4dh morphants exhibited the same sequence of phenotypes as LTA4H excess created by mRNA expression: initial decreased intracellular bacterial burden after mycobacterial infection degenerated within a few days into increased bacterial burdens associated with cording morphology characteristic of macrophage lysis and extracellular residence (Fig. 1d-f and Fig. 3a). Morpholino inhibition of TNF in the background of ltb4dh morphants restored wildtype infection burden and removed the cording phenotype, showing that TNF is a key mediator of susceptibility as it is with LTA4H excess [2] ( Fig. 1g and Fig. 3b).
If the hypersusceptibility of ltbd4h morphants is due to excess LTB 4 , then it should be possible to rescue this phenotype pharmacologically by blocking LTB 4 activity. Accordingly, a LTB 4 receptor antagonist reversed the cording-associated hypersusceptibility of ltb4dh morphants while restoring excess tnf to wildtype levels (Fig. 1g,h and Fig. 3c). Conversely, interventions that compromise production of anti-inflammatory lipoxins should have a detrimental effect in ltbd4h morphants, as they do in states of LTA4H excess [2]. A known 15-lipoxygenase inhibitor produced the expected worsened bacterial burden accompanied by a further increase in tnf (Fig. 4). Together these results provide functional evidence that both excess LTA4H and reduced LTB4DH result in increased LTB 4 activity, which in turn produces mycobacterial hypersusceptibility through TNF-mediated hyperinflammation. These findings suggest that, despite a degree of co-regulation of LTB 4 production and inactivation, LTB4DH levels can independently modulate control of infection. Importantly, the detrimental effects of LTB4DH deficiency can be countered by pharmacological antagonism of LTB 4 activity or by directly targeting the resulting excess TNF.
To probe further if LTB4DH can function as an independent resistance factor in humans, we used a dataset from an existing microarray analysis of human HapMap lymphoblastoid cell lines (LCLs) [14] to examine whether the correlation between lta4h and ltb4dh expression observed in the zebrafish was conserved in humans. We interrogated the collection of Asian cell lines, in which we had previously observed an effect on transcriptional activity of a common LTA4H promoter polymorphism [2]. As in the zebrafish, we found a direct correlation between LTA4H and LTB4DH RNA levels; individual cell lines with higher LTA4H transcript levels had higher LTB4DH expression (Fig. 5a). The level of correlation (r 2 of 0.2, P = 0.0003) suggests that there is ample space for additional genetic and non-genetic factors to influence LTB4DH expression in humans. Thus, the detrimental effects of LTA4H excess may be dampened only to a limited extent by compensatory induction of the inactivating enzyme, This idea is supported by our zebrafish findings that LTB4DH reduction is an independent and pharmacologically correctible source of hypersusceptibility.
We asked if increasing LTB4DH expression levels in vivo might ameliorate the LTA4H-high susceptibility phenotype, thus providing additional potential targets for intervention. Overexpression of LTB4DH mRNA was sufficient to rescue the LTA4H-high phenotype (Fig. 5b). In contrast, LTB4DH overexpression did not worsen infection of wildtype animals (Fig. 5b), consistent with previous data where pharmacological inhibition of LTB 4 receptor did not increase susceptibility [2]. The lack of a protective role for LTB 4 in tuberculosis is in contrast to the case of other infections where a protective role has been noted for this inflammatory mediator [15][16][17]. In tuberculosis, LTB 4 may mediate only pathogenesis, as it does in a variety of inflammatory diseasesasthma, atherosclerosis, rheumatoid arthritis, gout, obesity and cancer [18][19][20][21][22][23][24]. Accordingly, we find that excess LTB 4 resulting  Fig. 1g with cording at 4 dpi among sibling wildtype, ltb4dh morphant and ltb4dh/tnf double morphant animals. **P = 0.004, *P = 0.043; Fisher's exact test of contingency table comparing wildtype and ltb4dh/tnf double morphant each to ltb4dh morphant. c) Percentage of animals in Fig. 1h with cording at 4 dpi among sibling widtype, ltb4dh morphants treated with 10 mM U75302 and ltb4dh morphants in vehicle (0.5% DMSO). ***P,0.0001, **P = 0.01; Fisher's exact test of contingency from two independent deviations in its metabolic pathway produces hypersusceptibility to mycobacterial infection.
In conclusion, these findings implicate a downstream inactivating enzyme of a pro-inflammatory eicosanoid as an important controller of mycobacterial resistance. Despite finding some degree of co-regulation of LTB4DH and LTA4H in both zebrafish and humans, ltb4dh serves as an independent susceptibility locus in the zebrafish. Similarly, human variation in LTB4DH may influence TB susceptibility. Our work suggests that pharmacological interventions can compensate for the consequences of LTB4DH reduction, providing new approaches to titering eicosanoid balance. LTB 4 -mediated inflammation may have relevance to oncogenic transformation, as LTB 4 and components of its synthetic pathway are induced in different cancers [23][24][25]. Particularly intriguing from a therapeutic perspective is the ability of gallic acid and a purified compound from Radix astragali to induce LTB4DH expression and limit oncogenic transformation [26]. Specific pharmacological inducers of LTB4DH expression, besides serving as novel anti-inflammatory therapies, may provide a new route to addressing the known hypersusceptibility to tuberculosis of individuals with high LTA4H expression [2].

