Platelet Activating Factor Receptor and Intercellular Adhesion Molecule–1 Expression Increases in the Small Airway Epithelium and Parenchyma of Patients with Idiopathic Pulmonary Fibrosis: Implications for Microbial Pathogenesis

Background: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung fibrotic disorder of unknown cause. It has been reported that bacterial and viral co-infections exacerbate disease pathogenesis. These pathogens use adhesion molecules such as platelet activating factor receptor (PAFR) and intercellular adhesion molecule-1 (ICAM–1) to gain cellular entry, causing infections. Methods: Immunohistochemical staining was carried out for lung resections from IPF patients (n = 11) and normal controls (n = 12). The quantification of PAFR and ICAM–1 expression is presented as a percentage in the small airway epithelium. Also, type 2 pneumocytes and alveolar macrophages were counted as cells per mm2 of the parenchymal area and presented as a percentage. All image analysis was done using Image Pro Plus 7.0 software. Results: PAFR expression significantly increased in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Similar trend was observed for ICAM–1 expression in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Furthermore, the proportion of positively expressed type 2 pneumocytes and alveolar macrophages was higher in IPF than in normal control. Conclusions: This is the first study to show PAFR and ICAM–1 expression in small airway epithelium, type 2 pneumocytes and alveolar macrophages in IPF. These findings could help intervene microbial impact and facilitate management of disease pathogenesis.

Studies over the years have discovered that IPF patients suffer from respiratoryassociated infections that contribute to worsened disease pathogenesis [15][16][17].It has been reported that IPF patients have a higher bacterial load in bronchoalveolar lavage (BAL) fluid than healthy or chronic obstructive pulmonary disease (COPD) cohorts [15,16].The most common bacterial genus in IPF is Streptococcus, Hemophilus, Pseudomonas, Neisseria, and Veillonella [15,16].Moreover, research has also found that some viruses, namely the human rhinovirus (HRV) and influenza virus, are involved in affecting patients [15,18].A synergy between bacteria and virus has been documented, detailing that it causes superinfections that exacerbate IPF progression, reducing lung function and lowering the survival rate compared to uninfected patients [15].Similarly, SARS-CoV-2 receptor angiotensinconverting enzyme 2 (ACE2) has been shown to upregulate in patients with IPF, increasing their susceptibility to COVID-19 [15,19].With this knowledge, we wanted to investigate the presence of other potential microbial receptors, such as platelet-activating factor receptor (PAFR) and intracellular adhesion molecule-1 (ICAM-1), and their possible involvement in microbial entry into host cells [20][21][22][23].An increase in such microbial receptors is bound to make the patients susceptible to infections that further drive unwarranted inflammation and remodeling changes [24,25].
Platelet-activating factor (PAF) functions as a known mediator which causes platelet aggregation, blood vessel dilation, inflammation, allergic reactions, and shock [26,27].PAF is produced by many cells, such as platelets, endothelial cells, fibroblasts, macrophages, monocytes, mast cells, and some immune cells, such as neutrophils and eosinophils [26,27].PAF is mainly involved in inflammatory responses [26].Under normal conditions, PAF binds to PAFR, a G-protein-coupled seven-transmembrane receptor that activates several pathways and synthesis of some inflammatory mediators such as prostaglandins and cytokines [e.g., tumour necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8)] [26,28].However, in terms of bacterial involvement, a study on COPD found that upregulated PAFR in epithelium helps Streptococcus pneumoniae to interact and colonize epithelial cells with the help of phosphorylcholine (ChoP) [23,29,30].The ChoP is a molecular mimic of PAF found in S. pneumoniae walls and on some strains of Hemophilus influenzae, Pseudomonas aeruginosa, and Acinetobacter baumannii [23,31].As a result, bacteria with ChoP binds to PAFR, outcompeting PAF (natural ligand), leading to bacterial transmigration across the host cells [30].
In COPD, we have previously reported significantly increased PAFR expression in small airways and lung parenchyma, especially in smokers, compared to normal tissue [39].It indicates that irritants causing inflammation upregulates microbial receptors in sites such as small airways and lung parenchyma, enabling microbial attachment [26,32].We found similar changes for ICAM-1too in smokers and patients with COPD [36].Therefore, we wanted to explore if this activity is evident in IPF and potentially provide a better understanding of microbial implications in this disease.
We hypothesize that PAFR and ICAM-1 expression is upregulated in IPF, which act as adhesion sites for bacteria/viruses, increasing the vulnerability to infection in IPF patients.This study aims to determine the expression of PAFR and ICAM-1in small airways and lung parenchymal areas mainly in type 2 pneumocytes and alveolar macrophages of patients with IPF compared to normal lungs to explain if IPF patients are more susceptible to infections.

