TLR Tolerance Reduces IFN-Alpha Production Despite Plasmacytoid Dendritic Cell Expansion and Anti-Nuclear Antibodies in NZB Bicongenic Mice

Genetic loci on New Zealand Black (NZB) chromosomes 1 and 13 play a significant role in the development of lupus-like autoimmune disease. We have previously shown that C57BL/6 (B6) congenic mice with homozygous NZB chromosome 1 (B6.NZBc1) or 13 (B6.NZBc13) intervals develop anti-nuclear antibodies and mild glomerulonephritis (GN), together with increased T and B cell activation. Here, we produced B6.NZBc1c13 bicongenic mice with both intervals, and demonstrate several novel phenotypes including: marked plasmacytoid and myeloid dendritic cell expansion, and elevated IgA production. Despite these changes, only minor increases in anti-nuclear antibody production were seen, and the severity of GN was reduced as compared to B6.NZBc1 mice. Although bicongenic mice had increased levels of baff and tnf-α mRNA in their spleens, the levels of IFN-α-induced gene expression were reduced. Splenocytes from bicongenic mice also demonstrated reduced secretion of IFN-α following TLR stimulation in vitro. This reduction was not due to inhibition by TNF-α and IL-10, or regulation by other cellular populations. Because pDC in bicongenic mice are chronically exposed to nuclear antigen-containing immune complexes in vivo, we examined whether repeated stimulation of mouse pDC with TLR ligands leads to impaired IFN-α production, a phenomenon termed TLR tolerance. Bone marrow pDC from both B6 and bicongenic mice demonstrated markedly inhibited secretion of IFN-α following repeated stimulation with a TLR9 ligand. Our findings suggest that the expansion of pDC and production of anti-nuclear antibodies need not be associated with increased IFN-α production and severe kidney disease, revealing additional complexity in the regulation of autoimmunity in systemic lupus erythematosus

照顧者實用資訊手冊

手冊源起 香港面對人口高齡化，醫療和福利系統無可避免面臨持續的挑戰。照顧者因壓力問題而引發的家庭悲劇時有發生，反映照顧者自身亦極需要社會的重視和支援。 現時照顧者人數眾多，當中不乏配偶、父母、子女、女婿、媳婦，對長者照顧及社會無酬的付出，確實值得社會的認同和肯定。然而，儘管本港為長者及照顧者提供不少支援服務，由於不同的原因，照顧者似乎未有善用現有的服務，常常需要一力承擔所有照顧的責任，這無疑增加了他們的壓力。 照顧者需求多樣性 照顧者有不同種類，所需要的服務或支援亦有所不同。例如：「新手照顧者」剛開始接觸照顧工作，需要盡快提升日常護理和相關的疾病知識，以及獲得合適的離院/復康服務支援。「隱蔽照顧者」一直承擔照顧工作但沒有接受任何支援，因此，他們需要的是服務提供者/鄰里的主動接觸，並被轉介合適的照顧服務，以分擔他們部分照顧工作。「高危照顧者」幾乎長時間被照顧的工作佔據了其生活，他們需要的是喘息空間和暫託服務，甚至可能是情緒支援。即使「資深的照顧者」也可能需要喘息或暫託的支援服務去減輕壓力；而結束了照顧工作的「畢業照顧者」，無論是情緒的支援，還是重新規劃財務與就業的安排，都有非常實際的需求。基於照顧者的不同需要，照顧者的支援和服務也是多樣化，如何讓不同種類的照顧者能夠輕易了解現有的社區支援服務、可考慮甚麼因素選取相關服務、如何獲取所需的支援包括申領經濟支援或預早準備長期護理開支、如何自強增值等，都是這本手冊希望解答的問題。 手冊的目的 編製這本手冊的目的就是要減少照顧者四處尋找資訊的麻煩，提供一個整全且易於瀏覽的資訊來源。本手冊主要有兩大目標：其一，幫助照顧者認識不同的服務和資源，積極尋求援助；其二是一站式提供豐富的資訊服務，提升照顧者的能力和平衡身心健康。 手冊的意義 此手冊的社會意義是期望提高大眾對照顧者需要的關注，並引導更多的行動以支援照顧者。如果您的親友是照顧者，又或您認識的朋友甚至鄰居正面對照顧家人的困難，您可以主動伸出援手，又或利用這本手冊作為啟示，向他們介紹相關的服務。 另外，這本手冊可以視作為支援照顧者的工具，讓照顧者明白在承擔照顧家人的重任時並不需要孤軍奮戰，也可以善用社區提供的各種資源和服務。 手冊架構 本手冊由四個主要部分組成，每部分都專注於特定的主題，旨在提供照顧者的全方位支援和資訊，幫助他們在面對挑戰時能夠有所依賴，並從中獲得力量： 第一部分主要提供基礎知識，其中包括不同種類的照顧者的介紹、四種長者常見疾病（包括認知障礙、中風、老年抑鬱和癌症）的病徵說明、以及照顧者可能面臨的壓力與「喘息服務」的重要性。第二部分專注介紹長者常用的社區及健康服務。我們按照地區將服務機構進行了分類，羅列「認知障礙、中風、老年抑鬱、癌症」常用的服務機構，此外，我們還提供家居安全、樂齡科技租賃服務，以及財務支援等相關建議。第三部分將焦點重新放回照顧者身上，提供了支援照顧者的社福機構介紹、照顧者同路人的心得分享，以及照顧者的培訓資訊，旨在幫助照顧者在面對困難時能自強不息。最後，在本手冊的結尾部分，我們特別邀請了四位專家學者就照顧者政策提出見解和建議，為加強照顧者支援服務提供新思路。 如果按持份者來分類，手冊的四個部分的重點如下： 第一部分：照顧者的基礎知識 ◎ 針對預備照顧者、新手以及隱蔽照顧者，讓他們了解照顧者的角色和需要；照顧體弱長者的基礎知識，並提供應對壓力的建議，包括自我照顧和喘息服務等。 第二部分：照顧者支援 ◎ 針對所有護老者，提供長者常用的社區服務、健康服務和支援計劃，包括暫託、住宿、護送及陪診、善別支援、情緒支援、復康用品津貼及租賃的支援計劃，以及財務策劃師提供的理財貼士。 第三部分：照顧者自強 ◎ 針對照顧者，資訊包括不同的照顧者支援組織、同路人和畢業照顧者的經驗心得、培訓課程，以及實用的電子資訊平台推介。 第四部分：對照顧者支援政策的看法和展望 ◎ 針對服務提供者、政策倡議者及制定者，提供專家學者對照顧者服務及政策的未來發展方向的看法和建議，以期引發更多關於「如何為照顧者提供更好支持」的討論。 給讀者的使用指引 本手冊內容十分豐富，讀者可以先瀏覽目錄，選擇與您相關的部分進行細讀。對於新手照顧者或剛接觸/關注護老服務的人士(例如：學生、社區人士)，我們建議您可以參考手冊第24-33頁「給照顧者的智慧錦囊」的個案，了解不同照顧者可能面對的情境，這可能會給您一點概念，讓您可以考慮尋求哪些支援服務，又或您身邊照顧者朋友有哪些需要，以及如何協助他們獲取所需要的支援。我們深知閱讀本手冊時，您可能會遇到一些不明白的地方，請不必感到困惑。您可以嘗試找身邊的朋友、鄰居或社會服務機構的工作人員一起閱讀，或者向本中心的職員進行查詢。 此外，為方便讀者可以隨時隨地閱讀而不需要攜帶整本手冊，我們為本手冊設立了一個專屬網頁，免費下載整本手冊或某些章節內容。 希望您在閱讀的過程中能找到有用的資訊，並能從中獲得實際的幫助。https://commons.ln.edu.hk/apias_guide/1009/thumbnail.jp

