extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Introduction
In 2015, it was estimated that 9 million people developed tuberculosis (TB) due to infection with Mycobacterium tuberculosis (M.tb) and 1.5 million died from TB infection according to the World Health Organization (WHO). [1] The widespread emergence of multi-drug resistant M.tb and extensively drug resistant M.tb poses a serious threat to public health, making the development of new antibacterial drugs to treat TB of great importance. [2] A key component of TB pathogenesis is that the causative bacilli survive, grow and replicate within host macrophages. This is due in part to their ability to inhibit the maturation of the

Synthesis of mannose-conjugated F127
Succinic anhydride (0.88 mmol) was added slowly at room temperature (rt) to a solution of F127 (MW~12,500 Da, 5 g, 0.4 mmol), dimethyl amino pyridine (DMAP, 0.4 mmol) and triethyl amine (TEA, 0.8 mmol) in CH 2 Cl 2 . The mixture was stirred at rt for 24 h and the solvent was removed under reduced pressure. The remaining crude products were precipitated by adding cold diethyl ether while stirring in an ice bath. The precipitates were collected, washed with ether 3 times and dried under vacuum to afford a white powder (P407-COOH, 4.8 g, 91%). The product was analyzed using 1 H-NMR (S1 and S2 Figs).
P407-COOH was activated with N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxy succinimide (NHS) in CH 2 Cl 2 at rt for 24 h. NHS (5 eq) was added to a solution of F127-COOH (0.047 mmol, MW~12600 Da) and DCC (5 eq) in CH 2 Cl 2 at rt. After stirring for 24 h, D-mannose (5 eq) and TEA (5 eq) in DMSO was added to the reaction mixture and stirred at rt for 24 h. The urea byproduct was filtered off, and then CH 2 Cl 2 was evaporated under reduced pressure. The remaining crude products were precipitated by addition of cold ether while stirring. The white powder was collected, washed with ether and dried (40%). The product was analyzed using 1 H-NMR (S1 and S2 Figs).

Synthesis of folate-conjugate F127
Folic acid (220mg, 0.5 mmol) and F127 (3.1g, 0.25 mmol) was refluxed for 4 h in presence of 23 mL of ammonium hydroxide. The obtained product was washed with water, dried and characterized by 1 H-NMR (S1 and S2 Figs).

Preparation and characterization of nanoparticles containing Ga(III) or rifampin
Nanoparticles containing Ga(III) or rifampin were formulated and manufactured using high pressure homogenizer (Avestin C5 high-pressure homogenizer, Avestin Inc, Ottawa, ON, USA) and characterized by dynamic light scattering (DLS, a Malvern Zetasizer Nano Series Nano-ZS, Malvern Instruments Inc., Westborough, MA, USA) and SEM, as reported before. [20] Cytotoxicity assay 1 × 10 5 THP-1 cells/well were placed in a 96-well plate and differentiated in RPMI 1640 containing 10% fetal serum, PMA (7.5 ng/mL), 10 mM sodium pyruvate, 50 μg/mL gentamicin and 10 mM HEPES (pH 7.0) at 37˚C in 5% CO 2 humidified atmosphere for 24. After washing with PBS buffer, the THP-1 macrophages were treated with nanoparticles (100, 300, and 500 μM) suspended in RPMI 1640 containing 10% fetal serum for 24 h. The cells were washed with PBS three times. Fresh media (100 μL) was then added to each well and their ability to reduce resazurin was measured.

Measurement of drug uptake by THP-1 cells
THP-1 cells (7.5 × 10 5 per well) were differentiated in RPMI 1640 containing 10% fetal serum, PMA (7.5 ng/mL), 10 mM sodium pyruvate, 50 μg/mL gentamicin and 10 mM HEPES (pH 7.0) at 37˚C in 5% CO 2 humidified atmosphere for 24. After washing with PBS buffer twice, THP-1 cells were treated with 300 μM nanoparticles in RPMI 1640 media for 24 h. Uptake of nanoparticles by THP-1 cells were determined at 1, 4, 8 and 24 h incubation. Adherent cells were washed with PBS buffer three times and collected by scraping into 1.5 mL of PBS at the designated incubation time. The cells were centrifuged at 14,000 × g for 10 min and the supernatants were discarded. Cell pellets were sonicated in 200 μL of methanol and centrifuged at 21000 × g at 4˚C for 15 min. Drug amounts were determined by ICP-MS for Ga(III) or HPLC/ UV spectroscopy for rifampin. Drug retentions in THP-1 cells were assessed at day 1, 5, 10 and 15 following nanoparticle treatment for 24 h. THP-1 cells were washed, collected, centrifuged, and drug amounts were determined as mentioned above.

Analysis of Ga(III) by ICP-MS
Gallium was measured on a Perkin Elmer NexION 300D inductively-coupled mass spectrometer (Perkin Elmer, Shelton, CT). Briefly, solutions were diluted 1:100 in 2% nitric acid and mixed. From this 50 μL were added to 3.65 mL of a solution of 1% nitric acid, 3 mM EDTA, 0.03% Triton-X100 1 , and 10 ppb terbium (Tb). The Tb was used as an internal standard. A five-point calibration curve was generated, against which the samples were compared for quantitation. The raw response of Ga from each sample was divided by the raw response for Tb and this ratio was used for quantitation. Quality assurance samples were prepared from stock solutions obtained from High Purity Standards, Charleston, SC. Different concentrations were spiked into serum samples and these were assayed over several months to obtain target values. All samples were tested in kinetic energy discrimination (KED) mode, as the method used also measures iron. All quality assurance samples had adequate recovery and the internal standard counts were stable throughout the run. The r 2 for the calibration curve for all runs was >0.990.
Preparation of human monocyte-derived macrophages (MDM) and loading with drug-containing nanoparticles Human monocytes were purchased from the UNMC Department of Pharmacology cell core facility. The monocytes were purified by counter-current centrifugal elutriation, as previously reported. [23] The differentiation of monocytes was carried out as described before. [24] Briefly, monocytes (7.5 x 10 5 cells/well/mL in a tissue treated 24 well plate) were incubated in DMEM supplemented with 10% heat-inactivated pooled human serum (Innovative Biologics, Herndon, VA, USA), 10 ng/mL MCSF, 50 μg/mL gentamicin (to prevent replication of extracellular bacteria), 10 mM HEPES (pH 7.0), and 10 mM sodium pyruvate at 37˚C in 5% CO 2 humidified atmosphere. Half of the media was replaced on the 5 th day of incubation and then every 2 days thereafter, a complete media change occurred until day 10 of incubation. At the 10 th day, the differentiated MDMs were treated with DMEM supplemented with 1% human serum. On day 11, MDMs were incubated with drug (GaTP or rifampin, 300 μM) or nanoparticle (300 μM) for 24 h at 37˚C in 5% CO 2 humidified atmosphere. The nanoparticle loaded-MDMs were washed with PBS and incubated for up to an additional 5 to 15 days prior to infection.

Determination of M.tb growth residing in macrophages
For pretreatment experiments, drug (300 μM Ga nanoparticle) [20,24] treated or control (no drug or nanoparticle treatment) MDMs were infected with M.tb (H37Rv, MOI = 0.2) for 24 h, at days 5, 10 or 15 following drug or nanoparticle treatment (using media without gentamicin). After infections, MDMs were washed with PBS to remove extracellular M.tb and then placed in media containing gentamicin. MDM were then lysed 2 days after infection for determination of MDM-associated CFU. Briefly, 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 MDM 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 × g for 15 min and the pellets were resuspended, serially diluted in sterile PBS and plated onto 7H11 agar plates with colonies counted 2-3 weeks later. For treatment post-M.tb infection experiments, THP-1 macrophages were infected with H37Ra (MOI = 1) for 1 h and incubated with the drugs for 24 h. After washing with PBS to remove drugs, the macrophages were incubated in RPMI 1640 containing 1% FBS and 50 μg/mL gentamicin. The macrophages were lysed to determine CFU at day 3 and 6 post-infection as described above.

Synthesis of fluorescein-conjugated Ga(III) or rifampin nanoparticles
Fluoresceinamine was used to prepare dye-labeled nanoparticles as previously described. [20] In brief, DCC and N-hydroxysuccinimide (NHS) was added at room temperature to a solution of poly(lactic-co-glycolic acid) (PLGA) in dichloromethane. The mixture was stirred overnight at rt. After activating the carboxylic acid, fluoresceinamine in DMSO was added to synthesize fluorescein-labeled PLGA. The labeled PLGA was mixed with non-labeled PLGA in 1:3 ratio. Fluorescent Ga nanoparticles were prepared using single emulsion technique. To a PLGA solution in CH 2 Cl 2 was added Ga(III) meso teteraphenyl porphyrine chloride or rifampin. This organic solution was slowly added to 1% polyvinyl alcohol cooled in an ice, and sonicated at 20% amplitude for 10 min. The suspension was stirred overnight to remove CH 2 Cl 2 and centrifuged at 12,000 × g for 20 min. The obtained pellet was washed with water three times by centrifugation at 12,000 × g for 20 min. Finally, the pellet was resuspended in deionized water, lyophilized and stored at -80˚C until needed.