Patient Demographics
Surgically resected human lung tissue from informed consent patients with end-stage IPF (n = 11) were obtained from Alfred Health (Ethics approval ID: 336-13) at the time of lung transplantation.Normal controls (NC, n = 12) were provided by James Hogg Lung Registry (Ethics approval ID: H00-50110).The NC lung tissues were from patients who had died of a cause other than respiratory disease (Table 1).

Small Airway and Lung Parenchyma Quantification
Images were taken using a Leica DM500 microscope and Leica ICC50W camera (Leica, Macquarie Park, NSW, Australia), and analysis was performed using Image Pro Plus 7.0 software (Media Cybernetics, Rockville, MD, USA).The observer was blinded to patients and diagnosis.Small airway epithelium and lung parenchyma areas were captured at 40× and 20× magnification, respectively, and strictly avoiding any overlapping images.Then, eight images were randomly selected using online random generator software for measurement.The quantification of PAFR and ICAM-1 expression on the small airway epithelium is presented as a percentage of the epithelial layer.Also, type 2 pneumocytes and alveolar macrophages were counted as cells per mm 2 of the parenchymal area and presented as a percentage.

Statistical Analysis
Following normality check, non-parametric analysis of variance were performed using the unpaired one-tailed Mann-Whitney Test; specific group differences without correction for multiple comparisons were assessed using a two-way ANOVA test with Fisher's LSD test.The statistical analysis was completed using GraphPad Prism V9.1 (GraphPad Software Inc., La Jolla, CA, USA), with a p-value ≤ 0.05 considered statistically significant.

Quantification of PAFR Expression in Small Airway (SA) Epithelium, Type 2 Pneumocytes and Alveolar Macrophages
PAFR expression was prominent on the apical surface of small airway epithelial cells in IPF tissue, and mildly covering the whole perimeter of the cells (Figure 3A).There was negligible PAFR staining on the normal controls with patterns illustrated (Figure 3A).We further observed the expression of PAFR in the lung parenchyma, which increased mainly in type 2 pneumocytes and alveolar macrophages in IPF tissue compared to NC tissue (Figure 3A).
PAFR expression was prominent on the apical surface of small airway epithelial cells in IPF tissue, and mildly covering the whole perimeter of the cells (Figure 3A).There was negligible PAFR staining on the normal controls with patterns illustrated (Figure 3A).We further observed the expression of PAFR in the lung parenchyma, which increased mainly in type 2 pneumocytes and alveolar macrophages in IPF tissue compared to NC tissue (Figure 3A).The percentage expression of PAFR significantly upregulated in the small airway epithelium in IPF lung tissue (median 8.33%, range 3.80-42.7%)compared to NC lung tissue (median 1.43%, range 0.352-5.85,p < 0.0001) (Figure 3B).In addition, the PAFR positive expression in alveolar type 2 pneumocytes of IPF showed a significantly high percentage (median 78.1%, range 61.2-96.0%)compared to NC (median 40.1%, range 15.2-58.2%,p < 0.0001) (Figure 3B).The number of PAFR positive type 2 pneumocytes per mm 2 in alveolar area is significantly higher in IPF (median 498,525 cells per mm 2 , range 189,449-1,463,564 cells per mm 2 ) compared to NC (median 227,150 cells per mm 2 , range 34,483-463,444 cells per mm 2 , p = 0.0017) (Figure 3C), and in contrast, the number of PAFR negative type 2 pneumocytes is significantly higher in NC (median 334,189 cells per mm 2 , range 158,333-445,101 cells per mm 2 ) compared to IPF (median 96,519 cells per mm 2 , range 20,930-825,189 cells per mm 2 , p = 0.0017) (Figure 3D).

