Influence of total western diet on docosahexaenoic acid suppression of silica-triggered lupus flaring in NZBWF1 mice.

Lupus is a debilitating multi-organ autoimmune disease clinically typified by periods of flare and remission. Exposing lupus-prone female NZBWF1 mice to crystalline silica (cSiO2), a known human autoimmune trigger, mimics flaring by inducing interferon-related gene (IRG) expression, inflammation, ectopic lymphoid structure (ELS) development, and autoantibody production in the lung that collectively accelerate glomerulonephritis. cSiO2-triggered flaring in this model can be prevented by supplementing mouse diet with the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA). A limitation of previous studies was the use of purified diet that, although optimized for rodent health, does not reflect the high American intake of saturated fatty acid (SFA), ω-6 PUFAs, and total fat. To address this, we employed here a modified Total Western Diet (mTWD) emulating the 50th percentile U.S. macronutrient distribution to discern how DHA supplementation and/or SFA and ω-6 reduction influences cSiO2-triggered lupus flaring in female NZBWF1 mice. Six-week-old mice were fed isocaloric experimental diets for 2 wks, intranasally instilled with cSiO2 or saline vehicle weekly for 4 wks, and tissues assessed for lupus endpoints 11 wks following cSiO2 instillation. In mice fed basal mTWD, cSiO2 induced robust IRG expression, proinflammatory cytokine and chemokine elevation, leukocyte infiltration, ELS neogenesis, and autoantibody production in the lung, as well as early kidney nephritis onset compared to vehicle-treated mice fed mTWD. Consumption of mTWD containing DHA at the caloric equivalent to a human dose of 5 g/day dramatically suppressed induction of all lupus-associated endpoints. While decreasing SFA and ω-6 in mTWD modestly inhibited some disease markers, DHA addition to this diet was required for maximal protection against lupus development. Taken together, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO2-triggered lupus flaring in NZBWF1 mice, even against the background of a typical Western diet.

To better parallel human food consumption in rodent feeding studies, the total Western diet (TWD) was formulated to emulate typical American intakes of macro-and micro-nutrients on an energy density basis for rodents [32,33]. The TWD is based on 50 th percentile intakes reported in the Centers for Disease Control National Health and Nutrition Examination Survey (NHANES) for 2007-2008, which were adjusted for differences in caloric intake. Overall, the TWD is not necessarily extreme in the level of any given nutrient, but rather reflects the overall U.S. dietary pattern. The TWD has fewer calories from protein and carbohydrate sources and twice that from fat as compared to the AIN-93G diet, the standard diet fed to rodents in nutritional studies to date. The new TWD diet contains more SFA and monounsaturated fatty acids (MUFAs), less PUFAs, more complex carbohydrates, and twice the level of simple sugars. As such, the TWD better represents typical U.S. nutrition intakes, making it very useful for studies employing animal models of human health and disease [34]. Here, we employed a modified TWD (mTWD) based on U.S. macronutrient intake to assess the impact of DHA supplementation with or without SFA and ω-6 PUFA reduction on cSiO 2induced lupus endpoints in NZBWF1 mice. The findings reported herein provide important new insights into the translatability of DHA's effects on lupus flaring in this novel mouse model.

Animals
Experimental animal procedures were reviewed and approved by Michigan State University's Institutional Animal Care and Use Committee (AUF # PROTO201800113) in accordance with the guidelines of the National Institute of Health. Female lupus-prone NZBWF1 mice were obtained at 6 wks of age from Jackson Laboratories (Bar Harbor, ME), housed 4 per cage, and allowed free access to feed and water. Only females were included in this study, as the incidence and severity of lupus symptoms is less pronounced in male NZBWF1 mice [35]. Animals were maintained under a 12-hour light/dark cycle with regulated temperature (21-24˚C) and humidity (40-55%). Mice were monitored daily by animal facility staff, observed for signs of distress such as rapid heart rate and lethargy, and weighed weekly in case of unexplained weight loss. Veterinary staff was alerted to any potential issues or advised for further monitoring. Following silica installation, animals were under observation for approximately 1 hour immediately following the procedure to identify any adverse events and observed again approximately four hours post-instillation.

