The Difference Between SP-A and KL-6 Levels Responses for Treatment of Interstitial Lung Disease

Background The highly variable clinical course of interstitial lung disease (ILD) makes it dicult to determine patients’ prognoses. Serum surfactant protein-A (SP-A) and Krebs von den Lungen-6 (KL-6) were known biomarkers as a monitor of the prognoses. However, the clinical or pathophysiological differences of those biomarkers are not well evaluated. Therefore, through the comparison of the changes of SP-A and KL-6 levels before and after treatment, we investigated the clinical or pathophysiological differences which are embodied by those markers. Methods This study included retrospective data for 71 patients treated for ILD at the First Aliated Hospital of Guangzhou Medical University between August 2015 and September 2019. Serum SP-A and KL-6 levels were measured before and after treatment. The patients were followed for at least 3 months. Changes in the serum biomarkers (Delta SP-A and Delta KL-6) were signicantly correlated (r S = 0.482, P < 0.001); Delta SP-A and Delta KL-6 were inversely correlated with changes in pulmonary function (P < 0.05). In a cluster analysis of delta SP-A and KL-6 levels, patients were classied into three groups. In the cluster analysis, in the group in which only SP-A levels decreased after treatment, 50.0% of patients recovered respiratory function and had a signicant reduction of serum LDH levels.


Introduction
Interstitial lung disease (ILD) comprises a group of acute and chronic lung diseases characterized by diffuse pulmonary parenchyma, alveolar in ammation, and interstitial brosis. ILD includes both common and rare clinical diseases. In most cases, the causes remain unclear. ILD can be life-threatening; its overall mortality rate is as high as 52% [1]. Appropriate diagnosis and treatment are essential for patients with ILD.
The present ILD diagnostic methods depend on pulmonary function tests (PFT), radiological and histological examinations, and high-resolution computed tomography (HRCT) [2]. These methods provide important information for treating ILD. However, ILD's pathophysiology is complex. Various biomarkers are used to support the diagnosis of ILD and prognosis evaluation, and these could potentially help Improved, and 21 (30%) as Unchanged groups.
The patients' clinical baseline characteristics are shown in Table 1. The patients ranged in age from 19 to 80 years, with a median age of 54 (46-64) years; 34 (48%) were male and 17 (24%) were smokers. The median BMI was 24 (22.02-26.29) kg/m 2 . The median follow-up time was 8.5 months. The median values for the lung function parameters (% predicted values for FVC, FEV1, and DLCO) were lower than the normal range. There were no signi cant differences in any of the parameters between the three groups.
Serum SP-A and KL-6 levels before and after treatment Pretreatment serum levels of SP-A and KL-6 were not signi cantly different among the Progressive, Improved, and Unchanged group (Table 1).
Comparing SP-A and KL-6 levels before and after treatment, in the Progressive group, there was a signi cant increase in median serum SP-A levels from 39.6 (33.4-81.4) ng/mL to 75. 1 (42.4-95.2) ng/mL (P < 0.05) and in median serum KL-6 levels from 1329 (980-2138) U/mL to 2409 (1174-4909) U/mL (P < 0.05). Serum levels of SP-A and KL-6 were above the cutoff level in 47.6% and 95.2% of patients before treatment; those ratios became 76.2% and 100.0% after treatment (Table 1 and Figure 1 A, B).
Clinical and laboratory parameters before and after treatment Summarizing the pretreatment and post-treatment values in the three groups of WBC count, NEUTP ratio, MONO ratio, EOP ratio, CRP, LDH, and the PFT parameters (Figure 1), MONO and EOP ratios showed a signi cant decrease in the Improved group (P=0.024 and P = 0.005, respectively; Figure 1E and F, respectively). The three pulmonary function parameters (% predicted values of DLCO, FVC, and FEV1) signi cantly improved in the Improved group, and DLCO and FVC signi cantly reduced in Progressive group. These deteriorations and improvements were not statistically signi cant in the Unchanged group. Correlations between changes in Delta KL-6 and Delta SP-A and changes in pulmonary function Figure 2 shows the results of the correlation analysis of the relationships between pretreatment and posttreatment changes in the serum biomarker levels (Delta SP-A and Delta KL-6) and predicted PFT parameters (Delta DLCO, Delta FVC, and Delta FEV1). Delta SP-A showed signi cant inverse correlations with Delta DLCO (r S = -0.491, P < 0.001), Delta FVC (r S = -0.441, P < 0.001), and Delta FEV1 (r S = -0.354, P = 0.003). Similarly, Delta KL-6 showed signi cant inverse correlations with Delta DLCO (r S = -0.520, P < 0.001), Delta FVC (r S = -0.592, P < 0.001), and Delta FEV1 (r S = -0.610, P < 0.001). We also tested the correlation between Delta SP-A and Delta KL-6. This showed a signi cant positive correlation (r S = 0.482, P < 0.001; Figure 3). However, in Improved patients, cases with decreased SP-A and KL-6 were inconsistent.
