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. 2026 Sep 18;32:174. doi: 10.1186/s10020-026-01647-6

Dopamine alters macrophage metabolism promoting intracellular Mycobacterium tuberculosis survival

Radheshyam Modanwal 1, Sharmila Talukdar 1, Gaurav Kumar Chaubey 1, Rahul Dilawari 1, Anupam Jain 2, Ashish Arora 2,3, Chaaya I Raje 4, Manoj Raje 1,5,✉
PMCID: PMC13637417  PMID: 42834360

Abstract

Background

Dopamine (DA) is thought to aggravate progress of bacterial infections and enhance risk of intracellular bacterial infection like tuberculosis (TB). Though TB patients are known to present with high dopamine levels the causal link between them has remained unexplored. Mycobacterium tuberculosis (M. tb), the causative agent of TB, requires a copious supply of iron and carbon for survival and proliferation within host cells.

Methods

In vitro and in vivo administration of dopamine induced an increase of non-transferrin bound iron delivery into macrophages along with and lipid accumulation and foamy macrophage formation. The resultant intracellular milieu provided an abundant source of carbon and iron for invading M. tb pathogen. Binding of dopamine to iron was characterized by NMR spectroscopy implicating its role in intracellular trafficking. Using iron sensor M. tb strain, we observed elevated iron levels in intraphagosomal bacteria residing in dopamine treated cells while CFU analysis indicated enhanced M. tb survival and proliferation.

Results

We found that DA modulates macrophage metabolism resulting in creation of iron and lipid nutrient rich intracellular micro-environment favorable for survival of M. tb.

Conclusions

Survival of M. tb bacilli in dopamine treated macrophages was significantly enhanced demonstrating that dopamine leads to creation of an intracellular milieu that provides a conducive and nurturing environment for invading pathogenic bacilli.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s10020-026-01647-6.

Keywords: Dopamine, Mycobacterium tuberculosis, Iron, Lipids, Macrophage, Infection

Introduction

Tuberculosis (TB) caused by Mycobacterium tuberculosis, remains one of the most important infectious diseases worldwide. Despite being preventable and curable, TB continues to cause substantial morbidity and mortality. According to the WHO Global Tuberculosis Report 2025, TB remains one of the world’s deadliest infectious diseases, causing over 1.2 million deaths and affecting an estimated 10.7 million people in the previous year. WHO also reported that 8.3 million people were newly diagnosed and accessed treatment in 2024, representing about 78% of those estimated to have developed TB disease.

Mycobacterium tuberculosis is one of the most successful intracellular pathogens in terms of survival and multiplication within macrophages (Clemens and Horwitz 1995; Schlesinger 1996; Sturgill-Koszycki et al. 1994; Xu et al. 1950). During its intracellular residence it requires a continued and copious supply of iron for survival and multiplication. Studies have established a direct link between M. tb pathogenicity and iron accumulation in human subjects (Gangaidzo et al. 2001). In response to invasion of macrophage by microbes the host cell invokes a nutritional immune response that attempts to restrict the availability of essential nutrients, particularly iron, to the invader. The most well researched mechanism by which this is sought to be accomplished, is the down regulation of the transferrin receptor 1 (TfR1) dependent transferrin mediated iron uptake and increase in iron export (Thom et al. 2012; Wessling-Resnick 2015; Cellier et al. 2007; Zhong et al. 2001). Despite the host cells efforts to deny iron, successful intracellular pathogens like M. tb and some Salmonella species have evolved mechanisms to overcome these defensive maneuvers. These pathogens have developed sophisticated strategies to maintain iron supplies available within the host cell, thereby ensuring their continued survival and replication inside mammalian cells. Recently our group had reported one such counter measure employed by invading M. tb wherein it hijacks the host multifunctional molecule glyceraldehydes-3-phosphate dehydrogenase (GAPDH) to continue the traffic of mammalian iron carrier proteins transferrin and lactoferrin to intraphagosomal M. tb by a TfR1 independent pathway (Patidar et al. 2022). However, protein carrier mediated iron trafficking into cells only comprises a part of the total iron delivery processes. Cellular iron uptake through non-transferrin bound iron via small molecular weight iron carriers is also known to play a significant role especially in conditions of excess iron (Conrad et al. 2000; Kaplan 2002; Donovan et al. 2006; Sohn et al. 2012). Apart from iron, M. tb also requires an abundant source of carbon for its sustenance and replication within host cells. It has been earlier reported that lipid droplet (LD) formation occurs in macrophages upon infection. It is presumed that, as a mechanism for survival in a hostile foreign territory, M. tb specifically induces the formation of these intracellular lipid bodies to serve as a depot of host lipids that would constitute a readily available carbon source (Knight et al. 2018).

Catecholamines are a group of hormones and neurotransmitters, including epinephrine (adrenaline), norepinephrine (noradrenaline), and dopamine, that play a crucial role in the body's response to stress. They are released into the bloodstream in response to stress, causing a cascade of physiological changes. These small molecules have been reported to promote the growth of several species of pathogenic bacteria like Yersinia enterocolitica, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella (Beasley et al. 2011; Coulanges et al. 1997; Dichtl et al. 2019; Freestone et al. 2007). A recent study reported on the effects of catecholamine on enhancing M. tuberculosis biofilm formation (Lei et al. 2023). Classically thought to be associated with the central nervous system dopamine is now recognized to be widely present in most of the other body organs and plasma spanning a wide range in quantity from 10−3 M to 10−9 M (Matt and Gaskill 2020). Apart from neuronal cells DA has also been found to be produced and transported into cells of the immune system especially macrophages (Gaskill et al. 2012; Channer et al. 2023).

