Lipopolysaccharide (LPS) from Escherichia coli O111:B4, lactic acid (LA), sodium lactate (NaL), L-arginine hydrochloride (Arg-HCl), and L-arginine methyl ester dihydrochloride (Arg-ME) were purchased from Sigma Aldrich (Taufkirchen, Germany), whereas interferon γ (IFN-γ) and 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino] diazen-1-ium-1,2-iolate (DETA-NO) were obtained from Invitrogen (Darmstadt, Germany) and Cayman Chemical (Ann Harbor, MI), respectively. Hydrochloric acid (HCl) was purchased from Fisher Chemical (Schwerte, Germany). RPMI 1640, DMEM and PBS were purchased from Gibco (Darmstadt, Germany). Immunoblotting was carried out using the following antibodies: rabbit anti-Actin (A2066; Sigma Aldrich), mouse anti-HSP90α/β (sc-7947; Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-NOS2 (ADI-KAS-NO001; Enzo Life Sciences, Lörrach, Germany) and mouse anti-Arginase 1 (sc-166920; Santa Cruz). Either swine anti-rabbit HRP (P0399, Dako, Hamburg, Germany) or goat anti-mouse HRP (P0447, Agilent) were used as secondary antibodies.

L. major promastigote strain MHOM/IL/81/FEBNI was propagated in RPMI 1640 (10% fetal calf serum) on Novy-MacNeal-Nicolle blood agar slants for a maximum of five passages and used as described earlier (31, 32). L. major promastigotes were collected from blood agar slants. After washing with phosphate-buffered saline (PBS), 3 x 10⁶ parasites were used for infection. For in vitro infection of bone marrow-derived macrophages (BMDM), L. major were propagated in Schneider’s insect medium (Sigma Aldrich) for a maximum of five passages, washed with PBS and resuspended in RPMI microscopically 1640 complete medium.

BMDM were generated from wildtype C57BL/6NCrl (Charles River Breeding Laboratories, Sulzfeld, Germany) as described earlier (33). Briefly, BMDM were harvested from Teflon bags (FT FEP 100 C; Dupont; purchased via APSOparts, Fellbach, Germany) and infected with L. major promastigotes with a multiplicity of infection of 30 for 4 h in RPMI 1640 medium, as described earlier (32). Thereafter, cells were washed with PBS and Leishmania-infected BMDM were costimulated with 20 ng/mL LPS/IFN-γ each (unless indicated otherwise) in the presence or absence of 10 mM LA in RPMI 1640 medium for indicated period of time. After 72 h, infected BMDM were stained with Diff-Quik (Eberhard Lehmann, Berlin, Germany) and analyzed for determination of the percentage of infected cells. Per high power field up to 34 cells were counted.

Preparation of cell lysates, extraction of proteins, and immunoblotting were carried out as described earlier (32, 34). Proteins were separated on TRIS-glycine gels (7.5% for NOS2 and 12% for Arginase 1) and transferred to PVDF membranes. Staining with appropriate primary and secondary antibodies was followed by signal visualization using the Chemo Star Imager (Intas Science Imaging Instruments, Göttingen, Germany). Densitometry of signals was done using ImageJ (Version 1.52a; Rasband, W., ImageJ, National Institutes of Health, USA, https://imagej.nih.gov/ij/).

Accumulation of nitrite in cell supernatants was quantified by the Griess reaction, as described earlier (32). In brief, supernatants of infected and/or stimulated BMDM were mixed with equal amounts of Griess reagent 1 (1% sulfonamide in 5% phosphorous acid) and Griess reagent 2 (0.1% N-1-naphtyletylenediamine in H₂O). Sodium nitrite (Sigma) was used as standard. Absorbance was recorded at 540 nm using an iMark™ microplate absorbance reader (Bio-Rad, Feldkirchen, Germany).