Subcellular nanoparticle localization and immunocytochemistry analysis
Monocytes (5 x 10 4 cells/well) were differentiated to MDMs in 8-chamber plates (Lab-Tek 1 , U.S.A) as described above. After differentiation, MDM were treated with GaTP, GaTP nanoparticles (GaNP), or fluorescein-conjugated PLGA for 24 h at 37˚C, followed by fixation with 4% phosphate-buffered paraformaldehyde for 30 min. Permeabilization was performed in 0.1% Triton X-100 in PBS at rt for 10 min. After blocking with 5% BSA in PBS for 30 min, the slides were incubated with rabbit polyclonal Cathepsin D antibody (Millipore, USA) and rabbit polyclonal Galectin 3 antibody (Thermo Scientific, USA). Visualization of immunostaining was done incubating with Alexa Fluor 1 488 goat-anti-rabbit IgG (H+L) (Molecular Probes™). ProLong 1 Gold antifade reagent with DAPI was also added to wells.

Subcellular co-localization of FITC-labeled M.tb (H37Ra) with LysoTracker in THP-1 macrophages
The M.tb strain, H37Ra, was tagged with fluorescin isothiocyanate (FITC). The strain was grown to OD 600 nm of 0.4 in 7H9 medium supplemented with OADC (10%) and washed with 7H9 medium by centrifuging at 10000 × g for 15 min. The pellet was resuspended in 7H9 medium containing 100 μg/mL FITC and incubated at room temperature for 30 min. The FITC-labeled H37Ra was pelleted, washed with PBS three times and resuspended in RPMI 1640 supplemented with 1% FBS. GaNP-treated or untreated THP-1 macrophages were prepared and seeded on glass coverslips as described above and infected with the FITC-labeled H37Ra for 4 hours. The cells were washed with PBS three times and incubated overnight in the medium containing 50 μg/mL gentamicin. After washing with PBS twice, the cells were incubated with 100 nM LysoTracker Red DND-99 (Invitrogen Life Technologies, CA, USA) in RPMI 1640 supplemented 1% FBS for 1 hour followed by fixation with 4% paraformaldehyde in PBS at room temperature for 30 min. Coverslips were mounted by ProLong 1 Gold antifade reagent with DAPI. Confocal images were obtained using a Zeiss LSM 710 microscope. Percentage of FITC-labeled M.tb co-localized with LysoTracker was determined by analyzing 40~70 cells.

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 10 mM DTT. The lysates were collected and centrifuged at 4˚C at 12,000 × g for 10 min. The supernatants were collected and the 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 membrane was analyzed with rabbit polyclonal Cathepsin D antibody (Millipore, USA) and rabbit monoclonal Galectin 3 antibody (Thermo Scientific, USA). The bound antibody was detected using horseradish peroxidase-conjugated goat anti-rabbit IgG (Novex, MD, USA) and developed with a chemiluminescent substrate (Pierce Biotechnology, IL, USA). Image J was used for densitometry analysis of membrane blots.

Statistical analysis
All experiments were performed in triplicate, and data represent the mean ± standard error mean of triplicate samples (n = 3). Statistically significant variance for collected data was determined by Two-way ANOVA or Student's t test.

Preparation and characterization of nanoparticles
Mannose-or folate-conjugated F127 were synthesized by procedures outlined in S1 Fig. Mannose and folate groups were introduced to target the respective mannose and folate receptors on macrophages, with the goal of improving cell uptake and retention of drug content within cells. Nanoparticles were prepared from the mixture of free drug (Ga(III) or rifampin) and polymer (F127, P188 poloxamer, mannose/folate-conjugated F127 or dendrimer) by a high pressure homogenization technique. All of the nanoparticles manufactured differed by size, polydispersity index (PDI) and z-potential (Table 1). Relatively larger nanoparticles (698 nm for rifampin dendrimer nanoparticle (RifD) and 882 nm for gallium dendrimer nanoparticle (GaD)) were obtained when dendrimer was used. Smaller nanoparticles were manufactured using poloxamer F127 and P188. The measured z-potentials ranged from +35 for GaNP to -35.3 mV for rifampin nanoparticles (RifD). All nanoparticles manufactured with F127 polymer are positively charged, whereas dendrimer and rifampin nanoparticles are negatively charged. These positively charged nanoparticles that are made of block polymer should provide an advantage by penetrating cells better than anionic nanoparticles. [25] The physical morphology of the nanoparticles was visualized using scanning electron microscopy (SEM), which showed their shapes were dependent on the drug encapsulated. Morphologies of the two dendrimer nanoparticles, GaD and RifD, were different. RifD was a long rod shape, while GaD was small and rectangular. GaNP, RifNP, Ga mannose (GaM) and Ga folate nanoparticles (GaF) showed a similar rectangular shape (Fig 1), with different sizes than GaD. (Table 1)

Uptake of nanoparticles by macrophages
To determine the macrophage-loading efficacy of nanoparticles targeting the folate or mannose receptor of macrophages, THP-1 cells were treated with GaD, GaTP, GaM or GaF for 1, 4, 8 and 24 h (Fig 2). The amount of Ga(III) taken up by the THP-1 cells was measured by ICP-MS. Ga(III) content was 2.5 and 10 fold higher using GaF compared to GaM and free Ga (III) at 24 hour, respectively. The highest Ga(III) amount was observed after 2 hour using GaD, indicating that GaD was taken up faster than the other NPs by the THP-1 macrophages (Fig 2A). In addition, higher amounts of Ga(III) from nanoparticles were retained in THP-1 cells for up to 15 days, as compared with free drug treated THP-1 cells (Fig 2B). Among the F127 nanoparticles, folate-nanoparticles were taken up much better (Fig 2). However, the retention study ( Fig 2B) indicates that folate-conjugated nanoparticles (GaF) release Ga(III) slower than mannose-conjugated nanoparticles (GaM).  https://doi.org/10.1371/journal.pone.0177987.g002 (Fig 3). In order to examine the long acting potential of the nanoparticle for prophylaxis, MDMs were treated with nanoparticles before infection ( Fig 3A). All nanoparticles showed significant inhibition of M.tb for up to 15 days following drug loading of MDMs, suggesting prolonged drug retention and release from the nanoparticles within MDMs. In contrast, the free drug, GaTP, revealed no inhibition on the growth of M.tb 5 days after incubation of MDMs with the drug. These results are consistent with our prior data with M. smegmatis and an attenuated strain of M.tb (H37Ra). [20,21] Drug uptake and retention studies confirmed prolonged drug release and a high amount of drug uptake by MDMs (Fig 2). There was no significant difference in inhibition of growth of M.tb in MDMs between nanoparticles encapsulating Ga(III) or rifampin (Fig 3A). GaNP and GaM inhibitory activities were about 2-fold at day 15 compared to day 5, while GaD and GaF nanoparticles showed consistent inhibition against H37Rv throughout that period. This is likely due to the more sustained release of Ga over time by GaD and GaF nanoparticles. Interestingly, free drug rifampin itself did not show any inhibitory activity at the times studied after MDM drug loading. with specific nanoparticles. They were then incubated for an additional 3 or 6 days. Following that the MDM were lysed and viable intracellular M.tb determined by CFU. Data represent the mean ± SEM of triplicate samples (n = 3). Statistical differences were determined using Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with non-drug treated control. https://doi.org/10.1371/journal.pone.0177987.g003 Rifampin is known to penetrate cells effectively. [24] Hence, these results are probably due to failure of the MDM to retain adequate amounts of rifampin at day 5 and beyond when the drug is presented in this form ( Fig 3A).
Next, the inhibitory activities of Ga nanoparticles were tested against M.tb-infected THP-1 macrophages (Fig 3B). Consistent with our early results with GaNP and GaTP [21], all Ga nanoparticles showed significant growth inhibition of M.tb in THP-1 macrophages when they were lysed on days 3 and 6 after infection (Fig 3B). These Ga nanoparticles, regardless of their size and zeta potentials, showed 3-10 and 5-20 fold inhibition of M.tb growth compared to (+) control at days 3 and 6, respectively. Similar to pretreatment experiments (Fig 3A), the dendrimer nanoparticle GaD showed the most promising anti-tuberculosis activity, in spite of its larger relative size. These results support the concept that Ga nanoparticles are promising antituberculous agents for inhibition of the intracellular growth of M.tb.