Dopamine has a catechol like structure which is thought to function in a manner like siderophores that sequester and transport iron. Earlier studies have demonstrated that dopamine enhances iron levels in primary cultured astrocytes (Zhang et al. 2013) and causes intracellular iron accumulation via a non-transferrin mediated iron delivery process into cells (Dichtl et al. 2018). Other investigators have reported on the in vivo complexation of dopamine with Fe+3 at physiological pH leading to vesicular uptake of non-transferrin bound iron in neuronal cells (Porter et al. 2010). Dopamine mediated iron accumulation in mice associated with enhanced virulence was observed by Dichtl and co-workers (Dichtl et al. 2019). An earlier study revealed that DA levels are elevated in serum of TB patients (Aa et al. 1992). However there have been very limited investigations that explore the mechanistic role of this stress related hormone in patients diagnosed with active TB (Opolot et al. 2015) and no association has ever been made of dopamine induced macrophage micronutrient accumulation contributing to survival and proliferation of infecting M. tb bacilli. Based on this we proceeded to investigate if DA could alter iron and lipid homeostasis of human macrophages to modulate the intra cellular micro environment in favor of invading microbes. Our studies revealed that dopamine enhances iron availability to intraphagosomal M. tb. We also observed dopamine mediated lipid accumulation in infected cells. As dopamine was found to promote, both iron and lipid accumulation in macrophages our results could provide a possible explanation for the enhanced survival of resident M. tb in dopamine treated host cells.

Methods

Mice

Six to eightweekold C57BL/6 were acquired from ICARE experimental animal facility of CSIR-IMTECH, Chandigarh, India. All experimental protocols were approved by the statutory Institutional Animal Ethics Committee (IAEC) vide project No IAEC/20/01.

Media and cell lines

THP-1 and L929 cells were obtained from National Centre for Cell Sciences (NCCS), Pune, India and ATCC respectively. Cells were cultured in RPMI-1640 supplemented with 10% FCS. Before use, THP-1 monocytes were plated in tissue culture grade plastic wells and activated for 24 h with 25 ng/ml of phorbol 12- myristate 13-acetate (PMA) for differentiating into macrophages. Subsequently cells were rested for a further 24 h before use.

Mouse peritoneal macrophages (PMQ)

Thioglycolate-elicited (TG) peritoneal macrophages were obtained from the C57BL/6 mice as per previously standardized protocols (Chaubey et al. 2023; Chauhan et al. 2019). Briefly, 3–4 ml of autoclaved (TG) thioglycolate Medium Brewer Modified (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was injected intraperitoneally into mice. After 4 days mice were euthanized via cervical dislocation and peritoneal exudate cells were harvested using sterile lavage of the peritoneal cavity using ice cold RPMI media. The cells were centrifuged, cell pellets washed with ice cold media and further re-suspended in complete RPMI media (Media containing 10% FBS, 100 units/ml penicillin + 100 μg/ml streptomycin) (NacalaiTesque). Cells were incubated at 37 °C in a moistened incubator (5% CO2) for 24 h. Subsequently, the non-adherent cells were decanted and adherent macrophages were harvested using PBS-EDTA (2% of 0.5 mm EDTA in 1X PBS) and used for further experiments.

Bone marrow-derived macrophage cells (BMDM)

Bone marrow was isolated from femurs of 6–8 week old C57BL/6 mice. For harvesting cells, the bones were flushed with serum free medium (SFM) using a 26 G needle. Harvested cells were centrifuged at 900 g for 5 min. Erythrocytes present in the cell pellet were lysed by re-suspension in RBC lysis buffer (Dutton et al. 2018) followed by a wash with SFM. Macrophages obtained were then cultured in conditioned RPMI media (RPMI media containing 10% culture supernatant of L929 cells) for seven days at 37 °C, 5% CO2 before use (Trouplin et al. 2013). Subsequently cells were harvested for further experiments.

Dopamine treatment

For in vivo studies C57BL/6 mice were injected intraperitoneally with 5 mg/kg Dopamine (DA-Dopamine hydrochloride sigma H8502-5G) in 200 µl PBS every 12 h for 3 days (Matalka et al. 2011), macrophages were isolated for analysis as described above. For in vitro experiments involving THP-1 derived or mouse primary macrophages in culture, dopamine was dissolved in culture media (DMEM or RPMI) and the mixture used for incubating cells for given time periods as per experimental design described below.

M. tb strains and culture

M. tb H37Ra GFP, M. tb H37Rv and M. tb H37Rv GFP strains were as described previously (Patidar et al. 2022; Boradia et al. 2014). The H37Ra bfrB GFP iron sensor strain which has GFP under the regulation of the bactoferritin (bfr) promoter has been described earlier, the expression of the GFP reporter is enhanced when the cells accumulate iron (Talukdar et al. 2024). All M. tb strains were cultured in Middlebrook’s 7H9 broth supplemented with 0.1% Tween 80, 10% OADC and 0.2% glycerol. Strains harboring fluorescent constructs were maintained in culture media as described above that was supplemented with hygromycin (50 µg/ml). To study growth kinetics of planktonic bacilli in response to DA treatment a growth curve was plotted of M. tb H37Rv. Cells were inoculated in Sauton’s media (Chen et al. 2003) at an OD600nm of 0.025 with 25 μM DA and incubated at 37 °C with continuous shaking for a duration of 15 days with OD600nm being recorded every alternate day.

Infection of macrophages and mice with M. tb

THP-1 derived macrophages and murine macrophages were infected with different M. tb strains (H37 Rv wild type, H37 Rv GFP and with M. tb H37Ra bfrB GFP) using a multiplicity of infection (MOI) of 1:20. Cells in culture plates were permitted to ingest M. tb for 4 h, after which time the non-phagocytosed bacteria were detached by five washes with serum free media (SFM) and the macrophages were cultured for 24 h in complete media, at 37 °C, 5% CO2 (Patidar et al. 2022). Control cells wherein infection with M. tb was omitted (non-infected) were processed in parallel. In vitro experiments involving the reporter strain M. tb H37Ra bfrB GFP included a set of infected THP-1 cells incubated with 100 μM FeCl3 as positive control of cellular iron loading (Sheokand et al. 2014). For in vivo experiments, C57BL/6 mice were treated with DA (5 mg/kg DA in 200 μl PBS every 12 h for 3 days). Control animals received only PBS. At the start of the last 36 h of DA treatment, the animals were inoculated intraperitoneally with M. tb H37Ra bfrB GFP (3 × 108 bacilli in 200ul PBS).

