Multidrug-Resistant Pseudomonas aeruginosa Evokes Differential Inflammatory Responses in Human Microglial and Retinal Pigment Epithelial Cells.

Poonam Naik,Ashok Kumar,Inderjeet Kaur,Vivek Pravin Dave,Sushma Vishwakarma,Joveeta Joseph,Sukhvinder Singh

doi:10.3390/microorganisms8050735

Abstract

Increasing incidences of multidrug-resistant (MDR) pathogens causing endophthalmitis threaten our ability to treat this condition, and the modulation of inflammatory responses by MDR bacteria is not known. In this study, using human microglia and retinal pigment epithelial (RPE) cells, we compare the inflammatory responses of sensitive (S-PA) and multidrug-resistant (MDR-PA) clinical isolates of Pseudomonas aeruginosa. Infected cells were subjected to qPCR analysis, enzyme-linked immunosorbent assay (ELISA), and immunostaining to assess the expression of inflammatory mediators. Both microglia and RPE cells, challenged with S-PA and MDR-PA, induced a time-dependent expression of inflammatory cytokines. Significant differences were observed in expression levels of Toll-like receptors (TLR) TLR4, TLR5, and TLR9 in microglia cells challenged with MDR-PA vs. S-PA. Similarly, mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, Interferon (IFN)-γ, and matrix metalloproteinase (MMP)-9 were also higher in MDR-PA-infected cells. At protein levels, upregulation was observed for IL-10 (p = 0.004), IL-8 (p = 0.0006), IL-1β (p = 0.02), and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (p = 0.0006) in cells infected MDR-PA versus S-PA in both microglia and RPE cells; however, the response was delayed in RPE cells. Heatmap and STRING analysis highlighted the existence of a cross-talk between the inflammatory and cytokine-mediated signaling pathways. Our study highlights a differential inflammatory response evoked by MDR vs. sensitive pathogens in retinal cells during endophthalmitis.

Highlights

Multidrug-resistant (MDR) bacterial endophthalmitis remains a highly fulminant inflammatory condition, with a worse visual prognosis than infection by sensitive bacterial pathogens [1]
Since microglia and retinal pigment epithelial (RPE) cells have the ability to phagocytose bacteria, we assessed the bacterial viability and intracellular proliferation of MDR P. aeruginosa (MDR-PA) and strain of P. aeruginosa (S-PA) strains in both cell lines
The enumeration of colony-forming units (CFUs) at various times post-infection (2–24 h) revealed a 1–3 log increase in bacterial burden in cells challenged with MDR-PA versus S-PA (Figure 1A,B), at the later time points (12 h and 24 h), with the intracellular bacterial burden being higher in cells challenged with MDR-PA vs. S-PA after inoculation (10.65 log CFU/mL vs. 8.66 log CFU/mL; p = 0.008, 12 h; 10.60 log CFU/mL vs. 8.99 log CFU/mL; p = 0.01, 24 h in microglial cells, and 10.99 log CFU/mL vs. 8.95 log CFU/mL; p = 0.009, 12 h and 11.23 log CFU/mL vs. 9.66 log CFU/mL.; p = 0.01, 24 h in RPE cells)

Summary

Introduction

Multidrug-resistant (MDR) bacterial endophthalmitis remains a highly fulminant inflammatory condition, with a worse visual prognosis than infection by sensitive bacterial pathogens [1]. Despite recognizing the importance of the role of the host’s immune defenses, most studies focus only on the pharmacokinetics (PK) of the pathogen and the particular antibiotic, while the role of host immune response remains inconclusive [4,5,6,7]. Recent studies have shown that the immune response against antibiotic, MDR, and susceptible bacterial strains of the same species may be significantly different [8,9]. This inevitably raises the question about the role of immune mediators, such as pro- and anti-inflammatory cytokines, along with Toll-like receptors (TLRs), during MDR bacterial infections

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Results

Conclusion