Ga(III) nanoparticles inhibit growth of both Mycobacterium tuberculosis and HIV and release of interleukin-6 (IL-6) and IL-8 in coinfected macrophages

Article abstract of DOI:10.1128/AAC.02505-16

Treatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis. Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis. Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis. Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-β/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections.

Full text of DOI:10.1128/AAC.02505-16

AAC Accepted Manuscript Posted Online 6 February 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.02505-16
Copyright © 2017 Choi et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Ga(III) Nanoparticles Inhibit Growth of Both TB and HIV
and Release of IL-6 and IL-8 in Co-Infected Macrophages
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Seoung-ryoung Choi1, Bradley E. Britigan1,2,3 and Prabagaran Narayanasamy1,*
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1Department of Pathology and Microbiology, 2Department of Internal Medicine, College of
Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198 and 3Research
Service, VA Medical Center-Nebraska Western Iowa, Omaha, Nebraska 68105.
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Corresponding author: p.narayanasamy@unmc.edu
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Abstract: Treatment of individuals co-infected with human immunodeficiency virus-1 (HIV)
and Mycobacterium tuberculosis (M.tb) is challenging due to prolonged treatment requirements,
drug toxicity and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are
one of the natural reservoirs for both HIV and M.tb. Here, the treatment of HIV and M.tb co-
infection was studied by preloading human macrophages with MP-targeted gallium (Ga)
nanoparticles to limit subsequent simultaneous infection with both HIV and M.tb. Ga
nanoparticles provided sustained drug release for 15 days and significantly inhibited the
replication of both HIV and M.tb. Addition of Ga nanoparticles to MP already infected with M.tb
or HIV resulted in a significant decrease in these infections, but was of lesser magnitude than
with nanoparticle preloading of the MP. In addition, HIV/M.tb co-infected macrophages loaded
with Ga nanoparticles showed reduced secretion of IL-6 and IL-8 for up to 15 days after drug
loading. Ga nanoparticles also reduced IL-6 and IL-8 secretion by ionomycin and LPS induced
macrophages, likely by modulating the IKK-β/NF-κB pathway. Delivery of Ga nanoparticles to
macrophages is a potent long acting approach for suppressing HIV/M.tb co-infection of
macrophages in vitro and sets the stage for the development of new therapeutic approaches for
these important infections.
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Human immunodeficiency virus-1 (HIV) and Mycobacterium tuberculosis (M.tb) are two major
infectious agents that cause high mortality worldwide. M.tb, the causative agent of tuberculosis
(TB), is one of the leading causes of death in the world. In 2015, the World Health Organization
(WHO) estimated that 9 million people developed TB and 1.5 million died from it. Of the 9
million people who developed TB, 13% were also HIV positive.(1) Furthermore, HIV-
associated TB infections and death in the world are increasing in frequency.(2)
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Numerous challenges to the treatment of the HIV/TB co-infected patient have emerged. Multi-
drug resistant TB and extensively drug resistant TB strains are a growing problem and often
occur in the setting of co-existent HIV infection.(3) In addition, treatment of TB associated with
HIV requires prolonged multi-drug treatment regimens that interact with some anti-retroviral
drugs, increasing the potential for drug toxicity.(4) Thus, an urgent need exists for simplified,
long acting and effective regimens to treat HIV/TB co-infection.(5)
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In the pathogenesis of M.tb infection, the bacillus invades and multiplies intracellularly within
monocytes and macrophages. The primary initial target of the bacillus is alveolar macrophages.
There the bacilli replicate until their growth is restricted by activation of the macrophages by
interferon-γ and other factors released from T-cells.(6, 7) The infection is contained within the
lungs by forming granulomas comprised of M.tb infected macrophages, dendritic cells and T-
cells.
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After years of dormancy, M.tb can begin to multiply and cause reactivation disease if host
immune function decreases. HIV infection amplifies the risk for developing active TB by
decreasing T cell-mediated immunity, resulting in reactivation of latent M.tb infection. HIV

