The balance between lung regulatory T cells and Th17 cells is a risk indicator for the acute exacerbation of interstitial lung disease after surgery

doi:10.21203/rs.3.rs-2364399/v1

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The balance between lung regulatory T cells and Th17 cells is a risk indicator for the acute exacerbation of interstitial lung disease after surgery

https://doi.org/10.21203/rs.3.rs-2364399/v1

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Purpose: Acute exacerbation of interstitial lung disease is the leading cause of 30-day mortality among patients with lung cancer in Japan. We assessed the characteristics of the lung immune environment before the onset of acute exacerbation of interstitial lung disease.

Methods: This retrospective matched case-control study comparing the immune phenotype of helper T cells in the lung from patients with and without acute exacerbation of interstitial lung disease after surgery was conducted in 135 patients who underwent surgical resection for lung cancer and were pathologically diagnosed with interstitial lung disease at our institute between 2009 and 2018. There were 13 cases of acute exacerbation of interstitial lung disease and 122 cases without acute exacerbation matched using propensity score analysis, and 12 cases in each group were compared. We evaluated the percentage of T helper (Th)1, Th2, Th17, regulatory T cells (Treg), and CD8 cells in CD3⁺ T cells and the Th1:Th2, Th17:Treg and CD8:Treg ratios in patients with acute exacerbation by immunostaining of lung tissue in the non-tumor area.

Results: We found a significant difference in the lung Th17:Treg ratio between patients with and without acute exacerbation (1.47 and 0.79, respectively; p=0.041). However, we detected no significant differences in the percentages of lung Th1 (21.3% and 29.0%), Th2 (34.2% and 42.7%), Th17 (22.3% and 21.6%), Treg (19.6% and 29.1%), or CD8⁺ T cells (47.2% and 42.2%) of CD3⁺ T cells between groups.

Conclusion: Th17:Treg ratios in the lung from patients with acute exacerbation were higher than in those without.

Surgery

Acute exacerbation

Interstitial pneumonia

Regulatory T cell

Th17

Acute exacerbation (AE) of interstitial lung disease (ILD) is a lethal respiratory deterioration resulting from an unidentified cause (Leuschner G et al. 2017; Collard HR et al. 2016; Chunrg A et al. 2011). Idiopathic pulmonary fibrosis (IPF) is the most common form of ILD (approximately 65%), and up to 46% of deaths in IPF are preceded by an AE. The median survival of patients with IPF who experience AE is approximately 3 − 4 months (Collard HR et al. 2016).

AE of ILD (AE-ILD) is also the main cause of 30-day mortality in surgery for lung cancer patients (Masuda et al. 2016; Kumar P et al. 2016; Goto T et al. 2003). A multi-institutional retrospective study conducted by the Japanese Association for Chest Surgery reported that AE-ILD occurred in 164 (9.3%) of 1763 postoperative patients with ILD, with a mortality rate of 43.9% (Sato T et al. 2014). Several reports have investigated the risk factors for AE-ILD (Sato T et al. 2014; Raghu G et al. 2011); however, prediction using clinical factors is difficult. Some patients with similar previously reported risk factors, such as male sex, low % volume capacity (VC), low % diffusing capacity of the lung for carbon monoxide (DLCO), and usual interstitial pneumonia (UIP) appearance (Sato T et al. 2014; Raghu G et al. 2011), have AE, while others do not. Therefore, we speculated whether the risk of AE could be determined by assessing differences in the immune environment of the lungs.

