Tian X et al. (2022), Strongylocentrotus nudus Eggs Polysaccharide En...

ECB-ART-50401

Front Pharmacol 2022 Jan 01;13:807440. doi: 10.3389/fphar.2022.807440.

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Strongylocentrotus nudus Eggs Polysaccharide Enhances Macrophage Phagocytosis Against E.coli Infection by TLR4/STAT3 Axis.

Tian X , Guo M , Zhang X , Guo L , Lan N , Cheng Y , Han Y , Wang M , Peng Z , Zhou C , Fan H .

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Antibiotics resistance is one of the most significant public health threats globally. Strategies that strengthen host defenses to control pathogen infection has become a hot research field. Macrophages are part of early host defense mechanisms, and are activated via host pattern recognition receptors (PRRs), such as Toll-like receptor 4 (TLR4), which then facilitates phagocytosis and elimination of invading pathogens. However, few activators of PRRs have been approved for clinical use because of their toxic effects. This study aimed to investigate whether Strongylocentrotus nudus eggs polysaccharide (SEP), a non-toxic extract from seafood, contributes to host defense against bacterial infection. Results showed that SEP promoted bacterial clearance by enhancing phagocytosis by macrophages during E. coli infection in vitro, but was inhibited by TLR4 specific inhibitor TAK-242, STAT3 inhibitor Stattic or blockade of CD64. In addition, SEP protected mice from E. coli induced mortality, reduced pulmonary inflammation and inhibited dissemination of bacteria to organs, while TAK-242 retarded the protection of SEP. Overall, SEP strengthened innate host defense and improved the outcome in bacterial infection, suggesting that SEP could be used as a potential immunomodulator in host-directed therapies.

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	FIGURE 1. SEP protects against E. coli infection by enhancing host defense. (A) Molecular structure of SEP. (B) The effect of SEP on macrophages phagocytosis of live E. coli by gentamicin protection assay. RAW264.7 cells were administrated with Cyt D (10 μM) for 2 h prior to E. coli infection. (C) The effect of SEP on macrophages phagocytosis by fluorescence intensity. RAW264.7 cells were fed with pHrodo-conjugated inactivated E. coli bioparticles for 1 h and phagocytosis was quantified by the fluorescence. (D) The experimental design of E. coli infection and SEP treatment in mice. Mice were pre-treated with SEP (25 mg/kg) or vehicle (saline) by i. v. every 3 days for three times, and infected with E. coli (9 × 108 CFU per mouse, i. p.) on the eighth day. Then the survival of mice (E) were evaluated. (n = 10). (F) Mice were pre-treated with SEP (25 mg/kg, i. v.) and infected with E. coli (3 × 108 CFU per mouse, i. p.) on day eight, then sacrificed at 18 h post-infection. (n = 8). CFU of E. coli in tissue homogenates of lung, spleen, liver, and peritoneal lavage fluid were detected. (G) Lung sections were stained with H&E. (H) Myeloperoxidase (MPO) value were detected in the lung tissue. (I) The expression of IL-1β, TNF-α and IL-6 in the lungs were measured by ELISA. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, **p < 0.001.
	FIGURE 2. SEP promotes macrophages phagocytosis via TLR4. RAW264.7 cells were pre-treated with TLR2 inhibitor C29 (50 μM) or TLR4 inhibitor TAK-242 (20 μM) for 2 h, then stimulated with SEP (100 μg/ml) for another 24 h. (A) Phagocytosis of live E. coli by macrophages were measured by gentamicin protection assay. (B) Phagocytosis of pHrodo-E. coli by macrophages were quantified by fluorescence intensity. (C) The positive cell rates for phagocytosis of pHrodo-E. coli by macrophage were detected by flow cytometry. All data are represented as mean ± SEM acquired in triplicate determinations. ****p < 0.0001. ns, not significant.
