Susceptibility to systemic autoimmunity rather than the presence of the HLA-DR4 peptide binding domain alone leads to severe inflammatory arthritis following inhalation of crystalline silica

Abstract

Background

The mucosal origins hypothesis of rheumatoid arthritis (RA) posits that inhalant exposures, such as cigarette smoke and crystalline silica (c-silica), trigger immune responses in the lungs that contribute to joint disease onset. However, the relationship between inhalants, lung inflammation, and inflammatory arthritis remains poorly understood.

Results

This study compared the development of inflammatory arthritis in two genetically susceptible mouse strains, the BXD2/TyJ (BXD2) and chimeric HLA-DR4-IE transgenic (DR4-Tg), following delivery of c-silica to the lungs via oropharyngeal aspiration. In BXD2 mice, c-silica exposure was associated with rapid arthritis development, marked by synovial cell hyperplasia, pannus formation, and severe erosion of cartilage and bone. These features were preceded by pulmonary inflammation characterized by lymphoid-like cell clusters lining vessels and bronchi which cell-specific immunofluorescent microscopy identified as organized lymphoid structures consistent with inducible bronchus-associated lymphoid tissue (iBALT). Inflammatory arthritis was also preceded by autoantibodies associated with RA and other systemic autoimmune diseases including anti-citrullinated protein autoantibodies (ACPA) and rheumatoid factor (RF) in bronchoalveolar lavage fluid (BALF). However, the most predominant autoantibodies in BALF were against extractable nuclear antigens (ENA). Anti-ENA were also prominent in serum and microarray autoantigen analysis confirmed the response as targeting components of Sm and RNP small nuclear ribonucleoproteins (snRNPs). In contrast, DR4-Tg mice had no signs of arthritis, milder lung inflammation lacking iBALT, and negligible autoantibody responses.

Conclusion

Genetic predisposition beyond HLA-DR4 alone is required for the immunological manifestations that lead to c-silica mediated inflammatory arthritis. These findings provide novel insights into the relationship between mucosal exposure and RA pathogenesis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12989-026-00662-z.

Background

Rheumatoid arthritis (RA) is a chronic autoimmune disease driven by complex interactions between genetic predisposition and environmental exposures, culminating in chronic inflammatory synovitis and joint destruction [1–3]. RA affects ~ 0.5–1% of the general population, with women being two to three times more susceptible [4]. Among the genetic factors, the HLA-DRB1 “shared epitope” (SE) alleles [5], including HLA-DRB1*0401 (DR4) [6], represent the most significant risk, contributing an estimated 30% of the total genetic component [7].

Environmental risk factors also play a pivotal role in RA pathogenesis with cigarette smoke and crystalline silica (c-silica) representing the most well-established triggers for anti-citrullinated protein antibody (ACPA)-positive RA [8, 9]. C-silica exposure alone is associated with a greater than two-fold increase in RA risk [10]. This ubiquitous mineral, found in the Earth’s crust, becomes airborne during occupational activities such as sandblasting, mining, or artificial stone fabrication [11, 12]. Inhalation of respirable c-silica particles causes silicosis, a debilitating nodular inflammatory fibrotic pneumoconiosis [12]. Beyond its impact on pulmonary health, c-silica exposure is associated with several systemic autoimmune diseases in addition to RA, including systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) [13–15].

Animal model studies [16–20] (reviewed in [21]) have shown that c-silica can provoke autoantibody production and features of systemic autoimmune diseases, such as glomerulonephritis (GN), in certain mouse strains. However, despite its established association with RA [22], no experimental evidence has demonstrated a direct causal link between c-silica exposure and the hallmark inflammatory arthritis of RA [23]. This gap has limited understanding of the molecular and cellular mechanisms linking inhalant exposures to RA pathogenesis.

Existing RA models have successfully replicated inflammatory and erosive arthritis pathology and are valuable for studying disease progression and evaluating therapeutics [24]. However, these models often rely on induction methods unrelated to known environmental triggers of human disease. To address this limitation, this study examined the effects of c-silica exposure in two arthritis-susceptible strains, a chimeric HLA-DR4-IE transgenic (DR4-Tg) and the BXD2/TyJ (BXD2) mouse. DR4-Tg mice, which express the RA-associated HLA-SE as part of a chimeric MHC class II complex [25], develop arthritis when immunized with citrullinated fibrinogen [26], reflecting the relationship between the HLA-SE, ACPA, and arthritis [27, 28]. Since silica nanoparticles elicit protein citrullination [29], it can be hypothesized that c-silica exposure would lead to ACPA production and subsequent arthritis. Contrastingly, BXD2/TyJ mice spontaneously develop generalized systemic autoimmunity characterized by antinuclear antibodies (ANA), rheumatoid factor (RF), GN, and erosive inflammatory arthritis [30]. As c-silica exposure aggravates disease in mice susceptible to systemic autoimmunity [20], it is further hypothesized this would accelerate and intensify arthritis in this strain. Thus, this study has sought to leverage c-silica exposure in genetically susceptible mouse models to elucidate the relationship between pulmonary inflammation induced by inhalant exposure and RA pathogenesis.

Methods

Mice

BXD2/TyJ mice (BXD2/TyJ; Jackson Labs Strain #000075) were obtained from The Jackson Laboratory (Bar Harbor, ME) (hereafter referred to as BXD2). DR4-transgenic mice, containing a hybrid MHC class II molecule consisting of the peptide binding domains of human HLA-DRA and HLA-DRB*0401 and the membrane proximal domains of mouse I-Eα^d and I-Eβ^d (H2-E) (Abb Knockout/Transgenic HLA-DR4; Taconic Model #4149), on the C57BL/6 (B6) background were obtained from Taconic Biosciences (Germantown, NY) (hereafter referred to as DR4-Tg). Mice were housed under specific pathogen-free conditions, with rooms at 20–26 °C, 30–70% humidity, and a 12 h/12 h light-dark cycle. Sanitized cages were replaced bi-weekly with fresh water and food (Teklad 2018, Envigo, Madison, WI, USA) ad libitum. Experiments were initiated in 8-12-week-old male and female animals. All procedures were approved by the Institutional Animal Care and Use Committee at The Scripps Research Institute (TSRI) (protocol #08-0150), and use of c-silica was approved by TSRI Department of Environmental Health and Safety.