Fatty acid analyses
Fatty acid concentrations in diets and tissues were determined by gas liquid chromatography (GLC) as previously described [30]. Erythrocyte fatty acids were measured by Omega Quant Inc. (Sioux Falls, SD). Table 1 summarizes formulations of the four isocaloric experimental diets used in this study. Since the focus of this study was on macronutrients, a modified version of the TWD (mTWD) was used as a basal diet for all experimental groups, with the modification being that micronutrients were provided by the standard AIN-93G vitamin and mineral mixes. Experimental diets were prepared as follows: 1) control diet (CON) was the basal mTWD with no other alterations; 2) DHA-supplemented diet ("DHA) was prepared by replacing 30 g/kg of olive oil in the mTWD (composed primarily of the ω-9 monosaturated fatty acid oleic acid) with 30 g/kg DHASCO microalgal oil containing 40% DHA (provided by Dr. Kevin Hadley, Martek Biosciences Corporation Columbia, MD); 3) reduced SFA and ω-6 diet (#SF.ω6) was prepared by replacing a portion of SFA and ω-6 in the mTWD with olive oil; and 4) #SF.ω6 diet supplemented with DHA (#SF.ω6"DHA) was prepared by supplementing with DHASCO microalgal oil as indicated above. GLC analysis confirmed the expected changes in amounts of ω-3 PUFAs, ω-6 PUFAs, and SFAs in the four experimental diets ( Table 2, Fig 1). Experimental diets were prepared fresh biweekly, flushed with nitrogen, vacuum sealed, and stored at -20˚C until use. Mice were provided fresh diet every day to prevent oxidation of the fatty acids. Palatability was assessed for the first 2 wk to ensure proper consumption. cSiO 2 (Min-U-Sil1 5, 1.5-2.0 μm average particle size, U.S. Silica (Mapleton, PA) was acid washed and oven-dried before addition of sterile phosphate buffered saline (PBS) [16]. Stock suspensions were prepared fresh in PBS prior to use, and suspensions were sonicated and vortexed for 1 min before intranasal instillation of each animal.

Experimental design
Fig 2 depicts the experimental design for this study. Briefly, groups (n = 8) of mice were fed one of the four isocaloric experimental diets beginning at 6 wk of age. 7 mice from the #SF. ω6"DHA diet group were analyzed, as one animal succumbed to an unrelated illness prior to termination of the experiment. After 2 wk, groups of mice were anesthetized with 4% isoflurane and intranasally instilled with 1.0 mg cSiO 2 in 25 μl PBS or 25 μl PBS vehicle (VEH) as described previously [16]. Body weights were monitored weekly and urine assessed biweekly for proteinuria using clinical dipsticks (Cortez Diagnostics, Calabasas, CA). cSiO 2 instillation and experimental diets did not affect body weight changes over the course of the study (Fig 3) and proteinuria was not detectable. At 22 wk of age (11 wk following the final cSiO 2 exposure), mice were euthanized by intraperitoneal injection with 56 mg/kg body weight sodium pentobarbital and exsanguination via the abdominal aorta. This time point was selected to capture ectopic lymphoid tissue neogenesis in the lungs following cSiO 2 exposure prior to and during onset of glomerulonephritis based on previous studies [29,30]. Blood was collected with heparin-coated syringes and centrifuged at 3500 xg for 10 min at 4˚C for separation of erythrocytes and plasma, which were then stored at -80˚C. BALF was collected from whole lungs as described previously [36] and stored at -80˚C for cytokine and autoantibody analysis. For each gene, the copy number relative to the average expression of Gapdh, Hprt, and Actb was calculated. First, the delta Ct was calculated for each gene by subtracting the mean delta Ct of the two housekeeping genes from the gene of interest. The relative copy number was calculated as previously described [37,38] using the following equation: Missing values were replaced with ½ the minimum RCN for each gene. For example, the minimum RCN of Zbp1 was 0.75, thus samples with missing values for Zbp1 were assigned an RCN ofof 0.37. Then, outliers within each treatment group were identified using robust outlier test (ROUT) with a Q value of 0.05%. After removing outliers, each gene was autoscaled by subtracting the mean expression of each sample and dividing by the standard deviation. The IFN score was calculated by summing the autoscaled expression for each gene within a given sample. All genes were given equal weight.

