Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future.

The protozoan parasite Leishmania infects phagocytes causing a spectrum of diseases from less severe cutaneous leishmaniasis to lethal visceral leishmaniasis (VL). Pathogenic L. donovani (PLD; AG83/MHOM/1983), a protozoan parasite, causes VL or Kala-azar, which is prevalent in tropical and temperate regions 1 . Leishmaniasis is endemic in 88 countries with an estimated yearly incidence of 1-1.5 million cases of cutaneous leishmaniasis and 500,000 cases of VL, of which 70,000 people die every year 2,3 . Treatment options for leishmaniasis include sodium antimony gluconate (SAG or SbV), Miltefosine, Pentamidine, and Amphotericin B which are drugs with serious limitations due to toxicity or resistance 4 . Development of vaccines to thwart leishmaniasis has been an objective for a century now. First efforts at vaccination, termed leishmanization have been largely terminated due to a variety of reasons. The first-generation vaccines based on killed parasites have substituted leishmanization, but evidence from studies in animal models has been found inadequate in field studies 5 . Second generation vaccines employing genetically modified parasites have not proved their competence so far 6 .

Augmentation of superoxide generation in ALS treated MΦs. It is well established that Leishmania
abolishes the superoxide generation in MΦ s for its survival. Studies showed that augmentation of superoxide generation inhibited Leishmania growth. Therefore we sought to monitor superoxide generation in ALS treated MΦ s. Raw 264.7 cells were incubated with different concentrations of ALS, viz. 5 × 10 6 , 1 × 10 7 , 5 × 10 7 and 1 × 10 8 cells per ml and then p-nitro blue tetrazolium (NBT) reduction assay was performed. The optimum increment in superoxide generation (P < 0.05) was observed (Fig. 1A) in MΦ s primed with 1 × 10 7 ALS.
To confirm the finding, we further checked the ROS and NO production in ALS treated MΦ s. The sharp rise in ROS and NO production was observed (Fig. 1B,C) in MΦ s treated with 1 × 10 7 ALS, (P < 0.05). In a time dependent study, increased NO generation was observed at 24 h (Fig. 1D), whereas a steady state gradual increase in ROS generation was noticed up to 24 h (Fig. 1E) (P < 0.05). Therefore, a promising microbicidal activity was anticipated due to the augmentation of superoxide generation, which is an essential attribute for combating pathogens.
Increased expression of iNOS, and gp91 phox in ALS primed MΦs. Among the three types of Nitric oxide synthases, iNOS is responsible for anti-microbial NO production while gp91 phox , a subunit of NADPH oxidases, is one of the key components of microbicidal oxidases in phagocytes 25 . Our flowcytometric data showed augmented expressions of iNOS (Fig. 1F) and gp91 phox (Fig. 1G) in ALS primed MΦ s (P < 0.05). These further validate the capability of ALS to induce respiratory burst in MΦ s. ALS treated MΦs showed enhanced phagocytic activity. ALS treated MΦ s showed enhanced phagocytosis of latex bead. Engulfment was found to increase with time ( Fig. 2A  (A-C) 1 × 10 6 RAW 264.7 cells/ml was incubated with or without 5 × 10 6 , 1 × 10 7 , 5 × 10 7 , and 1 × 10 8 ALS or LPS for 4 h, washed, fresh media was added, and assayed after 12 h. Dose kinetics of the levels of NBT (A), NO (B) and ROS (C) were measured. (D,E) 1 × 10 6 RAW 264.7 cells/ml was incubated with or without 1 × 10 7 ALS for 4 h and processed as before. Flowcytometric analysis of NO (D) and ROS (E) production was evaluated in a time dependent manner using DAF-FM and DCF respectively. (F,G) In ALS treated MΦ s iNOS (F) and gp91 (G) levels were observed. Change in MFI and percent positive cells was represented graphically for each of the above experiments. Data are representative as the mean ± SD and are the cumulative results of three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001. (A) RAW 264.7 cells were treated with or without 1 × 10 7 ALS for 4 h, then washed and incubated with fresh media containing fluorescence latex beads for 1 and 2 h. Phagocytosis was studied by measuring the fluorescence intensity of engulfed latex bead. (B) Graphical representation depicted the latex beads containing percent positive cells and mean fluorescence intensity of engulfed latex beads in ALS treated/untreated cells. (C) ALS treated MΦ s were incubated with GFPexpressing bacteria for 4 and 24 h and phagocytosis was studied under confocal microscope. (D) Change of mean fluorescence intensity due to phagocytosis of GFP-expressing bacteria was shown in above mentioned condition. (E) Cells were treated with ALS as before. After 4 h cells were again infected with CFSE labeled AG83 for another 4 h and uptake of CFSE labeled AG83 was observed under confocal microscope. Data are representative as the mean ± SD and are the cumulative results of three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001. All the confocal microscopic data were analyzed by using ImageJ software.
