Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention

Figures & data

Table 1. Formulation of experimental diets.

	Experimental Diet
	(g/kg total diet)
Macronutrient	CON	DHA
Carbohydrates
Corn Starch	398	398
Maltodextrin (Dyetrose)	132	132
Sucrose	100	100
Cellulose	50	50
kcal (%total)	63.2	63.2
Proteins
Casein	200	200
L-Cysteine	3	3
kcal (% of total)	19.7	19.7
Fats
Corn Oil	10	10
High-Oleic Sunflower Oil	60	35
DHA-enriched Algal Oil	0	25
kcal (% of total)	17.1	17.1
Other
AIN93G Mineral Mix	35	35
AIN93G Vitamin Mix	10	10
Choline Bitartrate	3	3
TBHQ Antioxidant	0.01	0.01

Values are shown as mass g/kg diet—diet composition based on manufacturer descriptions. Corn oil contained 612 g/kg linoleic acid and 26 g/kg oleic acid. High-oleic safflower oil contained 750 g/kg oleic acid and 140 g/kg linoleic acid. Algal oil contained 395 DHA g/kg and 215 g oleic acid g/kg. 10 g/kg diet is calorically equivalent to a human equivalent dose of 5 g/d.

Figure 1. Experimental design. Female NZBWF1 mice were obtained at 14 weeks of age and were initiated on either an AIN-93G (CON) diet or an isocaloric diet amended with 1% DHA (5 g/d human equivalent dose [HED]). At 16 weeks of age, mice were intranasally instilled with either saline (VEH) or 1 mg cSiO₂ 1/week for four consecutive week. Proteinuria was assessed weekly starting at 20 weeks of age to monitor disease progression. Six animals per exposure/treatment group were sacrificed at 20 week of age (1-week PI), and the remaining 6 mice per group/treatment were euthanized at 24 week of age (5 weeks PI). At both timepoints, BALF was collected for differential cell counts and AAb microarray; blood was collected for AAb microarray and fatty acid analysis; lung and kidney tissues were collected for histopathology, immunohistochemistry (IHC), morphometric analysis, NanoString, and cytokine multiplex array.

Figure 2. DHA supplementation skews long-chain PUFA profile in mature adult female NZBWF1 mice. In VEH/CON and cSiO₂/CON mice, total omega-6 fatty acids were more abundant than ω-3 fatty acids in RBC cell membranes at 1-week and 5-weeks PI. In cSiO₂/DHA mice, total omega-6 fatty acids were significantly reduced, whereas total ω-3 fatty acids were significantly increased at both time points. Letters: a, significantly different from VEH/CON for the specified endpoint (p < 0.05); b, significantly different from cSiO₂/CON for the specified endpoint (p < 0.05).

Table 2. Fatty acid content of RBCs at 1- and 5-weeks post-instillation.