Isolation of intra-phagosomal M. tb

THP-1 derived macrophages were plated (5 × 106 cells/90 mm petri dish) and infected with M. tb H37Ra bfrB GFP as described above and then subjected to different treatments for 24 h. Subsequently, cells were harvested with PBS-EDTA, washed with PBS and processed for isolation of intra-phagosomal bacilli as described earlier (Olakanmi et al. 2004; Chaubey et al. 2026). Briefly, macrophages were lysed in 0.1%SDS with 1,000 units/ml DNase containing SFM and centrifuged at 10,000xg for 10 min to remove lysed cellular debris. Bacteria containing pellets were washed with 0.01% SDS containing SFM. Samples were fixed with 4% PFA at 4 °C for 45 min, washed with PBS and fluorescent signal was analyzed using a Flow Cytometer (BD).

Total cell iron measurement

Macrophages were seeded in 100 mm petri plate and treated with DA for 24 h. Subsequently cells were harvested using PBS-EDTA. Whole cell lysate was prepared by adding iron assay buffer and sonication (30–40% amplitude, 5 s on and 15 s off with total time 45 s). Samples were held on ice for 10 min with intermittent vortexing. After centrifugation (16000xg for 10 min at 4 °C) the clear supernatant was collected and protein concentration determined by BCA method. Total cell iron estimation was performed using a sigma iron estimation kit (# MAK025).

Cellular Labile iron pool (LIP)

Calcein AM (C1359 sigma) used to evaluate the intracellular labile iron levels. Macrophages were harvested and incubated with 200 nm calcein AM in SFM at 37 °C for 20 min. After rinsing 2X with SFM and PBS the cellular fluorescence was evaluated using flow cytometry. In case of bacteria, 2 µm calcein AM was used for staining of bacterial pellets resuspended in PBS at 37 °C for 60 to 90 min (Modanwal et al. 2025).

NMR spectroscopy

For NMR characterization of the dopamine-iron association, a 10 mM solution of dopamine was prepared in 99.96% deuterium oxide D2O (Sigma-Aldrich). Ferric chloride hexa hydrate was from Sigma-Aldrich. A degassed solution of FeCl3 was prepared in D2O and was diluted into dopamine solution to yield a molar ratio of 10:1: dopamine: FeCl3, immediately before the NMR experiments. All NMR experiments were recorded on a Varian 600 MHz Innova spectrometer equipped with a triple nuclear probe, at 25 °C. The proton spectra (1H) were recorded with 16,384 complex points, 16 scans with a spectral width of 12 ppm. The spectrum recording took a total time of 64 s. The carbon spectrum (13C) was recorded with 32,768 complex points with a spectral width of 250.2 ppm with 8192 scans. 13C-1H-HSQC spectra were recorded with 1H and 13C spectral widths of 12 ppm and 200 pm, respectively, with 8 scans, and an acquisition time of 150 ms. 13C-1H-HMBC spectra were recorded with 1H and 13C spectral widths of 12 ppm and 240 pm, respectively, with 16 scans, and an acquisition time of 150 ms. The 1D NMR spectra were processed with TopSpin ver. 4.0.8, while the 2D experiments were processed with NMRPipe (Delaglio et al. 1995). All spectra were referenced to 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TSP). Assignment of the resonances was based on the analysis of 1H, HSQC, and HMBC spectra, and was found to be in agreement with previously published data (Burlamacchi et al. 1969).

Lipid body staining and enumeration by Oil Red O and DID staining

Lipid body accumulation in macrophages was quantified by Oil Red O staining, as described previously (Mahajan et al. 2012). THP-1 derived macrophages and peritoneal macrophages from mice were seeded in 24 well plates and subjected to DA treatment for 24 h. Subsequently cells were fixed with 10% formalin (RT) for 10 min at which time the formalin was discarded and replaced with 2 ml of fresh formalin solution and incubated for 1 h. Cells were stained with 0.5% Oil Red O (sigma #O0625-25G) for 30 min at 25˚C, washed with PBS and DDW (double distilled water) before imaging. Lipid bodies were enumerated by light microscopy using a 10 X objective lens. For quantification of staining, Oil Red O dye was eluted from cells by adding 1 ml of 100% isopropanol and further incubating for 10 min with gentle shaking. Optical density of the eluted solution was measured at 518 nm was measured using 100% isopropanol as blank. For DID staining THP-1 derived and mice peritoneal macrophages were seeded in 24 well plates and treated with DA for 24 h. Subsequently cells were harvested and incubated with DID dye (5ul/ml Vybrant™ Multicolor Cell-Labeling Kit V-22889), in serum free media at 37˚C for 20 min. Then cells were washed 2X wash with SFM and PBS and the cell fluorescence evaluated by flow cytometry.

Western blotting

Cells were harvested using PBS-EDTA (2% of 0.5 mm EDTA) and whole cell lysate prepared by adding RIPA lysis buffer (G bioscience) with 0.5 M PMSF to the samples. Samples were held on ice for 30 min with intermittent vortexing. After centrifugation (12000xg for 10 min at 4˚C) the clear supernatant was collected and protein concentration determined by BCA method. Aliquots were stored at −80 ˚C for further experiments. Proteins were resolved using 10% SDS-PAGE. Separated proteins were transferred onto nitrocellulose membrane and blocked using 2% casein. Blots were probed either with monoclonal anti-ferritin heavy chain antibody (Cell signaling technology CST 3998S) and monoclonal anti-Lox 1(Abcam ab60178) for 2 h at 25˚C or overnight at 4˚C. Primary antibodies were detected with anti-mouse/anti-rat peroxidase antibodies (Invitrogen Goat anti-Rabbit/anti-mouse IgG (H + L) Secondary Antibody HRP respectively # 31,460 and # 62–6520) after multiple washing with PBST (PBS + 0.05% Tween 20) blots were developed using Luminata™ forte HRP substrate (Merck Millipore).