B Cell Activating Factor (BAFF) and T Cells Cooperate to Breach B Cell Tolerance in Lupus-Prone New Zealand Black (NZB) Mice

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help

Functional Dissection of Lupus Susceptibility Loci on the New Zealand Black Mouse Chromosome 1

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a strong and complex genetic basis. To dissect the function of the lupus susceptibility loci on New Zealand black (NZB) mouse chromosome 1, the lab had previously generated congenic mice with an introgressed homozygous NZB chromosome 1 intervals extending from ~35 or ~82 to 106 cM on the C57BL/6 background. Although both mouse strains made IgG anti-nuclear antibodies (ANAs), ANA titres and cellular activation were significantly higher in mice with the longer interval. These studies suggest the presence of two susceptibility genes. In this thesis I have sought to further characterize the cellular abnormalities and underlying genetic polymorphisms that produce them in these mice. Using mixed hematopoietic chimeric mice, with a mixture of tagged-B6 and congenic bone marrow I demonstrate that there are intrinsic B and T cell functional defects in chromosome 1 congenic mice. I further show that an intrinsic B cell defect is required for efficient recruitment of B cells into the spontaneous germinal centres and differentiation of autoantibody producing cells in these mice. To more precisely localize the susceptibility loci, I produced and characterized a number of additional subcongenic mouse strains. This revealed surprising genetic complexity with the presence of at least four lupus susceptibility loci and a suppressor locus on chromosome 1, several of which appeared to impact on T cell function. Finally, I generated bicongenic mice carrying both NZB chromosome 1 and 13 intervals, hypothesizing that since these were two of the major intervals associated with autoimmune disease in NZB mice they would fully recapitulate the autoimmune phenotypes. Although this hypothesis was incorrect, several novel phenotypes developed including marked expansion of the plasmacytoid and myeloid dendritic cell compartments and increased BAFF and IgA autoantibody production. Although this expansion was associated with TLR hyper-responsiveness, disease severity remained mild, possibly due to the lack of IFN- production, which appeared to be inhibited in these mice. Thus, lupus arises from immune defects affecting several cellular populations, which are the product of multiple genetic polymorphisms that interact in a complex fashion to produce the autoimmune phenotype.Ph

TLR tolerance impacts on IFN-α production, but not B7.2 upregulation, in BMDC from B6 and c1c13 bicongenic mice after repeated TLR9 stimulation.