Exposure to crystalline silica

Crystalline silica (c-silica) (SiO₂, Min-U-Sil-5, average particle size 1.5–2.0 μm; U.S. Silica Company, Frederick, MD) was acid washed (1 M HCl, 100 °C, 2 h), then washed (3x) with sterile water, autoclaved (1 h, 121 °C), dried, and suspended in PBS [31]. Immediately prior to use, c-silica was disbursed by sonication, using a sonicator bath. Mice were exposed to a single dose of 5 mg (222 mg/kg body weight) c-silica in a volume of 50 µl PBS delivered to the lungs by oropharyngeal aspiration or under the skin by subcutaneous injection of 5 mg c-silica in a volume of 100 µl PBS [17]. Control animals were exposed to either 50 µl or 100 µl of PBS. The selected dose and route, 5 mg c-silica in a volume of 50 µl PBS by oropharyngeal aspiration, provides the most consistent response in the lungs [17, 32] and represents 60% of a human lifetime exposure to c-silica at the recommended NIOSH exposure limit [20]. A single instillation was used based on research indicating a correlation between the intensity of c-silica exposure and the onset of autoimmune responses [15].

Blood and tissue collection

Blood for serum was collected from the retro-orbital sinus pre-c-silica exposure and at intermediate time points (4, 8, 12 and 20 weeks) post-exposure. BXD2 mice were euthanized 1, 2, 6, 12 or 20-weeks post-exposure. DR4-Tg mice were euthanized 20 or 40 weeks post-exposure. Time points were chosen based on autoantibody responses in intermediate time point serum samples and visual assessment of arthritis development. During harvest, bronchoalveolar lavage fluid (BALF) was collected and blood for serum was collected from the heart. Spleen and tracheobronchial lymph node (TBLN) weights were determined. Spleen, kidney, TBLN, and lungs were fixed for 48 h in zinc formalin and then transferred to 70% isopropanol. Ankles and knees were fixed for 72 h in zinc formalin and decalcified in formic acid. Paraffin sections (4 μm) were prepared and stained as described below.

Lung inflammation and silicosis

Hematoxylin and eosin (H&E) stained paraffin sections (4 μm) of lungs and TBLN were prepared by the Microscopy and Histology Core Facilities of the La Jolla Institute for Immunology (LJI, La Jolla, CA), and slides scanned using a Leica Aperio AT2 whole slide scanner. Silicosis was scored under blinded conditions for the percent of each lobe affected by alveolitis, or peribronchial and perivascular inflammatory infiltrates (5 lobes, 500 maximum score each), as described previously [17]. The values for alveolitis, and for peri-bronchitis and -vasculitis were combined to give a total lung score (max 1,000).

Fluorescent immunohistochemistry

Unstained 4 μm lung and TBLN sections were deparaffinized with Pro-Par Clearant (Anatech Ltd, Battle Creek, MI), rehydrated with ethanol, and underwent antigen retrieval in citrate buffer before immunofluorescent staining. Slides were UV-bleached to quench autofluorescence, blocked with 5% normal donkey serum and 0.3% Triton X in PBS, then incubated with primary antibodies against B cells (B220), T cells (CD4), and non-alveolar macrophages (Iba1). After incubation with secondary antibodies, nuclei were counterstained with Hoechst #33342 (1:1000). Tissue sections were prepared and stained (LJI, La Jolla, CA) and digital images collected and analyzed by QuPath (https://qupath.github.io) [33].

Evaluation of arthritis and joint damage

Paw and ankle thickness was measured using a digital caliper (General Ultra Tech TM, No. 1433). Swelling was defined as the increase in thickness from baseline values recorded at the start of the experiment, as described by Mountz et al. [30]. In addition, mice were visually assessed for joint redness, gait and activity changes. For histological assessment of synovitis and bone erosion, paraffin sections (4 μm) of ankles and knees were prepared and stained with H&E (LJI, La Jolla, CA). Inflammatory arthritis was scored under blinded conditions for synovitis and bone erosion in joints, on a scale of 0–3, as described by Hayer et al. [34].

Evaluation of glomerulonephritis

Periodic acid–Schiff (PAS) stained paraffin sections (4 μm) of kidney were prepared (LJI, La Jolla, CA) and glomerulonephritis scored on a 0–4 scale under blinded conditions, as described by Koh et al. [35].

Immunoglobulin quantification

Total serum and BALF IgG and IgA were measured by ELISA as specified by the manufacturer (Immunology Consultants Laboratory, Portland, OR).

Antinuclear antibody (ANA) detection by indirect immunofluorescence

Serum antinuclear antibodies (ANA) were detected as previously described [36] using a 1:100 dilution of serum on HEp2 ANA slides (Inova Diagnostics, San Diego, CA). Digital images of ANA patterns were captured using a Leica DFC 365 FX camera and analyzed using Leica Application Suite AF software (Leica Microsystems, Buffalo Grove, IL).

Autoantibody quantification by ELISA

Quantitative measurement of ENA6-antibodies (anti-Sm, -RNP, -SS-A (60 kDa and 52 kDa), -Jo-1, -SS-B and -Scl-70), anti-double stranded DNA (anti-dsDNA), and anti-CCP3 was performed using ELISA (QUANTA Lite ELISA Inova Diagnostics, San Diego, CA) modified to detect murine samples using a goat-anti mouse IgG or IgA HRP-conjugated antibody (Invitrogen, Waltham, MA). The units for each sample were calculated as described previously [17]. IgM and IgA Rheumatoid Factor (RF) was measured using a sandwich ELISA, as described previously [17].

High-throughput protein microarray autoantibody profiling

High-throughput profiling of IgG autoantibodies in serum against various autoantigens (AAgs) was performed by the Microarray and Immune Phenotyping Core Facility, UTSW (Dallas, TX). Briefly, serum samples were pre-treated with a 1:50 dilution of DNAse I to remove free-DNA, and hybridized to antigen-coated protein arrays. Antibody-binding was detected with Cy3-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, Westgrove, PA). Fluorescent images were captured (Genepix 4200 A scanner, Molecular Devices, CA) and transformed to signal intensity values using GenePix 7.0 software. Images were background-subtracted and normalized to internal controls. The processed signal intensity value for each autoantibody was reported as antibody score (Ab-score), which is expressed based on the normalized signal intensity (NSI) and signal-to-noise ratio (SNR) using the formula: Ab−score=log2(NSI∗SNR + 1). Ab-score values were represented in a heatmap, organized by unsupervised hierarchical clustering generated using ClustVis online software (https://biit.cs.ut.ee/clustvis/). Significant differences between PBS- and c-silica-exposed mice were assessed using t-tests, accounting for multiple comparisons. Robust autoantibody responses were defined as Ab scores exceeding the PBS average plus one standard deviation (SD). See Additional file 1 for the list of antigens in the array.