Co-localization of nanoparticles and virulent M.tb within MDM
Fluorescein-conjugated nanoparticles and M.tb reside in the same macrophage. [26,27] However, in order to determine the distance between both, fluorescein-conjugated nanoparticles and virulent M.tb expressing GFP within MDM, confocal microscopy experiments were performed. The confocal photomicrographs showed that nanoparticles and virulent M.tb colocalized (Fig 4).  MDMs with nanoparticles does not cause degradation of the Ga nanoparticles and confirms their location within the MDMs (Fig 5a and 5b). Two proteins, galectin 3 (Gal3) and cathepsin D (CatD), important in macrophage phagosome maturation were visualized, indicating the presence of phagosomes within the MDMs (Fig 5). [28] We observed co-localization of the Ga nanoparticles and Gal3 (Fig 5A). Similarly, the co-localization of CatD and Ga nanoparticles was also observed (Fig 5B, S3 and S4 Figs). In the case of CatD, the confocal image is not able to distinguish mature CatD (34 kDa) from its precursors. In order to further understand how phagocytosis is affected by M.tb and Ga nanoparticles, western immunoblot analysis was carried out to monitor the level of expressions of CatD and Gal3. The negative control (noninfected, first lane in Fig 6) expressed mature CatD, as well as Gal3. GaTP or Ga nanoparticletreated MDMs (no infection, Lane 2 and 3 in Fig 6A) produced similar Gal3 compared to the negative control. Whereas, a form of mature CatD was reduced in both MDMs. Infection with H37Ra appeared to interrupt CatD maturation. However, the mature CatD was still detectable 4h after infection, while further incubation of infected MDMs stopped maturation of pro-CatD (lane 4 in Fig 6). All MDMs infected with M.tb H37Ra exhibited a significant decrease in expression of CatD regardless of treatments, but the Ga nanoparticle treated MDMs showed a slightly higher expression of CatD compared to H37Ra infected MDMs (Fig 6C). Likewise, expression of Gal3 decreased when MDMs were infected with H37Ra (lane 4 in Fig 6A and  6B). However, infection of Ga nanoparticle-treated MDMs did not decrease the expression of Gal3 (lane 6, Fig 6B).

GaNP enhances phagosome maturation inhibited by M.tb
An additional experiment was carried out to support the ability of Ga nanoparticles to limit the ability of M.tb to interfere with phagosome maturation in THP-1 macrophages. Lyso-Tracker was used to co-localize H37Ra in acidic compartments, including lysosomes. Ga nanoparticle-treated macrophages exhibited a higher co-localization of FITC-labeled H37Ra with LysoTracker (78%), whereas only 28% of H37Ra were co-localized with acidic compartments in macrophages that were not treated with Ga nanoparticles (Fig 7 and S6 Fig). These observations demonstrate that accumulation of M.tb in acidic compartments and phagosome maturation is promoted by Ga nanoparticles.

Discussion
Morbidity and mortality from TB is increasing in large part to the growing problems of M.tb antibiotic resistance (XDR and MDR) and HIV-M.tb co-infection. M.tb, successfully parasitizes alveolar macrophages, in part by its ability to limit phagosome maturation and grow/replicate in this normally microbicidal environment. In order to survive within the macrophage phagosome, M.tb must be able to acquire key nutrients. Iron is essential for metabolism of pathogenic bacteria and their virulence in humans. [18,29] Gallium has been shown to inhibit iron acquisition and metabolism of a number of pathogenic bacterial species and shown efficacy in murine models of infection with these organisms. [14,15,18,19,30] Gallium nitrate, a FDA approved drug for treatment of hypercalcemia of malignancy, inhibits growth of M.tb (Erdman, H37Ra, and an isoniazid and rifampin resistant isolate) and/or M. avium complex (MAC) within macrophages by blocking iron acquisition of intracellular mycobacteria. [13] We have also demonstrated that nanoformulations of Ga potently inhibit growth of M. smegmatis, and an attenuated strain of M.tb (H37Ra) within human macrophages, as well as replication of HIV in co-infected macrophages. [20] Antibiotics incorporated into long acting nanoparticles possess great potential as antimicrobial agents because of their ability for prolonged drug release and targeting to the site of infection using targeted-polymer design. [24] From our literature search, nanoparticles containing Ga (III) and poloxamer have not been tested against a fully virulent strain of M.tb strain. Thus, six different nanoparticles, including target receptor ligand tagged nanoparticles, were prepared and compared for macrophage targeted delivery and their ability to inhibit growth of the fully virulent M.tb strain H37Rv, as well as the attenuated H37Ra strain. Dendrimer or F127 block polymers conjugated with mannose or folic acid were used to create antimicrobial nanoparticles containing Ga(III) or rifampin. Various sizes and shapes of nanoparticles with different z-potentials were obtained (Table 1), which displayed similar inhibitory activities against the growth of virulent H37Rv residing within MDMs for up to 15 days after macrophage incubation with the nanoparticles (Fig 1). Dendrimers generated bigger nanoparticles, with a range of 691~800 nm, whereas poloxamers generated nanoparticles that were smaller in size,~300 nm. Dendrimers are a new class of polymers with a highly bio-degradable property and they are non-toxic. [31][32][33] All synthesized nanoparticles, at concentrations ranging from 100-500 μM, did not induce any detectable cytotoxicity in THP-1 macrophages. The free GaTP-treated cells displayed 95 and 90% viability at 300 and 500 μM, respectively. In general, cellular uptake and cytotoxicity of nanoparticles depends upon surface charge, size, hydrophobicity and shape of the nanoparticles. Surface properties play crucial roles in nanoparticle-cell interaction, which affects cell penetration, as well as cytotoxicity. In our study, we modified the particle surface with folic acid or mannose to improve nanoparticle uptake by macrophages through engagement of cell membrane receptors specific for these molecules. Folic acid conjugated GaF showed higher cellular uptake than mannose-conjugated GaM, probably due to higher levels of cell interaction and internalization by the more positively charged GaF (Fig 2A). Macrophages are known to have folic acid receptors, leading to increased folate-conjugated nanoparticle-cell interaction and cellular uptake. [34] Even though use of different polymers and drugs for nanoparticle construction changed their physical properties, there was no dramatic change in their ability to inhibit the growth of M.tb.
Our observations support that inhibiting iron acquisition by mycobacteria and the use of nanoparticle delivery is a promising strategy for reducing pathogenic mycobacteria infections. Indeed, Ga nanoparticles were as effective as rifampin nanoparticles in inhibiting intra-macrophage growth of M.tb under our experimental conditions. However, it is important to better define the mechanism(s) whereby these formulations exhibit their antimicrobial effect. In addition to direct toxicity for the bacteria, Ga might enhance the antimicrobial mechanisms of the macrophage usually subverted by M.tb. In phagosome biogenesis, the early phagosome contains markers such as early endosomal antigen 1 (EEA1), Rab5 (a marker for early endosome), and the classic marker, transferrin. [35] A small GTPase of the Rab family, Rab5, is recruited to early phagosome through interactions between early endocytic compartments. Phagosomes containing mycobacteria also contain Rab5. [24] However, maturation of mycobacterial phagosomes stops between the early and late endosome stage, as the late endosomal marker, Rab7, is not detected upon infection of macrophages with virulent M.tb. [36,37] These observations are in good agreement with the levels of expression of the lysosomal aspartic hydrolase, cathepsin D, which is targeted to phagolysosomes, upon infection (Fig 6).
Cathepsin D (CatD), a lysosomal aspartic protease, is synthesized in the form of preprocathepsin D on rough endoplasmic reticulum (ER). [38,39] It undergoes sequential proteolysis (loss of signal peptide, 20 aa) and two N-linked glycosylations, followed by translocation to the golgi and phosphorylation at position six of mannose (M-6-P). The M-6-P pathway delivers this pro-CatD (52 kDa) to the late endosome and then it undergoes proteolytic maturation steps as follows. First, pro-peptide (44 aa) is removed from pro-CatD to generate an intermediate 48 kDa single chain protein that is cleaved into two mature chain enzymes when CatD has reached lysosomes. These consist of light (14k) and heavy (34k) chains. However, after phagocytosis of pathogenic mycobacteria by macrophages, the normal maturation process of the phagosome is inhibited. [40][41][42] Consistent with this, we found that MDMs infected with M.tb exhibited reduced mature CatD (Fig 6).
In recent work, we reported that macrophage phagosomes containing M. smegmatis fuse with early endosome containing nanocarriers, as reflected by co-localization of fluorescentconjugated nanoparticles and Rab5. [24] In the present study, we found that Ga nanoparticletreated MDMs showed greater evidence of phagosome maturation when infected with H37Ra, as evidenced by the presence of mature CatD (Fig 6). MDM infected with H37Ra exhibited reduced levels of Gal3 relative to uninfected MDM. Interestingly, Gal3 expression also increased by 1.5 fold when MDMs previously treated with Ga nanoparticles were infected with M.tb compared to untreated control MDMs. However, no significant change in the levels of Gal3 expression by the macrophages was observed when they were treated with GaTP or GaNP in the absence of M.tb infection (Fig 6). Gal3, a β-galactoside-binding lectin (~34 kDa), is known to be involved in cancer progression and metastasis, although its mechanism of action remains undetermined. [43] However, Gal3 is abundant in macrophages, as seen in Fig  6, and highly upregulated when monocytes differentiate into macrophages, whereas downregulation is observed when they differentiate into dendritic cells. [44,45] Furthermore, a phagosome proteomic study in a mouse macrophage cell line revealed Gal3 as a major component [46] and depletion of Gal3 affects phagocytosis of IgG-opsonized erythrocytes and apoptotic cells in vivo and in vitro. [28] Thus, M.tb infection of macrophages may downregulate Gal3 expression (Fig 6) to prevent or delay phagocytosis that could enhance growth of M.tb within the phagosome.
Further, accumulation of M.tb in acidic compartments was studied by assessing the extent of co-localization of M.tb and LysoTracker-labeled compartments in M.tb-infected THP-1 macrophages. As shown in Fig 7, 78% co-localization of H37Ra with LysoTracker was observed when infected macrophages were treated with Ga nanoparticles. However, only 28% of H37Ra co-localized with acidic compartments, such as acidified phagosomes and lysosomes, in macrophages not treated with Ga nanoparticles (Fig 7). These results support the possibility that Ga nanoparticles promote phagosome maturation.
Similar observations were noted in our recent report using M. smegmatis. [24] Fluorescentconjugated nanoparticles containing traditional TB drugs or Ga(III) co-localized with M. smegmatis in phagosomes like Rab5, Rab7, Rab11 and Rab14 and it could be detected. This is consistent with the possibility that the nanoparticles may enhance the maturation process or slow down phagosome (endosome) maturation inhibition by the mycobacteria. It also appears that the Ga nanoparticles may enhance the fusion process between early endosome and mycobacteria-containing vacuoles and facilitate the maturation process of phagosome-lysosome fusion (Fig 8). These results are consistent with previous work in which Ga treatment also appeared to enhance phagosome-lysosome fusion in M. avium-infected murine bone-marrow-derived macrophages and that iron availability to mycobacteria is required for the organisms' ability to inhibit this process. [47]