Cytokine analyses
Cytokine levels in BALF and plasma were analyzed with Immune Monitoring 48-plex Procar-taPlex Mouse Luminex Bead-based Immunoassay (Thermo Fisher Scientific, catalog number EPX480-20834-901) according to the manufacturer's protocols at the Michigan State University Flow Cytometry Core using a Luminex 200.

Autoantibody ELISAs
Autoantibody ELISA kits from Alpha Diagnostic International were utilized as per kit protocols for IgG-specific anti-double-stranded DNA (Alpha Diagnostic, San Antonio, TX, dsDNA, Catalog number 5120), and total anti-nuclear antigens (IgG + IgM + IgA) (ANA/EN, Catalog

BALF cell quantitation and identification
Total viable cell numbers in BALF were determined by Trypan Blue exclusion. Cytological slides from BALF were prepared, allowed to air dry, and stained with Diff-Quick (Fisher Scientific). Differential cell counts for macrophages/monocytes, lymphocytes, neutrophils, and eosinophils in BALF were determined using morphological criteria from 200 total cells on cytological slides. Remaining BALF was centrifuged at 2400 xg for 15 min, and supernatant collected and stored at -80˚C. Feeding of experimental diets was begun in 6 wk old female NZBWF1 mice. At 8 wk of age, mice were intranasally instilled with cSiO 2 once per wk for 4 wk. Body weights were measured weekly, and urine was collected weekly from 18 wk of age onward to monitor the development of proteinuria. Animals were necropsied at 22 wk of age, 11 wk following the final cSiO 2 instillation. Plasma, BALF, and tissues were collected for analysis. https://doi.org/10.1371/journal.pone.0233183.g002

Immunohistochemistry and morphometry of lungs
Immunohistochemistry was performed on formalin-fixed, paraffin embedded, left lung lobe for identification of B and T cell infiltration using anti-CD45R (1:600 rat anti-CD45R monocloncal antibody from Becton Dickinson, Franklin Lakes, NJ, catalog # 550286)and anti-CD3 antibodies (1:250 rabbit anti-CD3 polyconal antibody from Abcam, Cambridge, MA catalog # ab5690), respectively, as described previously [16,30]. Slides were digitally scanned using a VS110 (Olympus, Hicksville, NY) virtual slide system. At least 100 images were then captured at 20X magnification using systematic random sampling with NewCast software (Visiopharm, Hoersholm, Denmark). Volume densities of CD45R + or CD3 + cells in the bronchial and perivascular areas of the lungs were estimated using a point grid over the randomly sampled images with the STEPanizer 1.8 Stereology Tool. The number of points landing directly on the CD45R + or CD3 + cells were counted and the volume density or percentage of CD45R + and CD3 + per reference area was calculated.

Kidney histopathology
Fixed kidneys were sectioned, paraffin-embedded, cut and stained with either H&E or Periodic acid-Schiff and hematoxylin (PASH), and evaluated for lupus nephritis by a board-certified veterinary pathologist using a modified International Society of Nephrology/Renal Pathology Lupus Nephritis Classification system [39]. Slide sections were graded as follows: (0) no tubular proteinosis and normal glomeruli; (1) mild tubular proteinosis with multifocal segmental proliferative glomerulonephritis and occasional early glomerular sclerosis and crescent formation; (2) moderate tubular proteinosis with diffuse segmental proliferative glomerulonephritis, early glomerular sclerosis and crescent formation; and (3) marked tubular proteinosis with diffuse global proliferative and sclerosing glomerulonephritis.

