Fig 1.
Characterization of AgNP by TEM.
TEM images of citrate compared with PVP stabilized AgNPs (20 nm and 110 nm) in (a—d) DI water and (e—h) incubated in pH 7 perchlorate buffer solution at 37°C for 24 hours (magnification at 20 x); (i—l) show the corresponding (i, a and e; j, b and f; k, c and g; l, d and h) size distribution histograms of AgNP in DI water (n = 200 repeat measurements).
Table 1.
Physicochemical properties of AgNP.
Fig 2.
Ag+ ion release from citrate and PVP-capped Ag20 and Ag110 NP incubated in perchlorate acid/ perchlorate buffer solutions (pH 5, and 7).
Fig 3.
Cytotoxic effects of AgNP in MDM.
MDM were exposed to 5, 10, 25 and 50 μg/mL of Ag20-cit, Ag20-PVP, Ag110-citrate, Ag110-PVP and the stabilizers PVP10 (0.2, 0.3, 0.7 and 1.7 μg/mL) and PVP40 (0.3, 0.6, 1.3 and 3.1 μg/mL) for 3, 6 and 24 hours at 37°C in a humidified 5% CO2 environment. MDM cultured in complete culture media without NP exposure (0) were used as unexposed controls. Cell viability (panels a, b and c) and cytotoxicity proportional to LDH leakage (panels d, e and f) were measured by MTS and LDH assays, respectively. Percentages of viable cells were calculated as ratios of ODs of NP-exposed MDM (after the subtraction of background) to the ODs of unexposed MDM (after background subtraction) x 100. Toxicity was defined as percent (%) LDH leakage from cells calculated from the ratios of ODs of NP-exposed MDM (after background subtraction) to the ODs of unexposed MDM (after background subtraction) x 100. Each data point represents the mean ± SD from three independent experiments. Statistical significance relative to unexposed control MDM are shown as * (p<0.05) or ** (p<0.01). Horizontal dashed lines represent the level of viable cells (a, b, and c) or LDH (d, e, and f) leakage in unexposed MDMs.
Fig 4.
Effect of AgNP on cytokine mRNA expression by MDM in the absence and presence of M.tb.
MDM from four healthy human blood donors were exposed to 0, 1, 10 and 25 μg/mL of Ag20-citrate, Ag20-PVP, Ag110-citrate, Ag110-PVP and 1.7 μg/mL PVP10 and 3.1 μg/mL PVP40 in the absence (a, b, c and d) or presence (e, f, g, and h) of M.tb (MOI 10) for 4 hours at 37°C in a humidified 5% CO2 environment. Following incubation, RNA was extracted and the abundance of mRNA encoding IL1B, IL8, TNFA and IL10 examined by qRT-PCR using gene-specific primer sets as described [28]. For primer sequences see Materials and Methods. Results are shown as fold-changes relative to controls (MDM in culture media without AgNP (0 μg/mL) in a-h. In panels e-h, 0 μg/mL data points represent MDM exposed to M.tb MOI 10 in absence of AgNP. Each data point (Y-axis) represents mean fold-changes ± SD from four independent experiments. Statistically significant changes relative to unexposed MDM are marked by * (p < 0.05) or ** (p <0.01).
Fig 5.
Effect of AgNP and CB on IL-1β production.
After differentiation for 7 days, MDM were harvested, counted, and plated at concentrations of 0.7–1 x 105 cells per well into 96-well plates. The next day, MDM were incubated with 0, 1 and 10 μg/mL of Ag20-citrate (a) or CB (b) in the presence of M.tb at MOI 10 at 37°C and 5% CO2 in a humidified environment. MDM cultured in complete media served as negative control. Culture supernatants were collected at 6 and 8 hours and analyzed by IL-1β ELISA as described in Materials and Methods. Each data point (Y-axis) represents pg/mL of IL-1β ± SD from three independent experiments. Statistically significant changes relative to M.tb-exposed MDM (0 on X-axes) were determined by two-tailed unpaired t-test and marked by ** (p < 0.01) or *** (p <0.001).
Fig 6.
Effect of Ag+ ions on M.tb-induced IL-1β expression.
MDM were exposed to AgNO3 corresponding to 0.125, 0.25 and 0.5 μg/mL of Ag+ ions in the absence or presence of M.tb at MOI 10. Culture supernatants collected at 4, 6 and 8 hours after infection with M.tb ± Ag+ ion exposures were assessed for IL-1β production. Results are expressed as means ± SD from four independent experiments. Statistically significant changes relative to M.tb-exposed MDM (0 on X-axes) were determined by two-tailed unpaired t-test and marked by ** (p < 0.01).
Fig 7.
Effect of AgNP on the viability of M.tb.
Ten μl of M.tb stock suspension (107 CFU/mL) was diluted to 1 mL with cell culture media (RPMI1640 + 10% PHS, reflecting the environment in which AgNP and M.tb interacted with the MDM in vitro) and incubated at 37°C on a rotating shaker in the presence of 0, 10 and 25 μg/mL of AgNP and 15.7 and 39.25 μg/mL of AgNO3. The amounts of AgNO3 corresponded to the amount of Ag+ ions present in 10 and 25 μg/mL of AgNP, respectively. M.tb culture samples (10 μL) were removed at 24 hours, diluted 10-fold by serial dilution, plated in triplicate onto 7H10 agar plates and incubated for 21 days at 37°C. CFU were determined as described in the Materials and Methods and plotted as a function of AgNP or Ag+ ion concentration. Mean values from two independent experiments ± SD are shown.
Fig 8.
Effect of AgNP on the internalization of M.tb by MDM.
