In recent years, altered lung microbiomes have been associated with IPF (Hewitt and Molyneaux, 2017; Puiu et al., 2024). Lung dysbiosis contributes to pulmonary inflammation by elevation of alveolar profibrotic cytokines (O’Dwyer et al., 2019) and modulation of the host immune response (Fabbrizzi et al., 2021). Pre-clinical studies of bleomycin-induced fibrosis in germ-free mice revealed that the lack of a microbiome attenuated mortality related to fibrotic injury. These results suggest that modification of lung microbiota could serve as a new approach for treating IPF (O’Dwyer et al., 2019; Fabbrizzi et al., 2021).

A Correlating Outcomes with biochemical Markers to Estimate Time-progression (COMET) study in IPF examined 55 BAL fluid samples (Han et al., 2014). DNA analysis revealed a positive association with Staphylococcus and Streptococcus genera and progression of IPF. The precise identification of bacterial species was impossible; nevertheless, the study effectively conveyed that certain Staphylococcus and Streptococcus operational taxonomic units (OTUs) are linked to worse IPF outcomes. Another study compared the bacterial burden in the BAL contents of 65 IPF patients with that of 44 controls (Molyneaux and Maher, 2014; Molyneaux et al., 2014). Not only did they find double the burden of bacteria in the BAL of the IPF patients, but they also found a strong association between patients with higher bacterial burden detected in their BAL and a decline in lung function and death. Furthermore, the study identified that the OTUs Streptococcus alongside Gram-negatives, Haemophilus, Neisseria, and Veillonella spp. were linked to IPF.

A separate analysis of BAL samples from IPF patients sought to correlate the microbiome with host immune response signaling pathways. Inflammation through fibroblast function and leukocyte phenotypes was assessed, revealing that some IPF patients exhibited changes in microbial diversity and that the lung microbiome is particularly associated with genes involved in the immune response, such as inflammation and tissue remodeling (Huang et al., 2017). Although causality was not established, the study suggests microbial influence on innate immunity and fibrosis. Other culture-independent studies have also shown an increased bacterial burden in the BAL of IPF patients (Spagnolo et al., 2019), particularly those experiencing acute exacerbation of the disease (Molyneaux et al., 2017; Invernizzi and Molyneaux, 2019). One study implicated the toll-like receptor 3 L412F polymorphism in dysregulating the lung microbiome and reducing the immune response to bacterial infection, leading to increased acute exacerbation associated-IPF death (McElroy et al., 2022).

DNA analysis reveals several distinct microbial signatures of IPF showed a characteristic abundance of Streptococcus, Pseudobutyrivibrio, and Anaerorhabdus. Microbial gene functionality related to ABC transporter systems (ATP synthase (ATP)-binding cassette transporters), biofilm formation, and two-component regulatory systems were more prevalent in the microbiome of IPF patients. ABC transporters are known to be involved in the efflux of antibiotics from bacterial cells (Seeger and van Veen, 2009). Since bacteria forming biofilms are encased in a matrix of extracellular polymeric substances, which render them significantly more antibiotic-resistant (Stewart and Costerton, 2001; Wu et al., 2015), it is evident that the IPF lung microbiome emphasizes key antimicrobial resistance pathways.

Beyond examining bacteria in BAL samples, a study by Kitsios et al. sought to directly analyze the microbiome of fibrotic lung tissue taken from 40 end-stage IPF patients (Kitsios et al., 2018). Contrary to previous findings, the authors found little bacterial DNA in the samples of patients with severe or acute exacerbation of IPF, therefore, comparable to controls. However, it is worth mentioning that the samples used in this study, which targeted subpleural lung tissues with significant honeycombing, originated from regions that may be unconducive to bacterial growth. Moreover, the disparity in findings may also be attributable to the end-stage sample population used in this study, compared with the early-IPF patients of most BAL studies. While the established associations between bacterial infection, progression, and severity of pulmonary fibrosis are promising, causal mechanisms have yet to be uncovered. As such, the efficacy of potential antimicrobial therapies and applications of the lung microbiome as a prognostic biomarker need to be further elucidated (Fastres et al., 2017; Ntolios et al., 2021).