Scientific RepoRts | 6:22335 | DOI: 10.1038/srep22335 (Fig. 2C,D) (P < 0.05). Similarly, treated MΦ s showed enhanced uptake of PLD as compared to untreated ones (Fig. 2E). While it was reported that increment of phagocytic activity was not a critical hallmark of classical activation of MΦ s, the present observation is important because PLD infection restricts the phagocytic capacity of MΦ s 26 .
Elevation of expression and nuclear translocation of NF-κB and c-JUN in ALS treated MΦs. We next sought to determine the probable factors working behind the production of microbicidal mediators in ALS treated MΦ s and also to monitor the expression of transcription factors that have been reported to associate with it. We observed the expressions as well as nuclear translocation of NF-κB and c-JUN, which are the key transcription factors associated with the production of pro-inflammatory mediators. Our Flowcytometric analysis revealed significant up-regulation (P < 0.05) of NF-κB p65 (Fig. 3G), c-Jun (Fig. 3H), and IKK-α (Fig. 3I) in ALS primed MΦ s. Nuclear accumulation of NF-κB p65 and c-Jun was also Increased (Fig. 3J-M) in MΦ s pre-exposed to ALS for 4 h (P < 0.05). Simultaneously, there was a decreased translocation of NF-κB p50 (Fig. 3J,K) (P < 0.05), which further ensured the functional activity of NF-κB 27 , the key factor regulating expression of TNF-α , IL-12 and iNOS 28 . Activation of MAPK in ALS treated MΦs. Since MAPKs constitute the central hub for a wide variety of cellular function, including the effect on different cytokine production in MΦ s, we decided to explore the upstream signaling event involved in ALS mediated MΦ activation. Several reports suggested that Leishmania infection alters the MAPK signaling 11 and low intensity signal from CD40 signalosome cause significant up-regulation of p44/42 MAPK that leads to augmentation of IL-10 production in Leishmania infected MΦ s 12 . In our study, although the expressions of p38 MAPK, p44/42 MAPK and JNK were unaltered by ALS treatment ( Interestingly, when these ALS pre-treated MΦ s were infected with PLD, no suppression of p38 MAPK and JNK ( Fig. 4C) was observed (P < 0.05). On the other hand, in ALS pre-exposed MΦ s, elevated p-p44/42 MAPK level was normalized after infection with PLD ( Fig. 4C) (P < 0.05).
For further confirmation, we used inhibitors of p38 MAPK, JNK, and p44/42 MAPK. Result showed that p44/42 MAPK is an important regulator of NO production in ALS treated MΦ s, while p38 MAPK is responsible for IL-12 production (P < 0.05) (Fig. 4D). However, the increment of TNF-α production after treatment with ALS was under tripartite regulation involving all the three MAPKs ( Fig. 4D) (P < 0.05). Altogether this indicated that p38 and p44/42 MAPK were essential for ALS mediated MΦ activation 13,29 . Phagosome maturation conferring leishmanicidal attribute to ALS pre-treated MΦs. We next sought to determine whether ALS pre-treatment has any effect on parasite viability within those activated MΦ s. For this ALS primed MΦ s were infected with CFSE labeled PLD and viability were observed by flowcytometry. In comparison to untreated MΦ s, ALS primed MΦ s were loaded with a higher number (Fig. 5A) of viable PLDs after 4 h of infection (P < 0.05). Noticeably, at 12 h there was a decrease in fluorescence intensity of CFSE labeled PLD, which indicates enhanced parasite killing (P < 0.05) as compared to untreated MΦ s (Fig. 5A).
However, since the intracellular pathogen Leishmania resides in the phagosome, it significantly hinders fusion of the phagosome with lysosome 15 . So, our next effort was to delineate the extent of phagosome-lysosome fusion in ALS treated MΦ s. We found higher co-localization (P < 0.05) between CFSE labeled PLD and lysosome ( Fig. 5B) which indicates increased phago-lysosome biogenesis, whereas Fusion of PLD and lysosome remained unaltered in untreated MΦ s throughout the time point. Involvement of ROS and NO in inducing phagosome maturation is well established 30 . Treatment of NAC and NMMA in ALS pre-treated MΦ s were shown to inhibit ROS and NO generation respectively ( Supplementary Fig. S1). In our system inhibition of ROS generation was found to prevent phagosome maturation (Fig. 5C), rather than does by inhibition of NO generation (P < 0.05). This is particularly striking because it was reported that NO plays a major role in overcoming the blockade of phagosome maturation by rupturing the peri-phagosomal actin formed in the periphery of Leishmania loaded phagosomes 30 .