Discussion
This is the first study reporting PAFR and ICAM-1 adhesion molecules in the small airway epithelium and lung parenchyma of patients with IPF.We found that PAFR and ICAM-1 expression increased in the small airway epithelium in IPF compared to normal tissues.In the lung parenchyma of IPF patients, we found elevated levels of PAFR and ICAM-1 positive type 2 pneumocytes and alveolar macrophages.Furthermore, the proportions of positively and negatively expressed cells showed more positively expressed type 2 pneumocytes and alveolar macrophages in IPF and fewer negatively expressed cells in normal tissue.It could imply that although alveolar macrophages innately recognize pathogens but, cell activation and molecular receptor expression may provide shelter to microbes against inflammatory molecules, contributing to increased infection [39] The above results suggest that high molecule expression in IPF could be a significant link to microbes 'anchoring' and gaining cellular entry, increasing the risk of infection.As IPF is a chronic pulmonary disease, postulated risk factors involved in disease pathogenesis include environmental factors, tobacco, genetics, and infectious agents [1,5].These irritants result in a chronic injury, and inflammation affecting the molecular and cellular mechanisms [3,40].In chronic disease, microbes infiltrate the exacerbated inflammation and fibroblast hyperproliferation, leading to excess collagen deposition [40,41].
Phosphorylcholine (ChoP, a molecular mimic of PAF) is an important element expressed on the outer surface of various microorganisms; the most commonly seen bacterial genus in the respiratory tract is S. pneumoniae [42,43], H. influenzae [44] and P. aeruginosa and A. baumannii, which can direct interaction with host cells through ChoP [23,29,39,45].
The attachment of bacteria that ChoP to the PAFR facilitates their adhesion to and invasion into human cells [46,47].The general mechanism of chronic bacterial colonization has been well documented in COPD but under-researched in IPF [39].In our study, with increased expression of PAFR in small airways indicates that these bacterial pathogens can adhere to small airway epithelium because the ChoP outcompetes the PAF (natural ligand to PAFR) and anchoring to PAFR may increase the risk of infection [15,16] (Figure 6A).HRVs contribute to over 50% of upper respiratory tract infections [48], and HRV infections can lead to life-threatening effects worsen chronic respiratory disease, such as COPD, asthma, or cystic fibrosis [49].The major signaling pathway for HRV cellular access occurs because pathogens use ICAM-1 as a receptor [32,50].Specifically, the pathogen binds to leukocyte function-associated antigen 1 (LFA-1) or the macrophage-1 antigen (Mac-1), natural adhesion ligands [32,50] (Figure 6B).ICAM-1 becomes a major catalyst for the eventual uncoating of the cell-invading virus, potentially exacerbating IPF pathogenesis [16,32].The role of ICAM-1 in COPD has been investigated by our research group and we have showed that the receptor was highly expressed in COPD smoking cohort [36].The expression patterns were prominent in the airways, especially on goblet cells and sub-mucosal glands, and could be a potential risk factor of infection by common respiratory viral and bacterial pathogens [36].These biochemical mechanisms documented in other diseases shine a light on our findings as we believe our data bridges the gap in this area by demonstrating the abovementioned microbial activity in IPF, but this warrants further research.HRVs contribute to over 50% of upper respiratory tract infections [48], and HRV infections can lead to life-threatening effects that worsen chronic respiratory disease, such as COPD, asthma, or cystic fibrosis [49].The major signaling pathway for HRV cellular access occurs because pathogens use ICAM-1 as a receptor [32,50].Specifically, the pathogen binds to leukocyte function-associated antigen 1 (LFA-1) or the macrophage-1 antigen (Mac-1), natural adhesion ligands [32,50] (Figure 6B).ICAM-1 becomes a major catalyst for the eventual uncoating of the cell-invading virus, potentially exacerbating IPF pathogenesis [16,32].The role of ICAM-1 in COPD has been investigated by our research group and we have showed that the receptor was highly expressed in COPD smoking cohort [36].The expression patterns were prominent in the airways, especially on goblet cells and sub-mucosal glands, and could a potential risk factor of infection by common respiratory viral and bacterial pathogens [36].These biochemical mechanisms documented in other diseases shine a light on our findings as we believe our data bridges the gap in this area by demonstrating the abovementioned microbial activity in IPF, but this warrants further research.
Furthermore, our research group previously reported that ACE2, Furin and Transmembrane protease serine 2 (TMPRSS2) receptors are upregulated in IPF facilitating SARS-CoV-2 infection [19].We have reported similar findings in smokers and patients with COPD.Small airway epithelium, type 2 pneumocytes and alveolar macrophages were highly positive for these markers [51].Subsequently the expression patterns of various microbial receptors discussed above could suggest that IPF patients are at a higher risk of infections compared to healthy people.
Further Moreover, an animal model study by Iovino et al. investigated the role of PAFR in pneumococcal disease.The study demonstrated that although the absence of the PAFR gene (Pafr-/-) mice had higher bacterial (S. pneumoniae) growth in the lungs at 24 h postinoculation, the wild-type (WT) mice had higher bacteremia after 48 h [53].In WT, bacteria dispersed throughout the body and the central nervous system (CNS) compared to restricted local infection in the pulmonary area of Pafr-/mice [30,54].
Associated animal model research on the role of PAFR in pneumococcal pneumonia found that all WT mice died earlier after infection [53].At the same time, the mortality rate was delayed and reduced in Pafr-/mice [30,53].These models show that Pafr-/mice had a lower chance of developing an infection, especially when using PAFR antagonism [30,53].Further, heavy inflammation was detected in WT mice lungs compared to Pafr-/-[53].The results highlight that PAFR influences disease severity and could support the idea that molecule antagonisms could intervene in microbial activity, but further research in human subjects is warranted.This study's strengths include using resected human lung tissues to show expression, providing new information on disease pathogenesis, and assisting with future treatment or management strategies.This research has a few limitations, with the main challenge being the small number of IPF disease tissues available but they are rare human IPF tissue.Future investigation should aim for large sample sizes for better distribution and correlation with measured parameters.Secondly, the age of the normal control group is relatively younger than that of the IPF group due to the limited accessibility of obtaining the tissue.Further, comparing our data with previous research was challenging as very little human clinical work is done on these receptors in IPF, hence this is a novel study in IPF.The future applications for this study include performing cell cultures to understand the cellular activity between IPF and NC.Finally, need to further investigate microbial adherence to the respiratory epithelium and inflammatory cells as well as molecular analysis to study the role of genetics and proteins in relation to disease pathogenesis and treatment.