Statistical analysis
Statistical analysis was performed using GraphPad Prism Version 8 (GraphPad Software, La Jolla California USA, www.graphpad.com). First, suspected outliers were verified using the ROUT with a conservative Q value of 1%, meaning that there was <1% chance of excluding a data point as an outlier in error. Data that did not meet the normality assumption as determined by the Shapiro-Wilk test (p<0.01) were analyzed using a the Kruskal-Wallis nonparametric test with Dunn's post-hoc test for selected multiple comparisons. Data that did not meet the equal variance assumption as determined by the Brown-Forsythe test (p<0.01) were analyzed by the Brown-Forsythe/Welch ANOVA with Dunnett's T3 post-hoc test for multiple comparisons. Otherwise, data meeting both normality and variance assumptions were analyzed using a standard one-way ANOVA with Sidak's post-hoc test for multiple comparisons. The groups that were compared were as follows: 1) VEH/CON vs. cSiO 2 /CON; 2) cSiO 2 /CON vs cSiO 2 /"DHA; 3) cSiO 2 /CON vs. cSiO 2 /#SF.ω6; 4) cSiO 2 /"DHA vs. cSiO 2 /#SF.ω6"DHA; 5) cSiO 2 /#SF.ω6 vs. cSiO 2 /#SF.ω6"DHA; and, 6) cSiO 2 /CON vs cSiO 2 /#SF.ω6"DHA. For samples where every individual in the VEH/CON group was undetectable, a one-sample t-test was performed on the cSiO 2 /CON group to confirm that cSiO 2 -induced changes were significantly different from the limit of quantification. Then, the appropriate statistical test was applied to compare the remaining groups, as described above. In all instances, a significant effect of the diet group was inferred when the adjusted p<0.05.

DHA intake increases ω-3 PUFA content in red blood cells and tissues
Previous studies have shown that the Omega-3 Index, obtained by measuring DHA and EPA in total red blood cell (RBC) fatty acids, is correlated with the ω-3 PUFA content in other tissues [40][41][42]. We assessed this, as well as correlations between RBCs and tissues for SFAs, MUFAs, and ω-6 PUFAs. Individual tissues showed unique total FA profiles, with varying degrees of similarity to each other and to RBCs. In general, correlations between RBCs and tissues were higher for ω-3 and ω-6 PUFAs than for SFAs and MUFAs (S1 Fig). This is in agreement with previous studies demonstrating high correlations between dietary and tissue levels of fatty acids that cannot be produced endogenously, such as essential ω-3 and ω-6 PUFAs [43][44][45][46]. The most consistent trend across all tissues was a concurrent decrease of the major ω-6 PUFA arachidonic acid (ARA) as DHA levels increased (Figs 4 and 5).
RBCs had higher levels of total PUFA than the tissues, totaling approximately 40% of RBC fatty acids. The PUFA pool in RBCs was composed primarily of LA, ARA, EPA, and DPA. Total ω-3 incorporation into RBCs was approximately three times greater in mice that consumed "DHA or #SF.ω6"DHA diets compared to those fed CON or #SF.ω6 diets ( Table 3, Fig 4A and 4B). Both DHA (22:6ω3) and EPA (20:5ω3) contributed to this increase in total ω-3 PUFA. The increase in EPA observed with DHA supplementation is consistent with published studies performed in vitro and in vivo [47,48].
In the RBCs, reducing dietary SFA and ω-6 PUFAs had only a minor impact on SFA content (approximately 4%) and reduced RBC total ω-6 levels by approximately 10%, with the greatest change observed in linoleic acid (LA, 18:2ω6). Alternatively, combining DHA supplementation with ω-6 reduction resulted in decreased RBC ω-6 content by over 50%. The decrease in ω-6 PUFAs with DHA supplementation was largely due to changes in ARA.
The lung was the only organ where a significant change in total FA composition was observed in response to cSiO 2 (S1 Table, Fig 5A and 5B). Here, cSiO 2 exposure induced an increase in SFA in the form of palmitic acid (C16:0) from~30% (similar to RBC levels) tõ 50%. Palmitic acid is the fatty acid moiety of dipalmitoylphophatidylcholine (DPPC), a major component of pulmonary surfactant. This increase in palmitic acid appears to be at the expense of stearic acid and oleic acid (OA, C18:1ω9). OA was decreased further by DHA supplementation, but increased slightly in the #SF.ω-6 diets. Trends within the PUFA fraction are similar to those observed in the RBCs, with ω-3 PUFAs significantly increased by DHA supplementation.
The liver appeared to be the organ most reflective of dietary fat intake. In the liver, plasma non-esterified fatty acids (NEFAs) obtained from adipose tissue lipolysis or from dietary fatty acids are packaged into lipoproteins to be distributed throughout the body or stored in lipid droplets as triacylglycerides (TAGs) [49][50][51]. Furthermore, TAGs present in the liver are highly similar to the dietary FA composition, especially when dietary fat remains consistent over time. MUFA levels were greatest in the liver (S2 Table, Fig 5C and 5D), with OA com-posing~40% of total liver fatty acids in the CON and "DHA diets and~60% in the #SF.ω6 diet and #SF.ω6"DHA diets. This is notable because OA is the major FA in olive oil, which replaced corn oil (composed primarily of LA) in the #SF.ω-6 diet. A reduction in SFA and ω-6 PUFAs (mainly in palmitic acid and LA, respectively) was observed in animals fed the #SF.ω6 diet. SFA in the liver was comparable to levels in the spleen and kidney (~15%) (Fig 5E and  5G).  Table 3. Different letters indicate statistically significant differences between treatment groups for individual fatty acids (p<0.05). https://doi.org/10.1371/journal.pone.0233183.g004