Rab5 is a well-known early endosomal marker 31 . We found that Rab5 increasingly dissociated from CFSE labeled parasites (Fig. 6A) (P < 0.05), indicating a loss of early markers of phagosome. It is established that Rab7 and Lamp1 are the two key markers of late phagosomal activity 32,33 . Though previous report suggested that Rab7 recruitment is delayed in Leishmania loaded phagosome 34 , in our study both Rab7 and LAMP1 molecules showed higher co-localization with CFSE labeled PLD after 12 h of infection in ALS primed MΦ s (Fig. 6B,C) (P < 0.05). Our previous results showed an up-regulation of IL-12, which was reported to induce p38 dependent Rab7 expression in MΦ s 35 and that may be the reason for greater Rab7 accumulation in PLD loaded phagosomes of ALS pre-treated MΦ s. Mature Cathepsin D is also a known non-oxidative marker for late phagosome 36 and Rab9 is recognized for its function related to recycling of the endosome that has already undergone phago-lysosomal maturation process; thus these molecules also serve as markers of late phagosomal activity 37 . We found that there is increased co-localization of CFSE labeled parasites with both these molecules (Fig. 6D,E) (P < 0.05), which further validated enhanced maturation of parasite loaded phagosomes that serve as the prerequisite for killing an intracellular pathogen. Another key marker of maturing phagosome is V-ATPase, which serves to acidify the lumen of the endosome 38 . In our study we observed increased co-localization of V-ATPase and CFSE labeled PLD in ALS pre-treated MΦ s as compared to their untreated counterparts (Supplementary Fig. S2). From these data,  Both V-ATPase and cathepsin D were reported to be excluded from the phagosome by Leishmania through manipulation of synaptotagmin-V dependent procedure 39 . In our study we observed that inhibition of only cathepsin D using pepstatin A did show some degree of inhibition in phago-lysosome fusion in ALS pre-treated MΦ s, whereas inhibition of V-ATPase by bafilomycin A1 significantly hampered the process (Fig. 7A) (P < 0.05). This indicated that induction of acidification of phagosomes loaded with PLD is much critical for phago-lysosomal biogenesis in ALS pre-treated MΦ s. p38 MAPK regulates acidification and maturation of PLD loaded phagosome. Interestingly, p38MAPK which induced pro-inflammatory molecules in ALS pre-treated MΦ s is also one of the key molecules that participate in phagosomal maturation 40,41 and attenuate Leishmania growth within the host MΦ s 40 . In order to ascertain the role of p38 MAPK in phago-lysosome fusion in our system, we used inhibitors of MAPK. While inhibition of JNK and p44/42 MAPK could not halt the fusion of Leishmania loaded phagosomes with lysosome, use of a p38 MAPK inhibitor almost fully hindered the phagosome maturation and acidification process (Fig. 7B) (P < 0.05). Moreover, inhibition of p38MAPK also significantly decreased the leishmanicidal activity (P < 0.05) of ALS pre-exposed MΦ s (Fig. 7C). Hence, p38 MAPK can be considered as the key signaling molecule, which induces leishmanicidal activity via modulation of phago-lysosomal biogenesis in ALS pre-treated MΦ s.

Discussion
Leishmania has an extraordinary propensity to survive in the hostile environment of the host and suppress the host defense system 9,30 . But the attenuated version of this parasite was reported to boost TNF-α and NO generation in murine MΦ s 14 . In line with this, our present study has described Th1 cytokine bias along with respiratory burst in ALS pre-exposed MΦ s. For the first time, this study demonstrates that ALS pre-exposure surprisingly induce phagosome maturation in PLD infected MΦ s. Augmentation of both NO and ROS have encouraged us to speculate about its leishmanicidal activity because it is well known that NO and ROS combine with peroxynitrite to produce the strongest microbicidal weapon in defense against Leishmania 42 . Moreover, it was also observed that the already internalized PLD failed to limit the uptake of further PLD in ALS treated MΦ s which is important for overall fitness for the survival of the parasites.
