Adjunctive Thymosin Beta-4 Treatment Influences PMN Effector Cell Function during Pseudomonas aeruginosa-Induced Corneal Infection

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.

Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis

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.

Discovery of Thymosin Beta-4 as a Human Exerkine and Growth Factor

Skeletal muscle is an endocrine organ secreting exercise-induced factors (exerkines), which play a pivotal role in inter-organ crosstalk. Using mass spectrometry (MS)-based proteomics, we characterized the secretome and identified thymosin beta-4 (TMSB4X) as the most upregulated secreted protein in the media of contracting C2C12 myotubes. TMSB4X was also acutely increased in plasma of exercising humans irrespective of the insulin resistance condition or exercise mode. Treatment of mice with TMSB4X did not ameliorate the metabolic disruptions associated with diet induced-obesity, nor did it enhance muscle regeneration in vivo. However, TMSB4X increased osteoblast proliferation and neurite outgrowth, consistent with its WADA-classification as a prohibited growth factor. Therefore, we report TMSB4X as a human exerkine with a potential role in cellular crosstalk.

Utilizing Developmentally Essential Secreted Peptides Such As Thymosin Beta-4 to Remind the Adult Organs of Their Embryonic State—New Directions in Anti-Aging Regenerative Therapies

Our dream of defeating the processes of aging has occupied the curious and has challenged scientists globally for hundreds of years. The history is long, and sadly, the solution is still elusive. Our endeavors to reverse the magnitude of damaging cellular and molecular alterations resulted in only a few, yet significant advancements. Furthermore, as our lifespan increases, physicians are facing more mind-bending questions in their routine practice than ever before. Although the ultimate goal is to successfully treat the body as a whole, steps towards regenerating individual organs are even considered significant. As our initial approach to enhance the endogenous restorative capacity by delivering exogenous progenitor cells appears limited, we propose, utilizing small molecules critical during embryonic development may prove to be a powerful tool to increase regeneration and to reverse the processes associated with aging. In this review, we introduce Thymosin beta-4, a 43aa secreted peptide fulfilling our hopes and capable of numerous regenerative achievements via systemic administration in the heart. Observing the broad capacity of this small, secreted peptide, we believe it is not the only molecule which nature conceals to our benefit. Hence, the discovery and postnatal administration of developmentally relevant agents along with other approaches may result in reversing the aging process.

Thymosin Beta 4 as an Early Biomarker in Sepsis Induced Acute Kidney Injury

Background: The incidence of sepsis is high among patients in the intensive care units (ICU) and acute kidney injury (AKI) is a common complication of sepsis that contributes to increased mortality. Thymosin beta-4 (Tβ4) is an actin-sequestering protein that can prevent inflammation and fibrosis in several tissues. However, its functions in septic AKI remain unknown.Methods: 98 consecutive hospitalized patients with confirmed sepsis were enrolled. Demographics, comorbidities, laboratory findings, and outcomes were collected and analyzed. Serum Tβ4 levels at ICU admission were measured and analyzed for evaluating the probability of AKI using the logistic regression. In addition, the effects of exogenous Tβ4 on kidney injury was also conducted in mice where a sepsis model was induced by lipopolysaccharide (LPS) intraperitoneal injection. Results: Of the 98 patients with sepsis, 47 (48%) developed AKI. Patients with hypertension, diabetes, higher body mass index (BMI) and Sequential Organ Failure Assessment (SOFA) score were more likely to develop AKI. Among patients with AKI, hemoglobin, and Tβ4 were significantly decreased. Multivariate analysis showed decreased Tβ4, high SOFA, and high BMI to be independent risk factors for AKI in patients with sepsis. The overall mortality rate of the 98 septic patients was 20.4%, and the mortality rate of those with AKI was 29.8%. Kaplan-Meier analysis demonstrated that patients with AKI had a significantly higher risk of death. In particular, increasing AKI severity was associated with an increased risk of death. Furthermore, exogenous Tβ4 could reduce renal apoptosis and attenuated renal dysfunction, as well as reducing systemic inflammatory response through the prevention of the activation of NF-κB pathway in the sepsis model.Conclusions: The combination of Tβ4, SOFA, and BMI could allow for timely detection of septic AKI. Exogenous Tβ4 could prevent kidney injury in sepsis.

Recombinant Human Thymosin Beta-4 Protects against Mouse Coronavirus Infection

Coronaviruses (CoVs) are enveloped and harbor an unusually large (30–32 kb) positive-strand linear RNA genome. Highly pathogenic coronaviruses cause severe acute respiratory syndrome (SARS) (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome (MERS) (MERS-CoV) in humans. The coronavirus mouse hepatitis virus (MHV) infects mice and serves as an ideal model of viral pathogenesis, mainly because experiments can be conducted using animal-biosafety level-2 (A-BSL2) containment. Human thymosin beta-4 (Tβ4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tβ4 regulates actin polymerization and functions as an anti-inflammatory molecule and an antioxidant as well as a promoter of wound healing and angiogenesis. These activities led us to test whether Tβ4 serves to treat coronavirus infections of humans. To test this possibility, here, we established a BALB/c mouse model of coronavirus infection using mouse CoV MHV-A59 to evaluate the potential protective effect of recombinant human Tβ4 (rhTβ4). Such a system can be employed under A-BSL2 containment instead of A-BSL3 that is required to study coronaviruses infectious for humans. We found that rhTβ4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host’s immune response, alleviating pathological damage, and promoting repair of the liver. These results will serve as the basis for further application of rhTβ4 to the treatment of human CoV diseases such as COVID-19.