Systematic Review and Meta-analysis on the Association of Occupational Exposure to Free Crystalline Silica and Rheumatoid Arthritis

Key messages • Occupational exposure to free crystalline silica and tobacco smoking are associated with an increased risk rheumatoid arthritis, with the evidence of an interaction in seropositive subjects. • Further studies in the field are needed to support such association We carried out a systematic search for all published epidemiological studies concerning the association between occupational exposure to free crystalline silica (FCS) and subsequent development of rheumatoid arthritis (RA). A meta-analysis was conducted on relevant studies. We searched PubMed and Embase, search engines, for original articles published (from 1960 to November 2019) in any language. In addition, we also searched reference lists of included studies manually for additional relevant articles. Finally, twelve studies were included in the meta-analysis (seven case-control cases and five cohort studies). The odds risks and 95% confidence interval (CI) were calculated using a random effect meta-analysis. A primary meta-analysis (using a random effect model)—regarding RA risk in subjects exposed to FCS—yelled to an overall OR of 1.94 (95% CI 1.46–2.58). We also conducted three further meta-analysis, taking into account the presence of autoantibodies (anti-RF or anti-ACPA) and smoking habits and found a significant association between FCS and RA in both seropositive and seronegative subjects (OR 1.74, 95% CI 1.35–2.25 and OR 1.23, 95% CI 1.06–1.4, respectively) and in seropositive subjects which were smokers (OR 3.30, 95% CI 2.40–4.54). The studies that have investigated the association between RA and occupatational exposure to FCS are still scarce and the heterogeneity between the studies remains high. Some critical limitations have been identified within studies, among which, the methods for assessing exposure stand out. Although with due caution, our results confirm the hypothesis of an association between occupational exposure to FCS and RA development. There was an interaction between FCS and tobacco smoking in RA seropositive workers. Supplementary Information The online version contains supplementary material available at 10.1007/s12016-021-08846-5.


Introduction
The aim of this review and meta-analysis is to investigate possible associations between occupational exposure to free crystalline silica (FCS) and rheumatoid arthritis (RA). Although in the last decades this association has been discreetly studied by epidemiological studies, to the best of our knowledge, this is the first systematic review and meta-analysis to date. The studies conducted to date have led to variable results, motivating our study to carefully seek consistent evidence on a possible association.
RA is a chronic inflammatory disorder that typically affects small and medium-sized joints. The primary lesion is synovitis, with the invasion of immune cells in a normally acellular synovium, leading to the formation of inflammatory "pannus." This hyperplastic tissue causes cartilage breakdown, bony erosion, and, ultimately, loss of function.
While the main target is represented by the joints, RA can also present itself with a great variety of systemic and extraarticular manifestations: assecondary Sjogren's syndrome (i.e., xerophthalmia, xerostomia), rheumatoid nodules, haematologic involvement, pericardial and pleural effusions, interstitial lung disease, ocular inflammation, skin vasculitis, etc. [1].
The prevalence in the general population is estimated to be approximately 0.5-1%, and women are two or three times more likely to be affected than men [2].
In the majority of patients with RA, circulating autoantibodies are detectable; the most represented are the rheumatoid factor (RF) and the antibodies to citrullinated protein antigens (ACPA) commonly identified by antibodies to cyclic citrullinated peptides (anti-CCP) [3,4]. Autoantibodies are part of the ongoing classification criteria, proposed in the 2010 by the collaborative initiative of the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) [5].
When one or more of these autoantibodies are detected in serum, RA is defined seropositive (RA+) and the disease might manifest with more severe symptoms such as erosive joint degeneration and rapid radiographic progression [1,6].
Several studies and multiple scientific evidences have focused in particular on ACPA autoantibodies considered more sensitive and specific for diagnostic and prognostic purposes as early indicators of joint destruction, persistent RA and extra-articular involvement [7,8].
The main pathogenetic hypothesis about RA development is that of a multifactorial disease, that arises from geneenvironment interactions. Among environmental risk factors, the main suspects are for cigarette smoke, FCS, and viral infections [1]. Although the molecular pathogenesis of RA remains to date not fully understood, an increasing number of studies hypothesize that the lung might be a possible starting site of immuno-molecular alterations, alternative to joints [9,10].
This assumption has been hypothesized in particular in ACPA-positive RA [3,10] that manifests with early inflammatory lung changes, even many years before joint inflammation, suggesting that autoimmunity starts right in the lung [9].
It has beenhypothesized that FCS is able tostimulate the immune response by inducing the expression of citrullinatedproteins in lungs. Such modified proteins are presented by MHC complexesexpressed on the surface of antigen presenting cells (APCs), that activate Tcells, which in turn will stimulate the maturation of B cells responsible forthe production of ACPAs [9,11].
The aim of this study was to evaluate all published literature investigating the association between occupational exposure to FCS and RA development.