Serum inflammatory markers

Sera were tested for Rantes/CCL5, M-CSF, G-CSF, IFN-γ, MIG/CXCL9, IL-2, IL-5, IL-6, IL-17A, and MIP-1β/CCL4 by MULTIPLEX Map assay according to the manufacturer’s instructions (Millipore Sigma, Burlington, MA). Z-scores of raw values (pg/ml) were represented in a heatmap, organized by unsupervised hierarchical clustering generated using ClustVis online software.

Exposure to TLR7 agonist R848

DR4-Tg mice are on the B6 background which show limited development of c-silica-induced autoimmunity unless they receive a secondary stimulus such as infection with a single-stranded RNA virus [16]. To overcome this limitation DR4-Tg mice were treated with R848 (Resiquimod), an agonist of TLR7, a receptor recognizing single-stranded RNA. DR4-Tg mice were exposed to 5 mg c-silica in 50 µl PBS or 50 µl PBS alone by oropharyngeal aspiration and one week later to weekly subcutaneous injections of 25 µg of R848 (Resiquimod) (InvivoGen, San Diego, CA) in 100 µl PBS or 100 µl PBS alone. Comparative groups received either c-silica or R848 alone. Mice were euthanized after 20 weeks to assess body weight, spleen weight, TBLN weight, BALF cell numbers, and lung and ankle joint histology. Serum ANA was determined at 12 and 20 weeks post c-silica exposure.

Statistical analysis

Data is expressed as mean ± SEM unless otherwise stated. Statistical analysis was performed using GraphPad Software V6 (San Diego, CA). Mann-Whitney U tests were used to compare PBS- and c-silica-exposed mice unless stated otherwise. p < 0.05 was considered significant. Levels of significance were further indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Results

C-silica exposure preferentially accelerates arthritis development over glomerulonephritis in BXD2 mice, but does not induce arthritis or glomerulonephritis in DR4-Tg mice

Arthritis in BXD2 mice was significantly exacerbated by oropharyngeal aspiration of c-silica, as evidenced by pronounced ankle joint swelling (Fig. 1A), synovial inflammation and hyperplasia (~ 3-fold vs. PBS mice) (Fig. 1B and F), and pannus formation with bone and cartilage erosions (~ 7-fold vs. PBS mice) (Fig. 1C and F) at 20 weeks post-exposure compared to PBS controls. In addition, bone marrow hyperplasia of normal bone marrow cells, consistent with lymphoid neogenesis, was observed (Fig. 1F). Minimal arthritis was observed at earlier time points, with no bone erosions detected. Two-way ANOVA showed no significant sex or interaction effects, indicating a similar response to silica in both sexes (Fig. 1D). Subcutaneous injection of c-silica failed to elicit evidence of inflammatory arthritis in BXD2 mice after 20 weeks suggesting that oropharyngeal aspiration preferentially exacerbates inflammatory arthritis. In contrast, DR4-Tg mice did not develop inflammatory arthritis at 20 or 40 weeks post-oropharyngeal aspiration of c-silica, as evidenced by very limited synovitis (Fig. 1E) and absence of bone erosions. Representative histological images of ankle (Fig. 1F) and knee and front paw (See Additional file 2) illustrate these differences.

Fig. 1.

Inflammatory arthritis and glomerulonephritis assessment in BXD2 and DR4-Tg mice

DR4-Tg mice are on the B6 genetic background which may explain their lack of inflammatory arthritis as wildtype B6 mice do not develop silica-induced autoimmunity unless infected with the single-stranded RNA virus LCMV [16]. To exploit this requirement, DR4-Tg mice were treated with R848 (Resiquimod), an agonist for the single-stranded RNA sensor toll-like receptor 7 (TLR7), however this did not lead to joint swelling or histological evidence of joint disease.

Kidney histology assessments at 2, 6, 12, and 20 weeks post-exposure revealed no significant differences in glomerulonephritis (GN) severity between c-silica- and PBS-exposed BXD2, though average scores were slightly higher with c-silica (Fig. 1G). Mild to severe GN was observed in both treatment groups (Fig. 1G, representative histology images in Fig. 1I). No sex effect was observed (Fig. 1H). No GN was observed in DR4-Tg mice.

Thus, oropharyngeal aspiration of c-silica to BXD2 preferentially exacerbated inflammatory arthritis and not kidney disease. Moreover, the DR4-Tg mouse model did not develop arthritis or GN.

Blue graphs represent BXD2 data, green graphs represent DR4-Tg data. (A) Ankles of c-silica-exposed BXD2 mouse with visual swelling of left ankle (arrow). (B) Ankle synovitis scores in c-silica- and PBS-exposed BXD2 mice, 6 (n = 4–5/group), 12 (n = 7–8/group) and 20 weeks (n = 17–21/group) post-exposure. (C) Ankle bone erosion scores in c-silica- and PBS-exposed BXD2 mice, 20 weeks post-exposure (n = 17–21/group). (D) Comparison of synovitis scores between female and male c-silica- and PBS-exposed BXD2, 20 weeks post-exposure (n = 5–12/group). (E) Ankle synovitis scores in c-silica- and PBS-exposed DR4-Tg mice, 20 (n = 3–4/group) and 40 (n = 3–9/group) weeks post-exposure. (F) Representative histology images of PBS- and c-silica-exposed BXD2 mice, 20 weeks post-exposure, and DR4-Tg mice, 40 weeks post-exposure. Arrowheads indicate synovial inflammation (synovitis) (white arrowheads) and bone erosion/pannus infiltration (gray arrowheads). (G) GN scores from c-silica- and PBS-exposed BXD2 mice at 2, 6, 12, and 20 weeks post-exposure (n = 4–24/group). (H) Comparison of GN scores between female and male c-silica- and PBS-exposed BXD2 mice at 20 weeks post-exposure (n = 8–15/group). (I) Representative kidney tissue images (PAS) of BXD2 mouse that received a GN score of 0 and a BXD2 mouse that received a GN score of 3. Comparisons were performed with Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

C-silica exposure induces differential humoral autoimmune features in BXD2 and DR4-Tg mice

To evaluate whether inflammatory arthritis was accompanied by an increase in generalized autoimmunity in BXD2 but not DR4-Tg mice, spleen weight, lupus and RA-associated autoantibodies, and autoimmunity-associated cytokines and chemokines were assessed.

