2024年1月12日发(作者：)
BASIC RESEARCH PAPER
PTPN22 regulates NLRP3-mediated IL1B secretion in an autophagy-dependent manner
PTPN22通过一种自噬依赖性方式调节NLRP3介导的IL1B分泌
Marianne R. Spalingera, Silvia Langa, Claudia Gottiera, Xuezhi Daib, David J. Rawlingsb, Andrew C. Chanc, Gerhard Roglera,d, and Michael
Scharla,d
aDivision of Gastroenterology and Hepatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland; 瑞士 苏黎世大学附属医院 消化内科和肝病科
bDepartment of Pediatrics, University of Washington School of Medicine, and Seattle Children’s Research Institute, Seattle, WA, USA; 美国华盛顿州 西雅图儿童研究所，华盛顿大学医学院儿科，
cDepartment of Immunology, Department of Translational Immunology, and Department of Pathology, Genentech, Inc., South San Francisco, CA,
USA; 美国加利福尼亚州南旧金山Genentech公司免疫学系，转化免疫学系和病理学系
dZurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland苏黎世人类生理综合中心
ABSTRACT
A variant within the gene locus encoding PTPN22 (protein tyrosine phosphatase, non-receptor type 22) emerged as an
important risk factor for auto-inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus and type
1 diabetes, but at the same time protects from Crohn disease, one of the 2 main forms of inflammatory bowel diseases.
具有编码PTPN22（蛋白酪氨酸磷酸酶，非受体型22）基因位点的突变体是一种自体炎性疾病（包括类风湿性关节炎，系统性红斑狼疮和1型糖尿病）的重要危险因素，但同时又可以预防克罗恩病（炎症性肠病的两种主要形式之一）。
We have previously shown that loss of PTPN22 results in decreased NLRP3 (NLR family pyrin domain containing 3)
activation and that this effect is mediated via enhanced NLRP3 phosphorylation. However, it is unclear how phosphorylation
of NLRP3 mediates its inhibition.
我们之前研究已表明，缺乏PTPN22会抑制NLRP3（含NOD样受体家族pyrin结构域蛋白3）的活化，且该作用是通过增强NLRP3磷酸化介导的。然而，目前还不清楚NLRP3的磷酸化如何介导其抑制。
Here, we demonstrate that loss of macroautophagy/autophagy abrogates the inhibitory effect on NLRP3 activation observed
upon loss of PTPN22.
在此，我们证明了缺乏大自噬/自噬时会消除因缺乏PTPN22对NLRP3活化的抑制作用。
Phosphorylated, but not nonphosphorylated NLRP3 is found in autophagosomes, indicating that NLRP3 phosphorylation
mediates its inactivation via promoting sequestration into phagophores, the precursors to autophagosomes.
磷酸化的NLRP3（而非未磷酸化的NLRP3）存在于自噬体中，这表明NLRP3磷酸化通过促进其进入吞噬泡（自噬体的前体）介导其失活。
This finding shows that autophagy and NLRP3 inflammasome activation are connected, and that PTPN22 plays a key role in
the regulation of those 2 pathways.
这一发现表明自噬和NLRP3炎症小体的激活有关，且PTPN22在这两条途径的调控中起关键作用。
Given its role in inflammatory disorders, PTPN22 might be an attractive therapeutic target, and understanding the cellular
mechanisms modulated by PTPN22 is of crucial importance.
鉴于其在炎症性疾病中的作用，PTPN22可能是一种有吸引力的治疗靶点，并且对理解由PTPN22调节的细胞机制至关重要。
ARTICLE HISTORY
Received 18 July 2016 Revised 25 May 2017 Accepted 7 June 2017
KEYWORDS
inflammasome; Lyp; NLRP3; NOD-like receptor protein; PEST-enriched phosphatase; SQSTM1; tyrosine phosphorylation
Introduction
Inflammasomes are multiprotein aggregates that form upon intracellular presence of damage-associated molecular patterns
(DAMPs), and typically consist of 3 core components: (1) a receptor protein that initiates inflammasome assembly upon
activation, (2) the adaptor protein PYCARD/ASC (PYD and CARD domain containing; not present in some inflammasome-complexes), and (3) the protease CASP1/caspase-1.