Host gene Expression using Quantitative RT-PCR

Total RNA from host cells was extracted from cells using Trizol reagent. A static amount (1000 ng) of total RNA from each sample was used for cDNA synthesis using cDNA synthesis kit (Thermo Scientific K1622). Total RNA was converted into cDNA using cDNA Synthesis Kit, as per manufacturer’s instruction. RT-PCR was set as per standard conditions and protocol using gene specific primers. The specific gene fold expression was calculated after normalizing with β-actin gene expression level for every set of treatments. The specific forward and reverse primers, designed with Primer-BLAST software, are summarized in Table S1. Data was plotted as mean fold change expression ± SD. Statistical significance and p value calculation was done using unpaired t-test with respect to untreated cells.

Intracellular dopamine staining

THP-1 cells were harvested using PBS-EDTA. Subsequently cells were fixed in 4% paraformaldehyde at 4 °C for 40 min, and permeabilized using saponin buffer (saponin 0.2% with 1%FBS in PBS) at 4 °C for 40 min. Cells were then washed with FACS buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.02% NaN3, and 5% FBS). After washing, cells were blocked with FACS block (FACS buffer supplemented with 5% Normal human serum and 5% Normal goat serum), at 4 °C for 30 min and incubated mouse anti-DA monoclonal antibody (Invitrogen MA1-26,966) for 45 min at 4 °C. Secondary antibody staining was with anti-mouse antibody A633 (Invitrogen # A21063) at 4 °C for 45 min. After a final wash with FACS buffer cellular fluorescence was evaluated by flow cytometry. Fluorescence signal from 104 cells in each FACS tube were analyzed by a Flow Cytometer.

Measurement of intracellular bacterial survival in macrophages

Bacteria were grown as described above. THP-1 and BMDM cells were plated in 12 well plates (3 × 105 cells/well) and infected with M. tb as described above. After 24 h the infected cells were lysed using PBST (0.05% Tween 100 ml of PBS) and repeated syringing through a 26G needle. The released bacilli were re-suspended in 7H9 media and centrifuged. Bacterial pellet was diluted in fresh medium and plated onto Middlebrook 7H11agar plates (Difco). CFU counts were determined after 21 days of incubation at 37˚C (Dhiman et al. 2023).

Statistical investigation

All statistical investigation was executed in experiments using student’s t-test.

Results and Discussion

Dopamine enhances iron in macrophages

To understand the effect of dopamine on of essential micronutrients within macrophages, we first evaluated intracellular iron accumulation in the human macrophage cell line THP-1 and mice peritoneal macrophages that had been incubated with DA. The cellular labile iron pool status was evaluated using calcein quenching assays. Titration of DA while monitoring the cellular labile iron pool revealed a significant increase upon treatment with 250 μM DA (Fig. 1A) and this concentration was utilized in all in vitro experiments. Similar results on enhanced LIP were obtained with PMQ’s treated in vitro (Fig. 1B) and in vivo rodent experiments wherein dopamine was administered to mice and the LIP evaluated in their macrophages (Fig. 1C). We also observed significantly enhanced expression of the intracellular iron storage protein ferritin (FTH1) in DA treated; THP-1 derived macrophages (Fig. 1D) as well as in macrophages obtained from dopamine treated mice (Fig. 1E). As ferritin is an intracellular iron storage protein, it implies that DA increases the iron stored within macrophages. Chemical assay of the total cellular iron (protein bound + labile pool) also confirmed that the iron content of DA treated, THP-1 derived and mouse macrophages was significantly increased (Fig. 1F-G). Dopamine treatment did not enhance the uptake of holo-transferrin by cells (Fig S1A) thus indicating that the observed increase in cellular iron was via a non-transferrin dependent pathway. This was further confirmed with the lack of increase in the cell surface expression of either its classical receptor (TfR1) or the non-canonical transferrin receptor GAPDH (Fig S1B&C).

Fig. 1.

Fig. 1

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Dopamine induces accumulation of iron in macrophages. A-C Cellular labile iron pool evaluated in macrophages using Calcein AM staining and flowcytometry. (A) THP-1 macrophages and (B) mice primary peritoneal macrophages (PMQ). THP-1 and PMQ were treated with different concentrations of dopamine for 24 h in culture. The intracellular labile iron pool is significantly increased upon incubation with 250 µM DA. Data is presented as MFI ± SD (upper panels). Statistical significance was evaluated using unpaired t-test (n = 3 *p < 0.05, **p < 0.01). All experiments were repeated three times and one representative data is presented. Lower panels depict one representative FACS overlay histograms plotted for data from n = 104 cells per histogram using flowjo software. C The labile iron pool is also significantly elevated in PMQ isolated from mice administered DA for 3 days. C57 BL/6 mice were injected i.p with 5 mg/kg DA in 200 ul PBS every 12 h for 3 days. Control represents cells obtained from mice injected with PBS only. Data is presented as MFI ± SD. Statistical significance was done using unpaired t-test (n = 3 to 4 mice per group). Lower panel depicts one representative FACS overlay histograms plotted for data from n = 10.4 cells per histogram. D-E Representative western blots of cell lysates revealed that DA treatment also causes an enhanced expression of the cellular iron storage protein FTH1 (ferritin) in both, (D) THP-1 derived macrophages and (E) PMQ isolated from C57 BL/6 mice injected i.p with 5 mg/kg DA in 200 ul PBS every 12 h in for 3 days. Lower panel depicts quantification by densitometry with normalization using ImageJ software and presented as relative expression of Ferritin/β-Actin ± SD, *p < 0.5, n = 2. F Total cellular iron (measured by colorimetric assay of cell lysates) is significantly elevated in both: 24 h DA treated THP-1 derived macrophages and (G) mice primary PMQ, Data is presented as mean total iron nanomole/µg ± SD (n = 2 and 3 *p < 0.05, ****p < 0.0001)