The systemic autoimmune response induced by c-silica, which was significantly more severe in BXD2 mice than in DR4-Tg mice, was further validated by the observation of c-silica-induced splenomegaly. This was evident in BXD2 mice at 12 and 20 weeks post-exposure (Fig. 2A) but only at 40 weeks in DR4-Tg mice (Fig. 2B). The combination of c-silica and TLR7 agonist R848 increased splenomegaly in DR4-Tg mice (See Additional file 3). Total serum IgG levels were significantly higher in c-silica-exposed BXD2 mice vs. PBS mice by 6 weeks post-exposure (Fig. 2C), whereas DR4-Tg mice displayed delayed and lower IgG levels compared to BXD2 mice independent of treatment (Fig. 2D). Serum ANA levels in BXD2 mice remained unaffected by c-silica exposure, as they were present prior to exposure and increased similarly across groups (Fig. 2E). DR4-Tg mice developed lower and delayed ANA responses (Fig. 2F). The addition of R848 resulted in a modest increase in the mean ANA response of c-silica exposed DR4-Tg mice compared to c-silica only (See Additional file 3).

Fig. 2.

Spleen weight and total serum IgG and serum autoantibodies upon c-silica exposure in BXD2 and DR4-Tg mice. Blue graphs represent BXD2 data, green graphs represent DR4-Tg data. Spleen weights from c-silica- and PBS-exposed BXD2 (n = 4–20/group) (A) and DR4-Tg (n = 3–14/group) (B) mice. Total serum IgG (mg/ml) in c-silica- and PBS-exposed BXD2 (n = 5–15/group) (C) and DR4-Tg (n = 3–7/group) (D) mice. Serum ANA scores for BXD2 (n = 4-5/group) (E) and DR4-Tg (n = 3–8/group) (F) mice at different time points post exposure to c-silica and PBS. ENA6 levels (units) in serum of c-silica- and PBS-exposed BXD2 (n = 8–17/group) (G) and DR4-Tg (n = 3–12/group) (H) mice. CCP3 levels (units) in serum of c-silica- and PBS-exposed BXD2 (n = 5–21/group) (I) and DR4-Tg (n = 6–12/group) (J) mice. IgM RF levels in serum (ng/ml) of c-silica- and PBS-exposed BXD2 (n = 10–17/group) (K) and DR4-Tg (n = 6–15/group) (L) mice. Groups were compared using Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Autoantigen array analysis performed to assess the diversity of serum IgG autoantibodies, revealed broad autoantibody repertoires in c-silica-exposed BXD2 and DR4-Tg mice. Principal component analyses (PCA) showed almost full separation by treatment in the BXD2 mice, but not the DR4-Tg mice (Fig. 3A). The PCA showed a clear separation by strain. C-silica not only augmented disease related autoantibodies, but almost all autoantibody specificities. Co-clustering grouped autoantigens into three main clusters (Fig. 3B; vertical axis). The first cluster comprised antigens predominantly targeted in DR4-Tg mice compared to BXD2 mice; the second cluster consisted of autoantigens primarily targeted in c-silica-exposed BXD2 mice; and the third, largest cluster, included autoantigens against which BXD2 mice had autoantibodies in both PBS- and c-silica-exposed conditions, with these responses further amplified by c-silica exposure. When looking at the strongest and most consistent responses, c-silica exposure of BXD2 mice led to preferential targeting of Sm/RNP antigens, vimentin and ribosomal (P0 and P2) proteins (Fig. 3C), while DR4-Tg responses were more diverse, including responses directed toward histones (Fig. 3D). These differences were not evident in PBS-treated mice, although DR4-Tg mice generally had lower responses than BXD2 mice (Fig. 3E). Lower autoantibody responses in DR4-Tg mice are consistent with what we have observed previously with wildtype B6 mice [16].

Fig. 3.

Autoantigen array analyses of serum autoantibodies upon c-silica exposure in BXD2 and DR4-Tg mice. A PCA plot and B heatmap with unsupervised clustering depict Ab-score values for IgG autoantibody responses against an array of autoantigens in serum samples of c-silica- and PBS-exposed BXD2 (n = 7–8/group) and DR4-Tg (n = 3–4/group) mice, 20 weeks post-exposure. C Ab-values of most consistent responses in the BXD2 mice. D Ab-values of most consistent responses in the DR4-Tg mice. E Ab-values of the responses significantly different between PBS mice of the two strains

To confirm the strong c-silica-induced response against Sm/RNP antigens, which are part of the extractable nuclear antigens (ENA), the ENA response was tested at different time points. Anti-dsDNA was also measured over time as this is a prominent feature of the generalized autoimmunity in BXD2 mice [30]. Additionally, as responses against citrullinated proteins and rheumatoid factor (IgM) are a hallmark of RA, and as those were not part of the antigen array, these responses were tested at different time points. Serum autoantibodies against extractable nuclear antigens (ENA6), containing a number of snRNP antigens, were dramatically higher in the c-silica-exposed BXD2 group at 12 weeks (Fig. 2G). Although c-silica-exposed DR4-Tg mice achieved elevated serum ENA6 responses at 40 weeks, levels remained significantly lower than in BXD2 mice (Fig. 2H). Serum anti-dsDNA antibody levels in BXD2 mice were not affected by c-silica exposure (See Additional file 4). Surprisingly, serum anti-cyclic citrullinated protein (CCP3) antibodies were significantly elevated at 20 weeks post c-silica exposure in BXD2 mice (Fig. 2I), but not in DR4-Tg mice (Fig. 2J). Serum IgM RF levels were unaffected by c-silica exposure in BXD2 (Fig. 2K) and DR4-Tg mice (Fig. 2L).