炎症小体是一类在细胞内存在损伤相关分子模式（DAMP）时形成的多蛋白聚合物，并且通常由3个核心组分组成：（1）活化时启动炎性体装配的受体蛋白，（2）配体蛋白PYCARD/ASC（含有PYD和CARD结构域；一些炎性复合体中无此结构），（3）蛋白酶CASP1/caspase-1。
Once assembled, inflammasomes mediate the cleavage of pro-CASP1 into its active form.
一旦装配后，炎性小体将介导pro-CASP1裂解成其活性形式。
Active CASP1 in turn cleaves the precursors of several inflammatory molecules, including IL1B (interleukin 1 β) and IL18
(interleukin 18) into their active forms.1
活化后的CASP1依次将几种炎性分子的前体裂解，包括IL1B（白细胞介素1β）和IL18（白介素18），形成它们的活性形式。
One of the best studied inflammasome receptors is NLRP3 (NLR family pyrin domain containing 3), which responds to
several damage- and pathogen-associated molecules, including bacterial products, such as muramyl dipeptide (MDP)2 or
Listeria toxin,3 particulate materials (e.g., silicium dioxide, titanium dioxide, monosodium crystals)4,5 as well as changes in
intracellular potassium levels and increased levels of reactive oxygen species.6,7
研究最多的炎性小体的受体之一是NLRP3（含有NLR家族pyrin域蛋白3），可对几种损伤相关分子和病原体相关分子作出应答，其中对包括细菌产物如胞壁酰二肽（MDP）或李斯特氏菌毒素，颗粒物质（例如二氧化硅，二氧化钛，单钠晶体），以及改变细胞内钾水平和增加活性氧水平。
Due to the potent pro-inflammatory response upon IL1B and IL18 release, inflammasome-inducing receptors and subsequent
formation of inflammasome complexes is tightly regulated.
由于IL1B和IL18释放后会出现强烈的促炎反应，炎症小体诱导受体和随后形成的炎性体复合体受到严格调控。
Since NLRP3 responds to such a wide range of activators, control of NLRP3 activation is of special importance.
由于NLRP3可对多种活化剂做出应答，所以NLRP3活化的控制特别重要。
In addition to its restricted expression, and polyubiquitination that mediates NLRP3 degradation via the proteasome,8 we have
recently found that NLRP3 activation is negatively regulated by tyrosine phosphorylation.9
除了其限制性表达和通过蛋白酶体介导NLRP3降解的多聚泛素化外，我们最近还发现NLRP3活化受到酪氨酸磷酸化的负调控。
However the exact mechanism by which phosphorylation of NLRP3 interferes with its ability to form inflammasome-complexes is still unknown.
然而，NLRP3磷酸化干扰其形成炎性复合物的具体机制仍然是未知的。
The phosphatase responsible for dephosphorylation and subsequently robust activation of NLRP3 upon the presence of danger
molecules is PTPN22/PEP/Lyp (protein tyrosine phosphatase, nonreceptor type 22).9当存在危险分子时，负责去磷酸化并在随后激活NLRP3的磷酸酶是PTPN22 / PEP / Lyp（蛋白酪氨酸磷酸酶，非受体类型22）。
While loss of PTPN22 results in decreased NLRP3-mediated IL1B secretion, presence of a variant in the gene encoding
PTPN22 promotes inflammasome activity.9
尽管缺乏PTPN22会导致NLRP3介导的IL1B分泌减少，突变体在编码PTPN22的基因中促进炎性小体的活性。
This variant has been associated with increased risk to develop several chronic inflammatory disorders, including rheumatoid
arthritis (RA)10,11 systemic lupus erythematosus (SLE)12-15 and type I diabetes, but at the same time is negatively associated
with the onset of Crohn disease (CD), one subform of inflammatory bowel disease (IBD).16,17
这种突变体与一些慢性炎症疾病的风险升高相关，包括类风湿性关节炎（RA）系统性红斑狼疮（SLE）和I型糖尿病，但同时与克罗恩病（CD）（一种炎性肠病（IBD）的一种亚型）的发作呈负相关。
PTPN22 is not only involved in inflammasome-activation, but also exerts other prominent functions such as regulating the
response to type I interferons,18,19 and negative regulation of T cell receptor signaling cascades.20-22
PTPN22不仅参与炎症小体激活，还发挥其他突出功能，例如调节对I型干扰素的应答，和T细胞受体信号级联的
负调节。
We have further shown that PTPN22 expression is decreased in intestinal tissue of CD patients and interferes with MDP-induced autophagy.23,24
我们的进一步表明，CD患者的肠组织内PTPN22表达降低，并干扰了MDP诱导的自噬。
Autophagy is an important cellular process that mediates bulk degradation of damaged/dysfunctional proteins and organelles
in the cytosol.25,26
自噬是一个重要的细胞过程，可介导细胞质中受损/功能障碍蛋白质和细胞器的大量降解。
In addition, autophagy is critically involved in degradation and removal of invading bacteria27-29 making it a pivotal player in
intestinal homeostasis.27,30-32
此外，自噬主要参与降解和清除入侵细菌，使其成为肠道内稳态的关键参与者。
Genetic variants in molecules involved in the autophagy machinery, such as ATG16L1 (autophagy-related 16 like 1)33 and
IRGM (immunity-related GTPase M)17,34 increase the risk to develop IBD.