NMR analysis of dopamine-iron interaction

The proton NMR spectrum of 10 mM dopamine in D2O is shown in Fig S1D.The chemical structure of dopamine with carbon atom numbering is shown in the inset and peak assignment in the S1D spectrum is indicated by the corresponding atom numbers. The protons are given the same numbering as that of the carbon atom to which they are attached. Three peaks are observed in the aromatic region with chemical shifts of 6.76, 6.85 and 6.91 ppm. While in the aliphatic region, two triplets are observed that are centred at 2.88 ppm and 3.23 ppm. The 13C NMR spectrum with peak labelling is shown in Fig S1E. Expansion of peaks centred at 119.3 ppm is shown in the inset. Peaks in the 13C spectrum were assigned on the basis of HSQC and HMBC spectrum. The HSQC and HMBC spectra of 10 mM dopamine in D2O are shown in Fig S1F&G, respectively. All carbon peaks have been unambiguously assigned and the chemical shifts are listed in Table 1.

Table 1.

13C and 1H chemical shifts of dopamine in D2O

S. No Atom numbering 13C Chemical Shift (ppm) 1H Chemical Shift (ppm)
1 1 145.79 -
2 2 146.99 -
3 3 119.35 6.85
4 4 132.06 -
5 5 124.00 6.76
6 6 119.30 6.91
7 7 43.44 3.23
8 8 34.79 2.88
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The reference proton spectrum of dopamine in D2O is shown again in Fig. 2A. Upon addition of FeCl3 solution to dopamine, all NMR peaks were broadened because of the paramagnetic effect of Fe3+, as can be seen from the spectrum shown in Fig. 2B. The colour of the sample changed from clear to dark green. However, upon letting the sample sit for some time, a green precipitate settled down. 1H-NMR spectrum recorded subsequently showed recovery of sharp peaks that approximately corresponded to those of pure dopamine, as shown in Fig. 2C. This is confirmed from the HSQC experiment performed on the dopamine-FeCl3 sample.

Fig. 2.

Fig. 2

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A Proton NMR spectrum of 10 mM dopamine hydrochloride in D2O. B Proton NMR spectrum of 10 mM dopamine hydrochloride in D2O right after the addition of 1 mM FeCl3. C Proton NMR spectrum of 10 mM dopamine hydrochloride and 1 mM FeCl3 in D2O after settling of the precipitate

Dopamine induces lipid accumulation in macrophages

DID dye-based staining coupled with flowcytometry revealed a significantly enhanced lipid content in DA treated THP-1 macrophages as well as mouse PMQ (Fig. 3A). This observation was also confirmed by oil Red O based staining and quantification of eluted dye (Fig. 3B). Microscopy revealed that DA promotes formation of intracellular lipid bodies (Fig. S2A). Finally, when mice were treated with DA, the macrophages obtained from them were also found to have accumulated lipid (Fig. 3C). The expression of LOX-1 a well characterized marker of foamy macrophage formation was observed to be significantly enhanced in DA treated macrophages both at the mRNA as well as protein level (Fig. 3D&E).

Fig. 3.

Fig. 3

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Dopamine induces accumulation of lipid in macrophages. DID (A) and Oil Red O (B) staining reveal significant increase in cellular lipid content. A THP-1 derived macrophages and PMQ (left and right panels respectively). Cells were incubated with DA for 24 h and DID staining was performed. In upper panels data is presented as MFI ± SD. Statistical significance was done using unpaired t-test (***p < 0.001, ****p < 0.0001 n = 3). Lower panels present one representative FACS overlay histogram plotted using flowjo software with n = 104 cells per histogram. Experiments were repeated three times and one representative histogram is presented. B Comparison of cellular lipid content evaluated by Oil red O staining of 24 h DA treated THP-1 derived macrophages and PMQ (left and right panels respectively). The amount of eluted stain was evaluated by measuring OD595nm. Statistical significance was done using unpaired t-test and data presented as mean OD595nm ± SD of eluted dye (**p < 0.01, n = 3). C DID dye staining and flowcytometry of macrophages isolated from dopamine treated mice. C57BL/6 mice were injected i.p with 5 mg/kg DA in 200 ul PBS every 12 h for 3 days. Peritoneal macrophages were isolated and stained with DID dye, Control represents cells obtained from mice injected with PBS only. Data is presented as bar graph of MFI ± SD.**p < 0.001, n = 4 mice (left panel) along with a representative FACS overlay histogram for one pair of mice, n = 10.4 cells (right panel). Dand E Dopamine induced change in LOX-1 expression in THP-1 derived macrophages (D). Data is presented as mean fold change of mRNA expression ± SD. Statistical significance was done using unpaired t-test (**p < 0.01, n = 3). Experiment repeated three times and one representative data is presented. E Western blot of representative samples also reveals increase in LOX-1 protein expression. Lower panel presents results of densitometry with normalization done using ImageJ software and plotted as mean LOX1/β-actin ± SD *p < 0.5, n = 2

Dopamine enhances iron availability for intraphagosomal bacilli in M. tb infected macrophages

As our studies had demonstrated that DA increases iron within macrophage cytoplasm we proceeded to investigate if this would also result in enhanced quantity of this vital strategic micronutrient within resident intracellular M. tb. When dopamine was administered to mice infected with the M. tb Ra bfrB GFP reporter strain (iron sensor strain) we observed a significant increase in the iron dependent GFP signal from resident intraphagosomal bacilli of infected mice (Fig. 4A). In vitro cell culture experiments with DA treated macrophages infected with the reporter strain also revealed that the intracellular bacteria present within DA treated cells had significantly elevated iron levels as compared to non-DA treated control cells. The increase in the reporter signal was comparable to incubation of cells in culture medium supplemented with 100 μM FeCl3 used as a positive control for iron accumulation (Fig. 4B). Dopamine treatment also resulted in a significant increase in intra macrophage iron accumulation as evidenced by the increased expression of iron storage protein FTH1 (ferritin) in infected cells (Fig. 4C). For iron to build up within a cell the export of iron also has to decrease or remain unaltered. We observed that the expression of Nramp1 (Natural resistance-associated macrophage protein 1) in infected cells was significantly decreased (Fig. S2B). We also checked the uptake of apo-transferrin, a mammalian host protein involved in cellular iron export (Sheokand et al. 2016), in infected cells. We observed that DA treated cells, which had been infected with M. tb H37Rv GFP, presented with a significant decrease of apo-transferrin uptake (Fig. S2C).