		Experimental group
		(% of total fatty acid)
		1-week PI			5-weeks PI
Common Name	Chemical Formula	VEH/CON	cSiO2/CON	cSiO2/DHA	VEH/CON	cSiO2/CON	cSiO2/DHA
Lauric Acid	C14:0	0.16 ± 0.01	0.16 ± 0.00	0.26 ± 0.01*^†	0.14 ± 0.00	0.16 ± 0.00	0.25 ± 0.01*^†
Palmitic Acid	C16:0	28.38 ± 0.06	28.02 ± 0.09	31.85 ± 0.26*^†	27.1 ± 0.07	26.37 ± 0.39	30.88 ± 0.35^†
Palmitelaidic Acid	C16:1ω7t	0.13 ± 0.03	0.09 ± 0.01	0.10 ± 0.01	0.10 ± 0.01	0.12 ± 0.02	0.08 ± 0.01
Palimitoleic Acid	C16:1ω7	0.51 ± 0.01	0.58 ± 0.02	0.53 ± 0.01	0.50 ± 0.01	0.58 ± 0.04	0.55 ± 0.30
Stearic Acid	C18:0	13.76 ± 0.21	13.45 ± 0.11	12.90 ± 0.16*	14.21 ± 0.02	14.12 ± 0.2	13.21 ± 0.14*^†
Elaidic Acid	C18:1t	0.37 ± 0.12	0.20 ± 0.02	0.23 ± 0.04	0.29 ± 0.06	0.24 ± 0.05	0.21 ± 0.02
Oleic Acid	C18:1 ω9	17.68 ± 0.44	17.56 ± 0.12	15.47 ± 0.15*^†	18.33 ± 0.11	18.57 ± 0.07	16.30 ± 0.10*^†
Linoelaidic Acid	C18:2 ω6t	0.13 ± 0.02	0.07 ± 0.01*	0.08 ± 0.01	0.12 ± 0.02	0.08 ± 0.01	0.08 ± 0.00
Linoleic Acid	C18:2 ω6	8.31 ± 0.08	9.16 ± 0.23*	10.94 ± 0.15*^†	7.59 ± 0.07	8.77 ± 0.62	10.40 ± 0.24*
Arachidic Acid	C20:0	0.13 ± 0.02	0.10 ± 0.00	0.10 ± 0.01*	0.11 ± 0.00	0.11 ± 0.00	0.10 ± 0.00*^†
Gamma-Linolenic Acid	C18:3 ω6	0.05 ± 0.01	0.05 ± 0.00	0.02 ± 0.00*^†	0.06 ± 0.00	0.06 ± 0.01	0.03 ± 0.00*^†
Eicosenoic Acid	C20:1ω9	0.32 ± 0.02	0.29 ± 0.00	0.19 ± 0.01*^†	0.32 ± 0.01	0.33 ± 0.01	0.19 ± 0.01*^†
Alpha-Linolenic Acid	C18:3 ω3	0.02 ± 0.00	0.03 ± 0.00	0.03 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	0.02 ± 0.00
Eicosadienoic Acid	C20:2 ω6	0.26 ± 0.01	0.26 ± 0.01	0.20 ± 0.01*^†	0.22 ± 0.01	0.22 ± 0.01	0.19 ± 0.01*^†
Behenic Acid	C22:0	0.10 ± 0.01	0.09 ± 0.01	0.07 ± 0.00*^†	0.12 ± 0.04	0.07 ± 0.00	0.08 ± 0.01
Dihomo-gamma-linolenic Acid	C20:3 ω6	1.23 ± 0.02	1.27 ± 0.03	0.99 ± 0.05*^†	1.27 ± 0.01	1.22 ± 0.03	1.11 ± 0.06*
Arachidonic Acid	C20:4 ω6	19.35 ± 0.40	19.34 ± 0.20	7.32 ± 0.24*^†	21.03 ± 0.13	20.84 ± 0.24	5.82 ± 0.20*^†
Lignoceric Acid	C24:0	0.18 ± 0.02	0.19 ± 0.01	0.18 ± 0.02	0.17 ± 0.01	0.17 ± 0.01	0.17 ± 0.01
Eicosapentaenoic Acid (EPA)	C20:5 ω3	0.25 ± 0.01	0.30 ± 0.02	3.65 ± 0.18*^†	0.09 ± 0.01	0.10 ± 0.01	4.45 ± 0.16*^†
Nervonic Acid	C24:1 ω-9	0.17 ± 0.03	0.17 ± 0.01	0.15 ± 0.02	0.18 ± 0.01	0.18 ± 0.01	0.19 ± 0.01
Docosatetraenoic Acid	C22:4ω-6	2.01 ± 0.02	1.82 ± 0.04*	0.42 ± 0.01*^†	2.23 ± 0.03	2.02 ± 0.13	0.20 ± 0.01*^†
Adrenic Acid	C22:4 ω-6	2.01 ± 0.02	1.82 ± 0.04	0.42 ± 0.01	2.23 ± 0.03	2.02 ± 0.13	0.2 ± 0.01
Omega-6 Docosapentaenoic Acid	C22:5 ω6	0.58 ± 0.02	0.55 ± 0.03	0.12 ± 0.00*^†	0.95 ± 0.02	0.92 ± 0.04	0.05 ± 0.00*^†
Ω-3 Docosapentaenoic Acid	C22:5 ω3	0.66 ± 0.01	0.65 ± 0.01	0.92 ± 0.02*^†	0.32 ± 0.01	0.29 ± 0.02	0.9 ± 0.01*^†
Docosahexaenoic Acid (DHA)	C22:6 ω3	5.25 ± 0.08	5.60 ± 0.09	13.28 ± 0.22*^†	4.53 ± 0.06	4.44 ± 0.11	14.52 ± 0.13*^†
Omega-3 Index: EPA + DHA		5.50 ± 0.09	5.90 ± 0.11	16.93 ± 0.36*^†	4.62 ± 0.07	4.54 ± 0.12	18.97 ± 0.20*^†
Σ SFA		42.71 ± 0.12	42.02 ± 0.13*	45.35 ± 0.18*^†	41.85 ± 0.09	41.0 ± 0.35	44.69 ± 0.25*^†
Σ MUFA		19.18 ± 0.65	18.90 ± 0.12	16.67 ± 0.18*^†	19.72 ± 0.14	20.03 ± 0.13	17.53 ± 0.13*^†
Σ PUFA (ω-3)		6.19 ± 0.10	6.57 ± 0.12	17.88 ± 0.36*^†	4.96 ± 0.07	4.84 ± 0.14	19.90 ± 0.20*^†
Σ PUFA (ω-6)		31.93 ± 0.43	32.52 ± 0.14	20.10 ± 0.39*^†	33.46 ± 0.14	34.13 ± 0.40	17.89 ± 0.29*^†