Conclusion
We have synthesized, characterized and tested six different nanoparticles containing Ga(III) or rifampin. Following ingestion by macrophages, these nanoparticles exhibited sustained drug release over long time periods and significantly inhibited the growth of virulent M.tb (H37Rv) in MDMs for up to 15 days following drug treatment of the MDM. Likewise, a single treatment of M.tb-infected macrophages with Ga nanoparticles reduced the growth of M.tb (H37Ra) for up to 6 days following infection. Targeting macrophages with nanoparticles and ligated nanoparticles for drug delivery showed promising results and warrant future in vivo testing. Changes in the expression of CatD suggest that M.tb infection impairs pro-CatD trafficking between the Golgi and lysosomes because of depletion of mature CatD. In addition, our observation indicates that Ga nanoparticles are able to interrupt M.tb-mediated downregulation of Gal3 and thereby the phagosome maturation process, a key component of mycobacterial pathogenesis. Hereby, the synthesized Ga nanoparticles are able to deliver drug to the macrophage, inhibit bacterial growth and reduce the inhibition of phagosome maturation. Studies to identify the exact mechanism whereby the Ga nanoparticles slow or block the ability of mycobacteria to inhibit phagosome maturation are currently in progress.


extfieldsofstudy: Biology Medicine

text:
Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation

Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

Introduction
Psoriasis is a common immune-mediated chronic inflammatory skin disease that affects the quality of life of 2%-3% of the global population. Psoriasis is typically associated with red, scaly, raised plaques resulting from a marked thickening of the epidermis induced by enhanced keratinocyte proliferation, leukocyte infiltrates in the epidermis and dermis, and inflammation [1][2][3][4][5]. Leukocyte infiltrates in psoriatic lesions primarily comprise dendritic cells (DCs), macrophages, neutrophils, and T cells. DCs generate multiple proinflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-23, that promote the development of psoriasis. TNF-α is a potent proinflammatory stimulus that promotes IL-23 production in DCs. IL-1β can activate IL-17 secretion from Th17 cells.
IL-6 protects cutaneous T cells from Treg suppression and promotes Th17 participation in inflammation. Together, these immune cells and cytokines promote the inflammatory responses that underlie the development of psoriatic lesions.
Psoriasis can result from an interplay between genetic factors and external factors, including microbial infections, skin injuries, immune disorders, environmental influences, weather, and stress [6][7][8][9][10][11][12][13][14][15]. Nevertheless, the molecular mechanisms underlying the pathogenesis of this disease are not yet fully understood. TLRs are a family of pattern recognition receptors (PPRs) that localize to the cell surface or intracellular vesicles and are responsible for recognizing pathogen-associated molecular patterns (PAMPs) associated with microbes and danger-associated molecular patterns (DAMPs) released from dead cells in damaged tissues. A group of intracellular TLRs referred to as endosomal TLRs contributes to the pathogenesis and development of psoriasis by sensing endogenous DNA and RNA released from dead cells. In this review, we discuss current knowledge on the mechanism underlying endosomal TLR activation and the link between endosomal TLR activation and the pathogenesis of psoriasis. This mechanism can inform the development of therapeutics for psoriasis that target endosomal TLRs. Synthetic antagonists of endosomal TLRs are currently being developed. Natural products from plants, fungi, and bacteria are promising candidate drugs in this context because of their diverse structures and bioactivities. Many natural compounds have demonstrated acceptable safety profiles and immunomodulatory activity [16,17]. We also discuss recently identified natural compounds that inhibit endosomal TLRs and reduce psoriatic inflammation via different mechanisms.

Endosomal Toll-Like Receptors
The ten human TLRs are divided into three phylogenetic subfamilies. The first subfamily comprises TLR1, TLR2, TLR6, and TLR10. The second subfamily comprises TLR4 and TLR5, and the third subfamily comprises TLR3, TLR7, TLR8, and TLR9 (Figure 2(a)). TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface. In contrast, TLR3, TLR7, TLR8, and TLR9 localize to the endoplasmic reticulum and are trafficked to the endosomal/lysosomal compartment where they initiate cellular responses upon their activation by PAMPs and DAMPs. Therefore, these four TLRs are referred to as endosomal TLRs [89,90]. In addition to their unique mechanism of ligand recognition, the four endosomal TLRs are also distinct from other TLRs with respect to protein length. Specifically, TLR7, TLR8, and TLR9 are composed of more than 1000 amino residues (Figure 2(a)), and their extracellular domain is longer than that of other TLRs. Most TLRs contains 19-25 LRRs arranged in a horseshoe-shaped solenoid structure that mediates ligand binding. TLR7, TLR8, and TLR9 have 25 LRRs and a unique undefined region/Z-loop between LRR14 and LRR15 (Figure 2(b)). Previous studies have shown that mouse and rat TLR8 has low activity levels and can only be activated by agonists in the presence of PolyT-ODN. The lack of a five-amino-acid motif in the undefined region/Z-loop is proposed to account for the weak activity of these two TLR8 homologues, suggesting that the undefined region/Z-loop plays a role in the activation of TLR7, TLR8, and TLR9 [91,92].  Figure 1: TLR ligands and signaling pathways. TLRs localize to the cell surface and to intracellular vesicles such as endosomes where they respond to their exogenous and endogenous ligands as shown. The TLRs utilize adaptor proteins of the MyD88 family, including MyD88, TRIF, TIRAP, and TRAM, to initiate downstream signaling pathways that induce the activation of various transcription factors, including NF-κB, AP-1, and IRF3/7, and the production of inflammatory cytokines and type I interferons.
Endosomal TLRs must be trafficked from the endoplasmic reticulum to specific cellular locations in order to be activated. The intracellular trafficking of endosomal TLRs is regulated by accessory proteins such as UNC-93 homolog B1 (UNC93B1) and specific adaptor proteins (APs). UNC93B1 directly interacts with endosomal TLRs in the endoplasmic reticulum, facilitates their transport to the Golgi apparatus via coat protein complex II (COPII) vesicles, and remains associated with them in endosomes. AP1 and AP2 are required for UNC93B1-mediated endosomal TLR trafficking. AP3 facilitates TLR9 trafficking from endosomes to lysosome-related organelles (LROs). In contrast to the effects of TLR9 activation at endosomes, TLR9 activation at LROs induces the production of type I interferons rather than proinflammatory cytokines [93][94][95].
After reaching the endosomal compartment, TLRs are cleaved by specific proteases, including asparagine endopeptidase (AEP) and cathepsins such as cathepsin B, cathepsin H, cathepsin K, cathepsin L, and cathepsin S [96][97][98]. For example, TLR9 is cleaved at its undefined region/Z-loop in endosomes, and this cleavage is a prerequisite for its activation. Interestingly, following proteolytic cleavage, the Nterminal and C-terminal portions remain associated with one another, and this association is required for protein function [98][99][100][101][102]. This raises the question of what the role of proteolytic cleavage in TLR activation is. Recent studies have shown that the cleaved and uncleaved receptors bind with equal affinity to their ligand [101,103]. Nevertheless, ligand-induced TLR dimerization requires proteolytic cleavage to relieve steric hindrance at the undefined region/Z-loop. This finding is consistent with the observation that endosomal acidification is required for endosomal TLR activation, as an acidic pH is required for the activation of cathepsins and most endosomal and lysosomal proteases [99][100][101].