Fig. 4.

Fig. 4

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Dopamine increases, availability of iron & lipids in M. tb infected macrophages resulting in enhanced survival of invading mycobacteria resident within host macrophages. A and B Fluorescence signal from M. tb H37RabfrGFP bacteria isolated from TB infected (A) mice peritoneal macrophages (in vivo dopamine treated) and (B) THP-1 derived macrophages. Infected in situ, peritoneal macrophages were isolated from mice. The resident intracellular bacilli were isolated and subjected to analysis of fluorescence intensity by flowcytometer. THP-1 cells were infected with M. tb in culture, treated with DA for 24 h and then the intracellular bacteria were isolated for analysis of fluorescence signal. Data is presented as MFI ± SD (left panels). Statistical significance was done using unpaired t-test. One representative FACS overlay histograms (right panels) were plotted using flowjo software where n = 104 cells per histogram. (****p < 0.0001, n = 4 mice per group and **p < 0.01, n = 2 for THP-1 culture experiments). C Intracellular iron accumulation in M. tb H37 Rv infected THP-1 cells is indicated by the significant increase in expression of the iron storage gene ferritin (FTH1). RT-PCR data is presented as mean fold change of mRNA expression ± SD. Statistical significance was done using unpaired t-test (**p < 0.01, ****p < 0.0001, n = 4). D-F Significant accumulation of lipid in M. tb H37Rv GFP or M. tb H37Rv infected macrophages. D Evaluation of cellular lipid content by DID dye staining and flowcytometry of infected PMQ. Fluorescence signal was captured from cells gated + ve for GFP signal in infected cells. Left panel: DID staining data presented as mean MFI ± SD (***p < 0.001, ****p < 0.0001, n = 3). Experiment was repeated three times and one representative data is presented. Right panel: One representative FACS overlay with histograms plotted using flowjo software, n = 104 cells per histogram. E Oil Red O staining and colorimetric assay confirms significant lipid accumulation in infected PMQ, data presented as mean OD595nm ± SD of eluted dye, (*p < 0.05, **p < 0.01, n = 3). F DID dye staining and flowcytometry of infected THP-1 cells demonstrating significant lipid accumulation upon M. tb H37Rv GFP infection. Fluorescence signal was captured from cells gated + ve for GFP signal in infected cells. Left panel: DID staining data presented as MFI ± SD (*p < 0.05, **p < 0.01, ****p < 0.0001, n = 3). Right panel: One representative FACS overlay with histograms plotted using flowjo software, n = 104 cells per histogram. Experiment was repeated three times, and one representative data is presented. G and H Dopamine enhances intracellular survival of virulent M. tb bacilli in macrophages. G THP-1 derived macrophages and (H) BMDM were infected with M. tb H37 Rv. Infected cells were then treated for 24 h with DA. Bacillary load in the cells was enumerated and is presented as mean CFU ± SD (*p < 0.05, **p < 0.01, n = 3). I Growth curve of planktonic M. tb H37Rv in response to DA. Data is presented as mean OD600nm  ± SD. (*p < 0.05, ***p < 0.001, n = 2). J Evaluation of macrophage dopamine in M. tb infected cells. THP-1 derived macrophages were infected with M. tb H37Rv GFP. After 24 h post infection the cellular dopamine was evaluated using immuno-fluorescence staining coupled with flowcytometry based quantitation. Left panel presents bar graph of the dopamine signal in: control, by-stander (cells present in the culture but do not harbor bacilli) and infected cells as MFI ± SD. Statistical significance was done using unpaired t-test (ns not significant, p > 0.05, **p < 0.01, n = 3). In right panel one representative flow-histogram is plotted using flowjo software where n = 104 cells per histogram. Experiment was repeated three times and one representative data is presented

Dopamine promotes lipid accumulation in macrophages infected with M. tb

Next, we evaluated the accumulation of intracellular lipids in DA treated macrophages infected with M. tb. Both, mice PMQ as well as THP-1 derived macrophages that had been infected with M. tb bacilli revealed the occurrence of significant lipid accumulation (Fig. 4 D-F, S2D). Confocal microscopy demonstrated that the lipid droplets were in close association with the infecting bacilli within macrophages (Fig.S2E).

Dopamine promotes survival of M. tb in macrophages

The above results suggest that DA enhances iron and lipid within infected cells which facilitates the provision of essential nutrients to resident bacilli. When we measured bacterial survival in dopamine treated macrophages, we observed a significant increase in bacillary survival (Fig. 4G&H). We also observed that in response to dopamine treatment, planktonic M. tb cells grown in culture have higher iron level (Fig S2F&G) and their growth is also enhanced (Fig. 4I).

Cells harboring intracellular M. tb have more innate dopamine

An analysis of innate macrophage dopamine content within M. tb infected cells in culture, using immune-fluorescence staining coupled with flowcytometry, revealed a significantly higher DA signal from M. tb infected macrophages as compared to control cells or by-stander cells (cells present in the same culture well as M. tb infected cells but not harboring any intracellular pathogen) (Fig. 4J).

In any war the primary maxim for an invader seeking to establish himself in occupied territory is to ensure seizure and accumulation of strategic minerals and other vital resources in a quest to create an environment that is conducive for one’s own sustenance, propagation, war effort and finally to utilize this advantage to launch further assaults leading to overall domination. This dictum applies whether the conflict is between nations or between pathogenic microorganisms and infected host organisms.