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The total FA composition of the kidney (S3 Table, Fig 5E and 5F) and spleen (S4 Table, Fig 5G and 5H) were nearly identical. Unlike RBCs and the liver, there was no clear change in OA in these tissues. Changes in SFA levels across all diets were also minimal. In both tissues, there was a clear decrease in LA with the #SF.ω6 diets. The increase in DHA in these tissues was evident, though not as dramatic as observed in the RBC, lung, and liver.  . (A,C,E,G) The distribution of SFA, MUFA, and PUFA was compared between tissues. Statistically significant differences are indicated in S1-S4 Tables. (B,D,F,H,J) The seven most abundant fatty acids in each tissue were expressed as percent of total. Different letters indicate statistically significant differences between treatment groups for individual fatty acids (p<0.05). https://doi.org/10.1371/journal.pone.0233183.g005

DHA supplementation suppresses cSiO 2 -induced IRG response in the lungs
The effects of dietary treatments on cSiO 2 -induced IRG expression in the lung were compared using an IFN score, encompassing 15 genes with known functions in response to IFN signaling. Expression of each of these genes was significantly induced (p<0.05) following cSiO 2 exposure. The IFN score was calculated for each sample by taking the sum of the autoscaled expression of each gene (see Methods). The IFN score was elevated significantly (p<0.05) in cSiO 2 -instilled mice fed CON but suppressed nearly to baseline in those fed the "DHA diet (Fig 6A). The IFN score in cSiO 2 -treated mice fed the #SF.ω6"DHA diet was reduced compared to those fed CON or #SF.ω6. The mRNA expression profiles for four representative genes, Irf 7, Cxcl9, Isg15, and Oas1 are illustrative of the expression pattern for individual IRGs (Fig 6B).

DHA intake suppresses cSiO 2 -induced cytokine elevations in the BALF
As we have observed in prior studies employing AIN-93G diet [16,17,30], cSiO 2 instillation of mice fed CON diet induced a range of cytokines in BALF that are associated with leukocyte

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infiltration and the inflammatory response (Fig 7). Levels of the monocyte chemoattractant proteins MCP-1 (Fig 7A) and MCP-3 (Fig 7B) tended to be lower in the BALF of cSiO 2treated animals fed "DHA, with significant suppression occurring in mice fed #SF.ω6"DHA.
Though not all changes were statistically significant, similar observations were made for TNFα (Fig 7C), IL-1α (Fig 7D), IL-6 (Fig 7E), and IL-18 (Fig 7F), which are known for their roles in inflammation and lupus development. Furthermore, T-helper cytokines IL-17A (Fig 7G) and IL-22 (Fig 7H), which recruit T H 17 cells and polarize macrophages toward a proinflammatory M1 phenotype, were upregulated by cSiO 2 in CON-fed mice and downregulated in mice fed #SF.ω6"DHA and "DHA, respectively. Finally, while cSiO 2 instillation induced elevation of B cell activating factor (BAFF) (Fig 7I) in the BALF of CON-fed mice, this response was not affected by feeding "DHA, #SF.ω6, or #SF.ω6"DHA. Overall, DHA supplementation with or without SFA and ω-6 PUFA suppression alleviated induction of many cytokines involved in lupus pathogenesis.

