Thymosin Beta 4: A Potential Novel Adjunct Treatment for Bacterial Keratitis

Thymosin beta-4 (Tβ4) is a naturally occurring polypeptide that promotes rapid corneal re-epithelialization and a reduction in corneal inflammation (Sosne et al., 2002; Sosne et al., 2005). With the increasing incidence of antibiotic resistance and contraindication for corticosteroid use in bacterial keratitis (BK), it is logical to consider Tβ4 as an adjunct therapy to antibiotic to prevent corneal perforation and accelerate wound healing. Tβ4 is a ubiquitous highly conserved across species, 43-amino acid acidic polypeptide with a molecular weight of 4.9 kDa. and is found in all tissues and cell types except red blood cells (Goldstein et al., 2015). Tβ4 is a multifunctional protein that promotes cell migration, stem cell recruitment and differentiation, protease production, and the expression of various regulatory genes, such as laminin-332, fibronectin, zyxin, VEGF, matrix metalloproteases, hepatocyte growth factor, and antioxidative enzymes (Sosne and Kleinman 2015). It inhibits inflammation, microbial growth, scar formation (by reducing the level of myofibroblasts), and apoptosis, and protects cells from cytotoxic damage, including glutamate neuronal toxicity (Sosne and Ousler 2015; Sosne et al., 2016; Magharious et al., 2011).

Tβ4 binds to G-actin, blocks actin polymerization, and is co-released with factor XIIIa by platelets, suggesting its importance in wound healing (Goldstein and Kleinman 2015; Sosne and Kleinman 2015). Tβ4 promotes full thickness dermal wound repair in normal, steroid-treated, and diabetic animals and following dermal injury, high levels of Tβ4 are naturally present in the wound fluid (Philp et al., 2003; Malinda et al., 1999). Tβ4 is also active for repair and regeneration in the eye, heart, brain, peripheral nervous system, and spinal cord and promotes angiogenesis in some tissues, but not when added topically to the wounded eye surface (Sosne et al., 2002). A number of active sites on Tβ4 have been identified for some of these activities.30,31 Amino acid fragments 1–4 is anti-inflammatory, 1–15 is anti-apoptotic and cytoprotective, and 17–23 is active for cell migration, actin binding, dermal wound healing, angiogenesis, and hair growth (Sosne et al., 2010; Philp et al., 2003). Surprisingly little is known about the potential receptors. Purinergic signaling pathways have been reported, but given the number of activities and active sites, one would expect several receptors (Freeman et al., 2011; Yang et al., 2020). Much is also unknown about the role of Tβ4 in the nucleus. Upon incubation with cells, it is rapidly (30 minutes) transported to the nucleus where it may function as a transcription factor (Sosne et al., 2007; Qiu et al., 2011; Kim et al., 2017).

In an animal model of dry eye, Tβ4 promotes corneal integrity in part through increases in laminin-332 production (Sosne et al., 2002; Sosne et al., 2004; Sosne et al., 2010). Such activities in “normalizing” the corneal epithelium are expected to contribute in part to the improved comfort of the corneal keratitis patients receiving Tβ4. Scar formation is a severe and debilitating consequence of PA keratitis that leads to blindness and Tβ4 also reduces scar formation/fibrosis in many tissues (skin, heart, lungs, kidney, and liver) with scarring due to varying underlying causes (Li et al., 2018; Conte et al., 2013; Hong et al., 2017). It has not yet been tested for reduction in scar formation in the eye. Tβ4 is present in both cells and body fluids including tears and has shown efficacy in treating surface ocular wounds in animal models and in patients (Goldstein and Kleinman 2015; Sosne and Kleinman 2015).

In various animal eye injury models including, scrape injury, alkali burn injury and infectious keratitis, topical Tβ4 drops have been demonstrated to promote wound healing and reduce corneal inflammation. Tβ4 is currently in Phase 3 human clinical trials for dry eye disease and neurotrophic keratopathy. We recently published the efficacy and safety of 0.1% Tβ4 in promoting the healing of persistent epithelial defects in patients with Stages 2 and 3 neurotrophic keratopathy (Sosne et al., 2022). No significant adverse effects were observed in the trial. Tβ4 promoted rapid healing of epithelial defects in neurotrophic keratopathy, improved ocular comfort, and was found to be safe for treating this challenging population of patients.