The Role of Endosomal Toll-Like Receptors in the Pathogenesis of Psoriasis
In recent years, significant progress has been made in our understanding of the molecular mechanisms underlying the pathogenesis of psoriasis and the role of endosomal TLRs in this process. As shown in Figure 3, in the initiation phase, external triggers such as microbial infections and skin injuries induce the release of the antimicrobial peptide LL37 from keratinocytes and the release of self-DNA and self-RNA from dying cells to activate endosomal TLRs [104][105][106]. These TLRs can typically distinguish pathogen-derived nucleic acids from self-derived nucleic acids. Nucleic acids derived from viruses during cytosolic replication can be transported into endosomes during the process of autophagy where they activate endosomal TLRs. However, the localization of endosomal TLRs to intracellular compartments prevents their activation by self-nucleic acids under physiological conditions, because self-nucleic acids from dead cells in damaged tissues are unable to passively enter other cells and endosomes [107,108]. Nevertheless, tolerance to self-nucleic acids can be evaded under some pathological conditions. For example, the antimicrobial peptide LL37 is upregulated and delivered to inflammatory sites in psoriatic skin where it forms complexes with self-nucleic acids to facilitate their entry into DCs and the subsequent activation of endosomal TLRs. These events render nonstimulatory self-nucleic acids into potent immune stimuli [104-106, 109, 110]. Endosomal TLRs are differentially expressed in different subsets of DCs. Plasmacytoid DCs (pDCs) express TLR7 and TLR9, and myeloid DCs (mDCs) express TLR7 and TLR8 [111]. Thus, LL37/RNA and LL37/DNA complexes can trigger the production of various proinflammatory cytokines, including TNF-α, IL-1, and IL-6, as well as type I interferons in pDCs, by activating TLR7 and TLR9. Cytokines produced by pDCs in turn promote the activation of mDCs. In addition, LL37/RNA complexes can directly activate mDCs via TLR7 and TLR8, thereby inducing the production of IL-12 and IL-23 in mDCs at psoriatic inflammatory sites ( Figure 3). These cytokines activate T cells into Th1, Th22, and Th17 cells, thereby further activating cytokines that promote keratinocyte activation and proliferation and the recruitment of inflammatory cells such as neutrophils and macrophages to psoriatic lesions [112,113]. Together, these events result in chronic cutaneous inflammation.

Evidence for the Involvement of Endosomal TLRs in Psoriatic Inflammation
The involvement of endosomal TLRs in the pathogenesis of psoriasis is supported by studies of imiquimod in mouse models of psoriasis. Imiquimod is a small molecular weight agonist of TLR7. Aldara™ is a 5% imiquimod cream approved for the treatment of genital warts and superficial basal cell carcinoma. In mouse models, consecutive topical application of Aldara cream to the ear or shaved back skin causes inflammation, and the responses to imiquimod in mice closely resemble symptoms of human psoriasis, including skin thickening and erythema. Aldara not only causes phenotypic changes consistent with psoriasis but it also induces leukocyte infiltration and activation of the IL-23/ Th17 axis, suggesting that the mechanism of imiquimodinduced pathogenesis is similar to the pathogenesis of human psoriasis [114][115][116]. Consistent with these findings, there have been reports of psoriasis associated with the clinical application of imiquimod in patients with basal cell carcinoma or actinic keratosis with or without a history of psoriasis [116][117][118]. Direct evidence that endosomal TLRs are potential therapeutic targets of psoriasis treatment stems from clinical investigation of TLR antagonists. In a phase 2 clinical trial in patients with moderate to severe psoriasis, immune modulatory oligonucleotide-(IMO-) 3100, an antagonist of TLR7 and TLR9, was associated with a reduction in Psoriasis Area Severity Index (PASI) score. In an animal model of psoriasis established by intradermal injection of IL-23 in the dorsum, IMO-3100 inhibited epidermal hyperplasia. IL-23 injection altered the expression of more than 5000 genes and upregulated the expression of genes associated with IL-17 signaling [119]. Treatment with IMO-3100 modulated the expression of 1900 of genes and downregulated the expression of IL-17-regulated genes. IMO-8400 is a second generation IMO that antagonizes TLR7, TLR8, and TLR9. Similar to IMO-3100, IMO-8400 inhibited symptoms of psoriasis; however, IMO-8400 had a broader effect on the expression of IL-23induced genes. In a phase 2a clinical trial evaluating the safety and efficacy of IMO-8400 compared with placebo in patients with moderate to severe plaque psoriasis, IMO-8400 did not cause any serious or severe adverse effects and it demonstrated clinical improvements. PASI-50 with IMO-8400 was  38% compared with 1% with placebo, and PASI-75 and PASI-90 with IMO-8400 were 17% and 2%, respectively, compared with 0% with placebo [119,120]. These findings support the hypothesis that blocking endosomal TLR activation is a promising therapeutic approach for the treatment of psoriasis.

Strategies for Blocking Endosomal TLR-Mediated Inflammation
The design of strategies to block inflammatory responses elicited by the activation of distinct endosomal TLRs can be based on their unique functions and signaling mechanisms. These strategies are (1) neutralizing cytokines that mediate the effects of endosomal TLRs, (2) blocking TLR ligand interactions using TLR antagonists, (3) blocking TLR ligand interactions by sequestering TLR ligands, (4) blocking TLR activation by inhibiting proteasomal activity and endosomal acidification, (5) downregulating TLRs and their downstream signaling molecules, and (6) inhibiting signal transduction downstream of endosomal TLR activation. The feasibility of these strategies to treat psoriasis and other autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), has been demonstrated by the action mechanisms of the biological drugs and synthetic compounds (shown in Figure 3 and Table 1) that are currently being used or investigated.

Biological Drugs That Neutralize Effector Cytokines.
Several biological drugs targeting cytokines that can be directly or indirectly generated by the activation of endosomal TLRs have been approved by FDA for the treatment of psoriasis. These biological drugs can be divided into three classes: TNF-α antagonists, IL-12/IL-23 inhibitors, and IL-17A inhibitors. The TNF-α antagonists adalimumab, etanercept, and infliximab have demonstrated strong efficacy in the treatment of moderate to severe psoriasis. Adalimumab is a fully humanized monoclonal antibody [121][122][123]. Etanercept is a recombinant fusion protein containing a TNF-α receptor ligandbinding domain and a human IgG Fc domain [124][125][126]. Infliximab is a chimeric monoclonal antibody [127][128][129]. TNF-α antagonists reduce inflammatory responses at psoriatic sites and downregulate the differentiation and function of Th17 cells. These findings suggest that TNF-α functions upstream of the IL-23/Th17 axis. Ustekinumab is a member of the class of psoriasis drugs that target IL-12/IL-23. It is a humanized monoclonal antibody that neutralizes the p40 subunit common to both IL-12 and IL-23, thereby preventing the binding of these cytokines to their receptors and the subsequent initiation of Th1-and Th17-mediated signaling pathways [130,131]. Ustekinumab demonstrated a superior  [132,133]. The human monoclonal antibodies ixekizumab and secukizumab are members of the class of psoriasis drugs that target IL-17A.
Multiple studies have shown that ixekizumab and secukizumab inhibit the expression of a wide variety of genes associated with Th17-and Th1-mediated inflammatory responses.
Although these biological drugs have demonstrated efficacy in the treatment of psoriasis, their use is limited by their high cost and the fact that they must be administered by injection. Therefore, synthetic chemical drugs and natural inhibitors continue to be investigated and developed.
The binding of these compounds to endosomal TLRs blocks their access to the agonists that trigger their activation. Although their therapeutic potential for the treatment of psoriasis has not yet been evaluated, these compounds have demonstrated immune inhibitory effects in preclinical and clinical SLE trials. CPG-52364 is another endosomal TLR inhibitor that blocks ligand-induced activation of TLR7, TLR8, and TLR9. Structurally distinct from other inhibitory oligonucleotides, CPG-52364 is a derivative of the small molecular weight chemical compound quinazoline. CPG-52364 has been reported to be well tolerated in clinical trials evaluating its effect in the treatment of several inflammatory autoimmune diseases, including psoriasis, RA, and SLE [137][138][139].
6.3. Compounds That Inhibit Endosomal Acidification, Proteasomal Activity, and Sequestering TLR Ligands. The antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine are derivatives of quinine, a natural alkaloid isolated from the South American cinchona bark tree. Although they are primarily used to treat malaria, these drugs have long been used for treating skin diseases and reducing inflammation in RA and SLE. More recent studies demonstrated that these compounds function as inhibitors of TLR7, TLR8, and TLR9 [140,141]. They are weak bases; therefore, their ability to inhibit endosomal TLR activation has been attributed to their ability to inhibit endosomal acidification. These antimalarial compounds have also been shown to directly interact with nucleic acid-based TLR ligands, thereby sequestering these ligands and preventing them from binding to endosomal TLRs. Bortezomib (Velcade) is one example of a drug that targets proteasomal activity. Bortezomib is a proteasome inhibitor approved for the treatment of multiple myeloma, and it has demonstrated inhibitory effects in several autoimmune disorders, including psoriasis, RA, and SLE, in animal models [142][143][144]. In addition, bortezomib has been shown to suppress the trafficking of TLR9 to endolysosomes, inhibit TLR9 activation, and reduce lupus-and psoriasis-associated inflammation [145,146].