DHA consumption suppresses cSiO 2 -induced pulmonary immune cell infiltration, including B and T lymphocytes and ELS neogenesis
Further consistent with prior investigations utilizing AIN-93G diet [16,30], intranasal instillation with cSiO 2 increased total cells (Fig 8A), monocytes (Fig 8B), neutrophils (Fig 8C), and lymphocytes (Fig 8D) in the BALF of mice at experiment termination. Notably, total cell and monocyte accumulation were inhibited in mice fed "DHA, #SF.ω6, and #SF.ω6"DHA diets. Similar trends were observed with the increase for neutrophils and lymphocytes, though not all were statistically significant. Histological assessment of H&E-stained lung tissue from cSiO 2 -instilled mice fed CON showed robust peribronchiolar and perivascular leukocytic infiltration (Fig 9A). Immunohistochemical staining further indicated cSiO 2 -induced development of ELS in the lung, as evidenced by the organized accumulation of B cells (CD45R + ) and T cells (CD3 + ) (Fig 9A). cSiO 2 -induced cell infiltration and ELS neogenesis were suppressed in mice fed "DHA and #SF.ω6"DHA diets, but unlike the observations in BALF, not affected in mice fed the #SF.ω6 diet. Morphometric analysis confirmed that cSiO 2 treatment of mice fed CON diets elicited accumulation of B cells (Fig 9B) and T cells (Fig 9C) in the lung, further suggestive of ELS neogenesis, and that this response was markedly suppressed in mice fed "DHA and #SF.ω6"DHA diets.

cSiO 2 -induced autoantibody production is attenuated by DHA supplementation
Elevations in anti-dsDNA and anti-nuclear autoantibodies are hallmarks of lupus flaring and progression. Mice in the cSiO 2 /CON group exhibited significant increases for both of these autoantibodies in both BALF and plasma compared to VEH/CON (Fig 10). Consumption of "DHA, #SF.ω6, or #SF.ω6"DHA diets significantly reduced cSiO 2 -anti-dsDNA responses in BALF (Fig 10A), whereas plasma responses were reduced only in mice fed the "DHA diet (Fig  10B). Anti-nuclear antibody responses followed similar trends (Fig 10C and 10D).

DHA intake protects against cSiO 2 -induced lesions in the kidney
Naïve female NZBWF1 mice typically display glomerulonephritis around 35 wk of age [52] and die of kidney failure within 1 year. Previous studies in our lab and others have shown that exposure to cSiO 2 accelerates this phenotype, with nephritis being first observed within 3 months after the final cSiO 2 instillation (i.e. age 22 wk) in mice fed the AIN-93G diet [16,29].
While proteinuria was not evident up to experiment termination, histopathological analysis indicated that multifocal segmental proliferative glomerulonephritis was more severe in kidneys of mice after cSiO 2 instillation fed CON and #SF.ω6 compared to mice fed DHA-supplemented diets (Fig 11A and 11B).

