Protective Role of 5-Lipoxigenase during Leishmania infantum Infection Is Associated with Th17 Subset

Visceral leishmaniasis (VL) is a chronic and fatal disease caused by Leishmania infantum in Brazil. Leukocyte recruitment to infected tissue is a crucial event for the control of infections such as VL. Leucotriens are lipid mediators synthesized by 5-lipoxygenase (5-LO) and they display a protective role against protozoan parasites by inducing several functions in leucocytes. We determined the role of 5-LO activity in parasite control, focusing on the inflammatory immune response against Leishmania infantum infection. LTB4 is released during in vitro infection. The genetic ablation of 5-LO promoted susceptibility in highly resistant mice strains, harboring more parasites into target organs. The susceptibility was related to the failure of neutrophil migration to the infectious foci. Investigating the neutrophil failure, there was a reduction of proinflammatory cytokines involved in the related Th17 axis released into the organs. Genetic ablation of 5-LO reduced the CD4+T cells producing IL-17, without interfering in Th1 subset. L. infantum failed to activate DC from 5-LO−/−, showing reduced surface costimulatory molecule expression and proinflammatory cytokines involved in Th17 differentiation. BLT1 blockage with selective antagonist interferes with DC maturation and proinflammatory cytokines release. Thus, 5-LO activation coordinates the inflammatory immune response involved in the control of VL.


Introduction
Visceral leishmaniasis (VL) is one of the most severe clinical manifestations of infection with Leishmania parasites and it is a major cause of human mortality and morbidity globally; VL is caused by Leishmania donovani and Leishmania infantum (World Health Organization, 2010).
The host protective response against Leishmania spp. is predominantly mediated by cellular immunity mechanisms, which are critical for parasite replication control and disease resolution. Initially, during infection, activated dendritic cells (DCs) modulate inflammatory leucocyte recruitment to the infection foci [1] and the development of the T CD4 + lymphocyte response characterized by robust IFNand IL-17 production [2,3]. The immune cell recruitment to Leishmania infection foci is managed by inflammatory mediators. Chemokines and cytokines have crucial roles in determining the outcome of leishmaniasis [4,5]. Lipid mediators such as leukotrienes (LTs) are another class of molecules involved in host defense [6].
Regarding leishmaniasis, LTB 4 displays leishmanicidal activity on macrophages [24] and neutrophils [25] during in vitro infection with L. amazonensis, through mechanisms dependent on nitric oxide (NO) and reactive oxygen species (ROS), respectively. In addition, the inhibition of the 5-LO pathway promoted high susceptibility to L. amazonensis infection, increasing footpad swelling and harbored more parasites in resistant and susceptible infected mice [26]. In in vitro macrophage infection with L. donovani parasites, the 5-LO enzymatic activity is enhanced, leading to increased amounts of arachidonic acid metabolites [27], and in vivo, L. donovani infection promotes an increase of cyclooxygenase and lipoxygenase activities in spleen cells [28]. It was recently reported that L. infantum in vitro infection inhibits the LTB 4 signaling pathway dependent on homologous DCSIGN (SIGNR3) during parasite recognition by macrophages [29], suggesting a protective role of LTB 4 during VL induced by L. infantum. Their potential in the recruitment of leukocytes that might be involved in parasite restriction is less well understood. We investigated the role of 5-LO activity in the control of experimental VL induced by L. infantum, focusing on the inflammatory immune response. We demonstrated that mice lacking 5-LO signaling displayed high susceptibility to L. infantum infection because of a commitment on the related Th17 axis released by CD4 T lymphocytes and neutrophil migration to the infection foci.

Mice.
Female wild-type 129/SvEv (WT) mice or mice genetically deficient in 5-LO (129/SvEv-5-LO −/− ), 18-22 g in weight, were housed in the animal facility of the Department of Biochemistry and Immunology of the School of Medicine of Ribeirão Preto at the University of São Paulo (Brazil) in temperature-controlled rooms (22-25 ∘ C); the mice received water and food ad libitum. The experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines on the welfare of experimental animals and with the approval of the Ethics Committee of the School of Medicine of Ribeirão Preto.

Parasite Culture, Infection, and Parasite Load Estimation.
L. infantum (isolate HU-UFS14) was cultured in Schneider medium with 20% heat-inactivated fetal bovine serum, 5% penicillin and streptomycin (from Sigma-Aldrich, Saint Louis, MO, USA), and 2% male human urine. The parasite virulence was maintained by serial passages in BALB/c mice. The mice were injected in the retroorbital plexus with 10 7 stationary-phase L. infantum promastigotes in 100 L PBS. The hepatic and splenic parasite burdens were determined using a quantitative limiting dilution assay.