Compounds That Downregulate Endosomal TLRs and
Inhibit TLR Signaling. Other small molecular weight chemical compounds that inhibit TLR signaling and TLRmediated inflammatory responses in autoimmune diseases include SM934, ST-2825, IRAK4 inhibitors, and IKK2 inhibitors. SM934 (β-aminoarteether maleate), a derivative of artemisinin, possesses potent antiproliferative and antiinflammatory properties. In a preclinical study, SM934 provided a significant protective effect in a mouse model of SLE. SM934 inhibits TLR activation by promoting the downregulation of TLR7, TLR9, and MyD88 mRNA expression and the inhibition of NF-κB phosphorylation [147][148][149].
Other compounds that block NF-κB activation and cytokine production by targeting molecules associated with TLR signaling have also been developed. For example, the peptide mimetic ST-2825 targets MyD88. ST-2825 interferes with MyD88-mediated recruitment of IRAK1 and IRAK4 to the TLR signalsome, thereby inhibiting TLR-mediated inflammatory responses. This compound has been shown to inhibit TLR9 activation and block the production of autoantibodies in B cells in SLE patients [150,151]. AS-2444697, PF-05387252, PF-05388169, and PF-06650833 target the kinase activity of IRAK4 and have been investigated in preclinical or clinical studies for the treatment of multiple inflammatory and autoimmune diseases, including gout, sepsis, AR, and SLE [152,153]. IKK2 is a subunit of IκB kinase, a protein that controls NF-κB activation and the production of TLRinduced inflammatory cytokines. The chemical compounds ML120B and PHA-408 inhibit IKK2 kinase activity and have exhibited anti-inflammatory effects in an animal model of arthritis [154][155][156].

Natural Inhibitors of Endosomal TLR-Mediated Psoriatic Inflammation
Many natural compounds derived from bacteria, fungi, and plants have long been known to exhibit immunomodulatory activity and have been used for the treatment of inflammation-related disorders [16,17]. For example, retinoids, vitamin D, and their corresponding analogs are used in topical treatments for psoriasis [157,158]. These compounds bind to their endogenous cellular receptors, thereby initiating the transcription of genes that suppress inflammation and inhibit cell proliferation. In addition, the following plant extracts and natural compounds have been reported to inhibit endosomal TLR-mediated psoriatic inflammation in animal models via different mechanism of actions shown in Table 1. These natural products have therapeutic potential in the treatment of psoriasis.
7.1. Mustard Seed. Mustard seed from mustard plant is a popular food seasoning worldwide, especially in Japan, India, and China. Mustard seed possesses several biological effects, including anti-inflammatory, antioxidant, and antitumor effects. These effects are mediated by multiple active components, including erucic acid, isothiocyanate, phenols, and phytin [159]. In mice, a diet supplemented with 5% mustard seed for three weeks attenuated imiquimod-induced psoriasis-like inflammation. Mustard seed inhibited the infiltration of various types of leukocytes, including DCs, macrophages, and T cells, into psoriatic lesions. In addition, PASI score significantly decreased in mustard seed-fed mice compared with control mice. Furthermore, NF-κB, IFN-α, IL-17, and IL-22 levels decreased in the psoriatic lesions of mustard seed-fed mice compared with control mice [160]. However, the specific component mediating this effect and its molecular target in this context remains unclear.
cinnamomea is a species of fungus commonly used in Asia as a medicinal herb. It possesses a broad range of biological effects, including anti-inflammatory, antioxidant, antifatigue, and antitumor effects [161][162][163]. A previous study evaluated the effects of orally administered lyophilized extract from the fruit body of A. cinnamomea on disease severity in an animal model of imiquimod-induced psoriasis. A. cinnamomea extract reduced psoriasis-like inflammation, infiltration of CD4+ T cells, CD8+ T cells, and neutrophils, and the expression of TNF-α, IL-17A, and IL-22 in imiquimod-induced psoriatic skin lesions [164]. Although these observations support a role for A. cinnamomea in the treatment of endosomal TLR-mediated psoriasis inflammation, the effective component mediating these effects and the molecular target of A. cinnamomea in this context remain unclear.

Curcumin.
Curcumin is a bright yellow powder obtained from the rhizome of several types of ginger plants. Chemically, curcumin is a diarylheptanoid belonging to the curcuminoid group. It has diverse bioactive effects, including anti-inflammatory, antioxidant, antitumor, and antiatherosclerotic effects [165][166][167]. Topical use of a curcumin-formulated gel has been shown to inhibit imiquimod-induced psoriasis-like inflammation. Curcumin treatment significantly inhibited imiquimod-induced epidermal hyperplasia and TNF-α, IL-1β, IL-6, IL17A, IL-17F, and IL-22 production in psoriatic lesions [168]. Curcumin is known to inhibit NF-κB activation by inhibiting IκB phosphorylation and degradation. NF-κB signaling mediates the production of the inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-23, which cooperatively induce the production of IL-17 cytokines and IL-22 from dermal γδ T cells. Therefore, curcumin might inhibit endosomal TLR-induced psoriatic inflammation by targeting NF-κB signaling. Consistent with this hypothesis, curcumin-mediated inhibition of NF-κB activation resulted in the downregulation of IL-17 and IL-22.

Resveratrol.
Resveratrol is a stilbenoid, which is a type of natural phenol found in grapes, berries, and nuts. It possesses anti-inflammatory and antioxidant effects and is used as a dietary supplement [169,170]. Oral administration of resveratrol diminished the severity of imiquimod-induced psoriasis-like inflammation in an animal model, and microarray analysis revealed that resveratrol treatment inhibited imiquimod-induced expression of IL-17A, IL-19, and IL-23p19. Resveratrol has been shown to inhibit LPS-and TNF-α-induced NF-κB activation [171]. Thus, resveratrol might inhibit imiquimod-induced psoriatic inflammation by directly or indirectly targeting signaling of NF-κB.

Thiostrepton.
Thiostrepton, an antibiotic derived from several strains of Streptomycetes, functions as an antagonist of TLR7, TLR8, and TLR9. It was identified using a connectivity map screen for functional analogs of bortezomib. Subsequent studies demonstrated that thiostrepton inhibited TLR7-, TLR8-, and TLR9-mediated NF-κB activation in a cell-based assay. It also reduced the production of TNF-α and IL-12/23p40 induced by R848 and LL37/RNA complexes (TLR7 and TLR8 ligands) and by CpG-ODN and LL37/DNA complexes (TLR9 ligands) in DCs. This natural antibiotic inhibited imiquimod-induced psoriasis-like inflammation in mice. Specifically, thiostrepton inhibited the accumulation of monocytes and DCs and the expression of TNF-α, IL-1β, and IL-8 in inflammatory lesions. Thiostrepton uses two mechanisms to block endosomal TLR activation. One mechanism depends on its proteasomal inhibitory activity, similar to the mechanism underlying bortezomib-mediated inhibition of endosomal TLR activation. The other mechanism depends on its ability to inhibit endosomal acidification [172].
7.5. Azithromycin. Similar to thiostrepton, azithromycin is an antibiotic isolated from Streptomycetes. Azithromycin possesses anti-inflammatory and immunomodulatory properties and is used to treat bacterial infections [173,174]. Azithromycin inhibited imiquimod-induced expression of costimulatory molecules (CD40 and CD80) and cytokines (TNF-α, IL-10, IL-12p40, IL-12p70, and IL-23p19) in bone marrowderived DCs (BMDCs), and topical treatment with azithromycin attenuated the severity of imiquimod-induced skin inflammation in an animal model of psoriasis. Azithromycin treatment inhibited keratinocyte hyperproliferation and the accumulation of DCs, CD4+ T cells, and CD8+ T cells in psoriatic lesions. The functional mechanism of azithromycin in this context is similar to that of thiostrepton. Azithromycin inhibited lysosomal acidification and the proteolytic processing of TLR7, thereby blocking imiquimod-induced NF-κB and IRF7 activation in DCs [175]. In addition, a clinical study demonstrated that long-term oral azithromycin treatment improved PASI score in patients with chronic plaque psoriasis, further confirming the antipsoriatic function of this antibiotic [176].
7.6. Andrographolide. Andrographolide is a labdane diterpenoid isolated from the stem and leaves of Andrographis paniculata. This natural compound possesses anti-inflammatory activity and is currently used as a prescription medicine in China for the treatment of laryngitis, diarrhea, and RA. Intragastric administration of andrographolide alleviated imiquimod-induced psoriasis, but not IL-23-induced psoriasis, in mice. The therapeutic effect was dose-dependent, and treatment with 10 mg/kg andrographolide was as effective as treatment with 10 mg/kg etanercept in improving clinical scores in mice with imiquimod-induced psoriasis. Andrographolide inhibited imiquimod-induced expression of the genes encoding CD80, CD86, IL-1β, IL-6, and IL-23 in BMDCs. Treating BMDCs with andrographolide promoted MyD88 degradation and blocked the recruitment of TRAF6 to form signalsomes. Inhibiting autophagic proteolysis in BMDCs using NH 4 Cl or deleting the gene encoding microtubule-associated protein 1 light chain 3 (MAP1LC3B) abolished andrographolide-induced MyD88 degradation [177]. These findings suggested that andrographolide induces autophagic proteolysis of MyD88, thereby reducing psoriatic inflammation by inhibiting TLR-mediated cytokine production.

Conclusion
As summarized in Table 2, the studies described in this review indicate that natural products from plants, fungi, and bacteria inhibit the activation of endosomal TLRs via mechanisms that block their functions in the initiation and development of psoriasis. Compared with biological drugs, these natural modulators can be more cost-effective and are administered via different routes. Therefore, they are promising candidate drugs for the treatment of psoriasis, and they might inform the development of multiple treatment options.

