All patients meeting the inclusion criteria and not meeting the exclusion criteria regardless of prior treatment history will be recruited to phase 1 of the study. The recruitment process duration is estimated at 12 months. The previous recruitment of 40 patients led to a pilot study [7], which impacted further study design. The maximum number of patients included in this phase will be 80 (33.3%) in the IPAF group, 80 (33.3%) in the CTD-ILD group, and 80 (33.3%) in the IIP group.

Only patients with newly diagnosed disease (eg, no prior diagnosis of ILD and, hence, no prior treatment) will be recruited. The estimated recruitment duration is 36 months. The number of patients to be included in this phase will be 20 (33.3%) in the IPAF group, 20 (33.3%) in the CTD-ILD group (only those with RA, SSc, PM/DM), and 20 (33.3%) in the IIP group (only those with IPF, NSIP, COP, LIP; optimally 10, 50%, patients in the IPF subgroup and 10, 50%, in the non-IPF subgroup). Patients who were previously recruited to phase 1 of the study and meet the inclusion criteria of phase 2 may also be included.

Please see Figure 2 for a flowchart of the study design.

A thorough medical history will be obtained using the Visit 1 questionnaire. During the first hospitalization, the following procedures will be performed:

Only patients who qualify for phase 2 of the study will have further visits (60 newly diagnosed patients). The following procedures will be performed after an average of 6 (SD 3) months from visit 1:

The following procedures will be performed after an average of 12 (SD 3) months from visit 1:

The following procedures will be performed after an average of 24 (SD 3) months from visit 1:

The following procedures will be performed after an average of 60 (SD 3) months from visit 1:

PFTs will include spirometry with a reversibility test (in the case of obstruction), body plethysmography, and diffusion capacity for carbon monoxide (DLCO). Both absolute and predicted values will be recorded: forced expiratory volume in 1 second (FEV₁), FVC, vital capacity (VC), FEV₁/VC ratio, total lung capacity (TLC), diffusing capacity for carbon monoxide measured by the single-breath method (DLCO SB), inspiratory thoracic gas volume (ITGV), and residual volume/total lung capacity (RV/TLC) ratio. Cough assessment will be conducted using the Leicester Cough Questionnaire and numeric scale. Dyspnea assessment will be performed using the Likert scale and the Modified Borg Scale.

Depression and anxiety assessment will be conducted using the Hospital Anxiety and Depression Modified Scale (HADS-M) and the 9-item Patient Health Questionnaire (PHQ-9).

Briefly, a vein will be punctured in order to take a blood sample, and 30 mL of blood will be drawn. Next, 15 mL of plasma and 15 mL of full blood will be stored separately at –80°C. The following will be analyzed: N-terminal prohormone of brain natriuretic peptide (NT-proBNP) biomarkers routinely used during rheumatological workup and potential IPAF biomarkers. For this, 15 mL of venous blood will be drawn and divided into 2 parts (10 mL and 5 mL), and no anticoagulant will be added to the samples.

The first sample (10 mL) will be sent to the local laboratory for routine analysis of NT-proBNP and rheumatological factor (RF) and for anti–cyclic citrullinated peptide (anti-CCP) and antinuclear antibody (ANA) screening tests. In the case of the confirmed presence of ANAs and the determination of a fluorescence pattern (eg, homogenous, speckled, peripheral, nucleolar), further tests will be ordered: anti–double-stranded DNA (anti-dsDNA), anti-Smith (anti-SM), anti-SSA/Ro, anti-SSB/La, anti–topoisomerase I (anti-Scl70), and antiribonucleoprotein (anti-RNP) antibodies and myositis-specific autoantibodies, such as anti–histidyl-tRNA synthetase (anti-Jo1), anti–threonyl-tRNA synthetase (anti-PL7), anti–alanyl-tRNA synthetase (anti-PL12), anti–Mi2 antigen (anti-Mi2), anti–human exosome complex (anti-PmScl), and anti–melanoma differentiation–associated 5 (anti-MDA5) antibodies.