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infection accelerates the rupture of granules, releasing active M.tb that is transported by
dendritic cells to the lymph nodes.(6)
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Both HIV and M.tb target and replicate in macrophages, in turn weakening human
immunological functions. Intracellular replication of M.tb or HIV eventually leads to cell death
and extracellular release of the pathogen.(8) Maintenance of the active infection within the host
requires the ability of the infecting pathogen to continually establish infection in newly arriving
susceptible cell types. Failure to do so would be expected to result in termination of active
infection.
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Macrophages are located in various tissues and play important roles in immunity by engulfing
pathogens, eliminating apoptotic cells and recycling nutrients. Upon encountering bacteria and
other inflammatory stimuli, macrophages secrete pro-inflammatory cytokines like tumor necrosis
factor (TNF), IL-1, IL-6, IL-8 and IL-12. Some cytokines are potent pyrogens that activate and
recruit other cells to sites of microbial invasion/infection. Forming of granulomas is a pathologic
hallmark of host response to M.tb infection. Several cytokines have been identified in
granulomas from BCG infection. Here, IL-6 is involved in the pathological functioning of M. tb
infection.(9) IL-8 also controls granuloma formation which follows leukocyte influx on M. tb
infection. High levels of IL-8 are observed in M. tb infected human tissue, plasma, pleural fluid
and bronchoalveolar lavage fluid. In addition in in vivo studies, pretreatment of anti-IL-8
inhibited mycobacterial granuloma formation.(10) Cytokines also play a pivotal role in
maintaining granulomas with the aid of CD4+ T cells.(11) However, some cytokines enhance
HIV replication in macrophages.(12)

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To survive in humans and to be pathogenic, M.tb has to be able to acquire critical nutrients.
Among these nutrients is iron (Fe), whose acquisition is required for the survival of
mycobacteria residing in human macrophages.(13) (14, 15) Iron’s ability to undergo redox
cycling between ferrous (Fe2+) and ferric (Fe3+) oxidation states allows it to function as an
electron transporter in many enzymatic systems, including those involved in DNA replication
and cellular energy production.(16) Thus, blocking Fe acquisition by M.tb is a potential way to
reduce the growth of M.tb within macrophages.(17)
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Gallium (Ga) is a trivalent cationic element with many features that are similar to Fe, making it
largely indistinguishable from Fe to many biologic systems. Ga can interact with biologically
important proteins that are involved in Fe metabolism, both interfering with Fe acquisition
mechanisms and the function of Fe-dependent enzymes, including catalases, Fe superoxide
dismutase and ribonucleotide reductase. Insertion of Ga(III) into the active site of these normally
Fe-centered enzymes renders them inactive, as, in contrast to Fe, Ga(III) cannot be reduced to
Ga(II) in biological systems.(18) Ga(NO3)3 is a FDA-approved treatment for hypercalcemia
associated with cancer and concentrates in activated macrophages.(19) We previously
demonstrated that Ga disrupts Fe acquisition by mycobacteria and iron metabolism, leading to
inhibition of the growth of these and other intracellular pathogens.(14, 20-24)
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Development of antimicrobial nanoparticles that target macrophages has great potential
advantages in drug delivery and clinical efficacy in the inhibition of intracellular pathogens. This
strategy was applied to develop a macrophage-targeted nanoparticle to inhibit virus
replication.(25, 26) Nanoparticles targeting MDM containing conventional antiretroviral therapy
have been reported to lengthen the activity and efficacy of the existing therapies against HIV-1
infection with less toxicity.(27, 28)

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In a recent study, we showed that a long acting Ga nanoformulation inhibited the growth of both
HIV and M. smegmatis residing within monocyte-derived macrophages (MDMs) by releasing
Ga(III) over 15 days after the cells were loaded with the drug.(8) No significant cytotoxicity
resulting from these Ga nanoparticles was observed.(8) The results encouraged us to explore the
potential of Ga nanoparticles for the treatment of HIV and M.tb co-infection (Figure 1).
Furthermore, we studied the impact of these Ga nanoparticles on cytokine release by
macrophages infected with M.tb and HIV-1.
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114 Materials and Methods
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Ethics Statement. The human cell samples were purchased from and provided in an anonymized
fashion by University of Nebraska Medical Center (UNMC) Department of Pharmacology and
Experimental Neuroscience cell core facility. Experiments with human samples are performed in
full compliance with the regulations of the National Institutes of Health. All participants
involved in this study provided informed written consent. The methods were carried out in
accordance with the approved UNMC ethical guidelines. Institutional Review Board (IRB) #
162-93-FB approved all experimental protocols with the title ‘Leukapheresis of normal donors
for use in studies of disease pathogenesis and therapy’.
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Preparation and characterization of gallium nanoparticles. Gallium(III) meso
tetraphenylporphyrine chloride (GaTP) was purchased from Frontier Scientific (Logan, Utah,
USA). Nanoparticles were formulated, manufactured using high pressure homogenizer and
characterized by DLS and SEM as per our previously reported procedure.(8, 29)

