Abstract
Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tβ4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tβ4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tβ4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tβ4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tβ4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tβ4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.
Highlights
Microbial keratitis is a sight-threatening infection of the cornea often resulting from ocular injury or extended contact lens wear [1,2]
These findings reveal that adjunctive Tβ4 regulates NETosis toto improvedisease diseaseoutcome outcomefollowing followingP.P.aeruginosa-induced aeruginosa-inducedcorneal cornealinfection
neutrophil elastase (NE) were while levels remarkably inhibited by the ciprofloxacin andProtein the adjunctive analyzed by Western blot as well (B)
Summary
Microbial keratitis is a sight-threatening infection of the cornea often resulting from ocular injury or extended contact lens wear [1,2]. The pathogenesis related to microbial keratitis involves both the host and bacteria [6] Regarding the latter, P. aeruginosa can produce various cell-associated and extracellular virulence factors including toxins and proteases to trigger and sustain the infection, leading to tissue damage [7]. Excessive levels of ROS can induce lipid peroxidation upon contact with membrane phospholipids resulting in the formation of toxic breakdown products that alter cellular function and cause tissue degradation [12]. Another more recently recognized powerful effector mechanism of PMNs is the release of neutrophil extracellular traps or NETs to capture and kill pathogens. There are at least three diverse mechanisms by which NETs are formed: the classical or suicidal NETosis, the noncanonical pathway, and the vital NETosis
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