Cluster analysis based on the serum levels of SP-A and KL-6 To explore the pathophysiological differences between KL-6 and SP-A, we classi ed the patients by cluster analysis and compared pathophysiological characteristics between the clusters (Figure 4). The patients were classi ed into three groups based on the changes in KL-6 and SP-A. In G3 and G1, the levels of SP-A and KL-6 changed in the same direction: G3, SP-A, and KL-6 all decreased; G1, SP-A, and KL-6 all increased. On the other hand, only SP-A levels were reduced by treatment in G2. (Figure 5A, B).
In G1, 56% of patients progressed disease activity. In G3, pretreatment KL-6 levels were signi cantly higher than G2. Post-treatment FVC and FEV1 were signi cantly improved, and 77.8% of patients improved disease activity. In G2, FVC was signi cantly improved, and half of patients improved disease activity ( Figure 5C-H). Immune suppressive treatment ratios did not differ signi cantly among the groups ( Figure 5I). Serum LDH levels and EOP ratios were signi cantly reduced in G2 ( Figure 5J, K). In G3, pretreatment EOP ratios tended to be higher than other groups but were not statistically signi cant ( Figure 5L). Discussion KL-6 is a mucin-like, high molecular weight glycoprotein, strongly expressed on type II alveolar pneumocytes cells. It was discovered in 1985 by Kohno et al. [24], and several studies have reported that it is a serum marker of ILD. SP-A is a lung-speci c protein produced by two types of epithelial cells in the peripheral airway: alveolar type II cells and Clara cells within the lung [21]. Type II lung cells are alveolar wall cells that proliferate during lung injury repair [7]. Elevated serum levels of SP-A and KL-6 re ect increased type II pneumocyte activity in the injured lung, with resultant back-leak into the blood [6,9,[18][19][20]. Serum KL-6 levels are known as a biomarker in the diagnosis, severity assessment, and prediction of outcomes for patients with ILD, and have been used in Japan [3,4,[18][19][20]25]. In patients with IPF, SP-A is a useful predictor of mortality and disease progression [15]. It was suggested that serum SP-A has a potential as a biomarker of the therapeutic outcomes of anti-brotic drugs [26]. However, although there are increasing reports about KL-6 and SP-A in the ILD context, there has been little investigation of the difference between SP-A and KL-6 as biomarkers. Therefore, this study investigated the characteristics of SP-A and KL-6 based on treatment responses, and we evaluated their value for disease monitoring.
We categorized a decline in FVC ≥10% or in DLCO ≥15% as an indicator of disease progression and an increase in FCV ≥10% or DLCO ≥15% as disease improvement. In light of previous classi cations, our criteria can be considered reasonable [27,28].
In this study EOP ratios showed a signi cant reduction after treatment in the Improved group patients. Eosinophil is one of the Type 2 immune components. As previously reported, T2 immunity is activated in some ILD patients [30,31]. Among the patients in this study, EOP ratios were within normal ranges both before and after treatment. However, there is a possibility that T2 in ammation of ILD-induced low-level eosinophilic in ammation was improved by the immune suppressive treatment of the Improved group patients.
In pretreatment, 92% of patients showed higher serum KL-6 levels than the previously determined cutoff levels, whereas in SP-A, only 51% of patients showed higher than the cutoff levels. The cut-off value of SP-A was de ned by IPF patients, and sensitivity was reported as 78.8% [30]. In the present study, the patients were not limited to IPF, which may affect the sensitivity of SP-A.
In the present study, pretreatment serum levels of SP-A and KL-6 did not relate to disease prognosis. It was reported that pretreatment KL-6 levels was signi cantly different depending on the response of pirfenidone therapy for IPF [32]. In the present study, pirfenidone was used on only 6 patients; the difference of treatment may affect this discrepancy. On the other hand, our study indicated the changes in serum levels of SP-A and KL-6 correlated signi cantly with changes in respiratory function, which re ects disease activity. These results are also consistent with other reports [6,10,11,26,28,32,33,[35][36][37][38]. Because of the di culty of respiratory function tests for patients of ILD, this evidence strongly supports the value of biomarkers in monitoring the activity of ILDs.