As autoimmunity is also accompanied by the development of chemokine and cytokine responses, and as they are known to be elevated by c-silica exposure in bronchoalveolar lavage fluid (BALF) and serum [17, 38], inflammatory markers relevant to systemic autoimmunity were measured in serum of BXD2 and DR4-Tg mice. Direct comparisons between treatments of the different inflammatory markers showed significant elevations of IL-6 (p < 0.01), MIG (CXCL9) (p < 0.05), and MIP-1β (CCL4) (p < 0.05) in BXD2 mice exposed to c-silica compared to PBS controls, 20 weeks post-exposure. Such differences are significant because, although there are similarities in cytokine and chemokine changes in autoimmune diseases, elevations in IL-6 and MIP-1β (CCL4) are important contributors to the development of RA [39]. In contrast, no significant increase in serum cytokine levels was observed in c-silica-exposed DR4-Tg mice, and levels were lower overall, even in PBS controls, compared to BXD2 mice. Despite these differences, heat-mapping with co-clustering showed only limited clustering based on treatment (c-silica/PBS) and strain (See Additional file 5).

Thus, humoral immune responses in c-silica-exposed BXD2 mice occurred earlier and were greater in magnitude than those of DR4-Tg mice, suggesting significant associations between humoral autoimmunity and arthritis development. Notably, c-silica led to more significant elevations of autoantibodies than cytokines in serum, suggesting that serum biomarker profiles may underlie distinct differences in mechanisms of systemic autoimmunity in these strains.

Pulmonary c-silica exposure triggers development of T- and B-cell containing tertiary lymphoid structures in BXD2 but not DR4-Tg mice

Oropharyngeal aspiration of c-silica results in pulmonary inflammation characterized by alveolar, peribronchial and perivascular inflammatory cell infiltrates and tracheobronchial lymph node (TBLN) enlargement [17]. These early inflammatory events can be strain-specific [40], but it is not known if they correlate with systemic responses such as arthritis.

In BXD2 mice, significant peribronchial and perivascular infiltrates were observed at 6 weeks post-exposure (Fig. 4A), while alveolitis was already present at 1 week (Fig. 4C). DR4-Tg mice exhibited similar alveolitis scores at 20 weeks (Fig. 4D) but significantly lower perivasculitis and peribronchitis scores at 20 and 40 weeks (p = 0.004, p = 0.0079; Fig. 4B). This resulted in higher total lung scores in c-silica-exposed BXD2 mice (Fig. 4E) compared to DR4-Tg mice (Fig. 4F). TBLN enlargement in c-silica-exposed BXD2 mice occurred by 2 weeks post-exposure, progressing over time (Fig. 4G), whereas TBLN weights of c-silica-exposed DR4-Tg mice showed less TBLN enlargement at 20 weeks (p = 0.0138; Fig. 4H) with delayed increases by 40 weeks. This less extensive pulmonary inflammation in DR4-Tg mice has also been previously observed in wildtype B6 mice [16]. These findings suggest that genetic predisposition to generalized autoimmunity in BXD2 drives heightened pulmonary inflammation after c-silica exposure.

Fig. 4.

C-silica-induced lung inflammation and iBALT in BXD2 and DR4-Tg mice. Blue graphs represent BXD2 data, green graphs represent DR4-Tg data. Lungs of c-silica- and PBS-exposed BXD2 (n = 4–9/group) and DR4-Tg (n = 3–8/group) mice were scored for alveolitis and peribronchitis and perivasculitis, and scores were added to give a total lung score. Peribronchitis and perivasculitis score in BXD2 (A) and DR4-Tg (B) mice. Alveolitis score in BXD2 (C) and DR4-Tg (D) mice. Total lung scores in BXD2 (E) and DR4-Tg (F) mice. Tracheobronchial lymph nodes (TBLN) were collected and weighed in BXD2 mice (n = 4–19/group) (G) and DR4-Tg mice (n = 3–13/group) (H). I Representative histology images of H&E-stained lung tissue of c-silica-exposed BXD2 mice, 12 weeks post-exposure, and of c-silica-exposed DR4-Tg mice, 20 weeks post-exposure, showing organized cell accumulations around vasculature and bronchi, present in a higher extent in BXD2 mice compared to DR4-Tg mice. J Immunofluorescent staining for CD4, B220 and Iba-1 in lung tissue of c-silica- and PBS-exposed BXD2 and DR4-Tg mice shows the formation of iBALT/lymphoid accumulations in BXD2 but not DR4-Tg mice. K Individual markers in lymphoid accumulations in c-silica-exposed BXD2 mice from region selected in J. L TLS formation began with accumulations of CD4+ T cells (purple) and small numbers of B220+ B cells (green) and Iba1+ macrophages (red) at 2 weeks, progressing to segregated CD4+ and B220+ zones with increasing scattered Iba1+ macrophages (red) by 6 weeks and larger, more organized structures resembling iBALT by 12 weeks. Statistical comparisons were performed with Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Chronic inflammation in non-lymphoid tissues can result in inducible tertiary lymphoid structures (iTLS) consisting of organized accumulations of lymphoid cells [41] that are often associated with more severe disease [42]. iTLS formation in the lungs, especially near bronchi, termed inducible bronchus-associated lymphoid tissue (iBALT), has been found in RA patients with pulmonary complications, suggesting a relationship with RA pathogenesis [43]. To identify iTLS, lung sections were assessed with both H&E and cell-specific immunofluorescent staining. BXD2 mice developed organized lymphoid accumulations resembling iBALT lining vessels and bronchi at 12 weeks post c-silica exposure (Fig. 4I and J). These lymphoid accumulations contained CD4+ T cells at 2 weeks, progressing to segregated CD4+ T cell and B220+ B cell zones with scattered Iba-1+ macrophages by 6 weeks and larger, more organized structures resembling iBALT by 12 weeks post-exposure (Fig. 4K and L). By contrast, DR4-Tg mice showed minimal to no iTLS formation even at 20 weeks (Fig. 4J). In BXD2 mice, iBALT formation coincided with increased perivasculitis and peribronchitis, TBLN enlargement, and autoantibody production (Figs. 2 and 4A and G), linking iTLS to severe systemic autoimmunity, including inflammatory arthritis.