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Cytotoxicity assay of GaNP. Cytotoxic effects of GaNP were determined on THP-1
macrophages using resazurin reduction assay. 3 × 105 THP-1 cells/well in a 48 well plate (0.75 ×
106 THP-1 cells/well, 24-well plate) were placed and differentiated in the presence of PMA (7.5
ng/mL) for 24 hours. The THP-1 macrophages were loaded with GaNP (25, 100, 300, 500 µM),
washed after 24 hours and placed in culture with RPMI 1640 media supplemented with 10%
heat-inactivated fetal bovine serum. After 24 hours or 15 days, the cells were washed thoroughly
with PBS (5 times) and fresh media was added. 40 µL of a resazurin solution (0.15 mg/mL in
DPBS, 100 µL for 24-well plate) was added to each well. After incubation at 37 °C for 3 hour,
fluorescence intensities were measured at 560 nm excitation/590 nm emission.
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M. tuberculosis (H37Ra) and HIV-1 co-infection of human MDM. Human monocytes were
prepared and purified by counter-current centrifugal elutriation from normal human donors were
purchased from the UNMC Department of Pharmacology and Experimental Neuroscience cell
core using an institutional IRB approved protocol. All samples were provided in a de-identified
fashion.
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To induce differentiation into MDM, the monocytes (0.75 x 106 cells/well/mL in a tissue treated
24 well plate) were incubated in DMEM media that was supplemented with 10% heat-
inactivated pooled human serum (Innovative Biologics, Herndon, VA, USA), 50 μg/mL
gentamicin (Mediatech Inc., Manassas, VA) to prevent replication of extracellular bacteria, 10
ng/mL MCSF (BioLegend, San Diego, CA), and 10 mM sodium pyruvate (Mediatech Inc.,
Manassas, VA) at 37 °C in 5 % CO2 humidified atmosphere. Half the media was replaced on the
5th day of incubation and then every 2 days thereafter until day 10 of incubation. At the 10th day,
the differentiated MDM were treated with DMEM supplemented with 1% human serum. On day
11, MDMs were incubated with GaNP (300 μM) for 24 h at 37°C in 5% CO2 humidified

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atmosphere. The drug loaded-MDMs were washed with PBS and incubated for up to an
additional 15 days prior to infection to evaluate the long-acting potential of the Ga nanoparticles.
Drug treated-MDMs were infected at desired time points (1, 5, 10, or 15 days post-drug loading)
with M.tb (H37Ra) by incubating them, for 4 h (MOI = 1) in media lacking gentamicin. For
studies of HIV infection only, macrophages were incubated with HIV-1ADA (MOI = 0.01) for 24
h. For studies of HIV-1/M.tb co-infection, the cells were incubated with HIV-1ADA (MOI = 0.01)
for 24 h after 4 h infection with M.tb (MOI = 1) at days 1, 5, 10, 15 following drug treatment
(again using media without gentamicin). After infection, MDMs were washed with PBS to
remove extracellular M.tb and/or HIV and the cells were cultured in the same media containing
gentamicin as described above to block extracellular replication of M.tb.
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Determination of M.tb growth residing in macrophages after treatment. Drug treated-MDMs
or control MDMs were infected with only M.tb, only HIV-1, or both M.tb and HIV-1 and lysed
for analysis of M.tb CFU in macrophages 2 days after infection(s). After removing the media
from the wells, iced sterile water (300 μL) was added to the wells followed by incubation on ice
for 10 min. The MDMs were treated with 1.2 mL of lysis buffer containing 55% of 7H9 broth,
20% of the 0.25% SDS and 25% of the 20% BSA in PBS. The lysed cells were centrifuged at
14,000 x g for 15 min and the pellets were resuspended in 200 μL of PBS, serial diluted in sterile
PBS and plated onto 7H11 agar plates. M.tb CFU were then counted after 3 weeks.
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M.tb (H37Ra) or HIV-1 growth inhibition in infected THP-1 macrophages by Ga
nanoparticles. THP-1 macrophages (0.75 x 106 cells/well/mL in a tissue treated 24 well plate)
were infected with either H37Ra strain (MOI = 1, 4 hour incubation) or HIV-1 (MOI = 0.01, 24
hour incubation) in RPMI1640 media that was supplemented with 10% heat-inactivated fetal
bovine serum (no antibiotics were used). After washing with PBS buffer, the cells were treated