From the second sample of 5 mL, after clot formation, approximately 2.5 mL of serum will be separated and then divided into 5 portions of 0.5 mL each and placed in Eppendorf tubes. The samples will be marked according to a system allowing for patient anonymization: medical center symbol (sample number - type of biological material - date of sample collection, eg, A12-S_10/04/18). The samples will be stored at –70°C. They will be then used for IPAF biomarkers’ identification. This includes testing for chemokine C-C motif ligand 18 (CXCL18), surfactant protein (SP)-A, SP-D, Krebs von den Lungen-6 protein (KL-6), and chitinase 1 (CHIT1).

For genetic testing, 15 mL of full blood will be collected and divided into 10 Eppendorf tubes containing 1.5 mL of blood each. Samples will be marked according to the aforementioned organizational pattern and stored at –80°C.

Before FOB, local anesthesia with lidocaine and conscious sedation with midazolam and optionally fentanyl will be performed according to detailed anesthesia protocols used in the endoscopy units of the respective medical centers taking part in the study. An intravenous cannula will be inserted prior to the procedure. During the endoscopy, the patient will be monitored according to safety protocols effective in the respective endoscopy units.

Bronchoalveolar lavage fluid (BALF) will be obtained from areas characterized by the greatest interstitial fibrosis involvement on HRCT. If such an area cannot be distinguished, BALF will be obtained from the middle lobe bronchus or the lingula on the side (B4, B5). Localization will be chosen based on HRCT results and will be recorded in the patients’ medical files. Next, 200 mL of sterile 0.9% saline solution, previously warmed to body temperature, will be inserted in parts through a 25 mL syringe to perform BAL. For the test to be deemed diagnostic, the protocol will target BALF salvage of at least 60%.

BAL will be performed according to the guidelines of the ERS [8] and the practical guidelines of the Polish Respiratory Society [9]. A differential cell count will be performed in every sample. BALF samples will be processed by an experienced laboratory technician based on Polish Respiratory Society guidelines [9].

Microscopic slide preparation, determination of cell viability, and determination of the dominant population will be performed according to standard laboratory procedures in the Polish Respiratory Society guidelines. The whole-cell count and cell viability will be measured in a sample derived from fluid that has undergone filtration or the first centrifugation. Samples will be prepared with the cytospin method (routinely 1200-2000 rpm for 10 minutes), and cellular smears will be assessed in cytospin samples stained according to the May-Grünwald-Giemsa method.

BALF samples will be secured for further tests, including testing for potential IPAF biomarkers. Briefly, 15 mL of BALF will be divided into 10 Eppendorf tubes containing 1.5 mL of BALF each and stored at –70°C. Low-speed centrifugal supernatants of BALF will be obtained from each sample. We will measure the concentration of potential biomarkers in each sample using the sandwich enzyme-linked immunosorbent assay (ELISA) technique.

Next, 5 mucosa samples will be obtained from the middle lobe bronchus for histopathological assessment later. Directly after sample collection, they will be put into dry Eppendorf tubes and frozen at –80°C. In addition, transbronchial lung cryobiopsy may be used to obtain material for histopathological assessment.

The submaximal exercise 6MWT, which entails the measurement of the distance walked over a span of 6 minutes, will be performed according to the detailed protocol used in the respective medical centers. The blood pressure (BP), heart rate (HR), and SpO₂ will be measured directly before and after the test. The following will be recorded: whether the patient completed the test, the distance walked (m), SpO₂, the SpO₂ nadir, and dyspnea sensation measured with the Modified Borg Scale and the Visual Analogue Scale (VAS). If the test is stopped or paused before 6 minutes, the reason (eg, dyspnea, high BP, cardiac arrythmia, limb pain, angina, or other) will be recorded.

The 6MWT will be performed according to Polish Respiratory Society guidelines [10]. Participants will be informed at 30-second intervals about the amount of time remaining. The predicted distance will be calculated based on the equation published in Polish Respiratory Society guidelines for the interpretation of the 6MWT as the most accurate for participants’ characteristics [10].

Pulse oximetry will be performed for all patients. If SpO₂<92% or there are clinical indications for oxygen therapy, ABG analysis will be performed. For ABG analysis, an artery (radial or femoral) is punctured in order to take a sample of arterial blood. The artery is then compressed in order to prevent bleeding/hematoma. The procedure is performed according to ERS guidelines [11].