MDM were infected with M.tb at MOI 10 in the absence or presence of 10 μg/mL of AgNP and the percentage of MDM with internalized M.tb determined after 2 (panel a) and 4 (panel b) hours of infection. A total of 300 MDM was examined in each experimental condition as described in Materials and Methods. Results are expressed as mean percentages of MDM with internalized M.tb ± standard deviations from four independent experiments. To assess the number of internalized M.tb in MDM, MDM infected with M.tb (MOI 1) alone, MDM infected with M.tb and exposed to Ag20-citrate (10 μg/mL) and MDM infected with M.tb and exposed to Ag110-citrate (10 μg/mL) were lysed following a 4-hour incubation. MDM lysates were then plated onto agar plates and CFU determined as described in Materials and Methods. Panel c shows the mean number of CFU ± standard deviation from three independent experiments.
Fig 9.
Comparison of AgNP-induced TLR signaling pathway-specific gene expression in MDM in the absence and presence of M.tb.
MDM from five donors were exposed to Ag20-citrate ± M.tb, Ag110-citrate ± M.tb, M.tb, or left unexposed for 4 hours at 37°C in a humidified 5% CO2 environment followed by RNA extraction. The final concentration of AgNP was 10 μg/mL and M.tb was used at a MOI 10. RNA was analyzed by TLR pathway specific RT2 profiler arrays (Cat. No. PAHS 018E, Qiagen Sciences, MD) [28]. Levels of cDNA were calculated with the relative quantitation method (ΔΔCt method) from the PCR array data using analysis software accessed from http://sabiosciences.com/pcrarraydataanalysis.php. Statistical differences in fold-mRNA expression levels between AgNP-exposed and unexposed and uninfected cells were calculated using the same software. (a) Non-supervised clustering of the entire dataset showing the overall pattern of expression of 84 genes across 30 MDM samples from five human subjects with each row and column representing a gene and samples, respectively. A dendrogram including all 30 samples are shown. (b) Mean fold-changes (≥ 2-fold) of mRNA from MDM exposed to 10 μg/mL of Ag20-citrate or Ag110-citrate relative to unexposed MDM are shown. Statistically significant changes (p < 0.05) relative to AgNP-unexposed MDM are marked by an asterisk (*). (c) and (d) M.tb-induced alteration of mRNA expression with mean fold-changes ≥2-fold and p≤0.05 relative to uninfected MDM were compared with that from MDM treated with M.tb + Ag20-citrate or M.tb + Ag110-citrate. Panel c shows mRNA fold-changes induced by M.tb alone that are ≥ 100-fold and panel d shows mRNA fold-changes induced by M.tb alone that are < 100-fold. Statistically significant changes (p < 0.05) relative to M.tb-induced expression are marked by an asterisk (*).
Fig 10.
Comparison of Ag20 and Ag110 exposure effects on M.tb-induced gene expression.
Scatter plots comparing the normalized expression of genes from M.tb infected MDM against that from MDM exposed to Ag20-citrate + M.tb (a) and Ag110-citrate + M.tb (b) are shown. The central lines represent unchanged gene expression. Upregulated and downregulated genes are indicated in red and green colors, respectively.
Table 2.
Modulation of M.tb-induced TLR pathway activation by AgNP.
Fig 11.
Validation of Hsp72 expression induced by AgNP in MDM.
RNA from the samples used for TLR arrays (Fig 7) was used to validate array results by qRT-PCR using primers corresponding to IL1B (a) and HSPA1 (b) (see Materials and Methods). Each data point (Y-axis) represents mean fold-changes ± SD from MDM of 5 independent subjects. Statistically significant changes relative to unexposed MDM are marked by * (p < 0.05) or *** (p <0.001). Unexposed (blue line) and Ag20-citrate exposed (10 μg/mL for 4 hours at 37°C at 5% CO2 in a humidified environment, red line) MDM were stained with anti-Hsp72 Ab or control IgG (grey line) (c). Data shown in (c) are representative of the FACS profile from one experiment, which was repeated twice using MDM from a total of three different donors. MDM exposed to 42°C heat shock for 1h followed by incubation at 37°C for 3h stained with anti-Hsp72 Ab served as a positive control (d). Blue and red lines represent the unexposed and heat shocked anti-Hsp72-stained MDM. Unexposed MDM stained with Isotype-matched IgG Ab are shown by gray lines in (c) and (d), representative of two independent experiments).
Fig 12.
Schematic of hypothetical mechanisms of AgNP-induced suppression of M.tb-induced TLR signaling and proinflammatory responses.
M.tb binds to TLRs and activates MAPK and NF-κB leading to the expression of cytokines such as IL-1β and TNF-α. IKK complex phosphorylates IκB which leads to its dissociation from the NF-κB complex followed by translocation of NF-κB into the nucleus where NF-κB binds and activates target genes. M.tb induces the cytoplasmic translocation of HMBG1 protein by chromosome region maintenance 1 (CRM1)-dependent transport and secretion. HMBG1 binds to TLR receptors and activates MAPK and NF-κB pathways [45]. AgNP, which are thought to enter cells via receptor-mediated endocytosis (dashed lines) upregulate the expression of HSPA1A that encodes Hsp72 via the activation and binding of heat shock factor (HSF), the major transcription factor that binds to the promoter of HSPA1A. Intracellular Hsp72 potentially suppresses the cytoplasmic transport and secretion of HMGB1 proteins as well as the activation of MAPK and NF-κB pathways [66] leading to the suppression of M.tb-induced cytokine expression in presence of AgNP. Hsp72 can be secreted and bind to TLR4 and activate MAPK and NF-κB pathways [88]. Red dots represent phosphorylation of HSF-1 and IκB proteins. The potential inhibitory effects of Hsp72 are shown with red solid lines. Signaling pathways that are reported to be induced by specific ligand receptor interactions and the more hypothetical pathways are shown with solid and dashed lines, respectively.