In hospitalized patients with IPF, the prevalence of bacterial pneumonia, pulmonary hypertension, and lung cancer was 9.5%, 4.6%, and 3.7%, respectively (Oda et al., 2018). Among patients with bacterial pneumonia, the two most common Gram-positive pathogens were Streptococcus pneumoniae (31.6%) and methicillin-resistant Staphylococcus aureus (MRSA) (18.4%). Respiratory comorbidities, especially bacterial pneumonia and lung cancer, influence mortality in hospitalized patients with IPF (Oda et al., 2018). Notably, a recent meta-analysis revealed that bacterial streptococcal infection occurred in 99.5% of patients with IPF (Mostafaei et al., 2021). This section will discuss the relevant research regarding bacterial infection in IPF.

Streptococcus pneumoniae infection has been shown to exacerbate lung fibrosis in mice via the release of the cytotoxin pneumolysin (Knippenberg et al., 2015); notably, fibrosis progression was mitigated when mice were given a protein-based vaccine presenting a non-cytotoxic pneumolysin derivative. This suggests that pneumolysin may, in the future, be a potential target for fibrosis treatment in human patients through a protein-based pneumococcal vaccination targeting key virulence factors like pneumolysin, which could have significant preventive effects on S. pneumoniae-induced fibrosis exacerbation. However, some IPF patients undergo treatment with corticosteroids and immunosuppressive agents, which have been shown to interfere with their response to the pneumococcal vaccine (Kuronuma et al., 2018). A recent study by Bormann et al. (Bormann et al., 2021) highlighted a Cox2-dependent anti-inflammatory role of prostaglandin E2 (PGE₂) in the progression of experimental pulmonary fibrosis in mice. Streptococcus pneumoniae-induced IPF progression was associated with increased expression of PGE₂, and intratracheal application of PGE₂ worsened fibrosis in mice with AdTGF-β1-induced lung fibrosis.

A recent study reported that Staphylococcus nepalensis is responsible for releasing a peptide, corisin, which induces apoptosis of lung epithelial cells (D’Alessandro-Gabazza et al., 2020). The study showed that mice exposed to corisin-harboring S. nepalensis experienced acute exacerbation, unlike control mice, who were either untreated or infected with corisin-free bacteria. Furthermore, human IPF patients with acute exacerbation have notably higher counts of lung corisin levels than IPF patients without exacerbation.

Further, mice infected with MRSA were found to have a more difficult time fighting off infection due to fibrosis (Warheit-Niemi et al., 2022). Specifically, the authors reported that fibrosis diminished neutrophil elastase release and oxidative radical production, inhibiting intracellular killing of MRSA by neutrophils. Not only did the fibrotic mice exhibit inhibited neutrophil activity, but lung macrophages were also shown to have a reduced capacity for phagocytosis of MRSA. Thus, the study provides evidence for impaired immune response in fibrotic lungs and proposes a mechanism for why bacterial infection in individuals with IPF increases morbidity and mortality. Overall, there has been growing evidence showing an association between Gram-positive bacteria and the development of IPF and specific mechanisms by which bacteria are responsible for exacerbating the disease and hindering the immune system’s ability to fend off infection.

Respiratory infections caused by Gram-negative bacteria in IPF patients are relatively frequent during hospitalization and are reportedly effective in predicting mortality (Yamazaki et al., 2016). In 2016, a retrospective study analyzed causative pathogens in 48 IPF patients who had been hospitalized for pulmonary infections (Yamazaki et al., 2016). The study found causative pathogens in 20/48 patients, the most common of which were H. influenzae (14.5%), P. aeruginosa (4.1%), Moraxella (Branhamella) catarrhalis (4.1%), and Klebsiella pneumoniae (4.1%). Intriguingly, the causative pathogens were primarily Gram-negative bacteria, in contrast to the perspective that infection with Gram-positive bacteria causes most cases of bacterial pneumonia. Moreover, the Pneumonia Severity Index (PSI) score upon admission was significantly correlated with 30-day and hospital mortality.