Mycobacterium tuberculosis is a highly successful intracellular pathogen. To survive in host cells, it requires a continuous and abundant supply of iron as an essential micronutrient (Tanner and McShane 2017). The importance of iron for M. tb can be gauged from the fact that at least 40 different enzymes encoded within the M. tb genome require iron as a cofactor. Earlier studies have revealed that 7 to 64 μg of iron/g of bacterial cell mass is essential for the proper growth of M. tb within host cells (De Voss et al. 1999). In order to combat tuberculosis associated morbidity, efforts across the globe are focused upon targeting physiological mechanisms that can affect the survival of M. tb in mammalian host. As in case of other pathogenic prokaryotes, iron has a vital role in mycobacterial physiology. Iron is not only required for survival of M. tb but its virulence is also dependent on the availability of this strategic mineral (Ratledge 2004). Iron limitation within the host is a nutritional stress for M. tb during the early and later phase of infection. Post infection with M. tb, there ensues an intense struggle between host and pathogen to withhold or acquire iron respectively. Iron limitation is an important primary host defense strategy to arrest the survival of the aggressor. This is primarily achieved by reducing transferrin mediated iron import and sequestration of intracellular iron into ferritin. Successful pathogens have developed alternative mechanisms so that they continue to forage iron from host sources (Patidar et al. 2022; Talukdar et al. 2024; Ali et al. 2017). In the current study we observed that DA increased iron content of macrophages in culture as well as when administered in vivo. This was reflected in the increase in levels of the iron storage protein ferritin. Earlier it has been demonstrated that mycobacteria can commandeer iron from host ferritin for its own intracellular survival (Dai et al. 2023). Thus, the action of DA in increasing cellular ferritin can result in building up a depot of iron for invading bacilli to exploit.

NMR spectra of dopamine showed characteristic peaks for the aromatic region and the aliphatic dimethylene groups. Upon addition of FeCl3, the 1H NMR spectrum was recorded in a short period of 64 s. In this short period, no event other than iron-dopamine coordination is expected to take place. The paramagnetic relaxation enhancement (PRE), which causes the observed broadening of peaks, is a direct outcome of the iron-dopamine coordination. However, this coordination is very dynamic and because of this, the PRE effect extends over all of the dopamine present in the solution. This further leads to oxidation of either dopamine or iron, or both. Subsequently, the oxidized iron-dopamine complex precipitates out of the solution. After all of the iron has been precipitated, further chemical transformation of the remaining dopamine is curtailed. Characterization of binding of iron to dopamine by NMR spectroscopy has been reported earlier by Burlamacchi et al. (Burlamacchi et al. 1969). Their study examined the effects of iron on the nuclear spin–lattice relaxation rate (PRR) of dopamine in aqueous solution at a molar ratio of 200:1. It was reported that the effect of iron on the PRR of C1 and C2 carbons was much stronger than C3, C6 carbons, followed by C4, C5 carbons. Further, in this work, EPR studies were also performed. No EPR spectrum attributable to free Fe3+ ions were observed by them, based on which it was concluded that iron was totally bound to dopamine. In our study, a much lower molar ratio of 10 (dopamine: iron) has been used. Due to the lower molar ratio, the formation of dopamine-iron complex and its precipitation has been more clearly characterized by NMR spectroscopy. In addition, in our study, complete and unambiguous assignment of peaks in the carbon spectrum of dopamine was achieved. At low molar ratios, partially chelated iron forms redox-active dopamine–iron complexes that generate reactive oxygen species (ROS) via Fenton chemistry, alongside dopamine oxidation to toxic quinones. This dual oxidative and electrophilic stress is particularly harmful in infected or inflamed cells. It is well established that levels of free or labile iron increase during infection, a trend we also observed in our study. Additionally, we demonstrated an elevation in innate dopamine levels. Together, these findings suggest that the presence of innate dopamine–iron complexes during infection creates a more inimical environment for the host, while potentially offering a survival advantage to invading bacteria.

To accumulate and have available more of this strategic mineral within the cell it is also necessary to decrease its outflow. Nramp1 is an antimicrobial peptide which exports iron from phagosomes to cytosol. It has been known for limiting intraphagosomal iron availability during infection (Wessling-Resnick 2015). We found that DA caused a diminution of Nramp1 expression along with decreased uptake of the iron clearing protein apo-transferrin (Sheokand et al. 2016). The combined effect of these two phenomena (increased iron uptake + decreased iron exodus) results in enriching intracellular iron. This leaves more of iron to remain trapped within cells and available for use by the invading bacilli in M. tb infected cells. These results were confirmed using an in vivo mouse model. DA caused an increase of both; the labile as well as stored iron pools in the macrophages thus providing an iron rich intracellular microenvironment from where this strategic mineral would be accessible to invading pathogen (Kotey et al. 2024). Our investigations revealed that DA enhanced iron in intraphagosomal M. tb. as well as planktonic M. tb bacilli. Using a reporter strain we observed that the occurrence of elevated iron levels within intraphagosomal M. tb in dopamine treated THP1- derived macrophages as well as murine peritoneal macrophages.

Recently it has been shown that iron excess in macrophages induces the cells to accumulate lipid droplets and form what are known as ‘foamy macrophages’ (Cai et al. 2020). Macrophage transform into foamy cells due to accumulation of oxidized low-density lipoprotein (LDL) particles and free fatty acids (Russell et al. 2009). LDL particles are modified into oxidized LDL when exposed to elevated levels of labile iron. LDL is endocytosed via LDL receptor (LOX-1 also known as scavenger receptors, a well characterized marker for foamy macrophage formation (Palanisamy et al. 2012). We found dopamine induced lipid accumulation within macrophages with the cells also demonstrating a significantly enhanced expression of LOX-1. Our results suggest that LOX-1 may be a potential contributor to dopamine induced lipid accumulation, although its direct mechanistic involvement remains to be established in future studies. Dopamine induced lipid accumulation was also observed to occur within M. tb infected cells. Lipid laden macrophages provide a favorable niche for; bacillary replication, survival and escape from anti-TB drugs. Lipid droplet formation is also known to transpire in macrophages infected with M. tb. as a survival response aimed at creating a depot of carbon source to fuel their intracellular residence (Knight et al. 2018). M. tb containing phagosomes are also known to occur in close association with lipid bodies and bacilli can also display intracellular lipid inclusions suggesting that they access and utilize these host cell accumulated lipids as a nutrient.