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with GaNP (300 μM) in media (1% FBS) for 24 hours. Extracellular nanoparticles were removed
by washing the cells with PBS buffer three times. The treated cells were lysed at day 3 and 5
following infection to isolate H37Ra and growth of M.tb was monitored by determining M.tb
CFU as described above. For determination of HIV-1 growth, media were saved at day 4 and 5
and reverse transcriptase activity was assayed as described in the manufacture’s protocol.
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Determination of M.tb growth residing in THP-1 macrophages treated with Ga(NO3)3 or
Fe(NO3)3. THP-1 cells (7.5 × 105 per well) were differentiated in RPMI 1640 containing 1%
fetal serum, PMA (7.5 ng/mL), 10 mM sodium pyruvate, 50 µg/mL gentamicin, 0.75% NaHCO3
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and 10 mM HEPES (pH 7.0) at 37°C in 5% CO2 humidified atmosphere for 24 hours. After
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washing with PBS buffer twice, THP-1 cells were treated with 300 μM GaNP, Ga(NO3)3 or a
combination of GaNP and Fe(NO3)3 in RPMI 1640 media for 24 h. The growth of M.tb was
determined as described above. In brief, drug treated-THP-1 macrophages were infected at
desired time points (5, 10, or 15 days post-drug loading) with M.tb (H37Ra, MOI = 1) for 4 h in
media without gentamicin. In a case of combination treatment of GaNP and Fe(NO3)3, Fe(NO3)3
was continuously added to the culture media until lysis for CFU counting.
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Determination of HIV growth in macrophages after infection. For determination of the
magnitude of HIV infection of MDM, MDM-containing wells infected with only HIV-1 or both
HIV-1 and M.tb (MOI = 1) were incubated for 11 days at 37 °C, replacing the DMEM every 2
days. The cells were harvested at day 11 post-infection and stored at –80°C until Reverse
Transcriptase (RT) activity determination. The RT assay (EnzChek) was performed as described
in the manufacture’s protocol.
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Quantitation of cytokines. Culture supernatants were analyzed using a multiplex kit from Life
Technologies to assess levels of the human cytokines (GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5,
IL-6, IL-8, IL-10, TNF-α) according to the manufacturer’s instructions. In brief, the 96 well
filter plate was pre-wetted, and then 25ꢀμL of the diluted bead suspension was added to each well
and washed twice. 100 μL of samples and standards were added to each well. The plate was
incubated at room temperature for 2 h on a shaker. Following washing, biotinylated detection
antibody was added and incubated for 1 h. After washing, streptavidin-RPE was added to each
well and incubated for 0.5 h. The plate was again washed, resuspended in 100ꢀμL of the washing
buffer, and read on the Luminex xMAP® system (Bio-Rad, AtheNA Multi-Lyte®, USA). All
samples were run in triplicate and standards were run in duplicate.
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Whole cell extracts and Western blot analysis. MDMs cultured in 6-well plates were washed
with ice-cold PBS buffer and cell proteins were extracted in Ripa buffer containing Halt™
protease inhibitor cocktail (Pierce Biotechnology, IL, USA) and Pierce™ Phosphatase inhibitor
(Pierce Biotechnology, IL, USA). The lysates were collected and centrifuged at 4 °C at 12,000 x
g for 10 min. The supernatants were collected and BCA assay was performed to determine
protein concentration. Protein samples were electrophoresed under denaturing conditions using
4-20% polyacrylamide gels (Bio-Rad, USA). After transferring to a PVDF membrane, the
membranes were analyzed with goat polyclonal IKKβ antibody (Santa Cruz Biotechnology, CA,
USA), rabbit monoclonal NF-κB1 and phosphor-Akt antibodies (Cell Signaling Technology,
MA, USA). The bound antibody was detected using horseradish peroxidase-conjugated goat
anti-rabbit IgG (Novex, MD, USA) or rabbit anti-goat IgG (Santa Cruz Biotechnology, CA,
USA) and developed with a chemiluminescent substrate (Pierce Biotechnology, IL, USA). Image
J was used for densitometric analysis of membrane blots.

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Statistical analysis. Statistical analysis was performed using Student’s t- test. Data are
represented as mean ± SEM. Data are considered significant at p < 0.05.
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In order to optimize macrophage-utilized drug delivery, various physical properties of Ga
nanoparticles were tested to increase the encapsulating potential, prolong drug release and
minimize cytotoxicity. This resulted in the development of gallium nanoparticles (GaNP) using
water-insoluble gallium tetraphenyl porphyrin (GaTP) and p407 pluronic polymer using a high-
pressure homogenization technique.(8) Excellent drug loading (up to 48%) was achieved and the
SEM image showed a rod like shape (Figure 1). The potential for cytotoxicity of various
concentrations (25 ~ 500 µM) of GaNP on THP-1 macrophages was assessed for up to 15 days
post loading of the cells with the nanoparticles (Figure 2 and S1). No toxicity, as assessed by
resazurin reduction, was detected. Uptake of Ga nanoparticles by MDMs was observed and led
to sustained Ga release for 15 days.(8)
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Pathogenesis of infection with M.tb and HIV requires the ongoing infection of uninfected host
cells migrating to the site of infection. Interruption of this process by increasing resistance of
uninfected cells to infection should terminate infection. Therefore, we assessed the susceptibility
of MDMs pretreated with Ga nanoparticles 1 day, 5 days, 10 days and 15 days earlier to
infection with M.tb and/or HIV compared to untreated MDMs. These studies were designed to
test the duration of protective efficacy of the gallium nanoparticle against the two pathogens.
Defining the duration of Ga drug availability after drug loading of macrophages is also critical to

