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Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification or scarring, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts.Pseudomonas aeruginosaandStaphylococcus aureusare the two bacteria most commonly associated with this type of infection. Risk factors include patients who are immunocompromised, those who have undergone refractive corneal surgery, and those with prior penetrating keratoplasty, as well as extended wear contact lens users. Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics. Bacterial clearance is of utmost importance yet does not guarantee good visual outcome. Clinicians are often left to rely upon the eye's innate ability to heal itself, as there are limited options beyond antibiotics and corticosteroids for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, and anti-inflammatory drops, do not fully accommodate clinical needs and have many potential harmful complications. To this end, treatments are needed that both regulate the inflammatory response and promote corneal wound healing to resolve visual disturbances and improve quality of life. Thymosin beta 4 is a small, naturally occurring 43-amino-acid protein that promotes wound healing and reduces corneal inflammation and is currently in Phase 3 human clinical trials for dry eye disease. Our previous work has shown that topical Tβ4 as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages) while enhancing bacterial killing and wound healing pathway activation in an experimental model ofP. aeruginosa-induced keratitis. Adjunctive thymosin beta 4 treatment holds novel therapeutic potential to regulate and, optimally, resolve disease pathogenesis in the cornea and perhaps other infectious and immune-based inflammatory disease. We plan to establish the importance of thymosin beta 4 as a therapeutic agent in conjunction with antibiotics with high impact for immediate clinical development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

Our previous work has shown that topical thymosin beta 4 (Tβ4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa -induced keratitis. This study aimed to mechanistically examine how Tβ4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa -induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tβ4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tβ4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tβ4 alone and adjunctive Tβ4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tβ4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.

Prior work has indicated that thymosin beta 4 (Tβ4) administered with ciprofloxacin markedly improves disease outcome for Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tβ4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tβ4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tβ4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tβ4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tβ4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.

With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tβ4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (ROS) levels were determined. Adjunct Tβ4 treatment resulted in a significant improvement compared to PBS, Tβ4, and most remarkably, ciprofloxacin, correlating with changes in mediators of inflammation and wound healing. Collectively, these data provide evidence that wound healing is an essential aspect in the development of new therapies to treat corneal infection. Use of adjunctive Tβ4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response. [ABSTRACT FROM AUTHOR]