DC Generation and Infection.
Generation of bone marrow-derived dendritic cells (BMDC) was performed as previously described [30]. The BMDCs (1 × 10 6 /mL) cultured in RPMI-1640 supplemented with 10% FBS were infected with L. infantum promastigote forms at a 1 : 5 ratio (cells/parasites) for 12, 24, 36, and 48 h. The supernatants were collected to measure LTB 4 by ELISA (BiotrakTm, Amersham Pharmacia Biotech, UK). In some wells, LPS (200 ng/mL) was added to the BMDC culture as the positive control group. The cells were harvested and their surface expression characterized by flow cytometry using antibodies against CD11c, MHC class-II, CD86, and CD40 conjugated to APC, FITC, PECy7, PerCP, and Alexa700, respectively, as well as the control isotypes. The cytokine releases were measured into the supernatant culture using commercial ELISA kits, according to the manufacturer's instructions (BD Biosciences, R&D Systems, Minneapolis, MN, USA). In some experiments, selective BLT 1 leukotriene B 4 receptor antagonist (U-75302, Sigma-Adrich) (10 M) was added 12 h before L. infantum infection.

Cytokine Release.
To assess the influence of LTB 4 on cytokine production, the liver tissue samples were harvested by a tissue trimmer, weighed, and tittered in 0.5 mL of PBS Complete (Roche Diagnostics, Mannheim, Germany) containing protease inhibitor cocktail. The levels of IFN-, IL-17, TNF-, IL-12p40, IL-23, IL-6, TGF-, and IL-1 were determined using commercial ELISA kits.

Cell Culture and Inflammatory Cells
Phenotype. Singlecell suspensions of spleen tissue samples from the 5-LO −/− or WT mice at 6th wpi were aseptically prepared, diluted to a concentration of 2 × 10 6 cells/mL, and dispensed into 48-well plates in a total volume of 500 L of complete RPMI-1640 medium (1 × 10 6 cells/well; Gibco) with or without soluble Leishmania Ag (5 g/mL). The cell culture supernatants were harvested after 72 h of culture at 37 ∘ C in 5% CO 2 , and the cytokine levels in the supernatants were determined by ELISA with commercial kits (BD Biosciences and R&D Systems). For the leukocyte identification, the inflammatory cells were gated based on their characteristic size (FSC) and granularity (SSC), and the T lymphocytes (CD4 + CD3 + ), dendritic cell activation markers (CD11c high CD40 + , CD11c high CD86 + , and CD11c high MHC-II + ), and neutrophils subsets: activated (Ly6G high CD11b high ) or inactivated neutrophils (Ly6G int CD11b int ) were identified individually. For the intracellular staining, the cells were previously cultured with PMA (50 ng/mL) and ionomycin for 4 h in order to obtain the maximum of cytokine production and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's guidelines and stained with anti-IFN-or anti-IL-17 conjugated to APC-Cy7 and Alexa700 and with anti-CD3 and anti-CD4 for surface staining with FITC and PerCP, respectively. Rat IgG2b and rat IgG2a were used as the isotype controls. All the antibodies were supplied from BD Biosciences and eBiosciences (San Diego, CA, USA). The cell acquisition was performed using a FACSort flow cytometer. The data were plotted and analyzed using the FlowJo software (Tree Star, Ashland, OR). The total leucocytes counts were determined by relative expression of leucocytes subpopulation stained with specific antibody obtained in 300,000 events acquired and proportional to the leukocytes number obtained in Neubauer chamber. The results from the individual experiments were not combined because they were analyzed individually. The means from the groups were compared by ANOVA followed by Tukey's honest significant difference (HSD) test. Statistical significance was set at < 0.05.