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a potential strategy of using drug-loaded macrophages to deliver Ga drugs to sites of infection.
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GaNP decreases the growth of M. tuberculosis in MDMs and THP-1 macrophages. We
assessed anti-mycobacterial activities of GaTP and GaNP on human MDMs infected with M.tb.
MDMs were incubated with GaTP or GaNP for 24 h, washed and then placed back into culture.
At defined time points after exposure to GaTP or GaNP (1, 5, 10, or 15 days), the cells were
incubated with M.tb (H37Ra) for 4 hours, washed free of extracellular bacteria, and then placed
back in culture. After an additional 2 days of incubation, the MDM were lysed and M.tb CFU
were determined. Even only 1 day post drug loading, the free drug GaTP showed minimal
inhibitory effect on M.tb, with CFU similar to that of untreated control MDM (Figure 3A). In
contrast, GaNP exhibited significant inhibition (more than 10 fold compared to GaTP) on the
growth of M.tb, even when infection of the MDM occurred as long as 15 days after drug loading.
CFU detected in drug treated and control MDM infected at 10 days post drug treatment and
beyond were less than those infected with M.tb following drug exposure (Figure 3A). GaNP
results, which are consistent with our prior data with M. smegmatis,(8) suggest that incorporation
of Ga into MDM-deliverable nanoparticles leads to sustained release of Ga into the intracellular
compartment at a level sufficient to inhibit growth of M.tb up to 15 days post drug loading of the
MDM.
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Iron (Fe) is a critical nutrient for the survival of mycobacteria residing in human
macrophages.(13) In earlier studies, gallium nitrate shown to significantly inhibit intracellular
mycobacterial growth. This appeared to occur via disruption of bacterial iron metabolism, as
addition of exogenous iron to the media decreased the antimicrobial effect of gallium nitrate.(24)
We compared gallium nitrate activity with GaNP. Five days after incubation of the macrophages

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with the two drugs, both gallium nitrate and GaNP showed similar reduction in M.tb growth
(Figure S3). However, GaNP continued to limit bacterial growth for 15 days after macrophage
drug loading (Figure 3A). To provide insight into the role of iron limitation in GaNP-mediated
inhibition of M.tb growth in MDM, GaNP in the presence and absence of FeNO3 were added to
THP-1 macrophages, followed by M.tb infection. Interestingly, we did not see any difference in
bacterial growth (Figure S2). The observed result might be due to higher intracellular
concentrations of Ga achieved with GaNP, differences in timing of M.tb infection and gallium
treatment compared to the prior study, effect of the nanoparticle among possible explanations. A
detailed mechanistic study will need to be carried out.
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HIV growth increases in the presence of M.tb/HIV co-infection of MDMs: In preparation for
extending studies of GaNP to MDM that were co-infected with M.tb and HIV, we determined the
effect of co-infection of the MDM on the growth rates of M.tb and HIV-1. The growth of M.tb
residing within the infected MDMs in vitro, as assessed by CFU, was not significantly different
in the presence of HIV over 2 days (Figure 4A). This lack of apparent difference may be due to
the fact that M.tb grows slowly and here, our determinations were made after only two days,
possibly masking a difference in growth at a later stage of co-infection. In contrast, in vitro co-
infection of MDMs with M.tb (H37Ra) and HIV-1 increased the levels of HIV-1 up to 2 fold, as
assessed by reverse transcriptase (RT) assay, over the course of 11 days in culture (Figure 4B).
The viability of MDMs were decreased by infection with M.tb and HIV, but the cells survived up
to 15 days as viewed by inverted phase microscopy.
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Ga(III) nanoparticle inhibited the growth of M.tb and replication of HIV-1 residing in
MDMs. As shown in Figure 3, GaNP treatment of MDMs resulted in decreased growth of M.tb
when infection was initiated up to 15 days after drug loading occurred. HIV-1 replication