Analysis of fatty acid profiles of RBCs from experimental groups 1- and 5-weeks post-instillation as determined by OmegaQuant Analytics, LLC. p < 0.05; * Significant compared to VEH/CON, ^† Significant compared to cSiO₂/CON.

Figure 3. cSiO₂-triggered pulmonary perivascular and peribronchiolar lymphoid cell infiltration and ELT neogenesis in mature female NZBWF1 mice are inhibited by DHA supplementation. (A) Light photomicrographs of hematoxylin and eosin-stained lung tissue sections from mice instilled with saline vehicle and fed CON diet (VEH/CON), mice instilled with cSiO₂and fed CON diet (cSiO₂/CON), and mice instilled with cSiO₂ and fed DHA-enriched diet (cSiO₂/DHA). cSiO₂ exposure resulted in perivascular and peribronchiolar infiltration of lymphoid cells at 1-week PI, with even greater infiltration (ectopic lymphoid tissue, ELT, asterisks) at 5-weeks PI. DHA attenuated ELT formation at both time points. b, bronchioles; v, blood vessel; a, alveolar parenchyma; solid arrows, proteinaceous debris; stippled arrows, inflammatory cell influx in alveolar parenchyma (alveolitis).(B) Graphic representation of semi-quantitative severity scores following assessment criteria of (1) minimal (<10%); (2) slight (10-25%); (3) moderate (26-50%); (4) marked (51-75%); and (5) severe (>75%) amount of tissue affected. (C) cSiO₂ instillation caused a significant increase in total leukocytes, monocytes, and neutrophils in the BALF. DHA significantly reduced total leukocyte and monocyte cellularity in the BALF but did not influence neutrophils. a, significantly different from VEH/CON group; b, significantly different from cSiO₂/CON group; p < 0.05.

Figure 4. cSiO₂-induced chemokine protein expression in lung tissue of mature adult female NZBWF1 mice is inhibited by DHA consumption. Proinflammatory cytokine production was measured in lung tissue homogenate using a multiplex cytokine discovery assay. Cytokine production significantly increased with cSiO₂ exposure and was attenuated with the DHA-enriched diets at either 1-week or 5-weeks PI. Letters: a, significantly different from VEH/CON for the specified endpoint (p < 0.05); b, significantly different from cSiO₂/CON for the specified endpoint (p < 0.05).

Figure 5. cSiO₂-induced lung infiltration of perivascular and peribronchiolar CD45R⁺ B-cells, CD3⁺ T-cells, and IgG⁺ plasma cells in mature adult female NZBWF1 mice is suppressed by DHA feeding. (A-O) Light photomicrographs of lung tissue from VEH/CON, cSiO₂/CON, and cSiO₂/DHA mice at 5-weeks PI. Tissue sections were histochemically stained with hematoxylin and eosin (H&E; low power, A-C; high power, D-E) identifying perivascular and peribronchiolar ectopic lymphoid tissue (ELT, solid arrows in B and E) or scant lymphoid cell aggregates (stipple arrows in C). Other sections were immunohistochemically stained for CD45R⁺ B lymphoid cells (G-I), CD3⁺ T-cells (J-L), and IgG⁺ plasma cells (M-O) in ELT. In cSiO₂/CON mice, silica instillation triggered conspicuous formation of ELT containing CD45R⁺ B-cells, CD3⁺ T-cells, and IgG⁺ plasma cells. Only scant amounts of lymphoid cells were present in the lungs of cSiO₂/DHA mice (C, F, I, L,O). DHA treatment markedly attenuated lymphoid cell infiltration. b, bronchioles; v, blood vessels; a, alveolar parenchyma. (P) Graphical representation of morphometrically determined density of CD45R⁺ B-cells, CD3⁺ T-cells, and IgG⁺ plasma cells in lung tissue of mice at 1 week and 5 weeks after instillation. a, significantly different from VEH/CON group; b, significantly different from cSiO₂/CON group; p < 0.05.