Pulmonary c-silica exposure leads to anti-ENA6 IgG autoantibodies in BALF of BXD2 but not DR4-Tg mice

Pulmonary inflammation and iBALT or iTLS are associated with autoantibodies in the sputum and BALF of RA patients [43, 44] and with autoantibodies in c-silica-induced autoimmunity in mice [20, 45]. IgA dominates humoral mucosal immunity [46], and elevated IgA levels of RA-related autoantibodies have been found in the sputum of RA patients, including RF and ACPA [47, 48], indicating localized production of autoantibodies in the lungs [49]. To determine if c-silica exposure leads to IgA autoantibodies in BALF, the levels of total IgA, and IgA anti-ENA6, anti-CCP3, and RF, were measured, and the ratios of individual autoantibodies to the total concentration of IgA in BXD2 mice were calculated. Levels of IgG (Fig. 5A) and IgA (Fig. 5B) increased in BALF over time in c-silica-exposed mice with little change in the ratio of IgA to IgG (Fig. 5C). Although levels of IgA anti-ENA6 (Fig. 5D), anti-CCP3 (Fig. 5F), and RF (Fig. 5H) were elevated, these responses were low and only showed elevations in RF when corrected for total IgA levels (Fig. 5E, G, and H).

Fig. 5.

IgA autoantibodies in BALF of c-silica- and PBS-exposed BXD2 mice. Total immunoglobulin (IgG and IgA) and autoantibodies (anti-ENA6, anti-CCP3 and RF) were measured in BALF of c-silica- and PBS-exposed BXD2 mice (n = 3–14/group). A Total IgG, B Total IgA, C Ratio IgA/IgG, D ENA6 IgA (Units), E ENA6 (Units/mg total IgA), F CCP3 IgA, G CCP3 (Units/mg total IgA),H RF IgA (ng/ml). Statistical comparisons were performed with Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

In contrast, c-silica exposure in BXD2 mice significantly increased BALF levels of IgG autoantibodies against ENA6, CCP3, and RF (IgM) compared to PBS controls (Fig. 6A, B and C). BALF IgG autoantibody levels increased over time paralleling the development of perivasculitis and peribronchitis, TBLN enlargement, iBALT, and serum autoantibodies (Figs. 2 and 4). By comparison, DR4-Tg mice exhibited negligible BALF IgG autoantibody levels and no significant changes after c-silica exposure (Figs. 5F and 6D and E), reflecting their reduced pulmonary inflammation and absence of iBALT (Fig. 4). These findings highlight the importance of the development of pulmonary inflammation and iTLS formation for the localized production of autoantibodies in the lung mucosa.

Fig. 6.

IgG anti-ENA6 and anti-CCP3 and IgM RF in BALF of c-silica- and PBS-exposed BXD2 (blue) and DR4-Tg (green) mice. Autoantibodies (anti-ENA6, anti-CCP3 and IgM RF) were measured in BALF of c-silica- and PBS-exposed BXD2 (n = 3–14/group) and DR4-Tg (n = 3–5/group) mice. ENA6 in BXD2 (A) and DR4-Tg (D) mice, CCP3 in BXD2 (B) and DR4-Tg (C) mice, and IgM RF in BXD2 (E) and DR4-Tg (F) mice. Autoantibodies as Units/mg of IgG were determined in c-silica- and PBS-exposed BXD2 mice (n = 3–14/group) for anti-ENA6 in serum (G) and BALF (H), and anti-CCP3 in serum (I) and BALF (J). Statistical comparisons were performed with Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

ACPA and RF are the most characteristic autoantibody responses in RA [50] and both are found in RA linked to c-silica exposure [9, 28, 51]. In contrast, antibody responses to extractable nuclear antigens (ENA) are common in both humans [52] and mice [17, 20] following silica exposure but are less common in idiopathic RA [53]. Although c-silica exposure led to elevations in ACPA and RF in BXD2 mice (Fig. 2), responses against ENA were much greater in magnitude. However, these assays did not correct for differences in IgG levels between serum and BALF. Anti-CCP3 IgG and anti-ENA6 IgG responses expressed as Units/mg of IgG (Fig. 6G-J) confirmed anti-ENA6 IgG as being the predominant response in serum and BALF (Fig. 6G and H) but also revealed that anti-CCP3 responses as a fraction of IgG were no different between c-silica and PBS mice (Fig. 6I and J). Similar observations were made for anti-dsDNA IgG levels in BALF and serum of BXD2 mice at 20 weeks post-exposure (Additional file 4). These findings argue that IgA autoantibody responses in BALF after c-silica exposure are not as prominent as those of IgG especially for the anti-ENA6 response. This suggests substantial production of anti-ENA IgG antibodies in the lungs, which corresponds to the increased presence of iBALT over time (Fig. 4) and confirms that anti-ENA is the most prominent and most specific response to c-silica exposure (Figs. 2 and 3) [17].

Discussion

Here we describe findings demonstrating that c-silica exposure at an extra-articular site, specifically the lungs, can precipitate inflammatory arthritis in genetically susceptible hosts. Leveraging established models of genetic predisposition to arthritis and RA-associated autoantibodies, the present study provides new insights into the relationship between inhalant exposure and RA pathogenesis, substantiating the well-documented epidemiological association between occupational c-silica exposure and RA [22]. These findings further reveal that c-silica induced pronounced pulmonary inflammation in BXD2 mice, characterized by lymphoid cell accumulations at vascular and bronchial sites resembling iBALT. This was accompanied by a robust autoantibody response, notably against snRNP antigens, preceding the exacerbation of synovitis and erosive arthritis. In contrast, c-silica exposure in chimeric HLA-DR4-IE transgenic mice, a strain carrying a significant genetic risk for RA, failed to induce arthritis and exhibited less pulmonary inflammation, no iBALT formation, and limited autoantibody responses. These observations provide compelling evidence linking genetic susceptibility, pulmonary inflammation, iBALT, and autoantibodies triggered by inhalant exposure to the severity of inflammatory arthritis. These results are consistent with and thus lend strong experimental support to the mucosal origins hypothesis of RA, which posits that inflammatory events at mucosal surfaces, such as the lungs, lead to systemic autoimmunity including RA-related autoantibodies, which precede joint inflammation and erosive arthritis [44, 54, 55].

