By Gilang Cahya 
In preclinical models, the inhalation of a mixture of living Lactobacilli bacteria attenuated pulmonary inflammation and improved lung function and structure for the chronic lung diseases bronchopulmonary dysplasia and chronic obstructive pulmonary disease. This groundbreaking research, published in the esteemed journal Nature Communications, elucidates a novel mechanism by which a live biotherapeutic product, specifically a powder mixture of living Lactobacilli bacteria, effectively mitigates neutrophilic inflammation and a wide spectrum of inflammatory markers associated with both bronchopulmonary dysplasia (BPD) and chronic obstructive pulmonary disease (COPD).
The study was co-led by Charitharth Vivek Lal, M.D., a neonatologist at the University of Alabama at Birmingham (UAB), and Amit Gaggar, M.D., Ph.D., a pulmonologist also at UAB. Their findings offer a paradigm shift in understanding the progression of structural lung disease, identifying Lactobacilli as crucial regulators of lung protease activity. This activity is intrinsically linked to the destructive processes driven by matrikine generation, extracellular matrix turnover, and chronic neutrophilic inflammation, all of which contribute to the damage of lung air sacs.
Genesis of a Promising Therapeutic Avenue
The foundational insights for this research emerged in 2016 when Dr. Lal and his UAB colleagues observed a significant correlation between severe BPD in infants and a depleted presence of Lactobacilli in their airways. Concurrently, these infants exhibited an increased abundance of proteobacteria and elevated concentrations of proteobacterial endotoxin. This discovery suggested a potential protective role for Lactobacilli in the lung and hinted at their therapeutic potential for chronic lung diseases. The current study builds directly upon these observations, providing a robust mechanistic explanation for how Lactobacilli treatment can curb downstream disease development. Furthermore, it demonstrates the safety and efficacy of this live biotherapeutic approach in both a mouse pup model of BPD and three distinct mouse models of COPD.
Understanding the Target Diseases: BPD and COPD
Bronchopulmonary dysplasia (BPD) is a serious chronic lung disease that affects some extremely premature infants. It often develops as a consequence of lung damage incurred from the high oxygen concentrations or mechanical ventilation required to sustain their lives in the neonatal intensive care unit. The delicate lungs of these infants are particularly vulnerable to injury, and the treatments necessary for survival can inadvertently lead to long-term respiratory complications.
Chronic obstructive pulmonary disease (COPD), on the other hand, is a progressive and debilitating lung disease primarily affecting older adults, with a significant proportion of cases linked to smoking. COPD is characterized by persistent airflow limitation and respiratory symptoms, leading to significant morbidity and mortality. Globally, COPD is a leading cause of death, claiming approximately 3 million lives annually, with an additional 130,000 deaths occurring in the United States each year. The shared underlying pathology of chronic inflammation and lung tissue destruction in both BPD and COPD makes them compelling targets for a unified therapeutic strategy.
Unraveling the Mechanism of Action
The UAB research team hypothesized that mouse models of BPD would exhibit elevated levels of acetylated proline-glycine-proline (Ac-PGP). This peptide, derived from the extracellular matrix, had previously been identified in premature infants diagnosed with BPD. Their experimental results confirmed this hypothesis in the BPD mouse models.
Subsequent gain- and loss-of-function studies were conducted to precisely delineate the impact of Ac-PGP. The findings were striking: intranasal instillation of Ac-PGP led to an increase in neutrophilic inflammation and significant lung degradation. Conversely, when an inhibitor of Ac-PGP was administered alongside Ac-PGP, there was a notable decrease in markers of neutrophilic inflammation, and a corresponding improvement in lung structure. This established Ac-PGP as a key mediator in the inflammatory cascade leading to lung damage.
The researchers then investigated the efficacy of a proprietary blend of Lactobacilli, specifically L. plantarum, L. acidophilus, and L. rhamnosus. They demonstrated that this synergistic combination was highly effective in reducing the activity of matrix metalloproteinase-9 (MMP-9). MMP-9 is a crucial enzyme that facilitates the release of Ac-PGP from the extracellular matrix. Intriguingly, the supernatant derived from Lactobacilli growth medium also exhibited a similar capacity to reduce MMP-9 levels, suggesting that soluble factors produced by the bacteria play a significant role.
A pivotal discovery within this research was the identification of L(+) lactic acid as a key anti-inflammatory molecule. This lactic acid, produced during Lactobacilli fermentation, was found to directly reduce MMP-9 activity in vitro. The study revealed that when live Lactobacilli were present in the lungs, they provided a continuous, sustained release of L(+) lactic acid in a controlled and well-tolerated manner, offering a prolonged therapeutic effect.