In harmony with superoxide generation, there was clear enrichment of IL-12, IL-1β , and TNF-α in MΦ s after ALS treatment. One school of thought emphasizes that the promotion of Th1 over Th2 response in Leishmania infection obstructs disease progression 43 . Thus we hypothesize that ALS induced Th1 bias might inhibit survival of Leishmania in MΦ s. On the way to explore the molecular events which dictate the skewing of MΦ function to Th1 bias, we observed enhanced expression and nuclear accumulation of NF-κBp65 and c-jun in ALS treated MΦ s whereas NF-κB and AP-1 (a heterodimer of fos and jun or homodimer of jun) activity is hindered in Leishmania infected MΦ s 29 . In addition to this, there was decreased nuclear translocation of p50 which might be relevant in this context as because trans-activation capability of p65 is reduced during co-expression with p50, which has the competitive DNA binding activity with p65 27 .
Establishment of infection occurs due to the differential production of the counter regulatory cytokines IL-10 and IL-12 which are controlled by p44/42 MAPK and p38 MAPK respectively 12 . Though both these MAPK were up-regulated in ALS treated MΦ s, the increase in IL-12 production was much higher than that of IL-10. Surprisingly, the up-regulation of p-p44/42 MAPK which is responsible for IL-10 production was significantly inhibited when ALS pre-treated MΦ s were further challenged with PLD. On the other hand, the p-p38 MAPK, which is responsible for IL-12 production remained up-regulated in ALS pre-treated MΦ s even after PLD infection.
Heightened phosphorylation of p38 which is also effective in attenuating Leishmania virulence 40 substantiates the speculation of enhanced phagosomal maturation 40,41 in these macrophages. Moreover, Pro-inflammatory mediators have been reported to trigger phagosomal maturation process 30,44 ; whereas anti-inflammatory cytokines tend to dampen the process 45 . Based on these evidence we were first keen to find if these activated MΦ s are equipped with leishmanicidal activity. We ascertained that infection of viable PLD in ALS pre-exposed MΦ s leads to a collapse in a number of viable parasites with time which points towards enhanced leishmanicidal activity. Next, we want to monitor the phago-lysosome fusion which might be an interesting target to reduce the establishment of infection 46 . We observed significant interaction of CFSE labeled PLD with lysosome, and increased phagosomal acidification in ALS treated MΦ s. It is now well established that phagosome maturation continues with a loss of early endosome marker viz. Rab5 and subsequent acquisition of late endocytic marker viz. Rab7 31,47 . MΦ s with active mutant of Rab5 expression are shown to be not only 5-10% less efficient in killing Leishmania but also to restrict complete fusion of the phagosome with endosome 48 . In our study, loss of Rab5 positive phagosome containing PLD indicated the beginning of phagosome maturation in ALS pre-treated MΦ s. Simultaneously, Rab7 and LAMP1 showed increased co-localization with CFSE labeled PLD in ALS pre-exposed MΦ s which further emphasized phagosome maturation. In ALS treated MΦ s, Rab9 showed higher co-localization with CFSE labeled parasites, which is important because Rab9 is also involved in lysosomal trafficking of ingested particles 49 .
Acidification of phagosome is the most important readout because cathepsins become active only when phagosome attains sufficiently low pH 50 . V-type ATPases, which are central to the fall of pH, are kept aside from the Leishmania containing phagosome for up to 24 h 38,39 . Phagosome containing amastigotes that are devoid of LPG acquire several markers of late phagosome or phago-lysosome 39,51 . But unlike amastigotes, promastigotes cannot withstand at low pH and thus phagosome maturation is delayed which offers a timeframe during which they can differentiate to amastigotes. It is known that PLD containing phagosome has a pH of 5.5 which may rise up to 5.8 and exacerbate the spread of infection 52 . This is a much higher pH that makes PLD containing phagosomes incapable of acquiring active cathepsin D 51 . It has been reported that both V-type ATPase and cathepsin D were excluded from PLD loaded phagosome 39,51 . But in our study, CFSE labeled parasites were shown to co-localize with cathepsin D in ALS pre-treated MΦ s, a non-oxidative marker of phagosome maturation. Our results also provide the insight that probably the fall of pH is the key regulating factor for promastigote growth inhibition in ALS pre-treated MΦ s as because Inhibition of V-ATPase by bafilomycin A1 was found to have a major impact on the phagosome maturation as compared to pepstatin A, an inhibitor of cathepsin D. So, it may be postulated that V-ATPase is more important than cathepsin D in phagosome maturation process of Leishmania loaded ALS pre-treated MΦ s.
