Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc

Abstract

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.

Figure 1. Intradiscal Injection of rabbit lumbar discs

The IVDs of rabbits were dissected under sterile conditions within 12 hours after sacrifice for ex vivo organ culture model. En bloc intradiscal injections of LfcinB (200 µg per disc) were performed in rabbit lumbar spine discs using a 26-gauge needle (50 µL volume) (A), followed by isolation and dissection of each disc using an electronic microsaw (Stryker Biotech) (B).

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 2. LfcinB counteracts the catabolic effects of pro-inflammatory mediators IL-1 and LPS on PG accumulation in CM of NP cells

NP cells in alginate beads were treated with LfcinB (10 µg/mL), IL-1 (1 ng/mL), LPS (1 µg/mL), or LfcinB plus IL-1/LPS for 21 days. (A) NP cell pericellular matrix accumulation after 21-day culture was visualized using exclusion assay. A representative cell in each treatment group was photographed using an inverted phase-contrast microscope. The CM is defined as the area between the excluded erythrocytes and NP cell plasma membrane. (B) At the end of the 21-day culture period, the amount of PG in the CM was measured by DMMB assay, and normalized by DNA content. (C) Cell survival was measured by harvesting cells from alginate beads using Calcein AM to stain live cells (green) and ethidium bromide homodimer 1 to stain dead cells (red). Survival assay was conducted weekly (7, 14, and 21 days of culture) by counting at least 100 cells in triplicate for each data point. (D) NP cells were subjected to indicated treatment conditions for 7 days. At the end of the culture period, PG synthesis was measured during the last 4 hours of culture using ³⁵S-sulfate incorporation, and was normalized by DNA content. (E, F) Monolayer NP cells were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of aggrecan (2E) or biglycan and decorin (2F) induction. Gene expression was normalized by individual β-actin expression, and expressed as mean of fold-change ± S.D. (standard deviation) compared with control level. A value of p<0.05 indicates a significant difference in ANOVA.

Figure 3. LfcinB downregulates IL-1 and LPS-induced production of catabolic proteases in NP cells

Bovine NP cells in monolayer were cultured with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of (A) MMP-1, (B) MMP-13, (C) MMP-3, (D) ADAMTS-4, and (E) ADAMTS-5 gene expression. Level of gene expression was normalized by individual β-actin expression, and expressed as mean of foldchange ± S.D. (standard deviation) compared with control level. A value of p < 0.05 and p < 0.01 indicate a significant and a highly significant difference in ANOVA, respectively. To assess LfcinB-mediated catabolic protease repression at the level of protein, conditioned medium were collected and subjected to western blotting.

Figure 3. LfcinB downregulates IL-1 and LPS-induced production of catabolic proteases in NP cells

Bovine NP cells in monolayer were cultured with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of (A) MMP-1, (B) MMP-13, (C) MMP-3, (D) ADAMTS-4, and (E) ADAMTS-5 gene expression. Level of gene expression was normalized by individual β-actin expression, and expressed as mean of foldchange ± S.D. (standard deviation) compared with control level. A value of p < 0.05 and p < 0.01 indicate a significant and a highly significant difference in ANOVA, respectively. To assess LfcinB-mediated catabolic protease repression at the level of protein, conditioned medium were collected and subjected to western blotting.

Figure 3. LfcinB downregulates IL-1 and LPS-induced production of catabolic proteases in NP cells

Bovine NP cells in monolayer were cultured with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL) or LfcinB plus IL-1/LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses of (A) MMP-1, (B) MMP-13, (C) MMP-3, (D) ADAMTS-4, and (E) ADAMTS-5 gene expression. Level of gene expression was normalized by individual β-actin expression, and expressed as mean of foldchange ± S.D. (standard deviation) compared with control level. A value of p < 0.05 and p < 0.01 indicate a significant and a highly significant difference in ANOVA, respectively. To assess LfcinB-mediated catabolic protease repression at the level of protein, conditioned medium were collected and subjected to western blotting.