<p>(A) Experimental design to investigate TLR tolerance of BMDC to TLR9. BMDC were cultured for 24 hours in the presence or absence of CpG 2216, washed and then stimulated again with CpG 2216. Supernatants were harvested at 24 (before washing) and 48 hours, and cellular profiles were assessed by flow cytometry after 48 hours. (B) IFN-α production by stimulated BMDC was measured by ELISA and (C) the mean fluorescence intensity (MFI) for B7.2 expression was quantified in various DC subsets (CD11c⁺ DC, CD11c⁺B220⁺CD11b⁻ pDC and CD11c⁺CD11b⁺B220⁻ mDC) using flow cytometry. Each symbol represents the determination from an individual female B6, B6.NZBc1 (c1), B6.NZBc13 (c13) or B6.NZBc1c13 (c1c13) mouse. Horizontal lines indicate the mean for each population examined. The <i>p</i> values for significant differences between various congenic mice and B6 control mice are shown, <i>*p<0.05</i>, **<i>p<0.005</i>.</p

Increased activation of cells in the immature and mature pDC subsets of older bicongenic mice.

<p>Expression of the activation markers (A) B7.2 and MHCII was quantified by mean fluorescent intensity (MFI) on splenic pDC gated as CD11c+ B220+ CD11b− in 8 week old (young; Yng) and 8 month old (Old) B6 and bicongenic mice. pDC populations were further characterized with additional surface markers to remove contaminating IgM+ CD19+ ABCs and to determine the maturity of pDC. Immature (CD9+ SiglecH−) and mature (SiglecH+ CD9−) pDC were gated on the CD11c+ B220+ CD11b− IgM− CD19− splenic pDC population and the data expressed as a proportion of total live splenocytes (B) and as a proportion of total pDC (C). (D) PDCA-1 was highly expressed on mature (SiglecH+ CD9−) but not immature (CD9+ SiglecH−) pDC subsets. Elevated expression of activation markers (E) B7.2 and (F) MHCII was seen in older bicongenic mice in both pDC subsets gated from the CD11c+ B220+ cell population. Representative histogram plots for older B6 (grey dotted line) and bicongenic mice (solid black line) for (G) B7.2 and (H) MHCII were gated on the CD11c+ B220+ CD11b− cells population excluding ABCs, in both the CD9+SiglecH− and SiglecH+CD9− populations. Results from this figure show representative data from one of three independent experiments. The p values for significant differences between B6 and bicongenic mice are shown, *p<0.05.</p

Reduced IFN-α, but not TNF-α and IL-10 production following stimulation of splenocytes with TLR ligands in bicongenic mice.

<p>Freshly isolated splenocytes from 8 week or 8 month old female B6 or B6.NZBc1c13 (c1c13) mice were stimulated with (A) CpG 2216 or (B) CpG 1826 for 48 h. Stimulation with CpG 2216 control or CpG 1826 control did not show differences between B6 and c1c13 mice (data not shown). (C) CD11c⁺ splenic DC from B6 and c1c13 mice were stimulated with CpG 2216 for 24 h. Levels of IFN-α, TNF-α and IL-10 in the culture supernatants were determined using ELISA. Each symbol represents the determination from an individual mouse. The <i>p</i> values for significant differences between B6 and B6.NZBc1c13 mice are shown above bars.</p

Comparison of the splenic phenotype in 8 month old B6.NZBc1c13 bicongenic mice with B6.NZBc1 and B6.NZBc13 congenic strains.

<p>Results are mean ± SD as determined by weight or flow cytometry. Significance level for comparison of B6.NZBc1c13 mice with other mouse strains was determined by Mann-Whitney non-parametric test,</p>*<p>p<0.05,</p>**<p>p<0.005,</p>***<p>p<0.0005. Numbers of 8 month old mice examined in each group are shown on the top unless otherwise indicated in brackets. Numbers shown in bold indicate significant difference p<0.05 from B6 control mice.</p

Cytokines or other cellular populations do not inhibit IFN-α production by CpG 2216 stimulated splenocytes from older c1c13 bicongenic mice.

<p>Freshly isolated splenocytes from (A) 8 week or (B) 8 month old female B6 or B6.NZBc1c13 (c1c13) mice were stimulated with CpG 2216 for 48 h in the presence or absence of anti-IL-10 and anti-TNF-α blocking antibodies. Levels of IFN-α in the culture supernatants were determined using ELISA. Each symbol represents the determination from an individual mouse. (C) Freshly isolated splenocytes from 8 to 12 week (young) and 8 month (old) B6 or c1c13 mice were stimulated alone or mixed at a 1∶1 ratio with CpG 2216 for 48 h. Levels of IFN-α production were determined by ELISA. The dotted lines represent the average IFN-α production by the young and old splenocytes when stimulated separately by CpG 2216. Shown in the figure are representative mice from two separate experiments.</p