	FIGURE 3. TLR4/STAT3 signaling is involved in SEP induced phagocytosis. (A) The effects of SEP on protein levels of p-p65, p-ERK, p-JNK and p-mTOR were determined by western blot. (B) The effects of TLR4 signaling pathway inhibitors on phagocytosis of live E. coli were measured by gentamicin protection assay. RAW264.7 cells were pre-treated with NF-κB inhibitor BAY 11-7085 (2 μM), or JNK inhibitor SP610025 (5 μM), or MEK1/2 inhibitor PD184352 (5 μM), or PI3K inhibitor LY294002 (5 μM) for 2 h prior to SEP treatment. (C) Phagocytosis of pHrodo-E. coli by macrophages were quantified by fluorescence intensity. (D, E) The effects of SEP on protein levels of p-STAT1, p-STAT3 were determined by western blot. The effects of Stattic (5 μM) or Fludarabine (20 μM) on phagocytosis of pHrodo-E. coli were measured by flow cytometry (F), or phagocytosis of live E. coli by gentamicin protection assay (G), or phagocytosis of pHrodo-E. coli by fluorescence intensity (H). All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, ***p < 0.0001.
	FIGURE 4. CD64 is responsible for SEP induced phagocytosis via TLR4/STAT3 signals. (A,B) The effects of SEP on the relative mRNA expression of CD64, CD32 and CD16 were determined by q-PCR. (C,D) The effects of SEP on the protein levels of CD64 and CD16/32 were determined by flow cytometry. (E) RAW264.7 cells were pre-treated with SEP (100 μg/ml) for 24 h, and followed by incubation with 2 μg/ml anti-CD64 or anti-CD16/32 blocking antibodies for 1 h prior to pHrodo-E. coli infection. Then macrophage phagocytosis was quantified by the fluorescence. (F) The effects of blocking CD64 or anti-CD16/32 on phagocytosis of live E. coli by macrophages were measured by gentamicin protection assay. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.001,**p < 0.0001. ns, not significant.
	FIGURE 5. SEP promotes filopodia formation via TLR4/STAT3 axis. (A) The effects of SEP on the filopodia formation of macrophages. RAW264.7 cells were administrated with Src family inhibitor AZD0530 (10 μM) or ROCK1 inhibitor Y-27632 (20 μM) for 2 h prior to E. coli infection. The cellular filamentous actin were stained with rhodamine phalloidin and the cellular DNA with DAPI. (B) The effects of SEP on protein levels of p-Src and p-Lyn were determined by western blot. (C) The effects of AZD0530 or Y-27632 on SEP induced phagocytosis of pHrodo-E. coli were measured by fluorescence intensity, (D) or phagocytosis of live E. coli by gentamicin protection assay, or (E) phagocytosis of pHrodo-E. coli by flow cytometry. (F) The effects of TAK-242 or Stattic on SEP induced filopodia formation. (G,H) The effects of TAK-242, or C29, or Stattic, or Fludarabine on SEP induced protein levels of p-Src and p-Lyn were determined by western blot. *p < 0.001, **p < 0.0001.
	FIGURE 6. Inhibition of TLR4 abolishes the protection of SEP against E. coli infection. (A) The experimental design of SEP administration in mice. Mice were treated with SEP (25 mg/kg) or vehicle (saline) by i. v. every 3 days for three times, and sacrificed on the eighth day. The total protein and mRNA were extracted from mice peritoneal macrophages. (B) The protein levels of p-STAT3, p-Src and p-Lyn in the peritoneal macrophages were determined by western blot. (C) The relative mRNA levels of CD64, CD32 and CD16 in the peritoneal macrophages were determined by q-PCR. (D) The experimental design of TAK-242 combined with SEP administration in mice infection model. The TAK-242 group mice were pre-treated with TAK-242 (10 mg/kg, i. p.) 2 h before administration of SEP, once every 3 days for three times. Then mice were further challenged with E. coli (3 × 108 CFU, i. p) on day eight and sacrificed at 18 h post-infection. (n = 8). (E) CFU of E. coli was detected in tissue homogenates of lung, spleen, liver, and peritoneal lavage fluid. (F) MPO value in the lung tissue. (G) The protein levels of CD64 and CD16/32 in the peritoneal macrophages were determined by flow cytometry. (H) The experimental design of TAK-242 combined with SEP administration in mice. The TAK-242 group mice were pre-treated with TAK-242 (10 mg/kg, i. p.) 2 h before administration of SEP, once every 3 days for three times, and sacrificed on the eighth day. (I) The protein levels of p-STAT3, p-Src and p-Lyn in the peritoneal macrophages were determined by western blot. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, ***p < 0.0001.