Conflicts of Interest
The authors declare that they have no conflict of interests regarding the publication of this manuscript. Table 2: Key points of natural modulator in inhibition of psoriatic skin inflammation.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
A new rapid resazurin-based microdilution assay for antimicrobial susceptibility testing of Neisseria gonorrhoeae

Abstract Objectives: Rapid, cost-effective and objective methods for antimicrobial susceptibility testing of Neisseria gonorrhoeae would greatly enhance surveillance of antimicrobial resistance. Etest, disc diffusion and agar dilution methods are subjective, mostly laborious for large-scale testing and take ∼24 h. We aimed to develop a rapid broth microdilution assay using resazurin (blue), which is converted into resorufin (pink fluorescence) in the presence of viable bacteria. Methods: The resazurin-based broth microdilution assay was established using 132 N. gonorrhoeae strains and the antimicrobials ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and penicillin. A regression model was used to estimate the MICs. Assay results were obtained in ∼7.5 h. Results: The EC50 of the dose–response curves correlated well with Etest MIC values (Pearson’s r = 0.93). Minor errors resulting from misclassifications of intermediate strains were found for 9% of the samples. Major errors (susceptible strains misclassified as resistant) occurred for ceftriaxone (4.6%), cefixime (3.3%), azithromycin (0.6%) and tetracycline (0.2%). Only one very major error was found (a ceftriaxone-resistant strain misclassified as susceptible). Overall the sensitivity of the assay was 97.1% (95% CI 95.2–98.4) and the specificity 78.5% (95% CI 74.5–82.9). Conclusions: A rapid, objective, high-throughput, quantitative and cost-effective broth microdilution assay was established for gonococci. For use in routine diagnostics without confirmatory testing, the specificity might remain suboptimal for ceftriaxone and cefixime. However, the assay is an effective low-cost method to evaluate novel antimicrobials and for high-throughput screening, and expands the currently available methodologies for surveillance of antimicrobial resistance in gonococci.

Introduction
Neisseria gonorrhoeae is a very fastidious bacterium that causes the sexually transmitted infection gonorrhoea. Gonorrhoea is a public health concern globally 1,2 and N. gonorrhoeae has developed resistance to all antimicrobials introduced for treatment. 3 Accordingly, enhanced surveillance of antimicrobial susceptibility in N. gonorrhoeae is imperative globally. 1 Ideally, this surveillance should be performed using methods determining the MICs of relevant antimicrobials. MIC-based methods are also valuable to directly inform treatment after laboratory results are available and evaluate in vitro efficacy of novel antimicrobials.
Owing to the lack of any appropriate broth medium for MIC determination, MIC-based susceptibility testing of N. gonorrhoeae has been limited to disc diffusion, Etest and the agar dilution method (gold standard). Essential agreement with the agar dilution method is defined as +1 doubling dilution and should ideally be .90% for diagnostic purposes where the same resistance breakpoints are applied. 4 Etest has shown excellent agreement with the agar dilution method in many settings. [4][5][6][7] However, discordant results have been found, particularly when different growth media were used. 8 A multicentre international study revealed that the categorical agreement between Etest and agar dilution was !88%, but was very poor for disc diffusion. 9 Unfortunately, all these methods are relatively slow ($24 h), subjective, require expertise and/or are expensive. Faster methods that allow results to be obtained on the same day have been developed in the past for other bacteria, 10,11 but are not available for N. gonorrhoeae.
For many bacterial species, broth microdilution is the reference method due to accuracy, low costs and high versatility. 12,13 Several attempts have been made to develop a broth microdilution method also for N. gonorrhoeae, but none of these has been particularly accurate and suitable for routine use. [14][15][16] It is difficult to synchronize the growth of different N. gonorrhoeae strains and effects such as autolysis occur when the bacteria enter the stationary phase. [17][18][19] Chemically defined Graver-Wade (GW) broth 20 supports the growth of phylogenetically diverse auxotypes and clinical isolates, and might be a suitable medium for susceptibility testing. 21,22 Unfortunately, MIC values based on doubling dilution series are left-, interval-or right-censored discrete data, which makes error statistics challenging. 23 The potency of drugs in pharmacology is frequently measured with dose-response curves (Hill models), as this allows the estimation of the effective concentration (EC) at a specified response level. 24 Furthermore, EC values on a continuous scale take the variability of the data into account by calculating CIs. In the field of toxicology the lower CI is defined as the non-toxic concentration. This so-called benchmark dose approach has largely replaced methods that rely on dense dose spacing because of its statistical superiority and reduction of animal use. [25][26][27][28] Furthermore, the shape of the doseresponse curve can provide additional valuable information on the compounds being tested. 24 The Hill coefficient can provide information about the pharmacodynamic properties of an antimicrobial and has been used in modelling studies of single and dual antimicrobial effects. 21,22,[29][30][31] However, the interpretation and significance of the Hill coefficient has been unclear in previous studies and laborious colony counting has limited these studies to a few strains.
The biological response to a compound can be measured using different readouts. Traditionally the MIC is defined as the concentration of an antimicrobial that inhibits visual growth, but methods to quantify the number of bacterial cells more objectively are available. Methods in which OD (at e.g. OD 600 or OD 450 ), resazurin (Alamar blue), MTT, luciferase (ATP levels) and lactate dehydrogenase are measured are widespread, with readouts that correlate with the number of cells. 32 Resazurin is a blue dye that is converted into pink-fluorescent resorufin in the presence of metabolically active cells. 33,34 Unlike OD, a measure of growth inhibition, it reflects the viability of cells and is potentially suitable for time-kill assays. Resazurin has an excellent signal-to-noise ratio and has been used previously in screening for toxicity testing, 35 highthroughput applications, 36 biofilm screening 37 and MIC testing. 33,[38][39][40] The aim of this study was to develop a resazurin-based broth microdilution assay for antimicrobial susceptibility testing of N. gonorrhoeae that is rapid, objective, scalable, quantitative and inexpensive. Three datasets were generated in this study. The 2008 WHO N. gonorrhoeae reference strains (n " 8) 41,42 were studied to ensure the reproducibility of the assay and to compare multiple measurement endpoints between 0 and 15 h. Training data consisting of 84 N. gonorrhoeae strains were used to develop a regression model for estimating the MIC from dose-response curves. Finally, a panel of 40 strains with blinded MICs was used for validation.

Bacterial strains, culture and broth microdilution assay
The variability and reproducibility of the assay were validated in eight WHO reference strains (three replicates). 41,42 Additionally, 84 gonococcal strains were used as training data to develop a regression model for estimating the MIC after 6 h of incubation time (one replicate). The assay was finally validated with 40 gonococcal strains with blinded MICs (one replicate). The blinded strains were selected to represent a wide variety of antibiograms. The strains were preserved in glycerol stocks at #80 C. All strains were subsequently cultured on Chocolate Agar PolyViteX (bioMérieux, Marcy-l' Etoile, France) at 37 C in a humid 5% CO 2 -enriched atmosphere for 16-18 h and then sub-cultured once for 16 h. A McFarland standard of 0.5 was prepared for each strain and 1 mL of bacterial suspension was further diluted to $1%10 7 cfu/mL in 15 mL of heated (37 C) GW broth. 20 A volume of 90 mL of this suspension was added to 96-well round-bottom microtitre plates (360 mL wells), with each well containing 10 mL of a previously prepared dilution series. Dilution series of the antimicrobials were prepared in GW medium. Positive control (GW medium containing 1% Triton X-100) and negative control (10 mL of GW medium) were added to the first and last well, respectively. The plates were incubated for 6 h at 37 C in a humid 5% CO 2 -enriched atmosphere. Detailed standard operating procedures, including Figure S1, are available as Supplementary data at JAC Online.

Resazurin readouts
Resazurin powder (Sigma-Aldrich, China) was diluted in PBS (pH 7.4) to a final concentration of 0.1 mg/mL. We ensured that the pH of the highest antimicrobial concentration was neutral in all samples to avoid artefacts. After incubation of the broth microdilution plates, 50 mL of the dye was added to each well and mixed using an electronic multichannel dispenser. The plates were incubated for 75 min at 37 C. Fluorescence was then measured at 560 and 590 nm excitation in a plate reader (Varioskan Flash, Thermo Scientific).

Dose-response modelling
The antimicrobial effect on the different bacterial strains was quantified with dose-response curves. We first subtracted the background fluorescence resulting from dead bacteria in the positive control wells from the resazurin readout. We then fitted a sigmoidal dose-response curve to the fluorescence data of each antimicrobial-strain combination: 43,44 where f(x) is the fluorescence, x is the natural logarithm of the antibiotic concentration, and u and l describe the upper and lower asymptote, respectively. The EC 50 is the antibiotic concentration at which the effect is half-maximal and H denotes the slope of the sigmoidal function, i.e. the Hill coefficient. Next, the data were divided by u to normalize all dose-response curves to 100% viability. Hill coefficient differences across antimicrobials were tested with pairwise t-tests. Hierarchical complete linkage clustering was used to compare antimicrobial similarity. 45 Foerster et al.
Samples were considered to be above the limit of detection, and therefore categorized as resistant, if the antibiotic, at its highest concentration, reduced viability by ,50%. This was the case for six samples in the training data (n " 588) and nine samples in the validation data (n " 280). Excluding samples that were above or below the limit of detection (including Etest MICs beyond the limit of detection) resulted in 571 evaluable samples in the training data and 266 samples in the validation data. Reference strain data were not included to avoid bias from replicate testing of these samples. The relationship between EC 50 and Etest was analysed for the training data by log-transforming both values and fitting a linear regression: where e is a normally distributed error. The slope and intercept of this regression were then used to predict the MIC from the EC 50 values for the blinded strains. 95% CIs for each predicted MIC were calculated using 10 5 bootstrap samples taking into account the uncertainty from the sigmoidal model and the linear regression model. The analysis pipeline, descriptive statistics and raw data are available from GitHub (https://github.com/sunni vas/ResazurinMIC).

