Idiopathic Pulmonary Fibrosis, or IPF, is a progressive lung disease with genetic and environmental causes, affecting 5 million people globally. Although pollutants have long been linked to a range of lung diseases, no conclusive evidence regarding their link to IPF has been sought thus far.
The research of Dr. Eun Joo Kim, a post-doctorate researcher at The University of Colorado Anschutz Medical Campus, defines the role of cilia in lung repair following injury, and details how cilia related genes may be responsible for the severity of symptoms.
Read the original article: https://doi.org/10.1165/rcmb.2021-0554OC
Image Source: Adobe Stock Images /natalia_mis
Hello and welcome to Research Pod! Thank you for joining us today for another podcast on recent scientific breakthroughs.
In today’s episode, we will be discussing the research of Dr. Eun Joo Kim, a post-doctorate researcher in the Division of Pulmonary Sciences and Critical Care Medicine at The University of Colorado Anschutz Medical Campus, United States. Dr. Kim’s work is to define the role of cilia in lung repair following injury in idiopathic pulmonary fibrosis, or IPF – a progressive lung disease with genetic and environmental causes, affecting 5 million people globally.
Her current research builds on Dr. Ivana Yang’s discovery of molecular subtypes of IPF defined by increased expression of cilium-associated genes , especially those relating to a gene called MUC5B, the strongest genetic risk factor for IPF. Her recent paper sets out the degree to which the cilium associated genes may be responsible for the severity of symptoms, and raises questions for how multi-ciliated cells could accelerate the pathogenesis of IPF.
Idiopathic Pulmonary Fibrosis (IPF) refers to a progressive form of lung disease. Fibrosis refers to the development of scar tissues in the lungs. As scar tissues keep filling the lungs, they lose their elasticity to pump oxygen to the bloodstream which eventually leads to death. The exact cause of such a condition has not been deduced so far , which is why it’s called idiopathic. However, environmental pollutants like tobacco smoke, inhaled particle pollutants, viruses, and bacteria have long been linked to a range of diseases, especially those affecting the lungs and respiration. They’ve also been the subject of multiple studies analysing lung tissues through cell staining and direct microscopy, but no conclusive evidence regarding their link to IPF has been sought thus far.
As a result, scientists have resorted to gene expression profiling in the lung airway cells responsible for gas exchange to gather genetic information regarding IPF and group patients accordingly.
These genetic studies have shown at least two main genetic profiles of IPF – one caused by a common variant in the airway mucin MUC5B (mucin 5B) gene, which is responsible for the secretion of mucus in the airways, and the other by common and rare variants in genes dictating the length and health of genetic components called Telomeres, whose degradation has linked to a host of diseases.
Previous research conducted by the Schwartz & Yang lab at The University of Colorado Anschutz Medical Campus showed that a mutation in a single DNA base, aka a single nucleotide polymorphism, or Snip, located in a MUC5B gene promoter site was present in subjects with IPF. This “gain of function” mutation is the dominant risk factor of IPF, present in over 50% of patients with IPF, and is also a primary risk factor for other types of usual interstitial pneumonia (or UIP for short).
Polymorphism refers to the presence of two or more mutant versions of a gene, where the genetic code responsible for making a protein is altered, just enough to keep having some of its usual functions, but to still stand out as a way of telling two different people samples apart. In more scientific terms, polymorphism refers to the presence of different alleles at the same locus of homologous chromosomes in a population. To explain the link between this mutation and disease onset in more detail, this MUC5B promoter polymorphism contributes to overexpression of mucus in the lung, impaired mucociliary clearance and retention of air pollutants and bacteria. This primarily leads to honeycombing which ultimately expands the scar tissue and leads to terminal damage. However, this is not the only reason behind IPF pathogenesis. Cilia are filamentous structures responsible for the movement of mucus and fluid across organs and this process of differentiation into multi-ciliated cells is known as multi-ciliogenesis.
In 2013, Dr. Yang had performed a transcriptome profiling study of the entire lung tissue which revealed two major molecular subtypes of IPF – the first one was associated with airway genes and the other was associated with alveolar genes. The airway genes comprise genes responsible for the synthesis of cilia and other structural components and transcription factors, mucins, and keratins. The expression of these ciliary genes is associated with the overexpression of the mucin gene MUC5B, as mentioned before, and another gene called KRT5.
KRT5 positivity is a marker of cells that are heading towards becoming mucus secreting cells and multiciliated cells in the lung tissue and is also associated with honeycombing in epithelial cells in IPF lungs. KRT5 flagged airway cells differentiate into multi-ciliated cells and this process of differentiation into multi-ciliated cells is known as multi-ciliogenesis. Motile cilia of multiciliated cells beat in a coordinated manner to propel inhaled contaminants trapped by the mucus layer out of the lungs via Mucociliary clearance. Hence overexpression of this KRT5 gene marker in the lung airway cells is also indicative of its involvement in IPF. These observations highlight the multifaceted disease mechanism in the airway subtype of IPF.
Dr.Kim recently published animal studies with a bleomycin-induced lung injury fibrosis mouse model. Bleomycin is most commonly employed as a chemotherapy drug, but induces fibrosis in mouse lungs as well as human lungs as a side effect. Their research mentioned that excess MUC5B secreted as a result of bleomycin-induced injury is associated with fibrosis.
