Ideal biomarkers should be tests that are widely available, easy to measure in accessible biological samples (such as blood, urine, saliva) with a standardized and affordable method and highly reproducible. The neutrophil to lymphocyte ratio (NLR) certainly fulfills these criteria and its significance as prognostic biomarker for different pathologies is currently under investigation. It is remarkable in how many clinical conditions a high NLR has been associated with an unfavorable prognosis. The most extensive studies were conducted for: the disease-free survival and overall survival rate in breast cancer¹ or in other forms of cancer;² acute episodes frequency in mood disorders;³ cardio-vascular disease severity and risk of major events;^4,5 sepsis,⁶ or treatment response in hepatitis B.⁷ In pulmonary disease, high NLR is a marker for unfavorable prognosis in lung cancer⁸ and COPD.⁹ The common link between this variety of illnesses could be the low-grade permanent inflammation, a process in which neutrophils are essential.

The role of the neutrophil within the mechanism of inflammation triggered by silica in the lung parenchyma has been underlined in many studies. After the innate cellular response led by the macrophages is initiated, the inflammasome activation occurs and the chemokines attract neutrophils. The binding of silica to macrophage scavenger receptors releases IL-1β and could even lead to apoptosis. IL-1β and the direct effect of silica on Th17 response amplifies the recruitment and the activation of the neutrophils.¹⁰It has been experimentally demonstrated that silica directly recruits neutrophils via leukotriene B4, produced by mast cells and macrophages,¹¹independent of the inflammasome mediated signals.

In view of the aforementioned mechanisms related to the involvement of neutrophils in the silica induced lung inflammation, we tested the hypothesis that a high NLR value could be associated with a more rapid evolution of silicosis. Industrial exposure to silica is the leading cause for silicosis in most of the recently diagnosed patients, and it is also known for its irritative effects on the airways and association with chronic bronchitis. In order to avoid as much as possible biases in our results, we have excluded from the analysis all records in which symptoms of chronic productive cough or an obstructive lung function pattern were present.

We conducted a retrospective study, reviewing the medical records of patients registered with silicosis in the Department of Occupational Medicine, Colentina Hospital, Bucharest, Romania. First, from the 271 patients diagnosed with silicosis mentioned in the clinic’s pneumoconiosis register between March 2017 and March 2018, 121 were excluded because they had either a pseudotumoral pneumoconiosis or because no recordings from 5 years before were found. Secondly, we excluded the 34 that were current smokers without an obstructive lung function pattern and the 62 that had chronic obstructive lung disease. Finally, data from 32 medical records were extracted and analyzed.

Data recorded in 2013 (T₀) and data recorded in 2018 (T₁) were extracted from the medical records: age, gender, occupation, diagnostic, stage of silicosis, comorbidities, exposure and retention time and blood cell count. The exposure time was defined as the number of years the patient has been exposed to dust containing silica; it reflects the exposure duration. The retention time is the number of years from the first occupational exposure and represents an estimate of the time silica particles have been retained in the lung. The stage of silicosis was classified according to the International Labour Organization standards. The average reference level of the NRL for silicosis patients was 1.86, with a range between 1.32-2.6, according to previously reported data.¹²

Spirometry was performed with a Jager/Viasys Pneumotachograph (CareFusion, Höchberg, Germany); if several spirometries were recorded during hospitalization, we selected only the values recorded at admission. Data were interpreted in comparison to the international reference values.

Progression of silicosis was considered a change from the initial radiological findings (from 5 years before, in 2013) to a more advanced stage of silicosis during this length of time (in 2018), as assessed by the Commission of Pneumoconiosis, which includes 3 independent national certified readers, in accordance with the International Labour Office guidelines.¹³ Stages of silicosis were classified based on the total length of involvement of the small opacities in respect to the projection of the lateral chest wall; stage I included small opacities with a total length up to one-quarter of the projection of the lateral chest wall, stage I/II small opacities with a total length that exceeds one-quarter but is less than one-half of this projection and stage II if the length of the small opacities is at least one-half of the projection of the lateral chest wall. The tendency of coalesce of the small opacities is coded "ax” and defines the silicosis stage II/III. Stage III silicosis (presence of large opacities) was an exclusion criterion and none of the patients included in the analysis were in this stage.