在与自噬机制有关的分子如ATG16L1（自噬相关的16如1）和IRGM（免疫相关GTP酶M）的遗传变异增加了发生IBD的风险。
Further, there is evidence that autophagy might be involved in the control of IL1B secretion.35-37
此外，有证据表明自噬可能参与了IL1B分泌的调控。
The aim of this study was to investigate whether PTPN22 interferes with cellular pathways influencing NLRP3 activation.
本研究的目的是探索PTPN22是否会干扰影响NLRP3活化的细胞通路。
Since PTPN22 controls autophagy, and autophagy controls IL1B secretion, the focus was to address whether PTPN22
mediates its control on NLRP3 activation in an autophagy-dependent manner.
由于PTPN22控制自噬和自噬控制IL1B分泌，所以重点在于解决PTPN22是否以自噬依赖性方式介导其对NLRP3活化的控制。
To confirm this finding, we next used nontargeting control siRNA or siRNA constructs specific for ATG16L1/Atg16l1, a
molecule essential for autophagy induction.
为了证实这一发现，我们接下来使用了非靶向性对照siRNA或特殊的ATG16L1/Atg16I1的siRNA构建体（ATG16L1/Atg16l1是自噬诱导所必需的分子）。
Again, inhibition of functional autophagy resulted in enhanced cleavage of CASP1 and enhanced IL1B secretion in both
PTPN22-competent and PTPN22-deficient cells (Fig. 2A, B and Fig. S2A, B, Fig. S3A).
此外，功能性自噬的抑制导致CASP1的裂解增强，且PTPN22-感受态细胞和PTPN22-缺陷型细胞中的IL1B分泌增加（图2A，B和图S2A，B，图S3A）。
Interestingly, the reduction in CASP1 cleavage and IL1B secretion observed upon loss of PTPN22 was no longer present
when autophagy was blocked (Fig. 2A, B, Fig. S2A, B, Fig. S3A).
有趣的是，当自噬被阻断时，在缺乏PTPN22时观察到CASP1裂解和IL1B分泌的减少的不再存在（图2A，B，图S2A，B，图S3A）。
Again, these effects were similar in cells treated with MSU (Fig. 2) or with MDP (Fig. S2, Fig. S3).
此外，用MSU（图2）或MDP（图S2，图S3）处理的细胞中存在类似的效应。
Similar findings were obtained when we silenced LC3B/Lc3b (Fig. 2C, D, Fig. S2C, D, Fig. S3B), although cells lacking
LC3B are not completely deficient in autophagy due to the presence of other LC3 isoforms.
虽然缺乏LC3B的细胞由于存在其他LC3同型体而不完全缺乏自噬，但我们沉默了LC3B/Lc3b后获得了类似的发现（图2C，D，图S2C，D，图S3B）。
However, silencing of LC3B/Lc3b clearly reduced autophagy as indicated by enhanced levels of SQSTM1/p62 (sequestosome
1), a molecule degraded via autophagy.
然而，沉默LC3B/Lc3b会显著减少自噬，是通过自噬降解的分子SQSTM1/p62（sequestosome 1）的增多体现出来的。
Taken together, these data strongly indicate a role for autophagy in the reduction of IL1B secretion and inflammasome
activation observed in PTPN22-deficient cells.