Technological Innovation for Targeted Delivery
A significant technological advancement reported in this study was the development of an inhaled Lactobacilli powder through advanced particle engineering. This process ensured that the particles were sufficiently small to penetrate deep into the lungs while crucially preserving the viability of the bacteria. This finely tuned live biotherapeutic product was then rigorously tested in the established BPD and COPD animal models.
In the COPD mouse models, the Lactobacilli blend demonstrated remarkable efficacy. It successfully reduced inflammation within the lung microenvironment, whether administered concurrently with injury or post-injury, showcasing its potent anti-inflammatory effects. The treatment led to a decrease in several pro-inflammatory markers and a significant elevation of the anti-inflammatory marker immunoglobulin A (IgA).
Comparative Efficacy and Safety Profile
A particularly noteworthy finding was the comparative performance of the live biotherapeutic product against an established therapeutic agent. The inhaled Lactobacilli blend not only reduced MMP-9 and other pro-inflammatory cytokines but, in some instances, performed comparably to or even surpassed fluticasone furoate. Fluticasone furoate is a United States Food and Drug Administration (FDA)-approved inhaled corticosteroid commonly used in combination therapies for COPD. This suggests that Lactobacilli-based therapies could potentially offer an alternative or complementary approach to current treatments, potentially with a different safety profile.
Rigorous safety and biodistribution studies were conducted in one of the COPD mouse models. These studies confirmed that the inhalation of the bacterial powder did not induce adverse reactions or exacerbate disease progression. Furthermore, the Lactobacilli were found not to translocate to distal tissues or accumulate abnormally in the lungs, indicating a favorable safety profile for this delivery method.
Future Directions and Clinical Translation
The implications of these preclinical findings are far-reaching. "Inhaled live biotherapeutic products show promise in addressing common pathways of disease progression that in the future can be targeted at a variety of lung diseases," stated Dr. Lal. "Preclinical animal data is suggestive, and safety of the potential drug in humans will be tested in a forthcoming clinical trial."
The research team plans to initiate human clinical trials to evaluate the safety and efficacy of this inhaled Lactobacilli therapy. The data gathered from adult safety trials in COPD patients is expected to de-risk the pathway for potential approval and use of the drug in infants suffering from bronchopulmonary disease. This strategic approach aims to leverage existing knowledge and expedite the translation of this promising therapy from bench to bedside.
A Collaborative Endeavor and Intellectual Property
The study, titled "A Lactobacilli-based inhaled live biotherapeutic product attenuates pulmonary neutrophilic inflammation," boasts a robust team of researchers. The co-first authors are Teodora Nicola and Nancy Wenger from the UAB Department of Pediatrics, Division of Neonatology.
Additional authors contributing to this significant work include Xin Xu, Camilla Margaroli, Kristopher Genschmer, and J. Edwin Blalock from the UAB Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine. Colleagues from the UAB Department of Pediatrics, Division of Neonatology, who also contributed significantly are Michael Evans, Luhua Qiao, Gabriel Rezonzew, Youfeng Yang, Tamas Jilling, Kent Willis, and Namasivayam Ambalavanan.
This research received crucial financial support from the National Heart, Lung and Blood Institute of the National Institutes of Health through grants HL141652, HL135710, HL166433, HL156275, and HL164156.
Further underscoring the translational potential of this work, a portion of the research is patented under the title "Inhaled respiratory probiotics for lung diseases of infancy, childhood and adulthood" (U.S. Patent No. 11,141,443 B2). This patent is held by the University of Alabama at Birmingham Research Foundation, an integral part of the Bill L. Harbert Institute for Innovation and Entrepreneurship, with Drs. Lal, Gaggar, and Ambalavanan listed as inventors.
To accelerate the commercialization of this proprietary product, UAB has established a startup company, Alveolus Bio, Inc. This innovative enterprise, with operational bases in Birmingham, Alabama, and Boston, Massachusetts, is poised to bring this groundbreaking therapy to patients.
At UAB, the Departments of Pediatrics and Medicine are part of the Marnix E. Heersink School of Medicine. Dr. Lal holds the position of Director of Clinical Innovation at the Marnix E. Heersink Institute for Biomedical Innovation and is an Associate Professor in the Division of Neonatology. Dr. Gaggar serves as a Professor in the Division of Pulmonary, Allergy and Critical Care Medicine. Dr. Lal is also the visionary founder of two other UAB startups: Alveolus Bio, Inc., and Resbiotic Nutrition, Inc., further highlighting his commitment to advancing biomedical innovation.
