Plasmacytoid dendritic cell distribution and maturation are altered in lupus prone mice prior to the onset of clinical disease.

Plasmacytoid dendritic cells (pDCs) and their production of type I interferons (IFN) are key pathogenic mediators of systemic lupus erythematosus (SLE). Despite the key role of pDCs in SLE, the mechanism by which pDCs promote disease is not well understood. The first objective for this study was to assess the number and maturation state of pDCs in pre-disease NZM2410 lupus prone mice compared to control mice. Second, we sought to identify mechanisms responsible for the alteration in pDCs in NZM mice prior to onset of clinical disease. We compared the number and percent of pDCs in the spleens and bone marrow (BM) of pre-disease NZM24010 (NZM) mice to C57BL/6 (B6) control mice. In the spleens of pre-disease NZM mice, pDC percent and number were increased. This increase occurs in parallel with a decrease in BM pDC number and percent in the NZM mice. The decrease in BM pDC number suggests the increase in spleen pDCs is a result of altered pDC distribution and not increased production of pDCs in the BM. To determine if pDC developmental potential is altered in lupus prone mice, we cultured BM from NZM and B6 mice in vitro. We found a reduced percentage/number of pDCs developing from the BM of NZM mice compared to B6 mice, which further supports that the increase in pDC number is a result of altered pDC distribution rather than increased pDC production. To better characterize the pDC population, we compared the percentage of mature pDCs in the spleens and BM of NZM mice to controls. In the NZM mice, there is a dramatic reduction in the number of mature pDCs in the BM of NZM mice, suggesting that mature pDCs exit the BM at a higher rate/earlier maturation time compared to healthy mice. We conclude that pDCs contribution to disease pathogenesis in NZM mice may include the alteration of pDC distribution to increase the number of pDCs in the spleen prior to disease onset.

Estrogen Receptor α Deficiency Modulates TLR Ligand-Mediated PDC-TREM Expression in Plasmacytoid Dendritic Cells in Lupus-Prone Mice.

Female lupus-prone NZM2410 estrogen receptor α (ERα)-deficient mice are protected from renal disease and have prolonged survival compared with wild-type littermates; however, the mechanism of protection is unknown. Plasmacytoid dendritic cells (pDCs) and type I IFN drive lupus pathogenesis. Estrogen acting via ERα enhances both pDC development and IFN production. The objectives for this study were to determine if ERα modulates pDC function and IFN activity in predisease NZM2410 mice as a possible protective mechanism of ERα deficiency in lupus-prone mice. We measured the effect of ERα deficiency on spleen pDC frequency, number, maturation, and activation state. ERα deficiency reduced type I IFN activity and the frequency of MHC class II(+) pDCs in the spleen without altering overall pDC frequency, number, or maturation state. Additionally, ERα-deficient NZM2410 mice had a significantly decreased frequency of pDCs expressing PDC-TREM, a modulator of TLR-mediated IFN production. After in vitro TLR9 stimulation, ERα deficiency significantly reduced the expression of PDC-TREM on pDCs from both NZM2410 and C57BL/6 mice. Thus, we have identified a significant effect of ERα deficiency on pDCs in predisease NZM2410 mice, which may represent a mechanism by which ERα deficiency protects NZM2410 mice from lupuslike disease.

Estrogen receptor alpha modulates Toll-like receptor signaling in murine lupus.

Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals.

N,N,-diethyl-m-toluamide (DEET) suppresses humoral immunological function in B6C3F1 mice.

N,N-diethyl-meta-toluamide (DEET) is a particularly effective broad-spectrum insect repellent used commonly in recreational, occupational and military environments. Due to its widespread use and suggested link to Gulf War Illness, this study examined the immunotoxicity of DEET. Adult female B6C3F1 mice were injected sc for 14 days with DEET at 0, 7.7, 15.5, 31, or 62 mg/kg/day. Due to differences in the dermal absorption of DEET between mice and humans, this study eliminated this confounding factor by utilizing sc injection and measured circulating blood levels of DEET to assess bioavailability from sc administration. Effects on lymphocyte proliferation, natural killer cell activity, thymus and spleen weight and cellularity, the antibody plaque-forming cell (PFC) response, and thymic and splenic CD4/CD8 lymphocyte subpopulations were assessed 24 h after the last dose. No effect was observed in lymphocyte proliferation, natural killer cell activity, thymic weight, splenic weight, thymic cellularity, or splenic cellularity. Significant decreases were observed in the percentage of splenic CD4-/CD8- and CD4+/CD8- lymphocytes but only at the 62 mg DEET/kg/day treatment level and not in absolute numbers of these cells types. Additionally, significant decreases in the antibody PFC response were observed following treatment with 15.5, 31, or 62 mg DEET/kg/day. Pharmacokinetic (PK) data from the current study indicate 95% bioavailability of the administered dose. Therefore, it is likely that DEET exposure ranges applied in this study are comparable to currently reported occupational usage. Together, the evidence for immunosuppression and available PK data suggest a potential human health risk associated with DEET in the occupational or military environments assuming similar sensitivity between human and rodent responses.