综上所述，这些数据表明一种自噬的作用——在PTPN22缺陷型细胞中可观察到的IL1B分泌和炎性体活化减少。
Figure 2. Knockdown of LC3B or ATG16L1 abrogates the reduction in IL1B secretion observed in PTPN22-deficient cells. BMDC from WT or ptpn22-/- mice were treated with nontargeting control siRNA or siRNA specific for
Atg16l1 (A and B) or Lc3b (C and D) 48 h before priming with ultra-pure lipopolysaccharide (upLPS) (16 h) and
subsequent activation with MSU (150 mg/ml) for 6 h. The graphs show (A and C): representative western blots from
cell lysates for the indicated proteins; and (B and D): IL1B ELISA and western blot analysis from cell culture
supernatants. Data are representative for 1 out of 3 independent experiments with 3 replicates (n=3). Numbers below
the western blot pictures show results of densitometric measurements. *=p<0.05, **=p< 0.01; one-way ANOVA with
Bonferroni correction.
图2. 敲除LC3B或ATG16L1消除了在PTPN22缺陷细胞中观察到的IL1B分泌的减少。 在用超纯脂多糖（upLPS）（16小时）引发之前48小时，将WT或ptpn22 - / - 小鼠的BMDC用非靶向对照siRNA或对Atg1611（A和B）或Lc3b（C和D） 用MSU（150mg / ml）活化6小时。 该图显示（A和C）：来自细胞裂解物的代表性蛋白质印迹，用于指示的蛋白质; 和（B和D）：来自细胞培养物上清液的IL1B ELISA和western印迹分析。 数据代表3次重复的3次独立实验中的1次（n = 3）。 低于蛋白质印迹图片的数字显示密度测量结果。
* = p <0.05，** = p <0.01; Bonferroni校正的单因素方差分析。
Results
Inhibition of autophagy results in increased IL1B secretion in both PTPN22 competent and PTPN22-deficient cells
抑制自噬后PTPN22感受态细胞和PTPN22缺陷型细胞中的IL1B分泌均增加
Because we have previously shown that loss of PTPN22 results in enhanced levels of autophagy,23 and autophagy has been
implicated in the regulation of IL1B secretion35-37 we first addressed whether autophagy plays a role in the reduction of IL1B
secretion observed upon loss of PTPN22.
因为我们先前已经证明PTPN22的缺失导致自噬水平增加，自噬与IL1B分泌的调节有关，我们首先确定了自噬是否参与了缺失PTPN22时抑制IL1B分泌作用。
Therefore, THP-1 cells expressing either control or PTPN22-targeting shRNA, were treated with the autophagy inhibitors 3-methyladenine (3-MA), bafilomycin A1, or the phosphatidylinositol 3-kinase inhibitor wortmannin before treatment with
MDP, a pathogen-associated molecular pattern (PAMP) that induces autophagy as well as the secretion of IL1B.
因此，在用MDP（一种病原体相关分子模式（PAMP），可诱导自噬以及IL1B的分泌）处理前，先用自噬抑制剂3-甲基腺嘌呤（3-MA），巴弗洛霉素A1或PI3K抑制剂渥曼青霉素处理表达对照或靶向PTPN22的shRNA的THP-1细胞。
As expected, loss of PTPN22 resulted in enhanced autophagy as observed by increased MAP1LC3B/LC3B (microtubule
associated protein 1 light chain 3 b)-II accumulation, but impaired secretion of IL1B (Fig. 1A, B). 正如预期的那样，缺乏PTPN22会增强自噬，如通过增加MAP1LC3B / LC3B（微管相关蛋白1轻链3b）-II积累，但会抑制IL1B分泌（图1A，B）。
Of note, loss of PTPN22 did not affect protein expression of pro-IL1B (Fig. 1A).
值得注意的是，缺乏PTPN22不影响pro-IL1B的蛋白质表达（图1A）。
Inhibition of autophagy using 3-MA, wortmannin, or bafilomycin A1 promoted secretion of IL1B in both PTPN22-competent
and PTPN22-deficient cells (Fig. 1B).