Bacterial Strains
WT strain M (ATCC #BAA-535) was transformed with plasmids containing transcriptional fusions of the gene encoding Wasabi to a constitutively-expressed Mycobacterium marinum promoter as described [27]. The erp mutant expressing msp-12::gfp [28] was used for quantitation of intracellular bacterial burdens in Fig. 1d. Bacteria were grown in 7H9 media supplemented with oleic acid-albumin-dextrose-complex and 0.05% Tween-80 unless otherwise stated.

Zebrafish Strains and Infections
All experiments were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee of the University of Washington. Zebrafish embryos of the AB line were injected with Mm or PBS (mock-injected) using phenol red as a visual marker at 30 hours post fertilization or 48 hours post fertilization (hpf) via caudal vein as described [29]. Hindbrain ventricle injections were performed as described at 24 hpf [30]. Innocula at injection were determined injecting the same volume onto selective bacteriologic plates and enumerating bacteria when colonies formed in 5-7 days. Bacterial burdens of larvae were determined by fluorescence pixel counts (FPC) as described in [27]. After images have been taken with

Morpholinos and RNA Injections
Morpholinos were obtained from Genetools (Eugene, OR). Control and tnf morpholinos were as previously described [31,32]. ltb4dh morpholino targeted with an injection volume of 2-4 nL of 1 mM morpholino targeting the start codon (sequence 59 CTTGAGCCATGACTCTTTTTTTGCA 39, with complement of ATG bolded). All morpholino infections were performed on paired needles; an equal number of morpholino and control animals were injected on each needle in an alternating sequence to reduce variations in dosage between groups.

RT-PCR
Quantitative RT-PCR was performed as previously described [2,32]. Total RNA obtained by Trizol extraction from infected or mock-injected embryos were used as templates for generating cDNA (Superscript II reverse transcriptase; random hexamer primers; Invitrogen) for quantitative real-time RT-PCR analysis. qRT-PCR assays were performed such that each 20 mL reaction contained either 250 nM of gene-specific primers or b-actin specific control primers. SYBR green PCR Master Mix (Applied Biosystems) was used at 1X. All qRT-PCR assays were performed in triplicate with an ABI Prism 7300 Real Time PCR System (Applied Biosystems). Data were normalized to b-actin (DDCT analysis). Primer sets used for ltb4dh were F 59 TCTTGGAT-GACTGGCCTCAT 39 and R 59 TCCTGGTTTGATGGCA-CATA 39.
lta4h and ltb4dh RNA was synthesized using the mMessage mMachine kit (Ambion) and the polyA Tailing kit (Ambion). 2-4 nL of RNA was injected at one-four cell stage at a concentration of 200 ng/mL to create LTA4H-high animals and to overexpress ltb4dh.

Pharmacological Interventions in Zebrafish
After infection, small molecules were applied via soaking. Solutions were changed daily. All conditions and controls were standardized to a final concentration of 0.5% DMSO. PD 146176 (BIOMOL) was applied at a final concentration of 500 nM 16 hpi. U75302 (BIOMOL) was applied at a concentration of 10 mM directly after infection.

Microscopy
Microscopy was performed on a Nikon E600 equipped with DIC optics, a Nikon D-FL-E fluorescence unit with 100 W Mercury lamp and MFC-1000 z-step controller (Applied Scientific Instrumentation) or, for whole animal images, a motorized Nikon inverted Ti-E microscope. Objectives used included 26 Apo Objective 0. 1

Statistical Analysis
Statistical analysis was performed with Prism (Graphpad Software) for all comparisons.