Polo-like Kinase 1 Protects Intestinal Epithelial Cells from Apoptosis During Sepsis via the Nuclear Factor-κB Pathway
Sepsis, a life-threatening condition characterized by dysregulated host responses to infection, often culminates in multiple organ dysfunction. The intestinal barrier, a critical defense mechanism, becomes compromised during sepsis due to epithelial apoptosis, leading to barrier failure and exacerbated systemic inflammation. This study investigates the role of the nuclear factor-κB (NF-κB) pathway and its interaction with polo-like kinase 1 (PLK1) in regulating intestinal epithelial cell survival during sepsis.
Experimental Models and Methods
The study employed an in vitro sepsis model using the human colorectal cancer cell line HT-29, treated with lipopolysaccharide (LPS) to simulate intestinal inflammation. Apoptosis was quantified via Annexin V/fluorescein isothiocyanate staining and flow cytometry. NF-κB activation was assessed through immunofluorescence to detect nuclear translocation of the p65 subunit and Western blotting to measure inhibitor of κB-α (IκB-α) degradation. PLK1 expression was modulated using its inhibitor BI2536, while NF-κB activity was suppressed with pyrrolidine dithiocarbamic acid (PDTC).
Clinical validation involved 21 patients with septic shock secondary to intestinal obstruction or perforation. Surgically resected necrotic intestinal tissues and adjacent morphologically normal tissues were analyzed via immunohistochemistry for PLK1, NF-κB p65, pro-caspase-3, and pro-caspase-9 expression.
LPS Activates NF-κB and Induces Apoptosis
LPS treatment in HT-29 cells triggered NF-κB activation, evidenced by reduced IκB-α levels (Figure 1A) and nuclear translocation of p65 (Figure 1B). Apoptosis increased dose-dependently with LPS, correlating with elevated caspase-3 cleavage. To confirm NF-κB’s role, cells were pretreated with PDTC, an NF-κB inhibitor. PDTC pretreatment significantly attenuated LPS-induced apoptosis, restoring pro-caspase-3 and IκB-α levels (Figure 1C). This demonstrated that NF-κB activation exacerbates apoptosis in sepsis-like conditions.
PLK1 Suppresses NF-κB Activity
Previous work by the authors revealed LPS-induced PLK1 downregulation in HT-29 cells. Here, PLK1 inhibition with BI2536 reduced IκB-α expression (Figure 1D) and enhanced p65 nuclear localization (Figure 1E), mimicking LPS effects. Conversely, PDTC-mediated NF-κB inhibition did not alter PLK1 expression (Figure 1F), indicating that PLK1 acts upstream of NF-κB. These findings suggest PLK1 normally restrains NF-κB activity, and its loss during sepsis unleashes pro-apoptotic NF-κB signaling.
Clinical Correlates in Human Sepsis
Immunohistochemical analysis of patient tissues revealed higher pro-caspase-3 and pro-caspase-9 levels in normal-appearing intestinal margins compared to necrotic tissues (Supplementary Figure 1A, 1B). Necrotic tissues exhibited nuclear NF-κB p65 localization, absent in normal regions, confirming NF-κB activation in sepsis-damaged epithelia. PLK1 expression was markedly lower in normal margins, aligning with in vitro data that PLK1 downregulation permits NF-κB-driven apoptosis.
Mechanistic Insights and Therapeutic Implications
The study positions PLK1 as a critical regulator of intestinal epithelial survival during sepsis. Under physiological conditions, PLK1 inhibits NF-κB, preventing excessive apoptosis. During sepsis, LPS suppresses PLK1, relieving this inhibition and enabling NF-κB activation. While NF-κB is traditionally associated with pro-survival signaling via anti-apoptotic genes, this work highlights its context-dependent role in promoting apoptosis during intestinal inflammation.
The dual role of NF-κB in sepsis—pro-inflammatory cytokine production versus apoptosis regulation—underscores its complex involvement. In the gut, NF-κB activation may initially aid in pathogen clearance but becomes detrimental when unchecked, driving epithelial loss and barrier failure. PLK1’s ability to modulate this pathway offers a potential therapeutic target. Restoring PLK1 activity or pharmacologically inhibiting NF-κB could mitigate intestinal apoptosis, preserving barrier integrity and improving sepsis outcomes.
Conclusion
This study delineates a PLK1-NF-κB axis critical for intestinal epithelial cell survival during sepsis. LPS-induced PLK1 downregulation unleashes NF-κB, accelerating apoptosis and barrier dysfunction. Both in vitro and clinical data corroborate this mechanism, highlighting PLK1 as a guardian of epithelial integrity. Targeting this pathway may provide novel strategies to combat sepsis-induced organ failure, emphasizing the need for context-specific modulation of NF-κB in therapeutic interventions.
doi.org/10.1097/CM9.0000000000000780
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