使用3-MA，渥曼青霉素或巴弗洛霉素A1抑制自噬可促进PTPN22感受态和PTPN22缺陷型细胞中IL1B的分泌（图1B）。
When autophagy was inhibited, PTPN22-deficient cells secreted as much IL1B as PTPN22-competent cells (Fig. 1B).
当自噬被抑制时，PTPN22缺陷细胞分泌的IL1B与PTPN22-感受态细胞一样多（图1B）。
The effects on IL1B secretion were due to differences in inflammasome activation: levels of cleaved CASP1 were decreased
in PTPN22-deficient cells, but enhanced in cells where autophagy was inhibited (Fig. 1A).
对IL1B分泌的影响归因于炎性体活化的差异：在PTPN22缺陷细胞中切割的CASP1水平降低，但在自噬被抑制
的细胞中水平增加（图1A）。
Similar results were obtained in bone marrow-derived dendritic cells (BMDC) from either wild-type (WT) or Ptpn22-deficient
(ptpn22-/-) mice (Fig. 1C, D), and when monosodium urate (MSU) or ATP were used as inflammasome activators (Fig. S1):
whereas PTPN22-deficient BMDC secreted reduced levels of IL1B compared with WT cells, this effect was abrogated upon
inhibition of autophagy using 3-MA, wortmannin or bafilomycin A1 (Fig. 1C, D, Fig. S1).
在来自野生型（WT）或Ptpn22缺陷型（ptpn22-/-）小鼠（图1C，D）的骨髓衍生树突细胞（BMDC）中获得类似的结果，并且当尿酸一钠（MSU）或ATP 被用作炎性小体激活剂（图S1）：与PTPN22缺陷的BMDC相比，WT细胞分泌的IL1B水平降低，这种作用在使用3-MA，渥曼青霉素或巴弗洛霉素A1抑制自噬后被消除（图1C，D， 图S1）
Figure 1. Reduction of IL1B secretion upon loss of PTPN22 is not observed in autophagy-deficient cells. PMA-differentiated THP-1 cells expressing either control, or PTPN22-specific shRNA (A and B), and BMDC from wild-type
(WT) or PTPN22 deficient (ptpn22-/-) mice (C and D) were treated with 3-MA (1 mM), (Wortm; 10 uM), or bafilomycin
A1 (Bafilo; 100 nM) to inhibit autophagy, 1 h before treatment with muramyl dipeptide (MDP, 100 ng/ml) for 24 h. The
graphs show (A)and (C): representative western blot pictures of the indicated proteins and (B and D): results from IL1B
ELISA. Data are representative for 1 out of 3 independent experiments with 3 replicates (n = 3). Numbers below the
western blot pictures show results of densitometric measurements. *= p < 0.05, n.s. = not significant, one-way ANOVA
with Bonferroni correction.
图1.在自噬缺陷细胞中缺失PTPN22后IL1B分泌不会减少。 将表达对照或PTPN22特异性shRNA（A和B）和来自野生型（WT）或PTPN22缺陷型（ptpn22 +/-）小鼠（C和D）的BMDC的PMA-分化的THP-1细胞用在用胞壁酰二肽（MDP，100ng / ml）处理24小时之前1小时，以抑制自噬作用的3-MA（1mM），（Wortm;10μM）或巴弗洛霉素A1（Bafilo; 100nM） 该图显示（A）和（C）：所示蛋白质的代表性WB图片，（B和D）：来自IL1B ELISA结果。 数据代表3次重复的3次独立实验中的1次（n = 3）。WB图片下边的数字表示密度测量结果。 * = p <0.05，n.s. =不显着，用Bonferroni校正的单因素方差分析。
NLRP3 is found in autophagosomes upon inflammasome activation
炎症小体活化后可在自噬体中发现NLRP3
We have previously shown that loss of PTPN22 results in enhanced phosphorylation of NLRP3, and that phosphorylation of
NLRP3 results in decreased inflammasome activity.
我们先前已经表明，缺乏PTPN22会导致NLRP3的磷酸化增强，而NLRP3的磷酸化会抑制炎性小体活性。
To test how autophagy might affect phosphorylated NLRP3, we analyzed whether NLRP3 is present in autophagosomes, and
whether this is dependent on its phosphorylation status.