Figure 6. SiO₂-induced inflammatory/autoimmune pathways in lung tissue 1- and 5-weeks post-cSiO₂ instillation in mature adult female NZBWF1 mice are quelled by DHA supplementation. Z scores of selected inflammatory/autoimmune pathways are presented as Tukey box plots for select pathways of interest for lung tissue. Within a tissue type, different letters indicate that the treatment groups are significantly different (p < 0.05), as described in materials and methods. cSiO₂ exposure significantly enriched MHC antigen presentation, NLR signaling, and Type I/II Interferon signaling pathways. DHA significantly reduced pathway Z scores for MHC antigen presentation and Type I/II Interferon signaling at 1-week PI and for NLR signaling at both 1- and 5-weeks PI. Letters: a, significantly different from VEH/CON for the specified endpoint (p < 0.05); b, significantly different from cSiO₂/CON for the specified endpoint (p < 0.05).

Figure 7. DHA feeding affects cSiO₂-induced IgG AAb responses in the BALF at 5-weeks PI in mature adult female NZBWF1 mice. (A) AAb-score values at 5 weeks PI in BALF for the expression of 120 IgG AAbs are illustrated in a heatmap using unsupervised clustering (Euclidian distance method). Scale bar values reflect the range of variance stabilized AAb scores centered across rows. (B) cSiO₂induced increase total IgG AAb the BALF in mice-fed control diet but not in mice fed DHA. (C) cSiO₂ exposure significantly increased various classes of autoimmunity-related AAbs detected in the BALF. A downward trend was observed in the same classes of cSiO₂-triggered AAbs when mice were fed DHA. Letter a indicates significantly different from VEH/CON for the specified endpoint (p < 0.05).

Figure 8. cSiO₂-triggered membranoproliferative glomerulonephritis and glomerular IgG deposition at 5-weeks PI in mature adult female NZBWF1 mice is attenuated by DHA feeding. Light photomicrographs of kidney tissue stained with periodic acid Schiff (PAS) and hematoxylin (A, B, C) and immunohistochemically stained for IgG with hematoxylin counterstain (D, E, F). VEH/CON (A, D), cSiO₂/CON (B, E), and cSiO₂/DHA mice (C, F). cSiO₂-exposed mice (B, E) had modest increases in glomerular size, cellularity, size, and IgG deposition. No histopathology was observed in VEH/CON mice (A, D) and DHA-fed mice (C, F). Individual kidney sections were semi-quantitatively scored based on the modified International Society of nephrology/renal pathology lupus Nephritis Classification system described methods for lupus nephritis score for hyperplasia/hypertrophy (G) and IgG deposition (H). cSiO₂/CON mice had significantly increased severity scores for hyperplasia/hypertrophy and IgG deposition at 5-weeks PI. DHA-enriched diets attenuated renal hyperplasia and hypertrophy. G, glomeruli; arrow, IgG proteinaceous material. Letters: a, significantly different from VEH/CON for the specified endpoint (p < 0.05); b, significantly different from cSiO₂/CON for the specified endpoint (p < 0.05).

Figure 9. cSiO2-triggered infiltration of T and B cells is greater in mature adult NZBWF1 mice compared to young adult NZBWF1 mice. Morphometric data from this study were compared to that previously reported in for young adult mice (Bates et al. 2019). (A) cSiO₂ exposure induced significant infiltration of CD3⁺ T-cells at 5-wk PI in mature adult mice but not in young adult mice. (B) cSiO₂-induced infiltration of CD45R⁺ B-cells at 5-weeks PI in mature adult mice was greater than that in young adult mice. DHA significantly reduced (A) CD3⁺ T-cell infiltration in marine adult cSiO₂-exposed mice and (B) CD45R⁺ B-cell infiltration in both mature adult and young adult mice. Letters: a, significantly different from VEH/CON for the specified endpoint (p < 0.05); b, significantly different from cSiO₂/CON for the specified endpoint (p < 0.05); c, significantly different from young adult mice within the same treatment group and specified endpoint (p < 0.05).