HRCT of the lung will be performed during hospitalization. If the patient provides a CT scan from up to 6 months prior to recruitment (eg, from the outpatient clinic), we will analyze both scans and the radiological descriptions. To ensure the patient’s anonymity, a CD containing the patient’s scans will be marked by the technician according to the aforementioned pattern. Radiological analysis will be performed using the OsiriXLite DICOM Viewer. The following densitometric values will be measured: mean lung attenuation (MLA), kurtosis, skewness, and deviation of lung radiodensity (SD_IR). Statistical analysis of the data will be performed using Statistica software. Data will be presented as the median value and IQRs. Differences between quantitative data will be analyzed using the Kruskal-Wallis test and the post hoc Dunn test.

TTE with a thorough right heart description will be performed. The following parameters will be noted in the patient’s medical file: main pulmonary artery diameter; acceleration time; pulmonary valve peak velocity, tricuspid annular peak systolic velocity (TAPSV), tricuspid annular plate systolic excursion (TAPSE), and tricuspid valve peak regurgitation velocity; right ventricular basal and right ventricular midcavity diameters; right ventricular outflow tract and proximal and distal right ventricular outflow; right atrial short- and long-axis, inferior vena cava, and superior vena cava diameters; and right ventricular wall thickness.

Rheumatological consultation will be provided to patients with IPAF and CTD-ILD to verify the diagnosis.

KL-6 (mucin 1) is a glycoprotein encoded by the MUC1 gene and is expressed on the outer surface of type II alveolar epithelial cells and the airway epithelium. It has been proposed as a biomarker of IPF [12]. KL-6 probably promotes fibroblasts’ migration, proliferation, and survival in the respiratory system [12]. High serum KL-6 levels have been found in patients with IPF, NSIP, and other ILDs [13-15], which is associated with an aggressive clinical course and could also serve as a predictor of acute exacerbation [16]. High KL-6 levels have also been found in the BALF of individuals with various ILDs, including IPF [17].

Another proposed ILD activity biomarker is CC-chemokine ligand (CCL)18. In a population of patients with idiopathic ILD and SSc, it was revealed that the BALF CCL18 level was negatively correlated with the TLC and the DLCO and positively correlated with BALF neutrophil and eosinophil cell counts. During the follow-up period of ≥6 months, a negative correlation was observed between the decrease in the predicted TLC and the increase in serum CCL18 levels [18]. We hypothesize that such correlation also exists in IPAF, and we will test the theory that patients with IPAF with autoantibodies associated with SSc (anti-PmScl) have higher serum or BALF CCL18 levels than those who test negative for these autoantibodies.

Surfactant proteins SP-A and SP-D are large hydrophilic molecules produced by Clara cells and type II alveolar epithelial cells. SP-A and SP-D play an important role in innate immunity and modulate the excessive inflammatory response on the alveolar surface [19-21]. SP-A and SP-D levels are higher in patients with IPAF than in healthy individuals, patients with infectious pneumonia, or patients with nonfibrotic lung diseases [22-24]. Moreover, serum SP-D levels are lower in the IIP non-IPF group than in the IPAF group [22]. SP-A levels cannot be used to differentiate between patients with IPAF and CTD-ILD [24].

According to Xue et al [22], a negative correlation of serum SP-A levels and the DLCO was revealed in the IIP group (which included 27/69 patients with IPAF, although this subgroup was not separately analyzed). A negative correlation was also revealed with FEV₁ and the FVC [22]. This conclusion was partially confirmed by Wang et al [23], who revealed a negative correlation between serum SP-A levels and changes in the DLCO, FEV₁, and FVC values after treatment. Additionally, Wang et al [23] suggested a prognostic role of SP-A: the pretreatment levels were found to be significantly lower than the posttreatment ones in individuals with progressive fibrosis during the course of IPAF. Moreover, a positive correlation was found between shifts in KL-6 and SP-A levels [23]. In a prospective study, Xue et al [24] observed a significant difference in serum SP-A levels measured at baseline and after 1-year follow-up. In individuals whose condition had exacerbated, SP-A levels were correlated with lung involvement scores on HRCT [24].

