TLR7 agonism accelerates disease in a mouse model of primary Sjögren’s syndrome and drives expansion of age-associated B cells

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by chronic inflammation of exocrine tissue, resulting in loss of tears and saliva. Patients also experience many extra-glandular disease manifestations. Treatment for pSS is palliative, and there are currently no treatments available that target disease etiology. Previous studies in our lab demonstrated that Myd88 is crucial for pSS pathogenesis in the NOD.B10Sn-H2b (NOD.B10) pSS mouse model, although the way in which Myd88-dependent pathways become activated in disease remains unknown. Based on its importance in other autoimmune diseases, we hypothesized that TLR7 activation accelerates pSS pathogenesis. We administered the TLR7 agonist Imiquimod (Imq) or sham treatment to pre-disease NOD.B10 females for 6 weeks. Parallel experiments were performed in age and sex-matched C57BL/10 controls. Imq-treated pSS animals exhibited cervical lymphadenopathy, splenomegaly, and expansion of TLR7-expressing B cells. Robust lymphocytic infiltration of exocrine tissues, kidney and lung was observed in pSS mice following treatment with Imq. TLR7 agonism also induced salivary hypofunction in pSS mice, which is a hallmark of disease. Anti-nuclear autoantibodies, including Ro (SSA) and La (SSB) were increased in pSS mice following Imq administration. Cervical lymph nodes from Imq-treated NOD.B10 animals demonstrated an increase in the percentage of activated/memory CD4+ T cells. Finally, aged-associated B cells (ABCs) were expanded in the spleens of Imq-treated pSS mice. Thus, activation of TLR7 accelerates local and systemic disease and promotes expansion of the ABC subset in pSS. C57BL/10 and NOD.B10H2b/J mice were treated with a sham cream (Con) or a cream containing 5% imiquimod (Imq) for 6 weeks beginning at 6 weeks of age. The cream was administered epicutaneously 3X/week by application to the ear. Animals were euthanized at 13 weeks of age and sera were harvested. Sera were harvested from BL/10 sham-treated ( = 6), BL/10 Imq-treated (n = 6), NOD.B10 sham-treated (n = 9) and NOD.B10 Imq-treated mice (n = 8). PBS was used as negative control for the array. Each gpr file includes the raw signal data for 16 arrays (samples). Independent samples are identified in the .gpr file with the "Block" column ID included on each sample page as a characteristic.

原发性干燥综合征（Primary Sjögren’s syndrome，pSS）是一种以外分泌腺慢性炎症为特征的全身性自身免疫性疾病，可导致泪液与唾液分泌缺失。患者还会出现多种腺体外疾病表现。目前pSS的治疗仅为姑息治疗，尚无针对疾病病因的有效治疗方案。本实验室此前的研究表明，髓样分化因子88（Myeloid differentiation primary response 88，Myd88）在NOD.B10Sn-H2b（简称NOD.B10）pSS小鼠模型的发病机制中发挥关键作用，但目前尚不清楚疾病中Myd88依赖通路的激活途径。鉴于其在其他自身免疫性疾病中的重要作用，我们推测Toll样受体7（Toll-like receptor 7，TLR7）的激活可加速pSS的发病进程。

我们对疾病前期的雌性NOD.B10小鼠给予TLR7激动剂咪喹莫特（Imiquimod，Imq）或安慰剂处理，持续6周；同时设置年龄与性别匹配的C57BL/10小鼠作为对照，开展平行实验。经Imq处理的pSS模型小鼠出现颈部淋巴结肿大、脾大，以及表达TLR7的B细胞扩增。经Imq处理的pSS小鼠的外分泌腺、肾脏与肺部可见显著的淋巴细胞浸润。TLR7激动剂还可诱导pSS小鼠出现唾液分泌功能减退，这正是该病的典型临床特征。经Imq给药后，pSS小鼠体内的抗核自身抗体（包括Ro（SSA）与La（SSB））水平显著升高。对Imq处理的NOD.B10小鼠的颈部淋巴结进行分析发现，活化/记忆性CD4+ T细胞的占比有所增加。最后，经Imq处理的pSS小鼠脾脏中，年龄相关性B细胞（age-associated B cells，ABCs）出现扩增。综上，TLR7的激活可加速局部与全身性疾病进程，并促进pSS中ABC亚群的扩增。

C57BL/10与NOD.B10H2b/J小鼠自6周龄起，分别接受安慰剂乳膏（对照组，Con）或含5%咪喹莫特的乳膏（Imq）处理，每周3次经皮涂抹于耳部，持续6周。所有小鼠于13周龄时实施安乐死并采集血清。血清样本分别来自BL/10安慰剂组（n=6）、BL/10 Imq处理组（n=6）、NOD.B10安慰剂组（n=9）以及NOD.B10 Imq处理组（n=8）。实验中以磷酸盐缓冲液（Phosphate Buffered Saline，PBS）作为芯片检测的阴性对照。每个.gpr文件包含16个阵列（样本）的原始信号数据。独立样本在.gpr文件中以"Block"列的ID标识，该ID会在每个样本页面中作为特征项列出。