The upregulation of macrophage activation pathways is involved in lung inflammation during AE of idiopathic pulmonary fibrosis (AE-IPF). The M1 pathway is similar to the pathology of acute respiratory distress syndrome (ARDS) (Marchioni A et al. 2018), leading to increased expression of interleukin (IL)-8 and CXCL1 and increased neutrophil recruitment via the CXCR2 receptor. The M2 pathway is characteristic of AE-ILD, is activated by type II alveolar epithelial cell damage, and is involved in fibrosis (Prasse A et al. 2006). The rapidly deteriorating cytokine profile of patients with IPF during AE-IPF has been reported to be closer to the cytokine profile of ARDS, which is predominantly characterized by inflammation through the M1 pathway, rather than accelerating the endogenous fibrosis process via the M2 pathway (Papiris SA et al. 2018). Recent studies have revealed that lung inflammation mediated by CD4⁺ T cells may contribute to ARDS pathogenesis (Yu ZX et al. 2015; Risso K et al. 2015). However, there are no reports regarding the balance between subgroups of T cells in human alveolar tissue before the onset of ARDS or AE-ILD.

Thus, the present study conducted a lung T-cell subset analysis to investigate whether the balance of T-cell subsets prior to the onset of the disease is associated with postoperative AE-ILD.

Patients and study design

This was a single-center, retrospective, matched case-control study that enrolled non-small lung cancer patients with ILD who underwent pulmonary resection at Juntendo University between January 2009 and June 2018. The data presented here are from an observational database of surgical candidates at Juntendo Hospital.

This study was approved by the ethics committee of Juntendo University School of Medicine (No. 2016095) and performed in accordance with the guidelines of the Declaration of Helsinki and its subsequent amendments. Informed consent was obtained in the form of opt-out on the website. This study was supported by JSPS KAKENHI (grant number 21K16523).

Definitions

Interstitial lung disease

In this study, 137 patients with ILD were pathologically diagnosed using lung resection for lung cancer. All specimens were fixed overnight in 15% buffered formalin and sectioned at 5–10 mm parallel intervals, including the lungs in areas where lung cancer was absent. Pathological ILD was diagnosed according to the 2002 American Thoracic Society/European Respiratory Society (ATS/ERS) (Raghu G et al. 2011; American Thoracic Society et al. 2002). The pathology of UIP is characterized by the inconsistent progression of lesions, interstitial inflammation, fibrosis, and honeycomb changes. The histologic findings of other ILD were also diagnosed according to the 2002 ATS/ERS international multidisciplinary consensus (American Thoracic Society et al. 2002). Cases that could not be classified for various reasons, including inadequate clinical or radiologic information and major discrepancies between clinical radiologic and pathologic findings, were diagnosed as unclassifiable interstitial pneumonia.

Clinically diagnosed ILD cases where the ILD site was not resected or where the pathological diagnosis was not confirmed were excluded from this study.

Acute exacerbation

The definition of AE-ILD in this study was based on criteria reported in 2011 (Raghu G et al. 2011) and 2016 (Collard HR et al. 2016). AE-ILD was defined as worsening of dyspnea and hypoxia from baseline within 30 days, including new ground-glass abnormalities and/or consolidation superimposed on a background reticular or honeycombing pattern. Findings with alternative causes, such as left heart failure, aspiration pneumonia, pulmonary embolism, or an identified cause of acute lung injury, were excluded from AE-ILD.

Control patients

In total, 135 patients were pathologically diagnosed with ILD. Patients were divided into AE (n = 13, 9.6%) and non-AE (n = 122, 90.4%) groups. Thirteen cases of AE-ILD and 122 cases of non-AE were matched using propensity score analysis, and 12 cases in each group were compared.

Immunohistochemical analysis

The subpleural lung without cancer was used in this study, which was collected for the diagnosis of ILD and stored in a paraffin block. We evaluated immunohistochemical staining for CD3 and dual staining for CD4 and CD8, and we also evaluated immunohistochemical staining for CD4 and dual staining for T-bet, GATA3, STAT3, FOXP3, and CD8 for all non-cancer specimens.