Most importantly, Dr. Evgenia Dobrinskikh in the Schwartz & Yang lab also highlighted the correlation of fibrosis and KRT5 in the fibrotic lungs of mice. In fact, KRT5 has been shown to be overexpressed in ectopic forms (known as KRT5 positive pods) in animal models in a pattern resembling honeycombing in human IPF lungs. Hence, there are now two key points in the pathogenesis of the airway subtype of IPF – aberrant multi-ciliogenesis depicted by an increased number of KRT5 positive pods resembling honeycombing, and impaired mucociliary clearance due to the polymorphism of MUC5B promoter that we mentioned earlier.
To put it in a simple way, when one breathes in polluted air, the pollutants are retained in the lungs passageways due to excess mucus secretion, causing inflammation. Then, the overexpressed cilia and ciliary proteins cannot clear the mucus. This results in progressive scarring of the lungs and eventually leads to fibrosis. This hypothesis shifts the understanding of the idiopathic nature of IPF to a mucociliary disease of the airway epithelium, exacerbated by aberrant multi-ciliation and marked by honeycombing and later by proliferation of fibrosis.
Returning to Dr.Kim’s May 2022 paper, it was hypothesized that defects in multiciliated cells were positively associated with the pathogenesis of IPF based on previous findings. This was proved using mice models with bleomycin-induced lung injury as well as human IPF lung tissues.
Dr.Kim’s team found Myb and Foxj1 transcription factors which are key markers of multiciliogenesis in the 500 genes with the strongest correlations to MUC5B in IPF airway cells isolated from biopsy:
Firstly, Myb, which is required for initiating multi-ciliogenesis,
and then Foxj1 , which is needed for complete multi-ciliogenesis
Next, they demonstrated that protein expression of both Myb and Foxj1 was also elevated in IPF tissues. The study also revealed defects in the ciliary structures which indicated an aberration in repair and regeneration contributing to impaired mucociliary clearance.
Now, combining the overexpression of MUC5B and ciliary structural defects, with the over-expression of multi-ciliogenesis markers, Dr. Kim’s team concluded that aberrant multiciliogenesis is one of the marker of IPF. The overexpression of genetic markers would lead to aberrations in ciliary structure or multi-ciliogenesis, eventually leading to impaired mucociliary clearance in the distal airways, retention of air pollutants over time and eventually to progressive fibroproliferative damage in IPF.
Dr.Kim’s team also studied impaired multi-ciliogenesis in mice models with bleomycin-induced lung injury. Within 1-3 weeks of intrathecal bleomycin treatment, the mice lungs showed loss of motile cilia – those which are able to move and clear mucus – and within 5 weeks of treatment, there were signs of multi-ciliogenesis to recover lost motile cilia and persisting up to 16 weeks – albeit in significantly high numbers than normal. Also, increased production of multiciliated cells was accompanied by enhanced mucin secretion and persisting fibrosis in the lung up to 16 weeks.
A closer look at these motile cilia showed that they lacked the normal structure and had some form of aberration, be it irregular length, abnormally swollen, or a weak microtubular structure. This became a common observation in both human and mouse airway cells that there was an association between defective ciliary development, multi-ciliogenesis and impaired mucociliary clearance in response to lung injury which led to IPF. This conclusion also implied that modulating the expression of Myb and Foxj1, the key markers responsible for multi-ciliogenesis, could also be a possible therapeutic option deserving further research.
Dr.Kim’s team did not stop there. They further investigated the correlation between this aberrant multi-ciliogenesis and the KRT5 pods owing to previously proven associations between KRT5+ markers and ciliary genes in the context of IPF. Both mouse injury models and IPF lungs include the ectopic presence of KRT5+ progenitor populations in the fibrotic areas, mostly as pods which form cysts that persist long term and generate honeycombing in humans with IPF, and cyst-like structures for mice. However, KRT5 pods are not typically observed in the regions of normal lung tissue.
A key gene associated with this process is the Intraflagellar Transport 88 ( or IFT88 for short) because it is required for ciliary assembly and is also suggested to contribute to fibrosis in IPF by activating a Sonic Hedgehog , or SHH, signalling pathway. As such, Dr.Kim used IFT88 as a potential target to reduce fibrosis and KRT5 pods in bleomycin-induced IPF mice.
The team showed that targeting of IFT88 in KRT5 positive IPF mice led to a reduction in the area and the number of KRT5 pods and the amount of fibrotic lung tissue. This reduction was especially significant by the end of the second and third weeks in comparison to the control model. Further analysis also showed an increase in the amount of collagen in the lung tissues. After this breakthrough, Dr.Kim’s next focus was studying the effect of the IFT88 deletion on the Sonic Hedgehog pathway.
To study the effect of IFT88 deletion on the Sonic Hedgehog pathway, Dr.Kim’s team studied the expression levels of two markers of the Sonic Hedgehog pathway activation and mediation – GLI 2 and GLI 3 – using nuclear staining methods. Upon careful observation, it was seen that while Gli3 was only localised in the nucleus of the IFT88-knock out mice, there was hardly any trace of Gli2.
This showed that IFT88 deletion had indeed dampened the Sonic Hedgehog pathway, helping in significantly reducing KRT5-induced honeycomb formation and multi-ciliogenesis, culminating in the reduction of fibrotic tissue.
All in all, the two key takeaways from Dr Kims work is that the over-production of MUC5B and cilia-associated genes were associated with the onset of IPF via their interaction with the airway epithelial cells, and 2. the modulation of Myb, and Foxj1, levels and the deletion of IFT88 could lead to therapeutic breakthroughs in the treatment and management of the airway subtype of IPF.
That’s all for this episode. Thank you very much for listening. Keep following Research Pod on social media channels and subscribe to us for your regular dose of scientific breakthroughs. Speak to you again, soon!