5-LO Activation Is Required for Experimental L. infantum Infection Control.
To determine whether Leishmania infantum drives the activation of 5-LO pathway, we performed a kinetic in the release of LTB 4 by bone marrow-derived dendritic cells after 12, 24, 36, or 48 hours of parasite infection. L. infantum induces significant amounts of LTB 4 by BMDCs at 12 hours postinfection, peaked at 24 hours, and persisted for 36 hours. At 48 hours, the heightened levels of LTB 4 production were significantly reduced and similar that produced for uninfected cells (medium stimuli) (Figure 1(a)).
To characterize the LTB 4 function, 5-LO −/− and littermate control mice were infected with L. infantum and the course of infection was monitored by parasite quantification into the organs by a limiting dilution. We observed that WT presented progressive parasite titers into the spleen (Figure 1 Based on their characteristic size (FSC) and granularity (SSC), we observed a significant reduction of cells when analyzed in the granulocytes gate from infected 5-LO −/− mice. Phenotyping the cells, we found that the frequencies of Ly6G + CD11b high were present in the spleen samples from the WT infected mice. The percentage of the influx of neutrophils was affected in the 5-LO −/− infected mice, which showed an approximately 30% reduction compared with that of the WT infected mice (Figures 2(b) and 2(c)). In terms of total numbers, the neutrophil reduction was ∼50% in the 5-LO −/− mice (Figure 2(c)). We also observed another neutrophils population, LY6G + CD11b interm (Figure 2(b)), features of inactive neutrophils since CD11b is upregulated under proinflammatory stimuli [31,32]. However, their frequency and total cells (Figure 2(d)) in the spleens were similar in both groups. These findings suggest that 5-LO activity participates in neutrophil recruitment to inflammatory foci and, under appropriated activation, might be required for parasite control during L. infantum infection.  for the control of parasite replication in the target organs of LV, we investigated whether these responses were generated in a 5-LO dependent manner. Spleen cells from WT and 5-LO −/− mice at 6th wpi or naïve were in vitro restimulated with polyclonal PMA plus ionomycin and the intracellular cytokine production was analyzed. There was no difference in the frequency and absolute number of the IFN--producing CD4 + T cells in the WT and 5-LO −/− mice (Figure 3(a)). The IL-17-producing CD4 + T cells were significantly impaired in the spleens of the 5-LO −/− mice (Figure 3(b)), where the Th17 cells reduction was approximately 50% of that in the WT mice.

5-LO Activity Is
Having determined that 5-LO activity participates in the development of the Th17 response, we measured the production of cytokines in the culture supernatant of the total splenic cells from the WT, 5-LO −/− naïve, or infected mice at 6th wpi and restimulated them in vitro with soluble Leishmania Ag (SLA). The stimulation with SLA did not induce significant amounts of IFN- (Figure 4 (Figures 4(a)-4(g)). Infection in the 5-LO −/− mice resulted in a reduction of cytokine release related to the Th17 axis such as IL-17, TNF-, IL-23, and IL-6 ( Figures 4(b)-4(e)); however, neither IFN- (Figure 4(a)), IL-1 (Figure 4(f)), nor TGF- (Figure 4(g)) productions were affected by a specific stimulus, compared to that obtained in the infected WT mice when stimulated with SLA. Additionally, proinflammatory cytokines in the liver involved in the Th17 axis such as IL-17 (Figure 4(i)), TNF (Figure 4(j)), and IL-12p40 (IL-23) (Figure 4(k)) were reduced in the absence of 5-LO. Corroborating to Figures 3(a) and 4(a), IFN-amounts were not altered in the deficient mice (Figure 4(h)). These data suggest that 5-LO activity is associated with Th17 response development, and this pathway might be involved in the neutrophils recruitment to inflammatory foci.  (Figure 6(c)). The absence of 5-LO did not alter the LPS-induced dendritic cell maturation (Figures 6(a)-6(c)).
Next, we evaluated the release of innate cytokines involved in Th17 axis differentiation by DCs. Thus, we determined the levels of TNF, IL-23, IL-1 , and IL-6 in the supernatants from the WT or 5-LO −/− BMDCs cultured with L. infantum parasites or medium. As the positive control, the cells were activated with LPS. The parasites induced significant production of TNF ( Figure 6 before L. infantum infection and the release of cytokines related to Th17 pattern was measured into culture supernatant by ELISA assay. As expected, TNF (Figure 6(h)), IL-23 ( Figure 6(i)), and IL-6 ( Figure 6(j)) were produced during infection. The ability of BMDCs infected with parasites to produce cytokines such as TNF (Figure 6(h)) and IL-23 ( Figure 6(i)), but not IL-6 ( Figure 6(j)), was inhibited by BLT 1 blockage, confirming that LTB 4 is associated with the release of cytokines involved in the Th17 axis. We do not rule out the possibility of others leukotrienes that may contribute to cytokine release, herein, that is, IL-6 release, but we undoubtedly evidenced LTB 4 participation in the control of VL.