Conclusions
In conclusion, this is the first study to show PAFR and ICAM-1 expression in small airway epithelium, type 2 pneumocytes and alveolar macrophages in IPF patients compared to normal controls.Following previous research in COPD and IPF, high expression of these adhesion molecules could bridge the gap on inflammation and microbial activity in IPF.The clinical significance of these expression patterns suggests that microbes (bacteria and viruses) use these molecules as a mode of 'anchor' to gain cellular entry during inflammatory response, exacerbating disease pathogenesis.These findings can potentially help with future therapeutic development that can halt the disease progression, increasing the survival rate and reducing the global burden.
PAFR and ICAM-1 positive expression in small airways (SA) epithelium and lung parenchyma in IPF and NC are shown in Figures1A and 2B.PAFR and ICAM-1 were showing strong positive expression in epithelium (in brown) in IPF compared to NC, and similarly more positive expression in type 2 pneumocytes and alveolar macrophages in IPF than in NC.Negative control staining is shown in Figures1B and 2B, which indicate that there is no false staining.

Figure 3 .
Figure 3. (A) Immunohistochemical (IHC) staining with primary antibody PAFR of normal control (NC) and idiopathic pulmonary fibrosis (IPF) tissue.The IHC staining compares (A i, ii) small airway (SA) epithelium in NC and IPF respectively, (A iii, iv) type 2 pneumocytes (positive-red arrows, negative-orange arrows) in NC and IPF respectively, and (A v, vi) alveolar macrophages (positive-green arrows, negative-bright pink arrows) in NC and IPF respectively.(B) Percentage

J 15 Figure 6 .
Figure 6.(A) S. pneumoniae and H. influenzae express phosphorylcholine (Chop) which binds to platelet acting factor receptor (PAFR), facilitating the adhesion, colonization and eventual transmigration into the human airway epithelial cells and type 2 pneumocytes, increasing the risk of infection in patients.(B) Human Rhinovirus (HRV) uses intercellular adhesion molecule-1 (ICAM-1) as a receptor to anchor onto the airway epithelial cells and type 2 pneumocytes followed by uncoating of cell-invading virus, worsening disease pathogenesis.MAC-1; macrophage-1 antigen and LFA-1; leukocyte function-associated antigen.

Figure 6 .
Figure 6.(A) S. pneumoniae and H. influenzae express phosphorylcholine (Chop) which binds to platelet acting factor receptor (PAFR), facilitating the adhesion, colonization and eventual transmigration into the human airway epithelial cells and type 2 pneumocytes, increasing the risk of infection in patients.(B) Human Rhinovirus (HRV) uses intercellular adhesion molecule-1 (ICAM-1) as a receptor to anchor onto the airway epithelial cells and type 2 pneumocytes followed by uncoating of cell-invading virus, worsening disease pathogenesis.MAC-1; macrophage-1 antigen and LFA-1; leukocyte function-associated antigen.

Table 1 .
Patient demographics and lung function parameters.
, Moghoofiel et al. & Mostafaei et al. investigated bacterial coinfection in IPF and its possible role in disease progression.Their results demonstrated that coinfections (bacterial and viral) significantly exacerbate disease progression, enhancing the risk of death in IPF patients [15,16].This investigation served potential usefulness in understanding the underlying mechanisms in IPF infections and could provide insights in future therapeutics.Similar views shared by Santos et al. that if interventions can decrease or prevent pathogen adherence to the epithelium, it could protect high-risk populations before the disease has progressed [52].