Based on this definition of the silicosis progression, the patients were divided into two groups: group 1 included patients who developed a progression of silicosis when comparing the T₀to the T₁ X-rays, and group 2 included those with no detectable signs of progression (apparently stable disease), as identified from the radiological signs.

Data were processed with SPSS and using the Statplus for Mac software, 2016, v6 (Walnut, CA, US). The variables with normal distribution are presented as mean, and the ones that did not show a normal distribution as median. Comparison between groups was done using Anova for normally distributed variables and with Mann-Whitney U test for variables that did not have a normal distribution. The Chi² test was used to compare the distribution of the qualitative variables. A threshold of 95% was selected for the statistical significance.

Among the 32 patients included in the analysis, 10 (31.25%) patients had a progression of the disease. According to occupation, all cases were the result of silica dust exposure from foundries. At baseline (T₀), only 4 patients (one in group 1 and 3 in group 2) continued to work in activities related to silica exposure.

At T₀, the median age was 46.5 years (IQR=7.25). The gender distribution was similar in the two study groups: there were 7 women and 3 men in group 1, and 9 men and 13 women in group 2 (p=0.550). The patients in group 1 were younger, had higher exposure time, but lower retention time than those in group 2. However, none of these variables reached statistical significance, when the two groups were compared (Table 1).

 Table 1. Descriptive statistics of the two groups at T₀

 

 

 

 

 

The classification of silicosis at T_o, was as follows: in group 1 we recorded 8 cases of grade I silicosis and 2 with grade I/II. Group 2 included 11 cases of stage I silicosis, 6 of silicosis I/II, 3 of silicosis II and 2 of silicosis II/III. At T₀, although more advanced silicosis cases were in the group 2, the difference in distribution did not reach the statistically significant threshold (p=0.326). At T₁, the case distribution of silicosis stage I, stage I/II and II in the 2 groups was also similar (p=0.732).

The spirometry values were in the normal range in all cases. No significant differences in the lung function parameters were found at 5 years from baseline (p>0.05 for both VC% and FEF1%).

All patients in group 1 had the NLR above the average value reported for silicosis and 4 patients had the NLR above the upper limit. They all progressed from stage I to stage I/II during this period of time. In group 2, 45.5% of the patients had a NLR higher than the average and 13.6% had the NLR above the upper normal limit. The median of the NLR of the whole group was 2.11 (IQR=0.85). The median NLR in group 1 was 2.94 (IQR=0.89) and in group 2 it was 1.7 (IQR=0.84).

The comparison of the medians of the NLR in the 2 groups found a significantly higher NLR value in the group presenting a progression of the radiological pattern after 5 years of follow-up (p=0.016, U=51, r=0.424) – Figure 1.

 

Figure 1. Neutrophil to lymphocyte ratio in the study groups

After 5 years from the baseline data registration, the average NRL was 2.09 for the whole group. In group 1 this average was 2.9 and in group 2 the average NLR was 1.77. The

difference was statistically significant (p=0.031, U=57, r=0.38).

Even if its incidence has decreased in the last decade, silicosis maintains a second place in the hierarchy of the new registered cases of occupational diseases in Romania, with around 150-300 new cases/year.^14,15 The usual evolution of uncomplicated silicosis is to progress, but at a slower pace by comparison to other lung fibroses; some cases have a more aggressive progression, although the mechanism that accelerates progression is not well known. An increased probability of progression may be related to a high level of exposure, continuing to work in the same environment and the extent of the radiological opacities at the initial diagnosis.¹⁶Respiratory tuberculosis increases the mortality risk.¹⁷ Biological markers such as L-selectin,¹⁸ Krebs von den Lungen-6 (KL-6), surfactant protein D (SP-D), matrix metalloproteinase-2,¹⁹ oxidative markers²⁰ that would anticipate the radiological findings or would provide prognosis information have been reported in research studies, but none of these biomarkers has been translated into clinical practice.