Figure 10. Mouse age influences cSiO₂-triggered inflammatory and autoimmune responses but not preventive effects of DHA. Diagrammatical summary of the severity of cSiO₂-triggered inflammatory/autoimmune endpoints and effects of DHA supplementation in (A) young adult female NZBWF1 mice at 5 weeks PI as reported in prior studies (Bates et al. 2015, 2016, 2019; Benninghoff et al. 2019; Gilley et al. 2020; Rajasinghe et al. 2020; Pestka et al. 2021) and (B) mature adult NZBWF1mice at 5-weeks PI as determined in the present investigation. (A) Weekly exposure of young adult mice to 1 mg cSiO₂ for 4 weeks led to modest pathological changes and ELT development in the lung and no observable glomerulonephritis, whereas (B) exposure of mature adult mice to the same cSiO₂ resulted in marked pulmonary pathology and with significant glomerular hypertrophy and IgG deposition in kidney consistent with glomerulonephritis demonstrating the age-dependent response to cSiO₂ in this experimental model.

Bates MA, Benninghoff AD, Gilley KN, Holian A, Harkema JR, Pestka JJ. 2019. Mapping of dynamic transcriptome changes associated with silica-triggered autoimmune pathogenesis in the lupus-prone NZBWF1 mouse. Front Immunol. 10:632. doi: 10.3389/fimmu.2019.00632.

 PubMed Web of Science ®Google Scholar

Bates MA, Brandenberger C, Langohr I, Kumagai K, Harkema JR, Holian A, Pestka JJ. 2015. Silica triggers inflammation and ectopic lymphoid neogenesis in the lungs in parallel with accelerated onset of systemic autoimmunity and glomerulonephritis in the lupus-prone NZBWF1 mouse. PLoS One. 10(5):e0125481. doi: 10.1371/journal.pone.0125481.

 PubMed Web of Science ®Google Scholar

Bates MA, Brandenberger C, Langohr II, Kumagai K, Lock AL, Harkema JR, Holian A, Pestka JJ. 2016. Silica-triggered autoimmunity in lupus-prone mice blocked by docosahexaenoic acid consumption. PLoS One. 11(8):e0160622. doi: 10.1371/journal.pone.0160622.

 PubMed Web of Science ®Google Scholar

Benninghoff AD, Bates MA, Chauhan PS, Wierenga KA, Gilley KN, Holian A, Harkema JR, Pestka JJ. 2019. Docosahexaenoic acid consumption impedes early interferon- and chemokine-related gene expression while suppressing silica-triggered flaring of murine lupus. Front Immunol. 10:2851. doi: 10.3389/fimmu.2019.02851.

 PubMed Web of Science ®Google Scholar

Gilley KN, Wierenga KA, Chauhuan PS, Wagner JG, Lewandowski RP, Ross EA, Lock AL, Harkema JR, Benninghoff AD, Pestka JJ, et al. 2020. Influence of total western diet on docosahexaenoic acid suppression of silica-triggered lupus flaring in NZBWF1 mice. PLoS One. 15(5):e0233183. doi: 10.1371/journal.pone.0233183.

 PubMed Web of Science ®Google Scholar

Rajasinghe LD, Li Q-Z, Zhu C, Yan M, Chauhan PS, Wierenga KA, Bates MA, Harkema JR, Benninghoff AD, Pestka JJ, et al. 2020. Omega-3 fatty acid intake suppresses induction of diverse autoantibody repertoire by crystalline silica in lupus-prone mice. Autoimmunity. 53(7):415–433. doi: 10.1080/08916934.2020.1801651.

 PubMed Web of Science ®Google Scholar

Pestka JJ, Akbari P, Wierenga KA, Bates MA, Gilley KN, Wagner JG, Lewandowski RP, Rajasinghe LD, Chauhan PS, Lock AL, et al. 2021. Omega-3 polyunsaturated fatty acid intervention against established autoimmunity in a murine model of toxicant-triggered lupus. Front Immunol. 12:653464. doi: 10.3389/fimmu.2021.653464.

 PubMed Web of Science ®Google Scholar

Data availability statement

Original NanoString normalized linear counts and statistical analyses from the NanoString autoimmune profiling panel, and a summary of statistical analyses are available at Dryad. https://doi.org/10.5061/dryad.2280gb5vx.