After deparaffinization, monoclonal antibodies against CD3, CD4, CD8, T-bet, and GATA3 were added to the autostaining device. Immunohistochemical staining was performed using a commercially available autostaining device (VENTANA BenchMark GX; Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s protocol. Monoclonal antibodies against STAT3 (LSBio, Seattle, WA) and FOXP3 (Abcam, Cambridge, UK) were used for staining. A dual-labeled cell was defined by the presence of cytoplasmic staining for CD4 (red chromogen), CD3, T-bet, GATA3, STAT3, FOXP3, and CD8 (brown chromogen), and nuclear staining for the interrogated transcription factor (blue chromogen). Slides were visualized using an Olympus AX73 (Olympus, Tokyo, Japan) microscope. The dual-labeled CD3⁺/CD4⁺, CD4⁺/T-bet⁺, CD4⁺/GATA3⁺, CD4⁺/STAT3⁺, CD4⁺/ FOXP3⁺, and CD8⁺ cells were counted in over three independent areas, with the greatest abundance of lymphocytes (e-Figure 1). T helper (Th) 1 cells, Th2 cells, Th17 cells, and Tregs were identified as CD4⁺ T-bet⁺ cells, CD4⁺ GATA3⁺ cells, CD4⁺ STAT3⁺ cells, and CD4⁺ FOXP3⁺ cells, respectively. Any visible pigment, including weak or pale staining, was considered positive. Cell counts were performed using Image J (National Institutes of Health, Bethesda, MD). MF, who was blinded to the clinical backgrounds and courses of the patients, performed the immunohistochemical evaluation, which was later checked against clinical data. If the difference from other data was more than 20%, the patient was re-evaluated. The mean number of cells counted per high-power field was used to calculate the percentages of Th1, Th2, Th17, Treg, and CD8⁺ cells.

Statistical analysis

Clinicopathological features of patients with ILD with or without AE after surgery were investigated. Continuous variables were compared using Student’s t-test, Welch’s t-test, and the Mann − Whitney U test. Categorical variables were compared using the chi-squared test and Fisher’s exact test. We used propensity score matching to control for clinical selection bias in the two groups when comparing the AE and non-AE immune samples. Propensity scores were calculated based on the distribution of ILD, UIP pattern, preoperative arterial oxygen partial pressure, %VC, %DLCO, and the surgical procedure as independent variables. Patients were matched 1:1 by nearest-neighbor matching (caliper width: 0.3) without replacement.

All statistical analyses were performed using SPSS Statistics for Windows version 25.0 (IBM, Armonk, NY). P-values < 0.05 were considered statistically significant.

Clinicopathological characteristics of the matched groups are shown in Table 1. There were no significant differences in clinical factors between the two groups before matching (e-Table 1), but the difference between the groups decreased after matching (Table 1).

Table 1

Patient characteristics after propensity-score matching
Variable
	AE	Non-AE	p-value
Number	12	12
Male (%)	10 (83.3%)	10 (83.3%)	1.000
Age	73.0 ± 6.3	73.1 ± 8.9	0.978
Smoking status	46.2 ± 20.1	46.5 ± 41.1	0.985
Diffuse and central distribution of IIP in CT	3 (25.0%)	2 (16.7%)	0.611
%VC < 80%	3 (25.0%)	2 (16.7%)	0.611
FEV1.0/FVC < 70%	3 (25.0%)	4 (33.3%)	0.639
%DLCO	4 (33.3%)	2 (16.7%)	0.338
Preoperative pO₂ < 70 mmHg	1 (8.3%)	2 (16.7%)	0.534
Surgical procedure			1.000
Pneumonectomy	1 (8.3%)	1 (8.3%)
Lobectomy	8 (66.7%)	8 (66.7%)
Segmentectomy/ wide wedge resection	2 (16.7%)	2 (16.7%)
Histology of lung cancer (sq)	3 (25.0%)	5 (41.7%)	0.375
p-stage I	4 (33.3%)	2 (16.7%)	0.407
Pathological UIP	6 (50.0%)	6 (50.0%)	1.000
Data are presented as the mean ± standard deviation unless otherwise indicated. AE, acute exacerbation; IIP, idiopathic interstitial pneumonia; CT, computed tomography; VC, volume capacity; FEV1.0, forced expiratory volume in 1 second; FVC, forced vital capacity; DLCO, diffusing capacity of the lung for carbon monoxide; UIP, usual interstitial pneumonia.