Our study aimed to identify an easy to measure biological marker that has been proposed to predict the inflammatory status and disease progression for other diseases. In 5 years of follow-up, we report a significantly higher NLR in chronic uncomplicated silicosis in patients with progressive evolution compared to those without progression, as reflected by the standard radiological examination. In both groups, there was no significant lung function deterioration observed between the baseline values and after 5 years. The fact that lung function showed no steep deterioration is in line with our previous results on other silica patient groups.²¹

This result is not influenced by the smoking habit, as all the patients included in the analysis were non-smokers when the first registration was done and continued to be non-smokers during the follow-up period. None of these patients had obstructive lung disease or chronic bronchitis, although higher than the general population exposure to irritants such as vapors, metal fumes and solvents was estimated from their occupational history. In the last 10 years, most cases of silicosis diagnosed in this clinic – and all those included in the analysis – occurred in current or former employees exposed to silica in industrial settings, from sand casting, cast shakeout, grinding or sanding. In this particular type of exposure, furans, resins, organic solvents (toluene, phenols and even benzene trances), ammonia, sulphuric acids are commonly encountered.

We did not have a large enough sample to stratify the risk according to the level of the NLR, as has been done for endothelial dysfunction and cardio-vascular disease, for example.⁵ But the patients with the highest NLR values (higher than the upper limit found in other groups of patients with silicosis) also had the most pronounced evolution.

During the 5 years of the follow-up period, the patients with low NLR did not show radiological progression, while the ones with high NLR accumulated visible lesions. This might be related to the inflammation process in silicosis, which is not constant, with periods in which it is more aggressive and periods when it is better controlled. The high NRL could be the marker of the active inflammatory process and might not necessarily be a permanent feature during the lifelong evolution of the disease; it might reflect the progression and not the stage.

The lung inflammation in the chronic forms of silicosis develops gradually, generally in decades. The continuity of the exposure, but also other factors such as the immunological reactivity of the patient, lifestyle habits and the number of infections accelerate the progression and the formation of fibrosis. Except for one patient, all others in the group with disease progression had stopped the occupational exposure. Therefore, we cannot consider the continuation of exposure to be the main factor for progression.

The intimate mechanism for the relatively slow evolution in many patients is unknown and even less characterized is the neutrophil contribution to it. In recent years, the roles of the neutrophil in human physiology and in several pathological states have been much expanded²²from their initial anti-infectious role; neutrophils secrete interleukins, (such as IL-17, which attracts lymphocytes), promote angiogenesis in hypoxemic tissues,²³ produce neutrophil extracellular traps (NETs) that influence vascular inflammation, probably have an anti-cancer effect, promote thrombosis and autoimmunity. The role of neutrophils in the pathogeny of silicosis seems to be very complex: on one side, neutrophils further activate the oxidative stress and the recruitment of other inflammatory cells and on the other side, silica stimulates NETosis,²⁴ the DNA and enzymes released from polymorphonuclear cells. As far as other particles are concerned, studies on uric crystals have revealed that NETosis is a defense mechanism to limit gout inflammation.²⁵ There is no clear evidence whether in silicosis this is a defensive mechanism or a deleterious one, contributing to the inflammation and the destruction of the lung tissue.

Although the patients included in this study had neither simple chronic bronchitis nor obstructive lung disease, it is important to mention that, in other studies, high NLR have been associated with an unfavorable prognosis in COPD; the putative mechanism was the tissue destruction from the elastase released from the activated neutrophils.⁹We cannot exclude that past exposure to other particles, metals, fumes or irritants, in these patients would have influenced their lung inflammation²⁶even without a clinically manifested bronchitis, but, as their exposure was fairly similar, there are no major arguments against an independent role of the silica particles themselves.

The strength of this study is that, through the exclusion criteria, we avoided the influence of the most prevalent conditions for chronic lung inflammation: smoking and COPD. Therefore, we can consider the results to be mainly influenced by the sole silicosis evolution process. The limitation of our study consists in its retrospective design and in the rather small number of cases, due to our strict exclusion criteria. But, nevertheless, the strong association with the progression of the disease supports the hypothesis that NLR has to be investigated as a possible biomarker for an unfavorable prognosis in silicosis patients.

If prospectively confirmed, the NLR could be important in the definition of the high-risk patients, who should imperatively stop exposure in early stages of the silicosis process and also for the candidates to the innovative antifibrotic treatments (e.g., tyrosine kinase inhibitors, synthetic oligodeoxynucleotides, mesenchymal stem cells), which have been only experimentally tested until now.^27-29

To the best of our best knowledge, this is the first study relating high NLR to the progression of chronic silicosis in non-smokers with normal lung function. It is an insufficiently large sample to lead to a conclusion, but, as NLR has the major advantage of being easily determined and affordable, our results suggest that NLR could be investigated for the identification of the high-risk chronic silicosis patients.

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