Methods
PRISMA checklist [14] can be found in the supplementary material (Table S3). An a priori protocol was defined, in which we established the criteria for inclusion and exclusion of epidemiological studies. We searched PubMed and Embase for original articles published between 1960 and November 2019. The bibliographic search has been extended also using the links "related article" of PubMed and the reference lists of some key studies. There were no restrictions regarding the language of the articles.
We performed the following literature search on PubMed and Embase (Table 1): PubMed search strategy was developed through the strings proposed by Professor S. Mattioli [12]  ). Subsequently, the following "search filter" was applied to the string: silica.
We performed an additional literature search on Embase.
To be included in the meta-analysis, studies had to fulfill the following criteria: (i) studies examining the association between occupational exposure to FCS and RA; (ii) studies in which a measure of association such as relative risk (RR), odds ratio (OR), standardized mortality ratios (SMR), or standardized incidence ratios (SIR) was either reported or could be derived from data reported in the article. Exclusion criteria were as follows: (i) by study design: experimental studies, case reports, and reviews; (ii) data incompleteness, e.g., presence of prevalence data in only one of the two samples; (iii) use of data from samples of subjects already used in previous studies (in order to avoid duplication of results); (iv) use of non-standardized methods for diagnosis and/or very high risk of inaccuracy in the assessment; and (v) Inadequacy of results for metaanalysis purposes, e.g., studies with insufficient relative risk and CI information.
Two authors (AM, IS) independently reviewed the studies retrieved from the databases, paying particular attention to the inclusion and exclusion criteria. Doubtful cases or disagreement situations were resolved involving a third author (SC).
The final selection of the articles included in the study was based on a careful reading and analysis of the entire texts.
The whole process followed during the systematic review is shown in Fig. 1. To draw up the flow chart, we used the model on the official PRISMA website [14]. The bibliographic search (through PubMed and Embase) gave rise to 178 articles; three further articles have been identified through other sources [21,28,29].
Following the title revision, we eliminated 52 articles and further 11 after abstract revision. Additional articles (32 Reviews, 21 Case Reports, 8 Experimental, and 3 not available) were excluded based on the reading of the full text and non-compliance with the inclusion criteria.
Finally, the studies included in qualitative synthesis were twelve, and all of them were eligible for the meta-analysis.
In order to perform the meta-analysis, we extracted the most relevant data from each study ( Table 2), including type of exposure, exposure assessment, no. of cases, and effect size (95% CI).
The quality of the studies was evaluated by applying the Newcastle Ottawa Scale (NOS) [15], that is available in the Supplementary material (Supplementary Data S1 and S2). Total final quality scores of individual studies are summarized in Supplementary Table S2.
The data were analyzed using the Comprehensive Metaanalysis v3.0 software from Biostat Inc. [16]. We chose to use the random effect model for the aggregate estimation of the association rather than the fixed effect model according to the hypothesis that it was likely to assume a high level of variability among studies. Heterogeneity among studies was assessed using the I 2 index. Heterogeneity was considered low if I 2 values were between 25 and 50%, moderate if between 50 and 75%, and high if higher than 75% [17].
The Funnel Plot was used to identify and estimate the amount of publication bias, whereas the statistical evaluation was carried out by Egger's test.

Study Characteristics
Population. In the case-control studies, the cases were classified as RA according to the ACR/EULAR classification criteria (American College of Rheumatology) [19,20,22] or the American Rheumatism Society (ARA) criteria [26].
In the case-control mortality studies [21,23], subjects were identified through the specific death cause code ICD-9 (International Classification of Diseases, 9th revision) in their respective death certificates.
With regard to cohort studies, when declared, subjects with RA were selected using the International Classification of Diseases, 10th revision (ICD-10) [25][26][27] with M05 codes for seropositive rheumatoid arthritis and M06 for seronegative.
Exposures Most of the population studied was dusty trade workers subject to moderate or high occupational exposure to FCS.
Controls Subjects were randomly selected and frequencymatched to the cases by the principal indicators (e.g., age, sex).
Outcome May the occupational exposure to FCS be a significant risk factor in the development of RA? [18-20, 22, 24-29]. May the RA mortality rate possibly be related to occupational exposure to FCS? [21,23]. The studies were published between 1986 and 2017.
Exposure to FCS concerns different jobs (mainly "dusty trades"). The examined populations are predominantly men. Smoking habit was recorded and studied in four of the studies included in the meta-analysis.

