Normalized linear counts from NanoString autoimmune profiling panel and summary of statistical analyses

Though dependent on genetic anomalies, clinical manifestations of the
human autoimmune disease systemic lupus erythematosus (lupus) can be
triggered by environmental exposures including inhalation toxicants such
as crystalline silica dust (cSiO2), tobacco smoke, and ambient air
particles.  Prednisone, a glucocorticoid (GC), is a keystone
therapy for managing lupus flaring and progression, however, long-term use
is associated with many adverse side effects. Here, we characterized the
dose-dependent immunomodulation and toxicity of prednisone in a
preclinical model that emulates onset and progression of cSiO2-triggered
lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control
AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15,
or 50 mg/kg diet which span human equivalent oral doses (HED) currently
considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high
(PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally
instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk.
The experimental plan was to 1) terminate one cohort of mice (n=8/group)
14 wk after the last cSiO2 instillation for pathology and autoimmunity
assessment and 2) to maintain a second cohort (n=9/group) to monitor
glomerulonephritis development and survival. Mean blood concentrations of
prednisone’s chief active metabolite, prednisolone, in mice fed PL, PM,
and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent
with levels observed in human blood ≤ 12 h after single bolus treatments
with equivalent prednisone doses. Results from the first cohort revealed
that consumption of PM but not PL diet significantly reduced cSiO2-induced
pulmonary ectopic lymphoid structure formation, nuclear-specific AAb
production, and inflammation/autoimmune gene expression in the lung,
splenomegaly, and glomerulonephritis in the kidney. Relative to
GC-associated toxicity, PM but not PL diet elicited muscle wasting, but
these diets did not affect bone density or cause glucosuria. Importantly,
neither PM nor PL diet influenced latency of cSiO2-accelerated death.
PH-fed mice in both cohorts displayed robust GC-associated toxicity
including body weight loss, reduced muscle mass, and hyperglycemia 7 wk
after the final cSiO2 instillation requiring their early removal from the
study. Taken together, our results demonstrate that while moderate doses
of prednisone can reduce certain pathological endpoints of cSiO2-induced
autoimmunity in lupus-prone mice, these ameliorative effects come with
unwanted GC toxicity and, crucially, none of these three doses extended
survival time.

尽管人类自身免疫性疾病系统性红斑狼疮（狼疮）的临床表现依赖于遗传异常，但其可由环境暴露触发，包括吸入性毒物如结晶二氧化硅粉尘（cSiO₂）、烟草烟雾及环境空气颗粒物。泼尼松作为一种糖皮质激素（GC），是控制狼疮发作和进展的核心疗法，但长期使用会带来多种不良副作用。在此，我们在模拟cSiO₂触发狼疮发生和进展的临床前模型中，表征了泼尼松的剂量依赖性免疫调节作用及毒性。将两组6周龄雌性NZBWF1小鼠分别饲喂对照AIN-93G饲料，或三种含泼尼松的AIN-93G饲料之一（剂量为5、15或50 mg/kg饲料），这些剂量分别对应目前认为的低（PL；5 mg/d HED）、中（PM；14 mg/d HED）、高（PH；46 mg/d HED）人类等效口服剂量（HED）。小鼠8周龄时，每周经鼻滴注生理盐水载体或1 mg cSiO₂，持续4周。实验计划为：1）在最后一次cSiO₂滴注后14周处死一组小鼠（每组n=8），进行病理学和自身免疫评估；2）维持第二组小鼠（每组n=9），监测肾小球肾炎的发展和生存情况。饲喂PL、PM和PH饲料的小鼠中，泼尼松主要活性代谢物泼尼松龙的平均血浓度分别为27、105、151 ng/ml，这与人类单次推注等效泼尼松剂量后≤12小时的血浓度水平一致。第一组的结果显示，饲喂PM饲料（而非PL饲料）显著减少了cSiO₂诱导的肺部异位淋巴结构形成、核特异性自身抗体（AAb）产生、肺中炎症/自身免疫基因表达、脾肿大及肾脏肾小球肾炎。关于糖皮质激素相关毒性，PM饲料（而非PL饲料）引发了肌肉萎缩，但这些饲料未影响骨密度或导致糖尿。重要的是，PM和PL饲料均未影响cSiO₂加速死亡的潜伏期。两组中饲喂PH饲料的小鼠在最后一次cSiO₂滴注后7周表现出显著的糖皮质激素相关毒性，包括体重减轻、肌肉量减少和高血糖，因此需提前退出研究。综上，我们的结果表明，尽管中等剂量泼尼松可减少狼疮易感小鼠中cSiO₂诱导自身免疫的某些病理终点，但这些改善效应伴随不必要的糖皮质激素毒性，且关键的是，这三种剂量均未延长生存时间。