Discussion
The TWD represents typical eating patterns in the U.S, making it highly appropriate for investigating how modulation of dietary lipids affects flaring and progression in preclinical models of lupus. The results presented herein indicate for the first time that translationally relevant DHA supplementation against the complex background of the Western diet is highly effective in protecting against cSiO 2 -triggered IRG expression, cytokine/chemokine release, leukocyte infiltration, ELS neogenesis, autoantibody production in the lungs as well as glomerulonephritis (summarized in Fig 12). While some disease endpoints were modestly attenuated by reducing SFAs and ω-6 PUFAs through increasing the ω-9 PUFA content with olive oil, further DHA supplementation to this diet was required for maximal protection against lupus development. Finally, consistent with the observed effects in the lung, consumption of DHA-amended diets prevented early onset of glomerulonephritis in cSiO 2 -exposed mice. Together, these findings suggest that ω-3 supplementation to a Western diet without substantial diet changes may be protective against lupus flaring.
The observation that DHA can suppress IRG responses is highly significant because IFN signaling has been centrally linked to lupus disease activity in preclinical and clinical studies.  In NZBWF1 mice, adenovector-mediated delivery of IFN-α, a major type I IFN, induces the development of lupus [53]. Gonzalez-Quintial, et al. [54] demonstrated in C57Bl6 mice, which are not genetically predisposed to lupus, that early-life virus exposure combined with adult exposure to cSiO 2 results in the production of lupus-like symptoms, including autoantibody production and glomerulonephritis. Likewise, the IRG signature is closely related to the flaring and pathogenesis of lupus symptoms in human patients [55][56][57]. Randomized, double-blind, placebo-controlled phase IIb clinical trials indicated that sifalimumab, an anti-IFNα monoclonal antibody [58], and anifrolumab, a type I IFN receptor antagonist [59], positively impacted lupus symptoms. Recently, a large, double-blind, placebo-controlled phase 3 clinical trial , fed a diet with (+) or without (-) DHA supplementation, and with (+) or without (-) SF.ω6 reduction. Lung sections were stained with H&E (first column), immunohistochemically stained for CD45R + B lymphoid cells (arrows; brown chromagen; second column), or CD3 + T lymphoid cells (arrows; brown chromagen; third column). Peribronchiolar and perivascular accumulations of B and T lymphoid cells (ELS; arrows) were present in the lungs of cSiO 2 -exposed mice fed diets without DHA supplementation (second and fourth rows). B or T cell accumulations were not observed in the lung of control mice intranasally instilled with saline and fed CON (row one). Minimal perivascular and peribronchiolar accumulations of B and T lymphoid cells were seen in the lungs of mice intranasally exposed with cSiO 2 and fed "DHA (row three) or #SF.ω6"DHA. (row five). Abbreviations: a-alveolar parenchyma, b-bronchiolar airway, v-blood vessel. Morphometric analysis was used to quantitatively determine the volume density of (B) CD45R + and (C) CD3 + area in the measured lung area. Values of p<0.1 are shown, with p<0.05 considered statistically significant.
https://doi.org/10.1371/journal.pone.0233183.g009 (TULIP-2) was finalized and documented that intravenous anifrolumab lowered overall disease activity, reduced skin disease, and enabled oral corticosteroid tapering [60]. Accordingly, the finding here that DHA supplementation of CON and #SF.ω6 diets lowered the IRG response is potentially relevant from a translational perspective. Our analysis of tissue fatty acid content confirmed that the ω-3 PUFA content of the RBC, presented both as the Omega-3 Index and as total ω-3 PUFA, is reflective of ω-3 PUFA levels in the diet [61]. We found here that Omega-3 Indexes for mice fed "DHA and #SF.ω6"DHA diets were three times higher than those fed CON or #SF.ω6 diets. These trends were consistent with %EPA+DHA levels in the tissues. Alternatively, the effects of feeding #SF.ω6 diet on the Omega-3 Index and the %EPA+DHA in tissues were minimal compared to CON-fed animals. This may explain why the #SF.ω6 diet provided minimal protection against the cSiO 2induced inflammatory response. Additionally, the #SF.ω-6 diet had a minor impact on tissue and RBC SFA levels and did not alter levels of long chain ω-6 PUFAs. Though there was a reduction in total ω-6 PUFA levels, this was accomplished primarily by reduction of LA (18:2ω6) rather than ARA (20:4ω6).