Lifetime exposure to trichloroethylene (TCE) does not accelerate autoimmune disease in MRL +/+ mice.

A genetically-prone murine lupus model was used to assess the developmental effects of trichloroethylene (TCE) exposure on disease symptom onset (e.g., autoantibody production and proteinuria), lymphocyte proliferation, splenic B-cell populations, and thymic and splenic T-cell populations. MRL +/+ mice were exposed to TCE (0, 1,400 or 14,000 ppb) via drinking water beginning on gestation day (GD) 0 and continuing until 12 months of age. With the exception of splenic CD4-/CD8-cells in female mice only, no alterations were observed in splenic T-cell populations, numbers of splenic B220+ cells, or in lymphocyte proliferation at 12 months of age. Furthermore, populations of all thymic T-cell subpopulations were decreased in male but not female mice following exposure to 14,000 ppb TCE. Autoantibody levels (anti-dsDNA and anti-GA) were assessed periodically from 4 to 12 months of age. Over this period, no increase in autoantibody levels as compared to control was detected, suggesting that TCE did not contribute to or accelerate the development of autoimmune disease markers following lifetime exposure. Not only does this study offer encouraging results, but it is the first study to approach the development of autoimmunity in a novel lifetime exposure paradigm, using an autoimmune prone model, at environmentally relevant exposure levels.

Suppression of humoral immunity in mice following exposure to perfluorooctane sulfonate.

Adult male and female B6C3F1 mice were exposed to perfluorooctane sulfonate (PFOS) daily via gavage for 28 days (0, 0.005, 0.05, 0.1, 0.5, 1, or 5 mg/kg total administered dose [TAD]). Following exposure, various immune parameters were assessed and serum PFOS concentrations were determined. Lymphocyte proliferation was not altered in either gender. Natural killer cell activity was increased compared with control at 0.5, 1, and 5 mg/kg TAD in male mice but was not altered in female mice. At these treatment levels, splenic T-cell immunophenotypes were minimally altered in females, but all T-cell subpopulations were significantly modulated in males beginning at 0.1 mg/kg TAD. The sheep red blood cell (SRBC) plaque-forming cell (PFC) response was suppressed in male mice beginning at 0.05 mg/kg TAD and in females at 0.5 mg/kg TAD. Serum trinitrophenyl (TNP)-specific IgM titers were also decreased by PFOS after TNP-LPS (TNP conjugated to lipopolysacharide) challenge suggesting that the humoral immune effects may be attributed to the B-cell rather than T-cell because both T-dependent (SRBC) and T-independent (TI) (TNP-LPS) antigens result in suppressed IgM production. Based on the PFC response, the low observed effect level (LOEL) for males was 0.05 mg/kg TAD (ED(50) = 0.021 mg/kg TAD) and for females was 0.5 mg/kg TAD (ED(50) = 0.59 mg/kg TAD). Measured PFOS serum concentrations at these dose levels were 91.5 +/- 22.2 ng/g and 666 +/- 108 ng/g (mean +/- SD), respectively. The male LOEL serum level was approximately 14-fold lower than reported mean blood levels from occupationally exposed humans and fell in the upper range of concentrations reported for the general population. Overall, this study provides a profile of PFOS immunotoxicity showing effects at levels reported in humans and identifies the B-cells as a potential target.

Suppression of humoral immunity following exposure to the perfluorinated insecticide sulfluramid.