Besides COPD cases in adults and more pronounced cases in infants and children with cystic fibrosis, a limited connection has been made between Bordetella pertussis and IPF, such as a 2019 case report showing that 2 patients with IPF had been diagnosed with acute exacerbation of the disease and an acute pertussis infection (Paddock et al., 2008; Bos et al., 2011; Hashemi et al., 2015; Karamooz et al., 2018; Hirai et al., 2019). Although pertussis is preventable by vaccine, neither patient had been previously vaccinated. Thus, the study suggests further investigation into pertussis as a factor in the exacerbation of IPF and the potential use of antibiotics to treat IPF patients with infection. A 2007 study investigated the role of Chlamydophila (Chlamydia) pneumoniae infection in exacerbating IPF (Tomioka et al., 2007). Twenty-seven IPF patients were tested for C. pneumoniae antibodies, IgG, and IgA, ultimately revealing that when patients presented with an acute exacerbation of IPF, C. pneumoniae was not typically present.

A recent study examined the significance of macrophage scavenger receptor 1- (MSR1)-positive cells in the progression of IPF by analyzing lung transplantation tissue samples via immunohistochemistry (Zheng et al., 2021). MSR1-positive macrophages correlated with reduced lung function and poor prognostic outcomes in IPF patients (Zheng et al., 2021). MSR1 upregulation was also significantly more common in smoking patients than in non-smoking patients but also that the expression of MSR1 was significantly elevated in IPF patients infected with K. pneumoniae, corroborating the potential role of Gram-negative bacteria in the progression of IPF. MSR1 has been suggested to play a vital role in inflammatory, innate, and adaptive immune responses, and silica-induced fibrosis (de Winther et al., 2000; Beamer and Holian, 2005).

Several studies have revealed the importance of cytokines in NTHi. For example, interleukin-17A, a pro-inflammatory cytokine, has been demonstrated to play a role in lung fibrosis in the context of bleomycin-induced fibrosis in mice (Wilson et al., 2010). In addition, murine model of bleomycin-induced lung fibrosis has found that dysregulated lung microbiota can promote the production of interleukin-17B (IL-17B), driving disease progression (Yang et al., 2019). Other studies utilizing similar models to simulate bacterium-induced acute exacerbation of IPF by administering bleomycin to mice and then infecting them with a strain of NTHi (Chen et al., 2022) have revealed that NTHi infection can cause acute exacerbation of IPF and that the IL-17 gene is key for the progression of IPF acute exacerbation and could serve as a novel therapeutic target for treating the disease.

The decline in pulmonary function and development of fibrotic lung tissue is heavily associated with localized lung tissue inflammation (Bringardner et al., 2008; Homer et al., 2011). Several other pro-inflammatory cytokines, such as interleukin-8, interleukin-1 beta, and chemokine (C-X-C motif) ligand 1, are known to drive localized inflammation (Harada et al., 1994; Dinarello, 2011; Donahoe et al., 2015; Sawant et al., 2015). NF-kB is a transcription factor that has multiple impacts on target cells, such as regulating inflammation, inducing apoptosis, and regulating cell growth (Liu et al., 2017). NTHi infection has been shown to upregulate NF-kB activation with tumor necrosis factor, a pro-inflammatory cytokine (Watanabe et al., 2004). NF-kB is documented to have pro-inflammatory effects on fibroblasts, cells responsible for secreting collagen and extracellular matrix proteins, through regulation of gene transcription (Htwe et al., 2015). Fibroblast stimulation to secrete collagen and extracellular matrix proteins is one of the primary mechanisms that lead to the development of pulmonary fibrosis (Hanmandlu et al., 2022). As fibroblasts secrete increased amounts of collagen and extracellular matrix proteins, lung tissue hardens and interferes with proper gas exchange. NF-kB has also been shown to stimulate fibroblasts to secrete pro-inflammatory cytokines such as interleukin-8, macrophage inflammatory protein-1-alpha, and transforming growth factor-beta (Htwe et al., 2015). Moreover, a 2017 study found that lung fibroblasts could internalize live NTHi, and that fibroblasts were responsible for activating IFN-γ and IL-17A cytokine production via autologous NTHi-specific lung CD4⁺ T cells. This suggests that human lung fibroblasts play a crucial role in mediating T helper (Th) cell responses to bacterial infection, and specifically in conjunction with NTHi (Hutton et al., 2017).