Based on our prior data, we hypothesized that we could extend the use of topical Tβ4 as an important clinical therapeutic agent in the setting of BK. Bacterial keratitis is a serious ocular infectious disease that can rapidly lead to severe visual disability including corneal scarring, endophthalmitis, and perforation. The severity of the corneal infection usually depends on the underlying condition of the cornea and the pathogenicity of the infecting bacteria. Many patients have a poor clinical outcome if aggressive and appropriate therapy is not promptly initiated. However, in the absence of predisposing risk factors, bacterial keratitis is rare. Corneal opacification or scarring, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts (Ting et al., 2021). Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics, although bacterial clearance alone without maximum corneal wound healing does not necessarily guarantee good visual outcome.

We previously demonstrated that topical 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 Pseudomonas Aeruginosa (PA)-induced keratitis (Carion et al., 2020; Carion et al., 2018; Wang et al., 2021; Wang et al 2021). Adjunctive Tβ4 treatment holds novel therapeutic potential to regulate and, optimally, resolve disease pathogenesis in the cornea and perhaps other infectious and immune-based inflammatory disease due to the fast action of 0.1% Tβ4 in healing the defect and normalizing the ocular surface. This is likely due to its multiple mechanisms of action beyond promoting corneal epithelial cell migration to repair the epithelial defect (Sosne et al., 2002). For example, in animal models topical Tβ4 drops have been shown to reduce inflammation which can cause pain, burning, itching, etc. as well as contribute to the cellular/stromal damage (Sosne et al., 2002; Sosne et al., 2005).

Usually corneal ulceration stems from a simple minor trauma or abrasion but can finally end up with severe ocular morbidity and often prolonged suffering. A corneal ulcer which is easily countered by early diagnosis and treatment in the Western world, is considered a “silent epidemic” in low-and-middle income countries (Whitcher and Srinivasan 1997). The main cause of corneal ulcers varies with geographical locations: in developed countries it is typically associated with contact lens wear while in developing countries, corneal ulcers are often due to injury which primarily afflicts laborers involved in agriculture, manufacturing and domestic sectors (Byrd and Martin 2022, Katara et al., 2013). WHO has estimated 1.5–2.0 million new cases of unilateral blindness each year worldwide from corneal infection and in developing nations, corneal bacterial infections are estimated to be 10 times more than in developed nations (Srinivasan 2017, Ezegwui 2010, Rathi et al., 2002). Preventing corneal abrasions from becoming ulcers requires quick and accurate diagnosis and treatment. Also, liberal use of antibiotics is known to cause the rise of anti-microbial resistance and may make future treatment challenging (Wong et al., 2012).

Until recently, most cases of bacterial keratitis were associated with ocular trauma or ocular surface diseases. However, the widespread use of contact lenses has dramatically increased the incidence of contact lens related keratitis (Stapleton et al., 2012). Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can induce bacterial keratitis, especially in contact lens users. In the past several years, there has been a steady increase in contact lens wearers. Subsequently, contact lens wear is now the major predisposing factor for corneal infection in the United States and Western Europe (Low and Agarwal 2010). Soft contact lenses have greatly increased the risk of bacterial keratitis, which is estimated to be 10–20 times higher with the use of extended wear disposable contact lenses (ibid). Many physio-pathological effects of contact lens wear have been reported, the most important of which is an induced hypoxia and hypercapnia of the cornea. History of ocular surface diseases represents the second most common cause of bacterial keratitis, accounting for 21% of cases (Ang and Efron 2010, Rivera and Polse 1996, Lin et al., 2018)).

PA and Staphylococcus aureus are the two bacteria most associated with this type of infection (Lin et al., 2018). 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. PA is a pathogenic, gram-negative bacterium that despite the recent development of silicone hydrogel lenses has remained the most frequent cause of contact lens-related microbial keratitis (Hilliam et al., 2020). Following initial binding, invasive clinical isolates of PA have the unusual ability to invade and replicate within surface corneal epithelial cells in animal models of infection (LD Hazlett 2004).

Commonly used anti-microbial agents such as aminoglycosides (gentamycin, tobramycin, amikacin) cannot penetrate the cell membrane that increases the likelihood of a more adverse outcome which has led to the increased usage of fluoroquinolones. Topical fourth-generation fluoroquinolones, namely, moxifloxacin and gatifloxacin, are good alternatives to combination of fortified antibiotics in the management of infectious keratitis and they are often used clinically as empirical therapy. However, further studies are warranted to compare the response of PA infections to these antibiotics before we can conclude that the new fluoroquinolones are as potent as the standard combination of fortified antibiotics in the management of infectious keratitis. Fluoroquinolones are synthetic broad-spectrum antibiotics that inhibit DNA gyrase (topoisomerase II) and topoisomerase IV enzyme, which are key enzymes involved in DNA replication and transcription and inhibition of these enzymes leads to bacterial cell death (Herbert et al., 2022).