The recombinant inbred (RI) BXD2 line derived from C57BL/6J and DBA/2J strains, exhibits a spontaneous autoimmune phenotype encompassing erosive arthritis, glomerulonephritis (GN), and autoantibodies including RF and anti-DNA [30, 56]. While naïve BXD2 mice develop RA-like features, their phenotype extends beyond RA to resemble “rhupus,” a hybrid of RA and lupus [57]. Despite this, the model faithfully recapitulates key RA characteristics, including inflammatory erosive arthritis and RA-like autoantibodies. Mountz et al. [30] reported increased incidence of arthritis in female BXD2 mice with 50% afflicted by 8 months and 90% after 12 months. In the present study, evidence of arthritis with visible signs of joint swelling, changes in gait, or erythema were not commonly observed in PBS- or c-silica-exposed mice examined at 6–7.5 months of age. However, severe histological features of arthritis were found in c-silica-exposed mice, including synovial cell hyperplasia, pannus formation, and severe erosion of cartilage and bone with male and female mice similarly affected. Although epidemiological studies are limited, current evidence suggests there is also no significant sex bias [13, 58] among human autoimmune diseases, including RA, associated with c-silica exposure.

The absence of arthritis in c-silica-exposed DR4-Tg mice suggests that apart from MHC class II genes, other genetic requirements for inflammatory arthritis were missing. This is supported by generally low levels of RA-related autoantibodies in serum and BALF. The lack of a c-silica-induced ACPA (anti-CCP) response was unexpected given that other strains that do not express transgenic HLA-DR4 are capable of making ACPA [59], and that c-silica exposure increases the ACPA response in (DBA/1JxB10.q)F1 mice with collagen-induced arthritis (CIA) [60]. Moreover, DR4-Tg mice can produce ACPA responses when immunized with citrullinated antigen [26, 61]. HLA-DR4 is thought to promote ACPA by enhancing T cell recognition of citrullinated proteins providing T cell help for B cells targeting citrullinated proteins [62]. However, c-silica exposure did not result in an ACPA response in DR4-Tg mice, nor in the development of arthritis, arguing that DR4 alone is insufficient for the development of silica-mediated arthritis. As the presence of TLR7 is known to exacerbate the severity of CIA [37], c-silica-exposed DR4-Tg mice were treated with repeated injections of the TLR7 agonist R848, but this added stimulus did not lead to noteworthy changes of systemic autoimmunity.

The failure of c-silica to elicit arthritis in DR4-Tg mice raises several caveats. A significant concern is whether the C57BL/6 (B6) is an appropriate genetic background to study the effect of HLA-DR4 on development of inflammatory arthritis following c-silica exposure. Wildtype B6 mice do not develop silica-induced autoimmunity without an additional stimulus [16], and they are not overly susceptible to CIA without modification of immunization protocol and a susceptible MHC class II genotype [63]. However, the approach taken to develop DR4-Tg mice using the B6 employed several features that should have resulted in a susceptible host. The DR4-Tg mouse used was made with a chimeric MHC class II molecule consisting of the peptide binding domains of human HLA-DRA and HLA-DRB*0401 and the membrane proximal domains of mouse I-E [25]. Mouse I-E was chosen for the membrane proximal domains because it was not known if mouse CD4 would interact with full length HLA-DR4 chains and because I-E is more homologous to HLA-DR4 than I-A [25]. Additionally, the use of I-E ensured species specific function of the cytoplasmic tail of MHC class II molecules which is important for numerous events including T-dependent B cell responses [64, 65]. This HLA-DR4 transgenic has also been used to study the T cell repertoire recognizing citrullinated epitopes as well as the molecular interactions between citrullinated peptides and cognate T cell receptors [66]. An additional reason to select this DR4 transgenic mouse to study silica-induced autoimmunity is that immunization with U1-70 kDa snRNP component leads to anti-RNP antibodies and interstitial lung disease but not nephritis [67]. However, the DR4-Tg mouse used here also failed to develop arthritis following exposure to organic dust extract with and without CIA, even though presence of the transgene enhanced post-translational protein modification and pulmonary inflammation including iTLS when compared to wildtype B6 mice [68]. An additional issue is the observation that the expression of I-E tends to prevent autoimmunity while I-A promotes autoimmunity [69–72]. It thus seems prudent to question whether a chimeric transgene containing HLA-DR4 linked with mouse I-E sequences is the best approach to study silica-induced systemic autoimmunity. Unfortunately, Taconic is the only commercial source of HLA-DR4 transgenic mice not on a NOD background. While numerous other HLA-DR4 variants have been created [73], one that bears consideration is the DR4.AE^o mouse. This model uses a HLA-DR4 (DRB1*0401) transgene in which the CD4 binding site has been mutated to more closely resemble H2-Aβ [74]. After crossing onto mice lacking endogenous MHC class II genes, it expresses only DR4 (EαDRB1*0401) and following immunization with chicken collagen produces both RF and anti-CCP and develops CIA with a female bias [75]. However, cigarette smoke suppressed CIA in DR4.AE⁰ mice and also failed to induce ACPA and RF even though anti-collagen type II antibodies were elevated [76]. Significantly, although cigarette smoke alone did not induce arthritis in DR4.AE⁰ or DQ8.AE⁰ mice, it did exacerbate CIA in DQ8.AE⁰ mice, suggesting an important role for HLA genes in aggravation of arthritis by inhalant exposure [76].

Although arthritis was significantly accelerated and exacerbated by c-silica in BXD2 mice, this was not the case with GN. Proteinuria in naïve BXD2 mice has been shown to increase at approximately 6 months of age [30]. In the present study, GN, based on kidney histology, was found as early as 3–4 months of age in some mice but the severity changed little over time or with c-silica exposure, arguing that c-silica does not significantly exacerbate GN in BXD2 mice. However, it is likely that GN occurred before or soon after c-silica exposure, making it challenging to assess early acceleration or exacerbation. C-silica does accelerate proteinuria and increases the severity of nephritis in lupus-prone NZBWF1 mice in a dose-dependent manner [20] and induces nephritis in a proportion of outbred mice [17]. This disparity in strain sensitivity to GN may be reflective of the fact that arthritis and proteinuria can be segregated in F2 mice generated by mating of the (BXD2 X DBA/2)F1 or (BXD2 X B6)F1 mice [30], suggesting a complex genetic control of the arthritis and nephritis phenotypes in BXD2 mice. This is supported by the recent finding of strain specific inflammatory arthritis or nephritis in Collaborative Cross Recombinant Inbred strains CC039 and CC061 respectively following c-silica exposure (bioRxiv preprint) [77]. Moreover, it is possible that c-silica-induced pulmonary inflammation promotes immunological and inflammatory factors that favor arthritis pathogenesis more than GN in BXD2 mice. Few of the cytokines typically associated with SLE, or anti-dsDNA antibodies, were elevated by c-silica exposure, possibly explaining the lack of c-silica-induced exacerbation of GN.