Discussion
In this study, we report 5-LO activity, and presumably LTB 4 , as an important mediator in controlling infection induced by Leishmania infantum. This eicosanoid that is released during infection may promote the activation of dendritic cells, which influence the release of mediators involved in the drive of naive CD4 + T lymphocytes to the Th17 profile. In the last instance, the Th17 subtype recruits neutrophils to the infection foci that might retrain the parasite restriction. Understanding the role of LTB 4 in the inflammatory process mediated by L. infantum might elucidate some of the effector mechanisms that control the replication of the parasites. We demonstrated that infection with L. infantum results in the production of LTB 4 by dendritic cells during in vitro infection. The absence of endogenous LTB 4 promoted higher susceptibility to infection. The genetic ablation of 5-LO harbored more parasites in target organs such as the spleen and liver, demonstrating its role in the control of infection. These results are consistent with those of previous studies that demonstrate the role of LTB 4 in the control of infectious processes [13,17,21], increasing the leishmanicidal activity of macrophages [24] and of neutrophils [25] by a nitric oxide (NO)-dependent mechanism and release of reactive oxygen species (ROS), respectively.
Several studies have demonstrated that LTB 4 is a potent inducer of neutrophils. During leishmaniasis, neutrophils are rapidly mobilized to the inflammatory site [1,35], where they eliminate the pathogen by the production of reactive oxygen species (ROS) and the release of peptides and antimicrobial proteases [36][37][38]. In our results, the high susceptibility observed in animal 5-LO −/− was accompanied by the failure of neutrophil migration. LTB 4 has a central role in controlling the migration of neutrophils to sites of inflammation through BLT 1 and BLT 2 (leukotriene receptors) [39], directly by inducing the expression of the CD11b and CD18 integrins [40] or indirectly by amplifying the production of inflammatory mediators such as cytokines and chemokines by others cells [41,42]. In fact, we observed a significant reduction of activated neutrophils expressing CD11b into target organs that were infected by parasites in the absence of 5-LO. Furthermore, LTB 4 enhances effectors mechanisms of neutrophils such as phagocytic capacity [43] and granules releasing and stimulates the enzymatic generation of ROS [14,44,45], including in vitro infection by L. amazonensis [25]. Thus, it seems that the protector role of LTB 4 during LV may be played by the recruitment and activation of neutrophils to the site of infection. The recruitment of neutrophils might be induced by cytokines such as IL-17 because they are potent granulopoietic factors [46] that induce the release of CXC chemokines [47]. We found that the absence of LTB 4 synthesis impaired the Th17 response, whereas the Th1 response was unchanged in the target organs. Consistently, the production of IL-17 by spleen cells in response to the specific stimulus (i.e., Leishmania antigen) and its detection in the liver of 5-LO −/− infected mice was inhibited, confirming the interference of LTB 4 in the release of IL-17. We have not evaluated whether LTB 4 participates in the control of Leishmania infantum through Th17-dependent manner; however, we believe that the administration of recombinant IL-17 may rescue the protective effect of leucotrienes in susceptible 5-LO deficient mice. In fact, administration of recombinant IL-17 or IL-23 in susceptible BALB/c mice infected with L. donovani controlled parasite replication, which was associated with increased iNOS activity [3]. Furthermore, exogenous LTB 4 is able to positively modulate the differentiation of Th17 cells from naive CD4 + T cells [48]. The induction of experimental autoimmune encephalomyelitis (EAE) in animals genetically deficient in BLT 1 presented clinical score signs attenuated because of impairment of the Th17 generated response. Infiltration of T cells, macrophages, and granulocytes into the spinal cord was reduced in the BLT 1 −/− mice [49], demonstrating the involvement of LTB 4 in the development of the Th17 response. LTB 4 is produced during inflammatory and infectious processes by several leucocytes [50], including activated neutrophils, macrophages, and T cells [51][52][53][54]. Among the cells able to synthesize LTB 4 , DCs play an important role in the initiation of immune responses because they are the main cells involved in pathogen recognition, triggering several proinflammatory mechanisms that bridge to adaptative immune responses [55][56][57]. According to our results, DCs are potential sources of LTB 4 during L. infantum infection. Given the importance of the role lipid mediators play in leucocyte activation, LTB 4 production by DCs is a major mechanism for the modulation of the effector function of other cell subsets during LV, for example, mediating the recruitment of neutrophils to inflammation sites. We do not rule out the possibility of others leukotrienes that may contribute to cytokine release, since that the ablation of 5-LO lacks not only LTB 4 but also cysteinyl leukotrienes including LTC 4 , LTD 4 , and LTE 4 activity [34]. However, the pharmacological blockage of BLT 1 prevented, at least in part, the release of cytokines by DC, evidencing LTB 4 association with Th17 axis, and in last instance, controlling parasite replication.