As stated, infectious keratitis is a major global cause of visual impairment and blindness, often affecting marginalized populations. Topical antibiotics remain the best treatment for bacterial keratitis, and a recent review found all commonly prescribed topical antibiotics to be equally effective. However, outcomes remain poor secondary to corneal melting, scarring, and perforation. Adjuvant therapies aimed at reducing the immune response associated with keratitis include topical corticosteroids. The large, randomized, controlled Steroids for Corneal Ulcers Trial found that although steroids provided no significant improvement overall, they did seem beneficial for ulcers that were central, deep or large, non-Nocardia, or classically invasive PA; for patients with low baseline vision; and when started early after the initiation of antibiotics (O’Callaghan et al., 1996). Therefore, novel therapies in addition to antibiotics are needed to promote optimal healing and recovery from corneal ulcers.

Recent studies have identified membrane lipid rafts as the key element mediating internalization of PA by surface corneal epithelial cells in both in vivo and in vitro test conditions (Yamamato et al., 2005). Rafts possess the unique and fascinating property of macromolecular aggregation to form large membrane platforms which have been implicated in mediating many cellular functions including membrane trafficking, signal transduction in T cell lymphocytes and leukocytes, as well as the regulation of integrin function (Zaidi et al., 2008; Deretic et al., 1991). Recent in vitro studies, under serum-free conditions, have also revealed that lipid rafts mediate internalization of PA in human lung epithelial cells (Curran et al., 2018). Internalization involves an endocytic process whereby aggregated raft platforms act as elevator gateways into the interior of the cells. Two sub-components of lipid rafts, GMI and cholesterol appear to be essential to the internalization phenomenon (Schiumarini et al., 2017).

Bacterial products released during PA keratitis may damage the cornea directly and induce host reactions that contribute to corneal scarring and eventual reduction in visual acuity (LD Hazlett 2004; Marquart and O’Callaghan 2013; O’Callaghan et al., 2019). The significance of protease IV as a mediator of virulence and corneal damage has led to multiple reports describing the therapeutic use of protease inhibitors to limit corneal damage caused by PA keratitis (O’Callaghan et al., 2019). Furthermore, the results provide a target for the development of new inhibitors which, in conjunction with antibiotics, could reduce or eliminate corneal damage. Inflammation contributes to corneal damage, especially the migration of polymorphonuclear leukocytes into the cornea and the subsequent release from these cells of damaging lysosomal enzymes and oxidative molecules (O’Callaghan et al., 2019). Despite these reports, there is not much new in the treatment of PA keratitis. Antibiotic and anti-inflammatory therapy can reduce, but not prevent, corneal damage and scarring. We believe that the future development of therapies capable of preventing corneal damage is dependent on the identification and inhibition of those bacterial products that directly damage the host and/or elicit damaging inflammatory reactions.

To date, there are only a few papers in the literature that have reported the effect of photodynamic therapy for collagen crosslinking (collagen CXL) in the management of infectious keratitis (Alio et al., 2013; Makdoumi et al., 2010; Martins et al., 2008). The results of these trials appeared promising and imply that this new treatment modality may be useful in the treatment of resistant infectious corneal ulcer or as an adjunct for standard antibiotic treatment. However, since all of the published studies regarding CXL as the treatment of infectious keratitis were either based on animals or small numbers of patients, larger scale randomized, controlled trials should be conducted to evaluate the additional beneficial effects of CXL in infectious keratitis on top of conventional topical antibiotics. Furthermore, more evidence is required before it will be advisable to use CXL as the first line treatment for infectious corneal ulcers.

All things considered, clinicians are often left to rely upon the eye’s innate ability to heal itself, as there are limited options for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, amniotic membrane grafts, and anti-inflammatory drops do not fully accommodate clinical needs and have 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 we propose the use of Tβ4 as a novel adjuvant therapy in the treatment of BK. We plan to establish the potential importance of Tβ4 as a therapeutic agent in conjunction with antibiotics and to translate these findings into clinical development. Part of the explanation could be due to the fast action of 0.1% Tβ4 in healing the defect and normalizing the ocular surface. This is likely due to its multiple mechanisms of action beyond promoting corneal epithelial cell migration to repair the epithelial defect (Sosne et al., 2002). For example, in animal models topical Tβ4 drops have been shown to reduce inflammation which can cause pain, burning, itching, etc. as well as contribute to the cellular/stromal damage (Sosne et al., 2002; Sosne et al., 2005). Further mechanistic laboratory studies are warranted to uncover the specific corneal pathways and molecules affected by adjuvant topical Tβ4 therapy. In turn, we believe that the evidence is mounting that human clinical trials will soon be indicated and necessary to translate these basic science findings into novel clinical applications.