Although there was a signi cant correlation between Delta SP-A and Delta KL-6, the correlation coe cient was not high, suggesting that each marker may represent a different pathophysiology. Ishii et al. [4] reported that serum SP-A levels were higher in usual interstitial pneumonitis and lower in nonspeci c interstitial pneumonia, but KL-6 levels were higher in both. Yoshikawa et al. also indicated that changes in serum SP-A levels re ected more strongly the outcomes of anti-brotic drug therapy than KL-6 levels [26].
SP-A is produced mainly in Clara cells and type II alveolar cells, whereas KL-6 is produced only in type II alveolar cells [10,14,24]. SP-A is a C-type lectin with a molecular weight of 26-38 kDa, whereas KL-6 is a mucin-like glycoprotein with a large molecular weight of 200 kDa [14,39]. Biological and biochemical differences between SP-A and KL-6 are expected to be associated with different pathological changes in ILD.
To elucidate the pathophysiological differences between SP-A and KL-6, the patients were divided into three groups (G1, G2, and G3), by cluster analysis, according to the values of Delta SP-A and Delta KL-6. Pathophysiological characteristics were compared between clusters. Both SP-A and KL-6 were elevated in the G1 group. In this group, 80% of the patients were categorized as in the Progressive or Unchanged groups. Despite the higher immune suppressive treatment ratio of this group, respiratory function decreased. This suggests that non-in ammatory mechanisms, such as brosis, may contribute to pathophysiology.
In the G3 group, the levels of both SP-A and KL-6 decreased, and the patients showed improved respiratory function. Interestingly, the pretreatment EOP ratio tended to be higher in the G3 patients than in the other groups. Around 77.8% of the patients overall were treated with immune suppressive treatment. Although not signi cantly different due to the small number of patients, the EOP ratio decreased in 7 of 9 samples. These patients might be sensitive to immune suppressive treatment due to dependence on Th2 in ammatory pathophysiology. In the G2 group, only SP-A levels decreased; this also corresponded to improvement of respiratory function. Interestingly, the LDH and EOP ratios of G2 were signi cantly reduced after treatment ( Figure 5J), despite the LDH levels of G3 not being changed. Previous reports suggested the serum levels of LDH have value monitoring disease activity and progression. It is considered to re ect pulmonary cell damage [40]. In the acute phase of ARDS that is caused by COVID-19 infection, it was reported that SP-A levels elevated from a relatively early stage of the pneumonia [41].
Reportedly, serum SP-A levels increased with acute exacerbations of ILD, whereas KL-6 levels were elevated in drug-induced pneumonia or CTD-ILD [13]. In addition, SP-A expression was reported to correlate negatively with brosis score [42]. Takahashi et al. [10] reported that SP-A levels were associated with reversible ground-glass opacity levels, but not with indicators of the end stage of brotic changes, such as honeycombing. In addition, they reported that SP-A levels were signi cantly lower in parenchymal collapse opacity-dominant type patients than in ground-glass opacity-dominant type patients [10]. Studies have shown that serum SP-A rises faster than KL-6 in the progressive group [43]. This suggests the G2 patients were not at the end stage of the disease, which might explain why their respiratory function improved with immune suppressive treatment. Compared to KL-6, the lower molecular weight of SP-A may provide an advantage in detecting it in blood serum at an earlier stage of disease.
In summary, although both SP-A and KL-6 are pathological markers that re ect the disease course with treatment of patients with ILD; in some patients, only SP-A levels responded to an improvement of the disease. It is, therefore, important to measure both SP-A and KL-6 levels simultaneously for the pathophysiological monitoring of patients with ILD.
This has several limitations. First, we included patients with de nite usual interstitial pneumonitis patterns on HRCT but without surgical lung biopsies; therefore, misclassi cation was possible. Second, this was a retrospective study, and the observation period differed among the patients. Finally, we were unable to compare the results of serum biomarkers to histopathological patterns because of the lack of surgical lung biopsies. In the future, we intend to investigate these biomarkers in a prospective study setting.

Conclusions
In conclusion, our results demonstrated that serum levels SP-A and KL-6 in patients with ILD signi cantly decreased in patients who showed disease improvement and signi cantly increased in patients who showed disease progression. However, the response of both markers was different according to their pathophysiological or biological characteristics; measuring both markers are important for understanding a patient's pathophysiological condition. The use of human serum samples was in accordance with relevant legislation in China and the wishes of donors, their legal guardians, or next of kin, where applicable, who had offered written informed consent to using the serum samples for future unspeci ed research purposes.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.    Comparison of clinical and laboratory parameters between pretreatment and post-treatment. The patients were allocated to the Progressive, Unchanged, and Improved groups according to the difference between