Macrophages have also been shown to respond to NF-kB to secrete pro-inflammatory cytokines (Liu et al., 2017). Given that macrophages are present within alveoli, this could be another driving mechanism for localized inflammation. NF-kB is also documented to stimulate fibroblast transformation into myofibroblasts, a fibroblast state that secretes increased amounts of extracellular matrix proteins (Liu et al., 2017). Mice treated with NF-kB inhibitors were significantly protected from lung fibrosis development compared to control mice (Thakur et al., 2022). Given the wide range of effects that NF-kB has been shown to induce in various cell types, future research regarding NTHi and NF-kB upregulation could provide valuable information for pulmonary fibrosis pathogenesis.

Numerous mechanisms that enable NTHi to not only establish itself in patients with IPF, but also to maintain a persistent infection for prolonged periods of time are discussed (Murphy et al., 2004). Future studies will further reveal the specific mechanisms of H. influenzae in driving pathogenesis in individuals with IPF, using previously established research on related illnesses such as COPD.

NTHi has adapted several strategies to avoid the host immune response and maintain a persistent infection. For instance, NTHi uses a variety of adhesins, which allow the bacteria to attach itself to and invade host cells (Duell et al., 2016). In essence, these adhesins are essential in permitting NTHi to colonize primary sites, allowing further infection in secondary sites and eventually leading to the formation of biofilms and mediation of key virulence mechanisms (Duell et al., 2016). Biofilms comprise an extracellular polymeric substance inhabited by a community of bacteria. NTHi produces these protective biofilms to maintain a reserve of bacteria which can go on to cause subsequent infections (Gunn et al., 2016). Thus, the formation of biofilms is a key mechanism by which NTHi and other pathogens can sustain persistent infections in the host.

Studies have revealed that NTHi does not actually bind to the cell surface directly, but rather adheres to host vitronectin by means of an outer membrane protein, protein-E. The protein-E-vitronectin interaction plays a role in the adherence and invasion of NTHi in bronchial epithelial cells. It could serve as a potential target for novel treatments and vaccines (Ikeda et al., 2015). Lysostaphin-like metalloproteases (LytM proteins), known to facilitate cell division by affecting cleavage and membrane composition, contributed to the pathogenesis and physiology of NTHi. Data suggests explicitly that the component of outer membrane and cell wall physiology, the murein hydrolase activator, EnvC protein, possessing a LytM domain, might impact bacterial surface protein composition via a mechanism in which EnvC facilitates the transport of periplasmic chaperones to the outer membrane. This is further supported by studies showing that an NTHi envC-defective strain has diminished capacity to adhere to epithelial cells and form biofilm, while also displaying a reduced resistance to the immune system (Ercoli et al., 2015).

Although NTHi is widely considered an extracellular pathogen, NTHi has also been spotted residing intracellularly (St Geme and Falkow, 1990). For example, NTHi lipooligosaccharide interacts with the platelet-activating factor receptor, enabling NTHi to adhere to and infiltrate human bronchial epithelial cells (Swords et al., 2000). Morey et al. proposed a unique mechanism by which NTHi can invade the airway epithelium and reside intracellularly, involving the assembly of microtubules, integrity of lipid rafts, and activation of phosphatidylinositol 3-kinase (PI3K) signaling (Morey et al., 2011). NTHi were found to primarily reside in acidic sub-cellular vacuoles with late endosome features, existing in a metabolically active yet non-replicative state. This NTHi-containing vacuole co-localizes with LysoTracker, lysosome-associated membrane protein 1 (LAMP1), LAMP2, CD63, and Rab7 and does not possess Golgi- or autophagy-related markers. It may modulate epigenetic changes like many known bacterial nucleomodulins or by altering host endosomal proteins mechanisms to evade host immune response and gain intracellular persistence (Morey et al., 2011; Cohen et al., 2013; Denzer et al., 2020; Fol et al., 2020; Wrede et al., 2023).