While these studies do not provide a causal mechanism explaining the importance of pulmonary inflammation in the development of synovitis and bone erosion, it can be concluded that localized inflammation in the lungs precedes development of autoantibodies and that the increasing severity of inflammation and autoimmunity is important in the subsequent acceleration of inflammatory arthritis. A significant observation is the severity of perivascular and peribronchial inflammation and the development of iTLS with features of iBALT in the lungs following c-silica exposure. Such well-defined structures have also been found in lupus-prone NZBWF1 mice exposed to c-silica [20] but have not been formally described in humans with c-silica exposure. TLS, also called tertiary lymphoid organs or ectopic lymphoid structures, can be the result of a response against antigenic stimuli such as infection or environmental insult [78]. iBALT has been described in patients with RA [43] and similar structures have been found in RA joints [79] and the kidney in SLE [80], suggestive of a significant role in disease pathology. It is unclear whether these structures amplify autoantibody responses or initiate autoantibody production, but it is clear that they are linked to the presence of autoantibodies, including ACPA and RF [43, 54, 78]. Pulmonary inflammation in RA is argued to lead to protein citrullination and local production of ACPA [54, 81]. Because of the close temporal relationship between pulmonary inflammation, TBLN enlargement, iBALT, and serum autoantibody levels following c-silica exposure, the present studies do not establish whether the initiation of the autoantibody response begins in iBALT or the TBLN. However, in naïve mice, the small size of the TBLN makes it difficult to locate but it rapidly increases in size following c-silica exposure and develops c-silica-containing nodules [17]. This suggests that the trafficking of antigenic material from the lung to the TBLN may lead to T and B cell activation, expansion, and migration to inflammatory sites in the lung. Analysis of the antigenic specificity of B cells in both locations over time is needed to establish when and where c-silica-induced autoreactive B cell responses begin.

An important biomarker of RA is the presence of disease-associated autoantibodies with ACPA and RF being the most significant [50], both of which are found in RA linked to c-silica exposure [9, 28, 51]. Although the presence of such antibodies can precede clinical diagnosis in idiopathic RA [82], their role in pathogenesis is unclear [81, 83]. Similarly, autoantibodies have been found in a cohort of 1227 workers exposed to silica containing artificial stone in the absence of clinical autoimmune disease [84]. ANA was found in 24.6% including disease relevant specificities of ENA (4.6%) and RF (2.6%) suggesting the possibility of future progression to systemic autoimmune diseases. Autoantibodies, including ACPA and RF, have also been described in induced and spontaneous models of RA [85]. C-silica exposure in BXD2 mice led to elevations of both ACPA (anti-CCP3) and RF in BALF and ACPA in serum while DR4-Tg mice had elevation of RF in serum only. Autoantibody responses were low in BALF, likely reflecting the lower levels of immunoglobulin in BALF compared to serum. However, unlike anti-ENA6, anti-CCP3 levels were also low in serum arguing that anti-CCP3 may originate in the lung. The most prominent autoantibody response in BALF and serum was directed against ENA6, a mix of Sm, RNP, SS-A (60 kDa and 52 kDa), SS-B, Scl-70, and Jo-1. Previous studies [16, 17] as well as the antigen array analysis used here, identify Sm and RNP as the major targets of the c-silica-induced autoantibody response. Using a similar antigen array, Rajasinghe et al. [86] found a diverse spectrum of responses against nuclear, non-nuclear, and tissue specific antigens in BALF and serum of lupus-prone NZBWF1 mice exposed to c-silica. While the present studies also found a diverse array of antigenic targets in BXD2 mice, the greatest discrimination between PBS controls and c-silica-exposed mice was against polypeptides that are part of Sm and RNP snRNPs. Similar autoantibody responses have been found in those occupationally exposed to c-silica dust including responses against centromeric proteins, Scl70 (topoisomerase I), as well as RNA binding proteins Ro(60 kDa)/SS-A and La/SSB and the snRNP component U1-RNP [52]. Thus, a common feature of the c-silica-induced autoantibody response in humans and experimental models is against nucleic acid, especially RNA, binding proteins. Although the reason for this is unknown, it may be related to the ability of RNA binding proteins from cell lysates to coat silica nanoparticles [87] which could potentially provide a source of antigenic material following phagocytosis of c-silica particles. However, little has been done to define the autoantibody profile in RA patients reporting occupational exposure to silica.

Conclusions

Compared to other rodent models that rely on direct joint manipulation or systemic immunization [60, 88], this study uniquely employs mucosal exposure to c-silica as the sole arthritis trigger, recapitulating key features of RA, including genetic susceptibility, mucosal inflammation, autoantibody production, and erosive arthritis [44, 54, 55]. This study establishes a novel experimental framework connecting occupationally relevant c-silica exposure to RA pathogenesis and bridges epidemiological data and mechanistic insights. Future research should explore the cellular and molecular pathways linking mucosal inflammation to distant joint pathology, further illuminating the enigmatic relationship between environmental exposures and autoimmune diseases.

Author contributions

**LMFJ: ** Conceptualization, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing. **CdO: ** Formal analysis, Investigation, Data Curation, Writing - Review & Editing. **DHK: ** Conceptualization, Formal analysis, Writing - Review & Editing, Supervision. **SR: ** Writing - Review & Editing, Supervision. **MG: ** Writing - Review & Editing, Supervision. **PHMH: ** Writing - Review & Editing, Supervision. **KMP: ** Conceptualization, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. **JMM: ** Conceptualization, Formal analysis, Investigation, Data Curation, Writing - Review & Editing, Supervision, Project administration. All authors read and approved the final manuscript.

Funding

This research was supported by NIH grants R01ES029581, R21ES031454, and R21ES035980 to KMP. LMFJ was supported by FWO (Research Foundation Flanders) grants V420121N and V412323N. PHMH was supported by KU Leuven [Internal Funding C2 (C24/18/085)].

Data availability

The data that support the findings of this study are available from the corresponding authors, [KMP, JMM], upon reasonable request.

Declarations

Ethics approval and consent to participate

All experimental animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of The Scripps Research Institute (TSRI) (protocol #08-0150). The use of crystalline silica was approved by TSRI Department of Environmental Health and Safety.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.