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	FIGURE 1. SEP protects against E. coli infection by enhancing host defense. (A) Molecular structure of SEP. (B) The effect of SEP on macrophages phagocytosis of live E. coli by gentamicin protection assay. RAW264.7 cells were administrated with Cyt D (10 μM) for 2 h prior to E. coli infection. (C) The effect of SEP on macrophages phagocytosis by fluorescence intensity. RAW264.7 cells were fed with pHrodo-conjugated inactivated E. coli bioparticles for 1 h and phagocytosis was quantified by the fluorescence. (D) The experimental design of E. coli infection and SEP treatment in mice. Mice were pre-treated with SEP (25 mg/kg) or vehicle (saline) by i. v. every 3 days for three times, and infected with E. coli (9 × 108 CFU per mouse, i. p.) on the eighth day. Then the survival of mice (E) were evaluated. (n = 10). (F) Mice were pre-treated with SEP (25 mg/kg, i. v.) and infected with E. coli (3 × 108 CFU per mouse, i. p.) on day eight, then sacrificed at 18 h post-infection. (n = 8). CFU of E. coli in tissue homogenates of lung, spleen, liver, and peritoneal lavage fluid were detected. (G) Lung sections were stained with H&E. (H) Myeloperoxidase (MPO) value were detected in the lung tissue. (I) The expression of IL-1β, TNF-α and IL-6 in the lungs were measured by ELISA. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, **p < 0.001.
	FIGURE 2. SEP promotes macrophages phagocytosis via TLR4. RAW264.7 cells were pre-treated with TLR2 inhibitor C29 (50 μM) or TLR4 inhibitor TAK-242 (20 μM) for 2 h, then stimulated with SEP (100 μg/ml) for another 24 h. (A) Phagocytosis of live E. coli by macrophages were measured by gentamicin protection assay. (B) Phagocytosis of pHrodo-E. coli by macrophages were quantified by fluorescence intensity. (C) The positive cell rates for phagocytosis of pHrodo-E. coli by macrophage were detected by flow cytometry. All data are represented as mean ± SEM acquired in triplicate determinations. ****p < 0.0001. ns, not significant.
	FIGURE 3. TLR4/STAT3 signaling is involved in SEP induced phagocytosis. (A) The effects of SEP on protein levels of p-p65, p-ERK, p-JNK and p-mTOR were determined by western blot. (B) The effects of TLR4 signaling pathway inhibitors on phagocytosis of live E. coli were measured by gentamicin protection assay. RAW264.7 cells were pre-treated with NF-κB inhibitor BAY 11-7085 (2 μM), or JNK inhibitor SP610025 (5 μM), or MEK1/2 inhibitor PD184352 (5 μM), or PI3K inhibitor LY294002 (5 μM) for 2 h prior to SEP treatment. (C) Phagocytosis of pHrodo-E. coli by macrophages were quantified by fluorescence intensity. (D, E) The effects of SEP on protein levels of p-STAT1, p-STAT3 were determined by western blot. The effects of Stattic (5 μM) or Fludarabine (20 μM) on phagocytosis of pHrodo-E. coli were measured by flow cytometry (F), or phagocytosis of live E. coli by gentamicin protection assay (G), or phagocytosis of pHrodo-E. coli by fluorescence intensity (H). All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, ***p < 0.0001.
	FIGURE 4. CD64 is responsible for SEP induced phagocytosis via TLR4/STAT3 signals. (A,B) The effects of SEP on the relative mRNA expression of CD64, CD32 and CD16 were determined by q-PCR. (C,D) The effects of SEP on the protein levels of CD64 and CD16/32 were determined by flow cytometry. (E) RAW264.7 cells were pre-treated with SEP (100 μg/ml) for 24 h, and followed by incubation with 2 μg/ml anti-CD64 or anti-CD16/32 blocking antibodies for 1 h prior to pHrodo-E. coli infection. Then macrophage phagocytosis was quantified by the fluorescence. (F) The effects of blocking CD64 or anti-CD16/32 on phagocytosis of live E. coli by macrophages were measured by gentamicin protection assay. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.001,**p < 0.0001. ns, not significant.
	FIGURE 5. SEP promotes filopodia formation via TLR4/STAT3 axis. (A) The effects of SEP on the filopodia formation of macrophages. RAW264.7 cells were administrated with Src family inhibitor AZD0530 (10 μM) or ROCK1 inhibitor Y-27632 (20 μM) for 2 h prior to E. coli infection. The cellular filamentous actin were stained with rhodamine phalloidin and the cellular DNA with DAPI. (B) The effects of SEP on protein levels of p-Src and p-Lyn were determined by western blot. (C) The effects of AZD0530 or Y-27632 on SEP induced phagocytosis of pHrodo-E. coli were measured by fluorescence intensity, (D) or phagocytosis of live E. coli by gentamicin protection assay, or (E) phagocytosis of pHrodo-E. coli by flow cytometry. (F) The effects of TAK-242 or Stattic on SEP induced filopodia formation. (G,H) The effects of TAK-242, or C29, or Stattic, or Fludarabine on SEP induced protein levels of p-Src and p-Lyn were determined by western blot. *p < 0.001, **p < 0.0001.
	FIGURE 6. Inhibition of TLR4 abolishes the protection of SEP against E. coli infection. (A) The experimental design of SEP administration in mice. Mice were treated with SEP (25 mg/kg) or vehicle (saline) by i. v. every 3 days for three times, and sacrificed on the eighth day. The total protein and mRNA were extracted from mice peritoneal macrophages. (B) The protein levels of p-STAT3, p-Src and p-Lyn in the peritoneal macrophages were determined by western blot. (C) The relative mRNA levels of CD64, CD32 and CD16 in the peritoneal macrophages were determined by q-PCR. (D) The experimental design of TAK-242 combined with SEP administration in mice infection model. The TAK-242 group mice were pre-treated with TAK-242 (10 mg/kg, i. p.) 2 h before administration of SEP, once every 3 days for three times. Then mice were further challenged with E. coli (3 × 108 CFU, i. p) on day eight and sacrificed at 18 h post-infection. (n = 8). (E) CFU of E. coli was detected in tissue homogenates of lung, spleen, liver, and peritoneal lavage fluid. (F) MPO value in the lung tissue. (G) The protein levels of CD64 and CD16/32 in the peritoneal macrophages were determined by flow cytometry. (H) The experimental design of TAK-242 combined with SEP administration in mice. The TAK-242 group mice were pre-treated with TAK-242 (10 mg/kg, i. p.) 2 h before administration of SEP, once every 3 days for three times, and sacrificed on the eighth day. (I) The protein levels of p-STAT3, p-Src and p-Lyn in the peritoneal macrophages were determined by western blot. All data are represented as mean ± SEM acquired in triplicate determinations. p < 0.05, p < 0.01, ***p < 0.0001.