Our results suggest that the balance of long chain ω-3 and ω-6 PUFAs in the cell membrane might be critical to promoting inflammation or resolution. One explanation for this observation is that ω-3 and ω-6 PUFAs are substrates for downstream bioactive lipid metabolites. It is well established that lipid metabolites derived from the "arachidonic acid cascade" have primarily inflammatory actions, especially in the case of acute inflammation [62]. Over the last two decades, many metabolites of ω-3 PUFAs have been identified as having anti- . Animals were fed the CON diet (1,2) or "DHA diet (3). Others were fed the #SF.ω6 without DHA (4) or with DHA supplementation (#SF.ω6"DHA, 5). No renal histopathology was evident in saline-instilled control mice (1) or cSiO 2 -instilled mice fed DHA (3,5). In contrast, cSiO 2instilled mice fed diets without DHA supplementation (2, 4) had renal histopathology characteristic of a membranoproliferative glomerulonephritis characterized by hypercellular glomeruli with thickened mesangial tissue, tubular proteinosis (solid arrows), and tubular epithelial hyperplasia (stippled arrows). Abbreviations: g-glomerulus, rt-renal tubules. (B) Quantification of renal histology score, based on the following scoring criteria: No proteinosis, normal glomeruli (0); multifocal segmental proliferative glomerulonephritis (1); multifocal segmental proliferative glomerulonephritis and occasional glomerular sclerosis and crescent formation (2); diffuse global segmental proliferative glomerulonephritis (3). Values of p<0.1 are shown, with p<0.05 considered statistically significant.
https://doi.org/10.1371/journal.pone.0233183.g011 The results presented here and in other investigations suggest that cSiO 2 promotes cell death in alveolar macrophages, resulting in the release of proinflammatory cytokines and chemokines that recruit and activate additional immune cells, including T cells and B cells. Accumulation of cellular debris results in uptake and presentation of autoantigens (AAg) to T-and B-cells. Among the cellular debris are host nucleic acids, which stimulate the production of Type I IFN from plasmacytoid dendritic cells. Type I IFN promotes cytokine release, antigen uptake, and maturation of B cells to plasma cells, which produce autoantibodies (AAb) against host antigens both locally and systemically. Upon binding their cognate AAgs, inflammatory and pro-resolving properties [63]. A recent study demonstrated that the plasma and RBC levels of ω-3 PUFA were highly correlated with the production of ω-3 PUFA-derived lipid mediators, many of which are involved in the resolution of inflammation. Similarly, supplementation with EPA and DHA led to a decrease in ω-6 PUFAs, namely ARA, as well as decreased ω-6-PUFA derived metabolites [64,65]. Shifting the membrane composition to favor long chain ω-3 PUFAs rather than ω-6 PUFAs, such as arachidonic acid, may enhance the pro-resolving phenotype promoted by ω-3 PUFA-derived lipid mediators.
Despite a limited number of studies, there is evidence that increasing levels of long chain ω-3 PUFAs in lupus patients leads is protective against inflammation, a process that likely involves the action of bioactive lipid metabolites. A recent study showed strong associations between dietary PUFA intake from fish and the ω-3 status in lupus patients. Further, the RBC ω-3 levels were negatively associated with levels of C-reactive protein [66]. Others have reported that lupus patients had lower levels of plasma ω-3 PUFAs [67] and plasma resolvin D1, an anti-inflammatory metabolite of DHA than healthy controls [68]. Multiple human studies in lupus and other rheumatic diseases have shown decreased disease activity in patients receiving ω-3 supplementation [24], but few studies have investigated the impact of modulating other dietary lipids, such as ω-6 PUFAs and SFAs. To date, there has been no extensive study of the membrane fatty acid content or plasma lipidome of lupus patients. Investigation in this area-both in pre-clinical and clinical settings-is necessary to elucidate potential benefit of ω-3 PUFA supplementation in individuals with lupus.
To summarize, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO 2 -triggered lupus flaring in NZBWF1 mice fed a Western diet. Future perspectives should include understanding how the TWD may impact the ameliorative effects of lower DHA doses in this model over time and how these responses are influenced by ω-6, SFA, and total fat content. Ultimately, well-designed clinical trials will be needed to confirm the value of ω-3 PUFA supplementation for the prevention and treatment of lupus in humans.
Supporting information S1