As described earlier (32), total RNA was extracted from stimulated cells after 24 h with TriFAST reagent (VWR International, Ismaning, Germany) and subjected to reverse transcription (high-capacity cDNA reverse transcription kit, Applied Biosystems, Darmstadt, Germany). Quantitative real-time PCR was performed on ABI Prism 7900 sequence detector (Applied Biosystems) using FastSTart Universal Probe Master (Rox) (Roche Diagnostics, Mannheim, Germany) and the following TaqMan probes: Hypoxanthine phosphoribosyltransferase 1 (Hprt1; Mm03024075_m1), Nos2 (Mm00440502_m1), and Arg1 (Mm00475988_m1). These probes were purchased from Applied Biosystems. The ΔΔcCT method was used for quantification. The ratio of target mRNA to control Hprt1 in non-stimulated (ns) specimen was set to 1.

NADPH/NADP⁺-ratio was determined according to manufacturer’s instruction using the NADP/NADPH-Glo™ Assay (Promega GmbH, Walldorf, Germany) which allows for quantification of NADP/NADPH using a single-reagent. Briefly, BMDM were costimulated with LPS/IFNγ in absence or presence of lactic acid for 24 h and then mixed with NADP/NADPH-Glo™ reagent which quantifies NADP/NADPH in a single-step. Cells were incubated for 30 min at room temperature and luminescence was recorded on a Viktor3™ multilabel plate reader (PerkinElmer, Rodgau, Germany).

BMDM were washed three times with PBS to remove remaining traces of cell culture medium. Then, 80% of cold aqueous methanol was added and samples were immediately frozen at −80°C. As described earlier for amino acid analysis (35), samples were thawed and 10 µL of an internal standard mix containing uniformly ¹³C- and ¹⁵N-labeled amino acids (MSK-CAA-1, Euriso-Top GmbH, Saarbrücken, Germany) and deuterated ornithine and deuterated hippuric acid were added. The samples were vortexed, and centrifuged at 9,560 x g for 5 min at 4°C. The supernatants were collected, followed by addition of 200 µL 80% methanol, vortexing and centrifugation to wash the pellets. The wash steps were repeated, but the samples were centrifuged at a higher speed (13,800 g). All supernatants were combined and dried in a vacuum evaporator (CombiDancer, Hettich AG, Bach, Switzerland) followed by reconstitution in 100 μL pure water. Amino acid analysis by HPLC-ESI-MS/MS (HPLC 1200 (Agilent, Waldbronn, Germany) with API 4000 QTRAP (AB SCIEX, Darmstadt, Germany) or ExionLC AD with Triple Quad 6500+, AB SCIEX) after derivatization with propyl chloroformate/propanol was performed using a 10 µL aliquot of the sample extract as described (36).

Intracellular amino acid amounts were normalized to total protein content, which was determined using an assay based on the fluorescent dye SERVA Purple (Serva, Heidelberg, Germany) as recently described (37). The relative arginine content was calculated in relation to the mean value of the LPS/IFN-γ costimulated cells of the respective experiment.

For determination of recombinant NOS2 enzyme activity, 2 units of recombinant enzyme (Biomol, Hamburg, Germany) were incubated in appropriate buffer following the manufacturer’s protocol. In short, 50 mM HEPES (Carl Roth, Karlsruhe, Germany) and 1 mM magnesium acetate (Sigma Aldrich) were adjusted to pH 7.4 or 6.0 by addition of hydrochloric acid (Carl Roth). Prior to buffer use, 0.15 mM NADPH (Sigma Aldrich), 4.5 µM oxyhemoglobin (Sigma Aldrich), 18 µM tetrahydrobiopterin (Sigma Aldrich), and 180 µM dithiothreitol (Sigma Aldrich) were freshly added. 1 mM Arg-HCl (Sigma Aldrich) was also freshly added where indicated. After incubation at 37°C, nitrite accumulation was quantified at distinct time points between 1 h and 8.5 h using the Griess assay (32).

pH levels in culture medium were measured non-invasively by using the PreSens technology (PreSens Precision Sensing GmbH, Regensburg, Germany), as described earlier (38). 0.5x10⁶ BMDM were seeded in 24-well Hydrodish HD24 plates in 1 mL RPMI 1640 medium under cell culture conditions for the indicated period of time. pH levels were continuously monitored using the SDR SensorDish^® Reader. Data were analyzed with SDR_v38 software package (Presens Precision Sensing GmbH, Regensburg, Germany).