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requires Fe-dependent protein ribonucleotide reductase, NF-кB, CDK9, elF5a and ABCE1 and
recent work from our lab has shown the ability of Ga to inhibit HIV replication in MDM.(8)
Therefore, we investigated the antimicrobial activities of GaNP on MDMs co-infected with HIV-
1 and M.tb (Figure 5).
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Consistent with results from the experiments shown in Figure 3, GaNP showed long-acting
inhibitory activity against M.tb, i.e. significant M.tb growth inhibition was observed up to 15
days after incubation of the MDM with GaNP.
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Consistent with the earlier data from our lab(8), MDMs treated with GaNP that were co-infected
with M.tb and HIV-1 exhibited significantly reduced replication of HIV (2 fold) compared to
non-treated MDMs. In contrast, free drug (GaTP) did not have a significant inhibitory effect
(Figure 5B). As with M.tb, HIV-1 growth inhibition persisted for 15 days post loading of the
MDM with GaNP (Figure 5B). Interestingly, GaNP reduced the growth of M.tb residing in
MDMs by 10 fold compared to non-treated MDMs while a 2-fold growth decrease of M.tb in co-
infected MDMs was observed. This finding may indicate that M.tb and HIV are competing over
Ga uptake under the limited Ga environment, resulting in less GaNP-induced killing of M.tb.
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The effect of GaNP administered after THP-1 macrophages were infected with M.tb and/or HIV
was also investigated. GaNP treatment resulted in significant reduction of bacterial growth, with
61% and 70% compared to their positive controls at day 3 and 5 post infection, respectively
(Figure 3B). HIV growth was also inhibited by 25% and 37% at day 4 and 5, respectively, by
GaNP post treatment (Figure 3C). The magnitude of effect of GaNP on M.tb load in the
macrophages appears to be greater with GaNP pretreatment (Figure 3A) versus post treatment
(Figure 3B and Figure 3C). Inherently, GaNP pretreatment appeared to decrease the initial HIV

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and M.tb infection of the cells compared to control (Figure 3). Thus, it seems possible that GaNP
is able to both block initial infection and further replication of M.tb and HIV, but is unable to
decrease the burden of infection of a cell beyond that when GaNP treatment begins.
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Secretion of cytokines by MDMs co-infected with HIV-1/M.tb. Once exposed to M.tb, a host
immune defense response is initiated by producing a variety of molecules, including cytokines
and, chemokines, and by developing a T-cell mediated immune response.(30) These T-cells
produce interferon-γ (IFN-γ), which plays an important role in activating macrophages to
produce reactive oxygen and nitric oxide species and release other cytokines and chemokines.(6)
IFN-γ, TNF-α and IL-12 appear to be key cytokines in controlling M.tb infection in mice and
humans.(31, 32)
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In parallel to our initial studies examining the effect of macrophage loading with GaNP on
growth of M.tb (Figure 3), we also determined the effect of GaNP loading on the release of IL-6
and IL-8 from M.tb infected MDMs. Interestingly, GaNP reduced the release of IL-6 and IL-8
from the M.tb infected MDMs (See supporting Information) in parallel to reducing the number of
M. tb. CFU (Figure 3).
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Co-infection of macrophages with M.tb and HIV may also alter the profile of cytokines produced
by macrophages. In order to investigate this possibility, the cytokine profile of culture
supernatants from MDMs co-infected with HIV and M.tb, were analyzed using a Luminex
system. As seen in Figure 6, of 10 cytokines assessed, two major interleukins (IL-6 and IL-8)
were detected in high quantity, with trace amounts of IL-1β, TNF-α, IFN-γ, MCSF and IL-4 (See
supporting Information). No significant amount of TNF-α (<3 pg/mL) was detected, which is in
good agreement with other studies on HIV/TB co-infection.(33) Also, TNF-α released by

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macrophages facilitates macrophage apoptosis in response to M.tb.(34-36) Patel et.al(33) also
observed that HIV infection reduced macrophage apoptosis in response to M.tb due to reduced
production of TNF-α.
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It has been suggested that IFN-γ and TNF-α can serve as excellent biomarkers for the diagnosis
of TB.(37) Recently, IL-6 was also suggested as a potent biomarker in mycobacterial infections
(H37Rv, H37Ra, M. smegmatis) of mouse peritoneal macrophages.(38) Although the reported
study was conducted with murine macrophages in vitro, the high level of IL-6 release is
consistent with a human macrophage study.(39) As shown in Figure 6A, a significantly higher
amount of IL-6 was found in culture supernatants from MDM co-infected with HIV and M.tb
compared to uninfected MDM, indicating that HIV infection does not influence IL-6 release by
M.tb-infected MDMs, supporting the potential for the use of IL-6 as a biomarker in
mycobacterial infection of HIV co-infected patients.
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Interestingly, IL-6 production by MDMs loaded with GaNP for 5 and 10 days prior to infection
with M.tb and HIV was 6 fold less than control cells that were not treated with Ga (Figure 6A).
When infection occurred at day 15 post-GaNP loading, the levels of IL-6 in treated or non-
treated MDMs were lower compared to those infected at day 5 and 10 post drug loading (Figure
6A). This was likely at least in part due to a decrease in viability of macrophages with longer
time in culture. However, GaNP treated MDM still showed a 6-fold lower level of IL-6 release
compared to the non-GaNP treated control (Figure 6A). In contrast to results with GaNP, MDM
that received free drug (GaTP) (Figure 6A) or nanoparticles not containing GaTP (not shown)
exhibited IL-6 responses similar to the non-Ga treated control cells.