These findings need to be validated in European populations. There is a need to determine whether there is a correlation between lung involvement and a decrease in pulmonary function and SP-A/SP-D levels in the bodily fluids of patients with IPAF.

CHIT1 is a member of the glycosyl hydrolase family 18. It is expressed predominantly in polarized and differentiated macrophages and acts as an innate immune mediator digesting cell walls of pathogens that contain chitin (eg, fungi). CHIT1, due to its dysregulated activity, is a proposed marker in granulomatous and fibrotic ILDs associated with interstitial inflammation and lung tissue remodeling.

A study by Bargagli et al [25] revealed elevated CHIT1 activity in the BALF of individuals with IPF in comparison with the healthy control group; however, no significant differences in the serum levels were noted. This suggests the role of CHIT1 expression in interstitial macrophages in tissue injury and remodeling. We hypothesize that CHIT1 may play an important role in the pathogenesis of other ILDs, including IPAF, especially with a progressive fibrosing phenotype.

Lee et al [26] revealed that in 2 different cohorts of individuals with SSc, CHIT1 activity and concentrations increased in the serum and BALF compared to demographically matched healthy controls. Individuals with SSc-ILD, in comparison with patients without lung involvement, were characterized by higher CHIT1 serum activity, which was also correlated with disease severity. The authors revealed that CHIT1 upregulates transforming growth factor beta 1 (TGF-β1) receptor expression and its signaling in fibroblasts, which makes CHIT1 a modulator of progressive interstitial lung fibrosis [27].

These findings raise the question whether CHIT1 or TGF-β1 and their interaction could be a potential target for SSc pharmacological therapy. As the etiopathogenesis of IPAF is still obscure, we plan to analyze both circulatory and lung activities of the enzyme to establish its potential role in the disease pathogenesis.

S100A9, also referred to as myeloid-related protein 14 or calgranulin B, is a member of the calcium-binding protein S100 family and is mostly expressed in neutrophils but also on vascular endothelial cells and mature macrophages. S1009A expression is enhanced by some chronic inflammatory factors [28]. It acts as an immunomodulatory, proinflammatory, and profibrotic agent [29]. It is mostly secreted in inflammatory lesions [30], and it has been suggested as a potential target for IPF treatment [31] and as a candidate biomarker to differentiate between IPF and other fibrotic ILDs [32]. However, convincing evidence from contemporary trials is yet scarce.

Elevated serum S100A9 levels have been reported in samples obtained from patients with RA, SLE, SSc, vasculitis, lung cancer, and various other diseases associated with chronic inflammation [33-35]. It has also been demonstrated that S1009A may promote atherosclerosis [36]. In an observational study by Lin et al [37], it was revealed that S100A9 levels were significantly elevated in BALF samples obtained from patients with severe IPF. It has also been demonstrated that S100A9 levels in the BALF of patients with IPF are positively correlated with the percentage of BALF neutrophils. This suggests a relationship between S1009A levels and disease activity. Some studies have reported that BALF S100A9 levels are significantly elevated in individuals with pulmonary involvement during the course of SSc [38,39].

CCL2 is a protein that attracts T lymphocytes, natural killer lymphocytes, and fibrocytes [40]. It is associated with inflammatory processes, cell migration and activation, fibrosis, and angiogenesis [41] through its 7-transmembrane-spanning G-protein-coupled CC-chemokine receptor 2 [42]. CCL2 plays a significant role in the pathogenesis of various diseases, such as tuberculosis [43], asthma [44], and RA [45].

A connection was established between CCL2 polymorphism and the occurrence of autoimmune disorders. The CCL2 gene (–2518G/A) single-nucleotide polymorphism has been shown to be associated with sarcoidosis [46]. Increased CCL2 levels have been found in the serum and BALF of patients with IPF [47]. In vitro and in vivo studies have revealed that CCL2 is associated with fibrosis by supporting monocyte-/macrophage-mediated inflammation, angiogenesis, collagen synthesis, myofibroblast cell differentiation, and fibroblast activation [48].

It remains unclear whether the chemokine profile of serum and BALF of individuals with IPAF, especially patients with progressive fibrosis, resembles that of patients with IPF.