It is well established that IL-12 is responsible for up-regulation of Rab7 in a p38 MAPK dependent manner 35 ; LAMP-1 expression is also reported to be up-regulated by IL-12 stimulation in NK cells 53 . It has been proved that supplying IL-12 to MΦ s infected with M. tuberculosis enhances phago-lysosomal biogenesis and acidification of phagosomal lumen 44 . Even p38 MAPK was involved in phosphorylation of Rab5 GDI to enhance endosome maturation 54 . So we hypothesize that IL-12 secreted by ALS pre-treated MΦ s may work in an autocrine manner to induce p38 MAPK, which can modulate the phagosomal event. Results of our experiments using MAPK Scientific RepoRts | 6:22335 | DOI: 10.1038/srep22335 inhibitors confirmed that p38 MAPK was solely responsible for maturation of PLD loaded phagosomes and its subsequent leishmanicidal activity. Whether heightened IL-12 production paves the way for this maturation process is yet to be determined but the role of p38 MAPK is established in this study. So, it will be fascinating to investigate in future whether participation of autocrine IL-12 alone or TLR2/4 axis during phagocytosis of ALS has any role in the induction of p38 MAPK as well as in phagosome maturation.
Though phagocytosis serves as the pre-requisite to eliminate the invading pathogens, the paradox is that it could also serve as a portal of infection and provide unintended benefits to intracellular pathogens. But it is established in our study that pre-exposure of ALS to MΦ s shows a promising pro-inflammatory attribute essentially by up-regulating p38 MAPK, which makes PLD infected MΦ s capable of driving phagosome maturation. These findings are likely to be beneficial for the short-term prophylactic response during a visit to endemic areas identified for Leishmania infection. Moreover, attenuation of phagosome maturation which is a hallmark of intracellular pathogen infection was surmounted by ALS treatment; which might be effective in combat against other intracellular infections. Parasite treatment. 1 × 10 5 -1 × 10 8 cells/ml ALS was used to treat macrophages. In case of infection of macrophages with PLD promastigotes, a ratio of 1:10 (MΦ s: parasite) was used. Each time MΦ s were incubated with ALS washed, and then either fresh media was added or treated with inhibitors and prepared for respective assays after the requisite time of incubation. To study the phagosome maturation or leishmanicidal activity, these ALS pre-treated MΦ s were subjected either to direct PLD infection or initially treated with inhibitors, then washed and further infected with PLD. Then additional incubation was done in fresh media and prepared for experiments. A schematic representation of the infection and treatment protocol was shown in Supplementary  Fig. S3.

Chemicals
Measurement of iNOS, gp91 phox , intracellular cytokine, NF-κB and c-JUN. For the determination of intracellular cytokines, cells were treated with 10 μg/ml of brefeldin A for 3 h. Then they were washed with PBS, fixed with methanol, perforated by saponin and finally labeled with primary and fluorochrome conjugated secondary antibody.
For iNOS and gp91 phox determination cells were washed with PBS, then fixed with methanol, perforated by saponin solution (0.2%) and labeled with primary and respective fluorochrome conjugated antibodies. For measurement of NF-κB and c-JUN, the above procedures were followed except that Triton-x 100 solution (0.5%) was used for perforation.
Cells were then analyzed in BD FACS Verse or BD FACS LSR Fortessa. Total 10,000 events were acquired and analyzed using the trial version of FLOWJO software. Nitric Oxide measurement. Nitrite level in the culture supernatant was measured using the Nitric Oxide Colorimetric Assay kit that utilizes Griess reaction 29 . Briefly, 1 × 10 6 cells were treated with 1 × 10 5 -1 × 10 8 ALS/ ml for 24 h and the supernatants were used to measure NO using the Griess reagent, NED (0.1% in distilled water) and Sulphanilamide (1% in 5% H 3 PO 4 ). DAF-2 DA was used to study intracellular NO as described elsewhere 56 and observed under flow cytometer. Briefly, macrophages were scrapped and incubated in PBS containing DAF-2DA (7.0 μM) at 37 °C for 30 min and then assayed by BD FACS LSR Fortessa.

Measurement of reactive oxygen species.
To monitor the level of reactive oxygen species, the cell permeable probe DCFDA was used as described elsewhere 57 and observed under Perkin-Elmer LS50B Spectrofluorometer or flow cytometer. Briefly, cells were treated with or without ALS of varying dose for indicated time, then scraped, washed and incubated in PBS containing DCFDA (20 mM) for 30 min at room temperature in the dark. Then fluorescence was measured using spectrofluorometer or flow cytometer.