Essential agreement with Etest
Essential agreement was defined as the percentage of strains with predicted MICs that did not deviate by more than +1 doubling dilution from Etest MICs. Deviations from the Etest MICs were calculated as log 2 differences from the predicted MIC (837 evaluable samples for training and validation data). Reference strain data were not included to avoid bias from replicate testing of these samples.

Categorical agreement with Etest
The strains were categorized as susceptible (S), intermediate (I) or resistant (R) to each antimicrobial in accordance with the EUCAST 2016 guidelines. 46 As previously described, 47

Dose-response modelling
The 2008 WHO reference strains (n " 8) were exposed to ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline or penicillin for a time course from 0-15 h ( Figure S2). After 6 h, the difference between dead and viable gonococcal cells was sufficiently pronounced to fit dose-response curves to the data. For this endpoint of 6 h, the coefficient of variation (CV) was calculated for the EC 50 of three independent experiments. The CV ranged from 1.7% to 87% and the intra-assay CV was 29% (n " 56) ( Figure S3). Dose-response curves were gradually shifted towards higher concentrations, indicating decreased potency of the antimicrobials against the intermediate and resistant strains compared with susceptible strains (Figure 1). There was a clear separation of susceptible and resistant strains for ciprofloxacin and spectinomycin. For the b-lactam antimicrobials ceftriaxone, cefixime and penicillin the Hill coefficients (slopes) were more heterogeneous than for the other samples ( Figure 1). The mean (+SD) of this parameter gradually increased from ceftriaxone (1.6+1.3) to cefixime (1.9+1.5), tetracycline (2.1+0.9), penicillin (2.5+1.7), azithromycin (2.6+1.5), ciprofloxacin (2.7+1.2) and spectinomycin (2.9+1.7). A pairwise t-test showed that the differences between the antimicrobials were significant (P , 0.005) when the distance between the means was .0.5 ( Figure S4a). Furthermore, hierarchical clustering showed a high similarity of the Hill coefficient for the b-lactam antimicrobials ceftriaxone, cefixime and penicillin compared with the other antimicrobials ( Figure  S4b). For the training data (84 strains), Pearson's correlation between the Etest MICs and EC 50 values of all antimicrobials was 0.93 (Figure 2a). Compared with the Etest values, the EC 50 values were systematically lower, with a median deviation of #1.68 doubling dilutions (Figure 2b). The regression parameters a (â ¼ 1:10; SDâ ¼ 0:048) and b (b ¼ 1:00; SDb ¼ 0:016) of the linear log-log regression were used to predict the 837 MICs of the training and validation data. The deviation of the predicted MIC from Etest followed a normal distribution with a median of #0.015 and 95% of the deviations ranged between #4.45 and 9.22. Outliers were mostly attributed to the b-lactam antimicrobials penicillin (e.g. overestimation in b-lactamase-producing strains) and cefixime and ceftriaxone (e.g. potentially biphasic or triphasic curves with large CIs). One example of a strain with biphasic curves for ceftriaxone and cefixime was studied in detail ( Figure S5). 49 The 75% quartiles for the deviations were larger for azithromycin, cefixime and ceftriaxone compared with ciprofloxacin, penicillin, spectinomycin and tetracycline (Figure 2c). The essential agreement between the Etest MICs and the predicted MICs was 53% for all antimicrobials, being lowest for cefixime (31%) and highest for penicillin (61%).

Categorical agreement
The Etest and predicted MICs (n " 868) were classified as susceptible, intermediate resistant and resistant according to the EUCAST 2016 resistance breakpoints 46 (Figure 3). The sensitivity of the assay was 97.1% (95% CI 95.2-98.4). Minor errors resulting from misclassifications of intermediate resistant strains were found for 9% of the data. For penicillin, spectinomycin and ciprofloxacin no major errors were identified. False-positive misclassifications (S misclassified as R), i.e. major errors, occurred for tetracycline (0.2%), azithromycin (0.6%), cefixime (3.3%) and ceftriaxone (4.6%) for a total of 9% of the data. One very major error (R misclassified as S) occurred for ceftriaxone (Etest MIC 0.19 versus 0.053 mg/L). Many predicted MIC values (16.5%) had 95% CIs spanning two categories. The overall specificity of the assay was 78.5% (95% CI 74.5-82.9).

Discussion
The developed resazurin-based broth microdilution assay was able to discriminate between resistant and susceptible strains relatively reliably, was faster ($7.5 h for results) than currently available MIC methods for N. gonorrhoeae and had an excellent sensitivity of 97.1% (95% CI 95. 2-98.4). The gold standard MIC-based method agar dilution and the Etest method are both based on subjective, visual readouts and are therefore limited to a relatively low Microdilution susceptibility assay for gonococci JAC throughput. Dose-response modelling allows the precise estimation of the EC 50 of antimicrobials from a continuous scale and provides CIs rather than having the precision limited by doubling dilutions. It is inherently difficult to apply resistance breakpoints that were designed for doubling dilution-based methods to doseresponse curve-based MICs. This was reflected by many categorical errors resulting from estimates that had CIs overlapping two S/ I/R categories. The performance of the assay was excellent for Foerster et al.
ciprofloxacin, penicillin and spectinomycin (no major errors) and acceptable for azithromycin (0.6% major errors) and tetracycline (0.2% major errors). The deviations of resazurin-based MICs from Etest MICs followed a normal distribution. Outliers were mainly attributed to the b-lactam antimicrobials and contributed to the suboptimal essential agreement and assay specificity of only 78.5% (95% CI 74.5-82.9). For penicillin, substantially higher MICs were measured with the resazurin assay, e.g. for b-lactamase-producing strains. For cefixime and ceftriaxone there were many false-positive results and consequently an overestimation of resistance was measured. The complex mechanism of action and evolution of resistance to these antimicrobials is not fully understood and involves several resistance determinants in multifaceted interactions (penA, penB, mtrR, factor X). 3,50 The correlation of EC 50 and MIC has been previously shown to be not strictly linear and was largely influenced by different PBPs in Streptococcus pneumoniae. 51 The binding kinetics of a mechanism involving several targets and/or resistance determinants can result in dose-response curves that are biphasic and potentially triphasic ( Figure S5). 22,49 In these cases the correlation between EC 50 and Etest MIC differs from those in dose-response curves with only one inflection point and can result in false-positive results.
Performing the regression analysis for the different antimicrobials separately might improve the assay specificity, particularly for the b-lactam antimicrobials. An endpoint of 6 h provided only a snapshot of the antimicrobial properties and examining many more timepoints, more starting inocula and a very large number of strains, covering a wide range of MICs and ideally including in vitroselected resistant strains, might also provide valuable data for Microdilution susceptibility assay for gonococci JAC improvements. Scaling up the assay to a robotic platform might be necessary for appropriate examination of all these parameters and strains. Despite these limitations, the developed rapid resazurinbased broth microdilution assay was highly objective (avoiding visual subjective readout) and employs a standardized algorithm reducing operator bias, which can be especially valuable in multicentre studies. These properties, and the low price of resazurin, are especially valuable when screening large libraries of new compounds, antimicrobials or antimicrobial combinations. Frequently, the question that needs to be answered is the potency of antimicrobials relative to each other rather than absolute numbers. The b-lactam antimicrobials cefixime, ceftriaxone and penicillin displayed significantly lower Hill coefficients than the other antimicrobials. Information about this parameter is useful for research questions beyond susceptibility testing, such as combination therapy and pharmacodynamic modelling.
In summary, the developed resazurin-based broth microdilution assay is a rapid, objective, high-throughput, quantitative and cost-effective new tool for studying N. gonorrhoeae in liquid culture. The Hill coefficient could be compared for a large number of strains, highlighting differences between antimicrobials. The new assay opens up avenues for high-throughput synergy testing, evaluation of novel antimicrobials and surveillance of resistance.

Funding
This study was funded through an Interdisciplinary PhD (IPhD) project from SystemsX.ch (The Swiss Initiative

Transparency declarations
None to declare.

Supplementary data
The standard operating procedures and Figures S1-S5 are available as Supplementary data at JAC Online. Major errors (S misclassified as R) were found for ceftriaxone (n " 40), cefixime (n " 29), azithromycin (n " 5) and tetracycline (n " 2) and are shown in red. One very major error (R misclassified as S) was found for ceftriaxone (red). A high number of estimates (n " 138) have CIs spanning two categories.
Foerster et al.