Further investigation has suggested that NTHi strategically positions itself intracellularly to avoid immune responses and maintain a persistent infection, and IgA proteases are essential to NTHi’s ability to do this. Epithelium and airway mucous membranes contain IgA as a part of the innate immune system to defend against pathogens; in the upper respiratory tract, IgA1 is the dominant form (Salvi and Holgate, 1999). H. influenzae is documented to produce three types of IgA1 proteases that can cleave the heavy chain of IgA1 antibodies and render the Fc portion dysfunctional. While most capsulated strains of H. influenzae produce only one of the three IgA1 proteases, NTHi strains produce any of the three types (Foxwell et al., 1998). Each of the three proteases play distinct roles in the pathogenesis of NTHi. While IgA protease is necessary for NTHi invasion, IgA proteases B1 and B2 are essential to the intracellular persistence of NTHi (Murphy et al., 2017). Not only do IgA proteases serve to protect NTHi from mucosal immunity by cleaving human IgA1, but they have also been found to facilitate intracellular survival by cleavage of LAMP1 at each stage of the endolysosomal pathway, including the plasma membrane, early and late endosome, and lysosome stages. In doing so, IgA proteases B1 and B2 facilitate NTHi’s ability to evade the endo-lysosomal pathway, though NTHi is ultimately destroyed in lysosomes after variable durations of intracellular survival (Clementi et al., 2014). NTHi has also been documented invading epithelial cells and residing in membrane-bound vacuoles inside epithelial cells (Foxwell et al., 1998). The mechanisms of NTHi pathogenesis are depicted in Figure 2 .

To ensure that NTHi persists in various hostile COPD environments, the bacteria use several mechanisms for genetic variation, including antigenic variation (Sethi et al., 2004), phase variation (Murphy et al., 2004), and epigenetic variation (Atack et al., 2015). Additionally, NTHi must adapt specific metabolic pathways to take in necessary nutrients. For instance, ferric uptake regulator (Fur)-regulated genes in NTHi modulate iron usage within the cell, ultimately facilitating more severe and persistent infections (Duell et al., 2016).

Because iron is in limited supply within the host, NTHi sequesters iron to ensure its survival. Recent dual RNA-Seq studies have further elucidated these metabolic adaptations, revealing that NTHi upregulates iron acquisition pathways and modifies metabolic processes to survive under intracellular conditions (Baddal et al., 2015). These studies also show that NTHi dynamically adjusts its transcriptional responses in real time, responding to the intracellular environment to maximize survival. Concurrently, NTHi modulates host immune responses, suppressing inflammatory signaling pathways to evade detection and maintain a permissive intracellular environment (Ackland et al., 2021). This dual role of metabolic adaptation and immune modulation enables NTHi to persist intracellularly, protecting itself from immune clearance and ensuring long-term colonization of the host.

Using the mechanisms outlined above, NTHi can establish a persistent infection in the lower airways, lasting months to years. Moreover, this wide range of unique virulence mechanisms allows NTHi to persist in its host and exacerbate pulmonary diseases, including but not limited to IPF. Therefore, NTHi is particularly interesting when evaluating possible concurrence and associations with IPF. Mechanisms discussed above could play a role in the progression of IPF in patients infected with H. influenzae.

Most preclinical lung fibrosis studies widely use the chemotherapy drug bleomycin sulfate, which is intratracheally administered in rats and mice to induce an inflammatory response progressing into lung fibrosis that mimics the pathology of IPF and airway remodeling (Polosukhin et al., 2012; Tanjore et al., 2013). Bleomycin-induced pulmonary fibrosis is highly reproducible, with key molecular pathways triggered by reactive oxygen species and inflammation that leads to excessive collagen deposition and fibrosis (Degryse et al., 2011; Polosukhin et al., 2012; Tanjore et al., 2013; Moss et al., 2022). The rodent models are also employed to examine the in vivo efficacy of drugs for preventive and therapeutic potentials for IPF treatment (Kolb et al., 2020). While these models capture many qualitative histological aspects of IPF, they may not completely replicate all the complexities of the human disease, given the chronic nature of the disease in IPF patients. On a histologic level, bleomycin-induced fibrosis lacks the basal and subpleural predominance characteristic of IPF and involves more limited alveolar epithelial cell remodeling (Moss et al., 2022). However, repetitive bleomycin dosing and dosing of aged mice show promising results that more closely approximate non-reversible fibrotic phenotypes and classic histologic presentations of IPF (Degryse et al., 2010; Weckerle et al., 2023). Interestingly, positron-emission tomography-computed tomographic scanning was employed in bleomycin-induced mice to closely mimic the diagnostic methods of IPF and evaluate pathologic changes with different modes of bleomycin administration. It was shown that repeated intravenous bleomycin delivery for seven consecutive days matched closer to the early pathologic features of idiopathic pulmonary interstitial fibrosis (Sgalla et al., 2016; Comes et al., 2020; Gul et al., 2023).