Risk of Bias Within Studies
The quality and risk of bias appraisal was conducted using the Newcastle Ottawa Scale (NOS) [15] by two independent evaluators (AM, IS). We used a modified version of this tool in order to better adapt it to the studies we reviewed (see Supplementary Data S1 and S2). An overview of the risk of bias for each of the studies included in this work is shown in Supplementary  [25], 7 [28], and 2 [29].
A few study in particular [21,23,27,29] scored low on NOS scale, with significant shortcomings in both case selection and exposure assessment.
We found that the exposure assessment in all the studies has some critical limitations; this can be attributed to the use of self-completed questionnaires or a face-to-face interview in which the patient is asked to remember events dating many years in the past.
A widely used tool is the Job Exposure Matrix (JEM) [30] to estimate exposure to silica-based on work tasks, exposure measurements, and information on the work process [21,23,24,27].
It should also be taken into account that personal Interviews might be subjected to recall biases. The exposure assessment in the case-control mortality studies [21,23] is particularly weak, as the job and duration was inferred from the death certificate. These are often incomplete and inaccurate. It is also conceivable that the reported employment on the death certificate is attributable to the last job or to that mainly carried out with the risk of misclassification.
Cases of the different studies can be defined as dusty trade workers affected by RA and defined according to the American College of Rheumatology criteria (ACR). In this regard, it is important to note that over years, the classification criteria have undergone different revisions: in 1987 [18][19][20]22] and in 2010 (ACR/EULAR criteria) [25].
The 1987 revised ACR classification criteria have been criticized for including late manifestations (e.g., rheumatoid None was excluded in quality assessment nodules and radiological damage) failing to identify patients with early inflammatory arthritis. Nevertheless, they are able to classify established RA with high sensitivity and specificity.
With regard to cohort studies and mortality studies, the diagnosis of RA was defined according to the International Classification of Diseases (ICD) 10th [26,27] and 9th Edition [21,27]. ICD-10 is a classification introduced in 2003 and with greater specificity as compared with ICD-9. The specific RA codes used in ICD-9 were 714.0, 714.1, and 714.2 and in ICD-10 M06.9, M05 for seropositive rheumatoid arthritis and M06 for seronegative rheumatoid arthritis.
A non-negligible limit of the studies that use the ICD classification is to define RA+ patients based on the ICD-M05 code without distinction between antibodies for rheumatoid factor or citrullinated peptide.
In one study [25], the diagnosis of RA was self-reported by cases during the telephone interview, therefore with a high risk of misclassification or reporting bias.
All the studies controlled for the main and potential confounding agents (age, sex, geographical area) with a few exception [21,29].
Several studies [32][33][34], reported a strong association between tobacco smoking and RA; however, only a few of the studies adjusted the OR for potential confounding from smoking [19,20,25,27].
Some studies have also investigated the correlation between ever/never smokers exposed to FCS and RA [19,22,25,27].
As usual, in these kinds of studies, participants were selected on a voluntary basis and this may introduce a selection bias.
Some main biases affected the identification of cases and controls; specifically, in the Schmajuk et al. study [25], patients were asked to self-report the diagnosis of their physicians and the possible intake of glucocorticoid treatment. In Yahya et al. study [19], both hospital and general population controls have been enrolled.
In the studies [21,24] enrolling mine workers for long periods, we are aware that such individuals have a greater chance of multiple diseases and therefore false associations are more likely to report.

Subgroup Analysis Results (By Smoking)
Finally, we performed a further meta-analysis on five studies [18-20, 25, 27] to investigate the relationship between smoking habits and occupational exposure to FCS and the effect on RA+ development (Fig. 5).

Risk of Bias Across the Studies (Funnel Plot)
We used the Funnel Plot, Egger's test, and Higgins index to detect possible biases between the studies included in the meta-analysis (Supplementary Figures S6-S9).
The visible asymmetry in the Funnel Plots confirms the presence of heterogeneity in the majority of the meta-analyses. The p-value of the Egger's regression test resulted significant (p < 0.05) for publication bias, in all the performed meta-analyses.

Summary of Evidence
To the best of our knowledge, the present study represents the first meta-analysis that analyzes the correlation between development of RA and occupational exposure to FCS.
Since the status of seropositive/seronegative RA subjects has assumed increasingly prognostic importance, we performed two additional meta-analysis, one for each subgroup.
Furthermore, some epidemiological studies [35][36][37] have shown that lifelong cigarette smoking is associated with an increased risk of RA, particularly for ACPA positive subjects [38]. According to the main pathogenetic hypothesis, smokers inhale a lot of irritant compounds [39], that may act as triggers of the immune system. FCS could act like smoking [20]; therefore, we performed a meta-analysis to investigate a possible synergy between both risk factors.