Figure 1 shows a typical lung section of postoperative AE-ILD (Fig. 1A, 1C) and ILD without AE (Fig. 1B, 1D). Dual-labeled cells (CD4⁺/STAT3⁺) were more prominent in AE patients (Fig. 1A) than in non-AE patients (Fig. 1B), but dual-labeled cells (CD4⁺/FOXP3) appeared less in AE patients (Fig. 1C) than in non-AE patients (Fig. 1D).

Table 2 shows immunohistochemical findings in the lung from the postoperative AE and non-AE groups. There was a significant difference in the lung Th17:Treg ratio between AE and non-AE (p = 0.41). However, we detected no significant differences between the AE and non-AE groups in the percentage of lung Th1 (21.3% and 29.0%), Th2 (34.2% and 42.7%), Th17 (22.3% and 21.6%), Treg (19.6% and 29.1%), and CD8⁺ T cells (47.2% and 42.2%) of CD3⁺ T cells. There was no difference in the lung Th17:Treg ratio between UIP or non-UIP, and the ratio of lung Th2 was predominant in UIP (e-Table 2).

Table 2

Immunohistochemical findings in the AE and non-AE groups
	AE	Non-AE	p-value
Th1:Th2 ratio	0.66 ± 0.21	0.69 ± 0.17	0.755
Th17:Treg ratio	1.47 ± 0.92	0.79 ± 0.34	0.041
CD8:Treg ratio	3.64 ± 2.80	1.72 ± 0.92	0.052
Th cell subtype in CD3-positive T cells, %
Th1	21.3 ± 10.5	29.0 ± 7.1	0.067
Th2	34.2 ± 18.1	42.7 ± 9.2	0.193
Th17	22.3 ± 8.6	21.6 ± 8.0	0.858
Treg	19.6 ± 12.9	29.1 ± 12.3	0.101
CD8	47.2 ± 15.4	42.2 ± 15.4	0.409
Data are presented as the mean ± standard deviation. AE, acute exacerbation; Th, T helper cell; Treg, regulatory T cell.

To our knowledge, this is the first study to show that the Th17:Treg ratio in the lungs of patients with ILD and postoperative AE is higher than that in those without AE.

There are few reports regarding AE-ILD and cytokines, but Senoo et al. (Senoo et al. 2021) reported a relationship between AE-IPF and cytokine levels. They reported that the levels of IL-17 and IL-23 in bronchoalveolar lavage fluid of AE-IPF patients were increased, and Th17 cells were the predominant source of IL-17A in mice with AE of IPF (Senoo et al. 2021). Moye et al. also reported a relationship between T cells and AE-IPF; Treg depletion in wild-type mice with established lung fibrosis significantly worsened infection-induced fibrosis exacerbation, and Treg expansion completely inhibited AE (Moye et al. 2020). In the present study, the percentage of lung Treg and Th17 cells among T cells was not significantly different between the postoperative AE and non-AE groups, but the lung Th17:Treg ratio was higher in the AE group, which supports previous findings in mouse models that Tregs suppress the onset of AEs and that Th17 promotes the onset of AEs.

There are numerous reports regarding the relationship between ARDS pathogenesis and cytokine levels. Imbalances in Th17 or other inflammatory subsets in ARDS promote an increase in autoimmune disease. IL-17 acts synergistically with IL-6 and tissue necrotic factor and rapid neutrophils (Lin S et al. 2018). With the elucidation of the involvement of cytokines, clinical trials using IL-6 antagonists for ARDS caused by coronavirus disease have been established clinically. The utility of IL-6 antagonists has been reported in a meta-analysis (Shankar-Hari M, et al. 2021). Although AE-ILD is a fatal and clinically important problem, its pathophysiology is difficult to elucidate because it occurs less frequently than ARDS. In this study, the involvement of lung Th17 cells before the onset of postoperative AE-ILD was suggested; this may be useful for future pathological elucidation and therapeutic research.