A randomized, placebo-controlled trial was carried out to determine the effects of co-trimoxazole in patients with IPF (Shulgina et al., 2013). Despite a previous trial indicating an improvement in forced vital capacity (FVC) (Varney et al., 2008), this extensive study showed no significant differences in FVC between the co-trimoxazole-treated patients versus the placebo groups who received co-trimoxazole treatment at four and half months later timepoint after decoding the study. There was, however, a decrease in overall mortality rates across the two groups and a reduction in subsequent respiratory tract infections, suggesting that delayed treatment could also help recuperate with a significantly improved MRC5 Point Dyspnoea Score (Study #1, Table 2 ). A 2013 study designed to evaluate the effect of co-trimoxazole on IPF was terminated due to changes in standards of care (Study #2, Table 2 ), while results from another clinical trial did not support the use of the drug to treat patients with moderate to severe IPF (Wilson et al., 2020). Similarly, a 2021 study investigating the clinical performance of antimicrobial therapy in IPF found that the use of co-trimoxazole or doxycycline did not significantly improve the time to non-elective respiratory hospitalization or death, concluding that these antibiotics are ineffective in treating the underlying disease (Anstrom et al., 2020; Martinez et al., 2021). Another study evaluating the efficacy of minocycline as a treatment for IPF was conducted, although the results were inconclusive (Study #3, Table 2 ). A trial assessing simtuzumab was terminated due to low efficacy (Study #4, Table 2 ). Similarly, a clinical trial in 2011 was terminated upon finding that ambrisentan was ineffective in treating patients with IPF and may even worsen the condition (Raghu et al., 2013).

An initial phase I trial involving healthy participants revealed that INS018_055, an AI-generated drug designed to treat IPF, was both safe and tolerable (Ren et al., 2024). The drug is currently being evaluated in IPF patients, with promising potential for treatment (Study #8, Table 2 ). Several other drugs that are currently being evaluated as potential treatments for IPF, as listed in Table 2 , include PMG1015, AGMB-447, ZSP1603, RXC007, Leramistat, Pirfenidone and Deupirfenidone, LTI-03, N-acetyl cysteine, and PLN-74809 (Studies# 9-14, Table 2 ). Interestingly, a crucial ongoing clinical trial is currently recruiting patients to evaluate the utility of human umbilical cord MSC injections in treating IPF (Study #16, Table 2 ). In a preclinical model, Moodley et al. showed that human umbilical cord mesenchymal stem cells (MSCs) could reduce fibrosis in a bleomycin-induced mouse lung injury model (Moodley et al., 2009). Since then, several studies have gone on to show the clinical safety of administering autologous adipose-derived stem cells residing in the stromal vascular fraction (SVF) of white adipose tissue (Tzouvelekis et al., 2013), intravenous allogenic human placenta-derived MSCs (Chambers et al., 2014), and a single infusion of allogenic human bone marrow-derived MSCs in patients with moderate to severe IPF (Glassberg et al., 2017).

The discovery of novel diagnostic measures to better mark the presence and progression of IPF remains an essential goal for future research. In addition, investigating the efficacy of antibiotics and other therapeutics could enhance the quality and number of treatment options available for patients with IPF. A 2018 pilot study utilized ultra-high-performance liquid chromatography and high-resolution mass spectrometry to search for distinct IPF metabolic profiles in exhaled breath condensate (EBC) samples (Rindlisbacher et al., 2017). Although this approach requires replication across larger sample sizes to confirm diagnostic utility, investigators discovered preliminary evidence for differential regulation of 26 metabolic features between IPF patients and health controls. Assuming such findings are not exclusive to patients with advanced disease, early IPF detection via altered metabolic activity detection could facilitate more prompt treatment and improve patient outcomes. Another study used similar methods to perform serum metabolic profiling of patients with IPF, identifying a lysophosphatidylcholine as a potential biomarker (Rindlisbacher et al., 2018). Lysophosphatidylcholine is a precursor to lysophosphatidic acid, an established mediator of fibrotic development. An ongoing project which began in 2005 aims to identify and characterize genetic and biological markers of IPF (Study #1, Table 3 ). After accessing and analyzing data collected from patients in this study as well as from the ARTEMIS-IPF trial (Study #5, Table 2 ), researchers found an association between elevated levels of lysyl oxidase-like 2, which facilitates the cross-linking of collagen within the pathological stroma, and increased risk for IPF disease progression and mortality (Chien et al., 2014). Successful identification of reliable biomarkers would supplement and reinforce existing diagnostic methods for IPF and enhance our capacity to monitor drug effects. More studies should be conducted to improve our understanding and treatment of the disease. Furthermore, a closer look into specific demographics disproportionately affected by IPF, such as that performed by an observational study conducted in Spain (Study #2, Table 3 ), could offer insight into how IPF manifests within societies. Using demographic information to identify individuals at higher risk of IPF could help direct screening efforts and promote early identification of those with the disease.