Figure 4. Inhibition of LfcinB on IL-1 and LPS-activated catalytic enzyme activity

Bovine NP cells were treated with IL-1 (10 ng/mL) or LPS (10 µg/mL) in the presence or absence of LfcinB (100 µg/mL) for 24 hours followed by assessments of enzyme activity using (A) zymography and (B) MMP-13 activity ELISA. Gelatin zymography was performed by loading equal volumes of the conditioned media sample on polyacrylamide gel. Band images were digitally captured and intensity of bands (pixels/band) was obtained using the ImageJ densitometry analysis software in arbitrary optical density units. MMP-13 activity was assessed by Active MMP-13 ELISA using a highly specific monoclonal antibody for the activated form of human MMP-13 (sensitivity, 7 pg/mL) in triplicates and human NP cells in monolayer (n=3). Data shown are cumulative of three experiments. OP values presented as mean ± standard deviation; data without a common letter differ, p < 0.01.

Figure 5. LfcinB downregulates inflammtion- and oxidative stress-related gene expression in bovine NP cells

NP cells in monolayer were treated with LfcinB (100 µg/mL), IL-1 (10 ng/mL), LPS (10 µg/mL), or LfcinB plus IL-1 or LPS for 24 hours in serum-free medium. Total RNA was extracted for real-time qPCR analyses targeting expression of (A) iNOS, (B) IL-6, (C) TLR-2, and (D) TLR-4. Gene expression was normalized by individual β-actin, and expressed as mean of fold-change ± S.D. compared with control level. A value of p < 0.01 indicates a highly significant difference between groups in ANOVA.

Figure 6. Histological assessments and cell viability (original magnification: X40)

Lumbar spine discs of rabbit [A] or mouse [B] were dissected after en bloc intradiscal microinjection with LfcinB at the concentration of either 100 µg for mouse or 200 µg for rabbit per disc using a 26-G (50 µL in volume) or 30-G needle (1.5 µL in volume), respectively. Injected discs were then separated and maintained for 14 days in DMEM/Ham’s F-12 medium supplemented with 1% mini-ITS in the presence or absence of catabolic cytokine IL-1 (100 ng/mL). Harvested discs were fixed in 4 % paraformalin, and then decalcified in EDTA followed by paraffin embedment. Serial disc sections of exactly 5-µm thickness were prepared for slides, and Safranin O–fast green staining was performed [A (a)-(f) for rabbit; B (a)-(c) for mouse, each treatment n=3]. [A (g)-(i)] After 14 days of ex vivo organ culture, rabbit lumbar disc cell viability was assessed by fluorescent microscopy using the LIVE/DEAD staining kit. Sample disc tissue was dissected and enzymatically digested followed by incubation in 10 µM calcein AM green and 1 µM ethidium homodimer-1 for 30 min followed by visualization. [A (j)] The % cell viability was calculated by counting at least 100 cells in triplicate for each treatment (live/dead cell counts, n=3 for each treatment).

Figure 6. Histological assessments and cell viability (original magnification: X40)

Lumbar spine discs of rabbit [A] or mouse [B] were dissected after en bloc intradiscal microinjection with LfcinB at the concentration of either 100 µg for mouse or 200 µg for rabbit per disc using a 26-G (50 µL in volume) or 30-G needle (1.5 µL in volume), respectively. Injected discs were then separated and maintained for 14 days in DMEM/Ham’s F-12 medium supplemented with 1% mini-ITS in the presence or absence of catabolic cytokine IL-1 (100 ng/mL). Harvested discs were fixed in 4 % paraformalin, and then decalcified in EDTA followed by paraffin embedment. Serial disc sections of exactly 5-µm thickness were prepared for slides, and Safranin O–fast green staining was performed [A (a)-(f) for rabbit; B (a)-(c) for mouse, each treatment n=3]. [A (g)-(i)] After 14 days of ex vivo organ culture, rabbit lumbar disc cell viability was assessed by fluorescent microscopy using the LIVE/DEAD staining kit. Sample disc tissue was dissected and enzymatically digested followed by incubation in 10 µM calcein AM green and 1 µM ethidium homodimer-1 for 30 min followed by visualization. [A (j)] The % cell viability was calculated by counting at least 100 cells in triplicate for each treatment (live/dead cell counts, n=3 for each treatment).