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We also found that HIV/M.tb co-infected MDM production of IL-8 was 20-fold higher than
uninfected control MDM, regardless of how many days they were in culture prior to infection
(Figure 6B). IL-8 is a pro-inflammatory chemokine produced by monocytes, macrophages,
endothelial cells and other types of cells. Its main role is to recruit neutrophils to sites of
infection, as well as T lymphocytes and monocytes.(40) The main sources of IL-8 are monocytes
and macrophages infected with pathogens, including mycobacteria.(41) Although higher levels
of IL-8 along with IL-6 are found in pleural fluid in TB or HIV/TB patients, there are no
significant differences in pattern or level of cytokines between two groups.(42) Recently, Krupa
and et. al. suggested in their study that IL-8 can bind to M.tb and this association may enhance
the immune response in patients with TB.(43) These studies are in good agreement with our in
vitro study of IL-6 and IL-8 secretions in response to M.tb/HIV infection of MDM.
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Similar to IL-6, GaNP treatment also significantly reduced the secretion of IL-8 by HIV/TB co-
infected MDM (Figure 6B). This effect was maximal in the cells that were infected at day 15
post loading with GaNP (Figure 6B), where IL-8 production was equal to the uninfected MDMs.
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The above data show that GaNP decreases M.tb/HIV induced IL-6 and IL-8 production, which
could be by inhibiting M.tb and/or HIV replication in MDMs. However, it is also possible that
the GaNP has an effect on the ability of MDM to produce IL-6 and/or IL-8 by altering an MDM
signaling pathway needed for production of these cytokines. Therefore, the effect of GaNP on
MDM release of IL-6 and IL-8 in response to lipopolysaccharide (LPS, 10 µg/ml), ionomycin (1
µM), and MCSF (10 ng/ml, See SI) was investigated. Interestingly, GaNP was able to
significantly inhibit MDM IL-6 and IL-8 production in response to LPS and ionomycin (Figure
7A). We also observed changes in the regulation of IKK-β/NF-κB by GaNP (Figure 7B). By
western blot we observed the increased expression of IKK- β in the presence of GaNP. In
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general, IKK-β is a NF-κB inhibitor and accordingly, we observed the down regulation of NF-κB
by western blot. We did not see any changes in phosphoAkt and pP38 expression by western blot
with GaNP (Figure S10 and S11). These results are consistent with the possibility that GaNP
decreases IL-6 and IL-8 by targeting IKK-β signaling pathway.
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In general, IL-6 and IL-8 production appear to have a negative effect on host defense against
M.tb.(9, 44, 45) IL-6 promotes M.tb growth and inhibits production of TNF-α and IL-1β that are
needed for maximal killing of M.tb.(9, 45) Similarly, IL-8 increases inflammation and
granuloma formation of M.tb.(44) Therefore, GaNP mediated inhibition of both IL-6 and IL-8
could further enhance host defense against M.tb beyond the drug’s ability to inhibit growth of the
organism through disruption of Fe metabolism. Ga-NP is able to reduce the M. tb growth for
longer period independently and when it is co-infected with HIV. In addition, it is also able to
regulate the secretion of both IL-6 and IL-8 by the MDMs, likely by interfering in the IKKβ-
NFκB cell signaling pathway.
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In summary, the effect of GaNP loading of human MDM on in vitro co-infection of these
macrophages with HIV-1 and M.tb was studied. Up to 15 days post-loading with the drug, GaNP
was able to reduce the growth of both M.tb and HIV-1. Given what is known to date about the
mechanism of action of Ga, this is most likely due to disruption of iron metabolism critical to
pathogen growth/survival, although this could not be confirmed in the present work.
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HIV-1/M.tb co-infected MDMs in vitro exhibited increased production of IL-6 and IL-8, but
negligible amounts of TNF relative to uninfected macrophages. GaNP treated MDMs showed
reduced IL-6 and IL-8 production. This could be due to inhibition of M.tb/HIV infection and/or