In the mouse model of fibrosis, administration of mannose-incorporated LNPs carrying siRNAs, which down-regulate the EMT-associated protein, G2 and S phase-expressed protein 1(GTSE1), resulted in a notable reduction in collagen accumulation and EMT-related proteins and functional recovery from pulmonary fibrosis (Jin et al., 2023). Moreover, intratracheal administration of luciferase mRNA LNPs led to targeted lung accumulation in bleomycin-induced lung fibrosis. Bioluminescence was detected in the lungs as early as 2 hours post-delivery and persisted for 48 hours, with LNPs associating with AEC2 cells and fibroblasts in vivo (Massaro et al., 2023). These studies underscore the potential of LNPs as a promising approach for treating pulmonary fibrosis by facilitating targeted delivery of RNA therapeutics in the lungs. More recently, inhalable LNPs delivering dual mRNAs were shown to restore AT2 cell function and promote alveolar regeneration in IPF in bleomycin-induced lung fibrosis. These LNPs corrected mitochondrial dysfunction, prevented premature AT2 cell senescence, and halted pathological epithelial remodeling and fibroblast activation, facilitating alveolar regeneration (Wang et al., 2024). Using the LNP-mRNA system, this study demonstrated robust protein expression in lung epithelial cells, effectively reversing alveolar collapse and improving survival in fibrosis mice, which further supports using LNP-mRNAs as potential treatments against IPF.

Just as LNP-mRNAs may target the fibrotic pathways of IPF directly, in vitro and in vivo mRNA transfections have been previously shown to enhance epithelial resistance to invading bacteria by increasing local antimicrobial peptide levels (Zou et al., 2013; Maruggi et al., 2017). Nevertheless, two mRNA vaccines, encoding PcrV and OprF-I, both components of Pseudomonas aeruginosa, were delivered via LNPs and tested in-vivo infection models. Both vaccines elicited strong immune responses and reduced bacterial burden and inflammation in infection models. While mRNA-PcrV showed superior antigen-specific responses and higher survival rates against PA strains compared to mRNA-OprF-I, the combined mRNA vaccine demonstrated the highest survival rate and outperformed protein vaccines (Wang et al., 2024).

More recently, a group created LNP-mRNAs to enhance anti-MRSA (multidrug-resistant Staphylococcus aureus) immunity via in situ programming of macrophages. The nanoparticles delivered MRSA-targeted chimeric antigen receptor (CAR) mRNA to enhance macrophages’ ability to recognize and attack MRSA and CASP11 siRNA, inhibiting a key MRSA intracellular evasion mechanism. This resulted in the creation of CAR-macrophages with enhanced bactericidal ability, which could efficiently phagocytose and destroy MRSA intracellularly, overcoming the bacterium’s immune evasion (Tang et al., 2024). Thus, these findings highlight the potential of LNP-mRNA formulations in patients, combatting pathogens like Pseudomonas aeruginosa and MRSA, and infection-related mechanisms of IPF pathogenesis.

The use of LNPs in delivering targeted mRNA therapies shows promise in treating other bacterial infections, which could provide new avenues for addressing infection-related mechanisms of IPF. Further pre-clinical and clinical research is needed to explore the efficacy of LNP-mRNA therapies with other intracellular pathogens such as NTHi. Thus, as we continue to uncover new strategies for creating high-efficiency LNPs, our understanding of how to treat illnesses with mRNA-based therapeutics will improve dramatically. To that end, the use of targeted LNP-mRNAs could drastically improve the treatment of patients with IPF. Therapeutic strategies using LNP-mRNAs in intracellular persistent infections such as NTHi in ILDs, including IPF are depicted in Figure 3 .