Perfluorinated hydrocarbons have been manufactured for over 40 yr and have numerous applications in industry. This group of compounds has recently generated much interest, as some of these compounds such as perfluorooctane sulfonate (PFOS) and perfluoroctanic acid (PFOA) are persistent in the environment and detectable in blood samples of both wildlife and humans. Studies show that these perfluorinated compounds induce peroxisomal proliferation, induce hepatomegaly, alter steroidogenesis, and decrease body weight, accompanied by a wasting syndrome; however, effects on immune function have not been addressed at length. This study examined sulfluramid, a perfluorinated pesticide that is currently available in the marketplace and is a representative member of this class of chemicals. Adult female B6C3F1 mice were exposed via gavage to either an oil carrier control or sulfluramid for 14 d (1, 3, 10, or 30 mg/kg/d) or 28 d (0.3, 1, 3, or 10 mg/kg/d). Although responses were normal in natural killer cell activity and lymphocyte proliferation, dose-responsive suppression was noted in the plaque forming cell (PFC) response at exposure levels as low as 3 mg/kg/d in the 14-d exposure and 0.3 mg/kg/d for 28 d. Dose-responsive increases in liver mass were observed following treatment with 1, 3, 10, or 30 mg/kg/d for 14 d and 0.3, 1, 3, or 10 mg/kg/d for 28 d. A significant reduction in body weight was observed at the highest dose level in each study. Novel findings in this study indicate that sulfluramid suppresses immunoglobulin (Ig) M production. Additional immunotoxicity studies are required to understand potential mechanisms of suppression and determine potential health risks associated with exposure to perfluorinated hydrocarbons.

Developmental immunotoxicity of trichloroethylene (TCE): studies in B6C3F1 mice.

This study assessed the developmental immunotoxicity of trichloroethylene (TCE) in B6C3F1 mice exposed via drinking water (0, 1,400, 14,000 ppb) from gestation day 0 (GD0) to either 3 or 8 weeks of age. Lymphocyte proliferation, NK cell activity, SRBC-specific IgM production (PFC response), splenic B220+ cells, and thymic and splenic T-cell immunophenotypes were assessed at 3 and 8 weeks of age. Delayed type hypersensitivity (DTH) and autoantibodies to ds-DNA were assessed in 8-week old animals only. Proliferation and NK cell activity were not affected at either age. Decreased PFC responses were noted in male offspring at both ages and both TCE treatment levels. PFC responses in female offspring were suppressed by treatment with 14,000 ppb TCE at both ages assessed and at 1,400 ppb TCE at 8 weeks of age. Splenic numbers of B220 cells were only decreased in 3-week old pups exposed to 14,000 ppb TCE. The most pronounced alteration in T-cell subpopulations were increases in all thymic (CD4+, CD8+, CD4+/CD8+, and CD4-/CD8-) T-cell types in 8-week old animals. DTH was increased in females at both TCE levels and in males at the high dose only. These results indicate that TCE may be an effective developmental immunotoxicant and suggests that additional studies are required to determine the health risks associated with developmental exposure to TCE.

Immunological function in mice exposed to JP-8 jet fuel in utero.

Immunological parameters, host resistance, and thyroid hormones were evaluated in F1 mice exposed in utero to jet propulsion fuel-8 (JP-8). C57BL/6 pregnant dams (mated with C3H/HeJ males) were gavaged daily on gestation days 6-15 with JP-8 in a vehicle of olive oil at 0, 1000, or 2000 mg/kg. At weaning (3 weeks of age), no significant differences were observed in body, liver, spleen, or thymus weight, splenic and thymic cellularity, splenic CD4/CD8 lymphocyte subpopulations, or T-cell proliferation. Yet, lymphocytic proliferative responses to B-cell mitogens were suppressed in the 2000 mg/kg treatment group. In addition, thymic CD4-/CD8+ cells were significantly increased. By adulthood (8 weeks of age), lymphocyte proliferative responses and the alteration in thymic CD4-/CD8+ cells had returned to normal. However, splenic weight and thymic cellularity were altered, and the IgM plaque forming cell response was suppressed by 46% and 81% in the 1000 and 2000 mg/kg treatment groups, respectively. Furthermore, a 38% decrease was detected in the total T4 serum hormone level at 2000 mg/kg. In F1 adults, no significant alterations were observed in natural killer cell activity, T-cell lymphocyte proliferation, bone marrow cellularity and proliferative responses, complete blood counts, peritoneal and splenic cellularity, liver, kidney, or thymus weight, macrophage phagocytosis or nitric oxide production, splenic CD4/CD8 lymphocyte subpopulations, or total T3 serum hormone levels. Host resistance models in treated F1 adults demonstrated that immunological responses were normal after challenge with Listeria monocytogenes, but heightened susceptibility to B16F10 tumor challenge was seen at both treatment levels. This study demonstrates that prenatal exposure to JP-8 can target the developing murine fetus and result in impaired immune function and altered T4 levels in adulthood.