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GaNP-mediated modification of macrophage signaling pathway(s) that result in production of
these two cytokines.
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Development of Ga (III)-based drugs that target human macrophages may be a potential
approach for the treatment of co-infection of HIV-1/M.tb and reduction of IL-6 and IL-8
generation. A similar approach could allow targeting of organisms present in other cellular
reservoirs. In addition, improvement of drug loading, cell targeting, encapsulation efficiency and
sustained drug release will also be important for optimizing treatment.
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Acknowledgement
This work was supported in part by a Nebraska Research Initiative award to PN and VA Merit
403
Review award to BEB.
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Disclosures
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The authors declare no competing financial interest
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Legends
Figure 1. Scheme for testing antimicrobial activities of gallium (III) in M.tb and HIV-infected infected MDMs.
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Figure 2. Toxicity of Ga nanoparticle (GaNP) against THP-1 macrophages. In a 24 well plate, 0.75 × 106 cells/well
were seeded. The wells were treated with various concentrations of GaNP for 24 hours, and then the wells were
washed with PBS buffer thoroughly. The GaNP-treated macrophages were further incubated for additional 15 days
with changing media for every 2 day and viability was determined using resazurin reduction assay.
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Figure 3. Antimicrobial activities of gallium (III). (A) MDMs were incubated with GaTP or GaNP for 24 hours,
following which the cultures were washed free of extracellular drug. At days 1, 5, 10, and 15 following drug
treatment, these MDM, as well as control MDM that had not been exposed to drug, were then infected with M.tb
(H37Ra, MOI = 1) for 4 hours and then the M.tb allowed to grow for 2 days. M.tb (B) and HIV-1 (C) growth
inhibition in infected THP-1 macrophages by Ga nanoparticles. THP-1 macrophages were infected with H37Ra
strain (MOI = 1) or HIV-1 (MOI = 0.01) and then incubated with GaNP for 24 hours. Growth inhibition was
monitored over time by determining CFU for M.tb and RT assay for HIV-1. Ga was used in the form of Gallium
(III) tetraphenylporphyrin (GaTP, 300 μM) or GaTP encapsulated nanoparticles (GaNP, 300 μM). Data represents
the mean ± SEM of triplicate (n = 3). Statistical differences were determined using Student’s t test: * p < 0.05
compared with non-drug treated control. *** p < 0.001, **** p < 0.0001.
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Figure 4. Quantitation of the growth of M.tb and the replication of HIV in MDMs co-infected with HIV-1/M.tb. A)
M.tb growth was determined by CFU, B) HIV-1 quantitation in human MDMs was determined by Reverse-
Transcriptase assay. MDMs were infected with HIV-1 (MOI = 0.01), M.tb (H37Ra, MOI = 1), or co-infected with
HIV-1 and M.tb at Day 8, 13 and 18 following differentiation. The co-infected MDMs and HIV-infected MDMs
were further incubated for 11 days with changing medium every 48 h. Supernatants from HIV-1/M.tb co-infected

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cells were harvested at day 11 for analysis of RT. Data represents the mean ± SEM of triplicate (n = 3). Statistical
differences were determined using Student’s t test: *p < 0.01 compared with HIV (+) in (B).
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Figure 5. Antimicrobial activities of gallium (III) in HIV-1/M.tb co-infected MDMs. Antimicrobial activities were
determined by (A) CFU for M.tb and (B) RT assay for HIV-1. Gallium (III) tetraphenylporphyrin (GaTP, 300 µM)
and gallium (III) tetraphenylporphyrin encapsulated nanoparticles (GaNP, 300 µM) treated MDMs were infected
with both HIV-1 and M.tb (H37Ra) at days 5, 10, and 15 following drug treatment of the MDMs. Data represents
the mean ± SEM for n = 3 or 6. Statistical differences were determined using Student’s t test: *p < 0.01 compared
with non-drug treated control in (A) and (B).
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Figure 6. Analysis of cytokines present in supernatants from HIV-1/M.tb co-infected macrophages. A) IL-6, B) IL-
8. MDMs were co-infected with HIV-1 and M.tb (H37Ra) at Day 5, 10 and 15 following MDM incubation with
(gallium (III) tetraphenylporphyrin (GaTP, 300 µM) and gallium (III) tetraphenylporphyrin encapsulated
nanoparticles (GaNP, 300 µM)). Following infections, supernatants were analyzed for the presence of cytokines
released from infected MDM day 11 after infection. Data represents the mean ± SEM for n = 3 or 6. Statistical
differences were determined using Student’s t test: *p < 0.05 compared with HIV-1 and TB positive control in (A)
and (B).
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Figure 7. A). Release of IL-6 and IL-8 by MDMs in the presence of LPS (10 µg/ml) or ionomycin (1 µM),
compared with GaNP treated MDMs exposed to the same agents. Pre-loading with GaNP significantly reduced IL-6
(*p < 0.01) and IL-8 (*p < 0.05) generation induced by LPS and ionomycin. B). Western blot analysis of IKK-β and
over-expression of IKK-β was observed in presence of GaNP. C). Western blot analysis of NF-κB and observed
down regulation in the presence of GaNP, confirming the relation with IKK-β. Statistical differences were
determined using Student’s t test: n = 3, *p values compared with non-drug treated MDMs.
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