Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β–lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons.

Background: We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein-2 (BMP2) and BMP4 micro-immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. Methods: We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization-related enzymes (matrix metalloproteinase-1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long-term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. Results: LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re-establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. Conclusions: LD BMP4 MI treatment shows anti-inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis. K E Y W O R D Sbone morphogenetic protein 4, gingiva, in vitro technique, periodontitis, bone morphogenetic protein 2

In the last years, several studies testing commercial periodontal gels that contain chlorhexidine (CHX) or other antibacterial agents, have raised concerns regarding their cytotoxicity in periodontal tissues. We aimed at comparing the biocompatibility but also the efficacy as regards to the antibacterial and wound healing ability of different commercial periodontal gels. In vitro human gingival fibroblasts (GF) and a 3D model of human tissue equivalents of gingiva (GTE) were used under inflammatory conditions to evaluate wound closure, cytotoxicity and gene expression. Antibacterial effects were also investigated on Porphyromonas gingivalis growth, viability and gingipain activity. In GF and in the bacterial study, we found cytotoxic effects on GF and a high inhibition on bacterial growth rate in gels containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol. Of the two gels that were non-cytotoxic, Syntoss Biogel (containing chondrontin sulfate) and Emdogain (EMD, containing amelogenin and propylene glycol alginate), EMD showed the best wound closure, with no effect on P. gingivalis growth but decreased gingipain activity. On the other hand, Syntoss Biogel reduced viability and gingipain activity of P. gingivalis, but lack wound healing capacity. In the 3D GTE, Syntoss Biogel and EMD showed a good biocompatibility. Among all the tested gels, formulations containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol showed high antibacterial effect but also showed high cytotoxicity in eukaryotic cells. EMD was the one with the best biocompatibility and wound healing ability at the conditions tested.