Essentially, quantification of intracellular pH in BMDM followed an earlier published protocol (39). BMDM were incubated with indicated treatments for 24 h in RPMI 1640 medium. Afterwards, cells were loaded with 10 µM carboxy SNARF-1 AM acetate (Thermo Fisher Scientific, #C1272) in 1 mL Hank’s balanced salt solution (HBSS, containing 2 g/L NaHCO3) for 30 min and subsequently incubated with or without 10 mM lactic acid for 10 min in the presence or absence of increasing concentrations of Arg-HCl. For calibration curves, an intracellular pH calibration buffer kit (Thermo Fisher Scientific, #P35379) was used. In brief, BMDM were incubated with a mix of pH-controlled buffers and valinomycin/nigericin according to the manufacturer’s instructions. Intracellular pH was assessed by flow cytometry. In detail, pH-dependent spectral shifts of SNARF-1 were recorded and the ratios of the emission wavelength at λ1, transmitted by a 585/42 BP filter, to the wavelength at λ2, transmitted by a 670 LP filter were calculated, as described earlier (39). Treatment induced changes were delineated from the calibration curve, performed for each experiment. FlowJo software version 10.7.1 was used for data analysis.

All animal experiments followed a protocol that had been approved by the Animal Welfare Committee of the local governmental authority (Regierung von Unterfranken, Würzburg, Germany). Mice were infected subcutaneously with 3 x 10⁶ stationary-phase L. major promastigotes (of low in vitro passage [≤ 5] in the right hind footpad in 50 µL PBS), as described earlier (32). 14 days post infection, mice were sacrificed and, immediately thereafter, footpad pH was determined by a micro fiber optic pH meter with needle-type housed pH microsensors (20/0.4) using a manual micromanipulator (PreSens Precision Sensing GmbH) essentially as described earlier (38). After overnight calibration according to the manufacturer’s protocol, the microsensor was inserted 3 - 4 mm into the footpad and pH values were recorded over a period of 1 - 3 min after insertion of the pH sensor with the software pH1-View (PreSens Precision Sensing GmbH).

Data is expressed as mean ± SEM (unless indicated otherwise). Statistical analysis was carried out using Prism v6.0 or v8.0 software (GraphPad). Outliers were identified using the ROUT test ( Figures 4A, D ). Comparing two groups, unpaired Student’s t-test ± Welch correction (if unequal variances were detected by the F-Test) was applied for datasets where the Kolmogorov-Smirnow test indicated normal distribution. Otherwise, the Mann-Whitney test was used. Multiple groups were tested either with Kruskal-Wallis test in combination with Dunn multiple-comparison test for not normally distributed data points or ANOVA followed by Bonferroni’s multiple-comparison test for normally distributed data. Unless indicated otherwise, p-values <0.05 were considered as significant.

In our experimental setup, C57BL/6 mice usually develop a clear clinical lesion at day 14 after infection. At this point, the cutaneous lesion has reached its maximum or it barely enlarges thereafter, before it heals over several weeks (32, 40). Lesional pH was determined 14 days after infection with L. major in cutaneous lesions and contralateral uninfected healthy skin tissue. The pH of uninfected tissue was 7.1, while the pH of Leishmania-infected lesions dropped to 6.7, indicating that lesional pH is acidic ( Figure 1A ).

In vitro, addition of 10 mM lactic acid to LPS/IFN-γ-costimulated macrophages resulted in an immediate steep decrease in extracellular pH. Compared with LPS/IFN-γ-costimulated macrophages, pH remained lower in cells additionally treated with lactic acid throughout the 72 hour observation period. At 24 hours after the addition of lactic acid, this simulated quite well the situation in vivo. ( Figure 1B ).

