In vitro Interaction of Pseudomonas aeruginosa Biofilms With Human Peripheral Blood Mononuclear Cells.

Esingül Kaya,Giuseppantonio Maisetta,Mariagrazia Di Luca,Arianna Benedetti,Carolina Pileggi,Lucia Grassi,Giovanna Batoni,Semih Esin

doi:10.3389/fcimb.2020.00187

Esingül Kaya, Giuseppantonio Maisetta + Show 6 more

Open Access

https://doi.org/10.3389/fcimb.2020.00187

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Abstract

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was <10% upon 24 h incubation of PBMC with P. aeruginosa biofilms. PBMC incubated for 24 h with preformed P. aeruginosa biofilms were significantly more activated compared to PBMC incubated alone. Interestingly, a marked activation of CD56+CD3− natural killer (NK) cells was observed that reached 60% of NK cells as an average of different donors. In the culture supernatants of PBMC co-cultured with P. aeruginosa biofilms, not only pro-inflammatory (IL-1β, IFN-γ, IL-6, and TNF-α) but also anti-inflammatory (IL-10) cytokines were significantly increased as compared to PBMC incubated alone. Furthermore, incubation of biofilms with PBMC, caused a statistically significant increase in the CFU number of P. aeruginosa, as compared to biofilms incubated without PBMC. In order to assess whether PBMC products could stimulate the growth of P. aeruginosa biofilms, we incubated preformed P. aeruginosa biofilms with or without supernatants obtained from the co-cultures of PBMC with biofilms. In the presence of the supernatants, the CFU count of biofilm-derived P. aeruginosa, was two to seven times higher than those of biofilms incubated without supernatants (P < 0.01). Overall, the results obtained shed light on the reciprocal interaction between human PBMC and P. aeruginosa biofilms. P. aeruginosa biofilms induced PBMC activation and cytokine secretion but, in turn, the presence of PBMC and/or PBMC-derived components enhanced the number of P. aeruginosa biofilm associated bacteria. This may indicate a successful bacterial defensive/persistence strategy against immune response.

Highlights

Pseudomonas aeruginosa is an environmental Gram-negative opportunistic pathogen involved in a large spectrum of infections, especially in immunocompromised and hospitalized patients (Driscoll et al, 2007)
To assess the exact number of initial bacteria used in each experiment, an aliquot was plated on Tryptone Soya Agar (TSA; Oxoid, Basingstoke, UK) plates for colony forming unit (CFU) count
The biofilm forming ability of P. aeruginosa ATCC 27853 in an eukaryotic cell-compatible medium was assessed by incubating stationary phase P. aeruginosa in complete RPMI 1640 medium

Summary

Introduction

Pseudomonas aeruginosa is an environmental Gram-negative opportunistic pathogen involved in a large spectrum of infections, especially in immunocompromised and hospitalized patients (Driscoll et al, 2007). The pathogenesis of many P. aeruginosa infections depends on a striking ability of the bacterium to form biofilms, complex bacterial communities adhering on a substrate, such as mucosal surfaces or invasive medical devices (TolkerNielsen, 2014). The EPS acts like a barrier that hampers the diffusion of antibiotics as well as host immune cells. In this regard, it has been reported that antibodies or phagocytic cells at most enter the water channels intercalating the micro-colonies that constitute a mature biofilm (Costerton et al, 2003), but hardly penetrate into the deep layers of the micro-communities, especially when biofilms are grown under static conditions (Leid et al, 2002)

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