Apart from sources of LTB 4 , DCs are the target of the action of lipid mediators as an important mechanism for modulating the immune response [58,59]. An impaired Th17 response might result from failed DC activation in the absence of 5-LO. This hypothesis might be supported by the following explanations. First, exploring the role of LTB 4 in DC activation, our data demonstrated that the maturation phenotype of DCs from animal 5-LO −/− was reduced during in vivo and in vitro infection. Consistently, the addition of LTB 4 in cultured BMDCs induces maturation of these cells to increase MHC-II expression. Blockage of 5-LO with NDGA protects cells from the effects of LTB 4 on DC maturation [60]. BMDCs migrate and are activated in response to LTB 4, and its effect is lost in cells that lack BLT 1 [61]. LTB 4 upregulates the expression of CCR7 and its ligand CCL19/ELC, which mediate the migration to lymphoid organs. Second, the impaired ability of DCs from animal 5-LO −/− to secrete cytokines is involved in the polarization of naïve CD4 T cells to the Th17 profile. Naïve CD4 + T lymphocytes are polarized to the Th17 subset through the combined pattern of the action of cytokines such as IL-1 , TGF-and IL-6 [62], whereas activation requires sustained stimulation with IL-23, which is predominantly produced by dendritic cells and TNF release [63]. Our data demonstrated that the production of TNF, IL-23, and IL-6 in vivo, at least, was compromised in the absence of 5-LO. Supporting our hypothesis, a significant reduction of IL-23, TNF, and IL-6 by BMDC was observed in the dendritic cells derived from animal 5-LO −/− . Consistently, Lefèvre and colleagues demonstrated that cytokines such as IL-1 , TGF-, and IL-6 are highly produced by macrophages infected in vitro with L. infantum [29]. The role of LTB 4 in the induction of innate cytokines related to the Th17 profile differentiation released by DCs is unprecedented. It is known that IL-1R signaling is dependent on the BLT 1 downstream pathway. The requirement for the BLT 1 signaling pathway is overcome by exogenous administration of IL-1 in LTB 4 −/− mice [64]. Moreover, BLT 1 expression is upregulated in Th17-differentiated T cells [49] and ex vivo studies have demonstrated that the production of TNF and IL-6 was impaired in the absence of BLT 1 −/− cells [65,66], confirming the role of LTB 4 in driving the Th17 response.
We do not investigate the molecular mechanisms by which 5-LO activity interferes with maturation process and subsequent activation of dendritic cells, but we believe that the initial response is dependent on TLR4 signaling. During parasite recognition through TLR4 pathway, the adapter molecule MyD88 is recruited and activates factors such as NF-B [67,68], leading to transcription of proinflammatory cytokines such as TNF, IL-6, and IL-23. MYD88 recruitment also activated 5-LO enzyme, promoting the synthesis of leucotriens, especially LTB 4 that, through BLT 1 pathway, amplifies the activation of NF-B which may induce cellular activation [66]. Interestingly, genetic deletion of 5-LO or pharmacological blockade of BLT 1 receptor interferes with the secretion of proinflammatory cytokines by DCs and their maturation phenotype. The 5-LO pathway may act in autocrine manner, increasing the activation and function of DCs, and greatly influence the magnitude response of Th17 cells as well. Thus, the amplification of the inflammatory response mediated by 5-LO activation during parasite recognition by DCs appears to play an important role in controlling parasite replication.

Conclusion
Our data demonstrated that 5-LO activity, and perhaps LTB 4 , plays a prominent role in controlling L. infantuminduced visceral leishmaniasis, which may be associated with the development of the Th17 response and the subsequent recruitment of neutrophils to the inflammatory site that is dependent on dendritic cell activation. Future studies might characterize which innate receptors on DCs are involved in the recognition of the parasite, leading to a subsequent synthesis of LTB 4 . The results show, for the first time, the role of LTB 4 in the development of the Th17 response in the context of an infectious disease.