The present study had several limitations. First, this was a retrospective study conducted at a single center. Therefore, this study may lack external validity. We would like to verify whether this can be demonstrated in more cases. Second, the lung parenchyma to be evaluated was defined in this study. This is a limitation because there were only a few residual specimens of non-tumor tissue. The specimens were subpleural lung parenchyma with typical ILD findings. AE-ILD often develops from the subpleural lung parenchyma, regardless of laterality or lobe; therefore, we believe that the specimen in this study is useful as an evaluation site. However, the distribution of T cells in the lungs may vary depending on the section being evaluated. Further prospective studies are required to confirm our findings.

In conclusion, there may be a difference in the balance of T-cell subgroups in lung tissue before the onset of AE after surgery. The lung Th17:Treg ratio was higher in the AE group than in the non-AE group.

Data availability: The data are available from the corresponding author on reasonable request. The data are not publicly available due to privacy reasons.

Acknowledgements

We thank the members of the Laboratory of Morphology and Image Analysis, Research Support Center, Juntendo University Graduate School of Medicine, for technical assistance with microscopy. We would also like to thank Drs. Sachiko Miyake and Asako Chiba from the Department of Immunology, Juntendo University School of Medicine, for providing recommendations for improvement. We wish to thank Drs. Kazunori Hata, Takuya Ueda, and Tomohito Takeshige for their advice regarding the immunostaining method. We would like to thank Editage (www.editage.com) for English language editing.

Funding: This study was supported by JSPS KAKENHI (Grant Number: 21K16523). The funders of the study had no role in the study design, data collection, data analysis, or data collection, interpretation, or writing of the manuscript.

Author information

Departments of General Thoracic Surgery, Juntendo University School of Medicine, Tokyo, Japan

Mariko Fukui, Kazuya Takamochi, Takeshi Matsunaga, Aritoshi Hattori, Kenji Suzuki

Departments of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan

Norihiro Harada

Departments of Human Pathology, Juntendo University School of Medicine, Tokyo, Japan

Takuo Hayashi

Departments of Human Anesthesiology, Juntendo University School of Medicine, Tokyo, Japan

Izumi Kawagoe

Authors’ contributions: MF and KS had full access to all study data and took responsibility for the integrity of the data and the accuracy of the data analysis, including any adverse effects. MF, NH, and KT contributed to the study concept and design. MF, NH, TM, AH, KT, IK and KS contributed to the data collection, data analysis, revision, and final approval of the manuscript.

Corresponding author: Mariko Fukui

Conflict of interst: The authors declare no conflicts of interest.

Ethics approval: This study was approved by the ethics committee of Juntendo University School of Medicine (No. 2016095) and performed in accordance with the guidelines of the Declaration of Helsinki and its subsequent amendments.

Consent to partivipate: Informed consent was obtained in the form of opt-out on the website.

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No competing interests reported.

supplmentalfigure.tif
Supplemental Figure 1. Hematoxylin and eosin staining and immune double-staining in one patient in the non-AE group. The patient was pathologically diagnosed with usual interstitial pneumonia. A dual-labeled cell was defined by the presence of cytoplasmic staining for CD4 (red chromogen), CD3, T-bet, GATA3, STAT3, FOXP3, and CD8 (brown chromogen), and nuclear staining for the interrogated transcription factor (blue chromogen). The dual-labeled CD3⁺/CD4⁺, CD4⁺/T-bet⁺, CD4⁺/GATA3⁺, CD4⁺/STAT3⁺, CD4⁺/FOXP3⁺, and CD8⁺ cells were counted in the high-power field. AE, acute exacerbation
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The balance between lung regulatory T cells and Th17 cells is a risk indicator for the acute exacerbation of interstitial lung disease after surgery

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Abstract

Figures

Introduction

Methods

Patients and study design

Definitions

Interstitial lung disease

Acute exacerbation

Control patients

Immunohistochemical analysis

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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