Macrophages play a critical role in fighting Leishmania [reviewed in: (20, 41)]. Therefore, we tested whether acidification of the extracellular microenvironment would influence the ability of LPS/IFN-γ-cotreated macrophages to produce leishmanicidal NO and to ward off Leishmania. Addition of 10 mM lactic acid to LPS/IFN-γ-costimulated macrophages curtailed their ability to produce NO ( Figure 2A ). Its sodium salt [sodium lactate (NaL)], in contrast, did not impact the release of NO from macrophages ( Figure 2B ). Extracellular acidification with hydrochloric acid (HCl) also impaired NO production of LPS/IFN-γ-coactivated macrophages ( Figure 2C ). Decreased NO release under acidic conditions was accompanied by a reduced ability of the macrophages to clear Leishmania ( Figures 2D, E ). Addition of NO donor 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino] diazen-1-ium-1,2-iolate (DETA-NO) restored not only NO levels ( Figure 2F ) but also leishmanicidal activity ( Figure 2G ) of activated macrophages exposed to 10 mM lactic acid.

Next, we wanted to elucidate the mechanism that underlies the reduced ability of macrophages to produce NO. Production of high-level NO in macrophages hinges on the ability of macrophages to induce the expression of the type 2 NO synthase (NOS2) [reviewed in: (19, 20)]. In contrast to only LPS-stimulated macrophages (28, 30), addition of lactic acid to LPS/IFN-γ-costimulated macrophages did not interfere with Nos2 expression at both the mRNA ( Figure 3A ) and protein ( Figure 3B ) level. From these findings we concluded that reduced NO production was not caused by impaired Nos2 expression.

Alternatively, addition of lactic acid might affect the availability of L-arginine and NADPH, both of which are required for NO production by NOS2 [reviewed in: (42)]. Extracellular acidification increased the NADPH/NADP⁺-ratio compared to controls suggesting that reduced NO production is not due to lack of NADPH ( Figure 4A ). Acidic conditions can induce the expression of arginases (43) and, thereby, limit the availability of L-arginine. Here, addition of lactic acid to LPS/IFN-γ-costimulated macrophages led to a minor, non-significant increase in Arginase 1 (Arg1) expression at both the mRNA ( Figure 4B ) and protein levels ( Figure 4C ), which nonetheless resulted in a significantly reduced intracellular pool of L-arginine ( Figure 4D ). Next, we tested the impact of L-arginine shortage on NO release from activated macrophages exposed to acidic conditions. For that purpose, we used cell permeable L-arginine methyl ester dihydrochloride (Arg-ME) to replenish the intracellular L-arginine pool ( Figure 4D ). In contrast to Nω-nitro-L-arginine methyl ester hydrochloride [reviewed in: (44)], Arg-ME did not inhibit NO production ( Figure 4E ). Of note, addition of Arg-ME to infected macrophages did not restore NO production by LPS/IFN-γ-costimulated macrophages exposed to acidic conditions ( Figure 4E ). This suggests that the reduced L-arginine availability is not limiting NO production under acidic conditions.

Extracellular acidification can trigger intracellular acidification (45). Therefore, we used the pH-sensitive dye SNARF-1 to monitor intracellular pH levels (46). In line with earlier findings in mouse dendritic cells (47), we found that coactivation of macrophages with LPS/IFN-γ resulted in a drop in intracellular pH ( Figure 5A ). More importantly, exposure of LPS/IFN-γ costimulated macrophages to acidic conditions resulted in a further significant decrease in intracellular pH ( Figures 5A, B ). Acidic pH is known to affect the activity of various enzymes [reviewed in: (48)] including NOS2 (29). Therefore, we tested whether the pH (pH 5.81 ± 0.19) encountered within LPS/IFN-γ-costimulated macrophages upon exposure to lactic acid ( Figure 5A ) was able to inhibit the enzymatic activity of recombinant NOS2 directly. These experiments showed that enzymatic NOS2 activity at pH 6.0 was substantially diminished compared to its activity at pH 7.4 ( Figure 5C ).

Infusion of arginine hydrochloride (Arg-HCl) lowers extracellular pH in rats, but increases intracellular pH levels (49). In line with this, we found that Arg-HCl treatment significantly elevated the intracellular pH levels in LA-exposed macrophages in vitro ( Figure 5D ). This was accompanied by a significant increase in NO production ( Figure 5E ) and leishmanicidal activity ( Figure 5F ) of LPS/IFN-γ-cotreated macrophages exposed to lactic acid. Taken together, these findings demonstrate that normalization of intracellular pH is able to restore NO production and leishmanicidal macrophage activity in acidic microenvironments.