Finally, we confirmed that DA enhanced the survival of M. tb within macrophages. Dopamine treated macrophages had more bacillary load in comparison to non-DA treated cells. In our studies we also observed that DA enhanced iron uptake in planktonic bacilli resulting in a faster growth compared to non-DA treated M. tb bacilli. However, because M. tb iron acquisition mutants were not tested under planktonic conditions, the DA associated iron uptake pathway remains unknown. Several reports suggest that dopamine levels are elevated in TB patients (Aa et al. 1992; Opolot et al. 2015). This has been postulated to be part of a stress induced secretory response at the level of the CNS (central nervous system) that affects body homeostasis and is geared towards influencing the course of any disease (Padgett and Glaser 2003; Kloet et al. 2005). In our present work we also found that M. tb infected macrophage cells produce higher amounts of dopamine. This could be due to reprogramming of the cell by invading pathogen so as to create a nutrient rich niche for its own survival in an otherwise alien hostile environment.

In case of Mycobacterium, there is only one prior study that has sought to explore a link between DA and survival of M.tb. This involved mutating the dopamine β-hydroxylase enzyme in mice leading to DA accumulation however the authors attributed the enhanced survival of the pathogen to be due to impaired T cell function (Alaniz et al. 1999). Our current studies provide a new insight into this phenomenon.

We surmise that DA enhances a non-transferrin mediated iron delivery into macrophages and intraphagosomal bacilli. This leads to creation of a micro environment within macrophages that is enriched with iron and lipids, both strategic materials vital for survival and replication of invading mycobacteria. While the accumulation of iron and lipids within infected macrophages is favorable for survival and proliferation of the pathogen, it is also deleterious for the host cell survival. Iron is a critical catalyst in production of reactive oxygen species (ROS) and an excess can result the overproduction of ROS, leading to oxidative stress, cell death and tissue damage (He et al. 2019). For a patient of tuberculosis, the most unfavorable progression of the ailment beyond infection and proliferation of bacilli within host macrophages is the manifestation of active tuberculosis where pathogen containment within host cell is lost. This results in the bacilli spreading rapidly from cellto cell within tissue and between organs leading to a case of disseminated disease. Previous studies have implicated necrotic death of M. tb infected macrophages as a major factor influencing this process. Necrosis is a mechanism used by bacteria to exit macrophage, evade the host defenses, and disseminate (Lerner et al. 2017). Recently, an additional pathway of regulated necrosis, referred to as ferroptosis, has been described that, interestingly, is triggered by iron overload. The dependence of ferroptosis on iron is highly relevant to M. tb infection, which has been shown to be influenced by the availability of this bioactive metal. Thus, increased iron levels have been reported to be associated with elevated risk of active pulmonary TB in patients (Amaral et al. 2019). In the regulated cell death associated with ferroptosis, iron overload triggers excessive ROS generation and lipid peroxidation resulting in plasma membrane disruption (Conrad et al. 2018).

Conclusions

In summary, our observations indicate that dopamine treatment favors the survival and growth of intracellular bacilli by trapping intracellular iron as well as promoting the accumulation of lipids in infected macrophages. This favors the initial survival and multiplication of intracellular M. tb in macrophages. The cellular iron loading coupled with lipid accumulation could subsequently trigger a necrotic cell death that aids in the dissemination of the pathogen.

Dopamine is routinely prescribed for the treatment of clinical depression. Studies exploring the co-morbidity of TB and depression have revealed a; higher rate of treatment failure, development of antimicrobial resistance and higher mortality rate (Cáceres et al. 2022). In case of TB infection, the intracellular environment is further skewed in favor of the invader due to increase in dopamine production by infected macrophages. These observations indicate the potential hazards of dopamine administration in patients of tuberculosis and suggest a possibility for anti-tuberculosis therapy that involves inhibition of bacterial micronutrient acquisition vital for its survival and replication within host.

Future studies could concentrate on elucidating the direct mechanistic interplay between DA and LOX-1 during infection and the association between dopamine and ferroptosis.

Supplementary Information

Supplementary Material 1. (2.4MB, doc)

Acknowledgements

Mr. Anil Theophilus & Mr. Randeep Sharma are acknowledged for technical assistance. Financial support of ICMR by way of Emeritus fellowship to MR is acknowledged. This is IMTECH communication No.052/2023.

Authors’ contributions

Chaaya Iyengar Raje and Manoj Raje conceptualized and planned the overall research work and finalized the manuscript. Radheshyam Modanwal planned most of the experiments, collected the data, performed, analysis and wrote the initial draft of paper. Gaurav Kumar Chaubey, Rahul Dilawari and Sharmila Talukdar collected and performed analysis of some of the data, maintained the cell lines, assisted in tissue culture and flow cytometry experiments. Anupam Jain and Ashish Arora performed, analysed and compiled the NMR experiments.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

Chaaya Iyengar Raje has patent Expression vector for constitutive and regulated expression of 8X-histidine tagged Mycobacterium tuberculosis (M. tb) proteins, Indian Patent application no. 202411063076 dated 21.08.2024 pending to Chaaya Iyengar, Vishant Boradia, Ajay Kumar, National Institute of Pharmaceutical Education and Research (NIPER). All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (2.4MB, doc)

Data Availability Statement

No datasets were generated or analysed during the current study.

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