Main Meta-Analysis
Six out of twelve studies included in this systematic review were performed in Sweden. Three [18,20,31] were based on the same extensive populationbased study EIRA (Epidemiological Investigation of Rheumatoid Arthritis) [40]. The others made use as well of national registers. We can speculate that the predominance of Swedish studies might be due to national regulations that require constant checkups for workers occupationally exposed to FCS and a valid National Register of RA patients.
All studies have long-running data sets conducted on workers mainly involved in dusty trades, including also iron foundries [26] and coal or gold mines [25,26].
The results of our study support the pathogenetic hypothesis that occupational exposure to FCS is a possible risk factor for RA. The final magnitude of the association obtained from the primary meta-analysis is statistically significant but we have found a high heterogeneity among studies ((OR 1.94 95% CI 1.46-2.58) I 2 = 95%). Such heterogeneity can be explained by different study designs, studied populations, and exposure assessment.
It is important to highlight that four studies [23,24,28,29] include silicotic patients. We believe that such studies are particularly valuable because they give us the certainty about occupational exposure to FCS. Silicosis is indeed a progressive and irreversible disease caused by inhaling large amounts of FCS, usually over decades (10-20 years) [41].
We specify that in the study Brown et al. [29] for the purpose of meta-analysis, we decided to extract the most conservative risk index. Furthermore, it is specified that the Sluis-Cremer et al., 1986 study [24] was also excluded from the meta-analysis (FCS-RA) due to the lack of an appropriate OR. The study was instead included in the subgroup meta-analysis since it provided

RA
Odds ratio and 95% CI Odds Lower Upper Relative ratio limit limit w eight   ORs for RF+ and RF− RA patients. We could not perform analyzes stratified by age since only one study [22] provided age groups.

Meta-Analysis of the Autoantibodies
With this further analysis, we investigated the association between exposure to FCS and seropositive/seronegative RA. Several studies have shown that autoantibodies may be originated in the lungs by autoimmune responses to citrullinated peptides. Moreover, it is well known that anti-CCP is more sensitive than RF and may appear earlier in the course of RA [42]. Only few studies have provided data about autoantibodies, and among these, few have specified the type of autoantibody: RF and ACPA [19,27], ACPA [20], and RF [24]. Cases were considered RA+ , if including subjects characterized by positivity for rheumatoid factor and/or anti-CCP (ACPA) antibodies. The corresponding OR observed in these meta-analyzes for subgroups were as follows: These results indicate that the association between occupational exposure to FCS and RA is higher for seropositive RA subjects. Nevertheless, such results are yet based on few studies and with limited numbers of cases to consider them as conclusive.

The Role of Tobacco Smoking
Tobacco smoking is considered a main risk factor for ACPA+ RA develoment. The pathogenic mechanism is still unknown, and it is not clear how the breakdown of immunological tolerance towards citrullinated proteins and the production of ACPAs occurs [43].
Since both FCS and tobacco smoke are inhaled through the airways and are deposited at the lung level, it has been speculated that both can act synergistically in the establishment of RA [10]. To perform this meta-analysis, we considered ever smokers/RA+ cases who had been occupationally exposed to FCS. The OR we obtained was 3.30 (95% CI 2.40-4.50). Such result reinforces the hypothesis of a synergistic interaction between cigarette smoking and occupational exposure to FCS. Nevertheless, the limited number of studies included in this metaanalysis as well as the presence of further unknown confounding agents implies caution in the interpretation of the results

Limitations
A major limitation of meta-analysis, and this one is not exempt, is that the investigated populations, the FCS exposure, and the outcome definitions are not the same across the studies.

Conclusions
To the best of our knowledge, the present study represents the first systematic review and meta-analysis performed, investigating such association. Our study, notwithstanding some limitations, expands knowledge about the FCS-RA association with greater strength than the single studies published so far.
The results of our meta-analysis confirm an increased risk of rheumatoid arthritis, especially if seropositive, among subjects with previous significant occupational exposure to FCS. With an additional meta-analysis, we proved a solid interaction between smoking habits and occupational exposure FCS, mostly in RA seropositive patients.
However, it must be remembered that to date, evidence has not conclusively demonstrated that silica is a causative factor of RA.
As discussed previously, some critical limitations have been identified within studies, among which, the methods for assessing exposure stand out.
More efforts are needed to collect more accurate exposure assessments and limit the heterogeneity among studies. If desirable, that further case-report and cohort studies with a sufficient number of subjects will be conducted in the near future. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.