为了检测自噬如何影响磷酸化的NLRP3，我们分析了NLRP3是否存在于自噬体中，以及它是否依赖于其磷酸化状态。
To this end, we enriched autophagosomes37,38 from MDP- or MSU-treated THP-1 cells or BMDC and probed for NLRP3.
为此，我们从MDP或MSU处理的THP-1细胞或BMDC细胞中富集自噬体，并检测NLRP3。
While in untreated cells only a faint NLRP3 band was detectable in the autophagosome-enriched fraction, NLRP3 was readily
detectable in autophagosomes of MDP- or MSU-treated cells (Fig. 3A, Fig. S4A, B).
虽然在未经处理的细胞中，经自噬体富集后仅检测到微弱的NLRP3条带，但在MDP或MSU处理的细胞的自噬体中很容易检测到NLRP3（图3A，图S4A，B）。
Of note, in cells lacking PTPN22, more NLRP3 was present in the autophagosome-enriched fraction upon NLRP3 activation
(Fig. 3A).
值得注意的是，在缺乏PTPN22的细胞中，当NLRP3活化时，在自噬体富集部分中存在更多的NLRP3（图3A）。
This was clearly an effect resulting from the lack of PTPN22 phosphatase function, because the same effect was observed in
cells that express a loss-of-function variant in PTPN2213 (Fig. S4B).
这显然是由于缺乏PTPN22磷酸酶功能导致的效应，因为在PTPN2213中表达功能丧失变体的细胞中观察到相同的效应（图S4B）。
We also detected PYCARD/ASC, but not CASP1 or IL1B in autophagosomes upon inflammasome activation (Fig. 3A, Fig.
S4B).
我们还检测了PYCARD / ASC，炎症小体活化时自噬体中的无CASP1或IL1B（图3A，图S4B）。
Localization of NLRP3 in autophagosomes was confirmed by confocal microscopy of cells costained for NLRP3 and LC3B.
NLRP3在自噬体中的定位通过NLRP3和LC3B所共有的细胞的共聚焦显微镜确认。
While in cells without inflammasome activation, NLRP3 was found all over the cytosol, activation of NLRP3 with MSU
resulted in NLRP3 aggregation.
而在没有炎性体活化的细胞中，细胞液发现很多NLRP3，用MSU激活NLRP3导致NLRP3聚集。
Some of these NLRP3 aggregates colocalized with LC3B puncta, indicating that they were recruited to phagophores (Fig. 3B;
left panel).
其中一些NLRP3聚集体与LC3B点共定位，表明它们被募集到吞噬泡（图3B;左图）。
NLRP3 was not only recruited into phagophores, but was also found in lysosomes upon NLRP3 activation, indicating that it
is degraded in autolysosomes (Fig. 3B; right panel).
NLRP3不仅被募集到吞噬细胞中，而且在NLRP3活化后也在溶酶体中被发现，表明它在自体溶酶体中被降解（图3B;右图）。
In ptpn22-/- cells, or cells expressing the loss-of-function Ptpn22 variant, more LC3B puncta were observed in nontreated
cells, and, upon activation with MSU, more NLRP3 aggregates colocalized with LC3B puncta.
在ptpn22-/-细胞或表达功能丧失的Ptpn22变体的细胞中，在未处理的细胞中观察到更多的LC3B点，并且在用MSU活化后，更多的NLRP3聚集体与LC3B点共定位。
Conversely, cells expressing an altered function Ptpn22 variant, which results in a gain of function in terms of NLRP3
regulation,9 NLRP3 was not present in autophagosomes (Fig. 3C).
相反，表达相反功能的Ptpn22变体的细胞，其导致NLRP3调节功能的增益， NLRP3不存在于自噬体中（图3C）。
Figure 3. Phosphorylated NLRP3 is found in autophagosomes. (A) BMDC were primed for 16 h with upLPS to
induce induction of NLRP3 and IL1B expression, before treatment with MSU for 6 h. The pictures show representative
western blots from the indicated proteins in cell lysates and autophagosome-enriched fractions as indicated. (B)
Confocal microscopy of THP-1 cells treated with LPS, or LPS and MSU, immunostained for NLRP3 (green) and LC3B
(red; left side of the figure) or immunostained for NLRP3 (green) and the lysosome marker LAMP1 (red; right side of
the figure); below and beside the image: z-stack at position of the arrows. scale bars: 10 mm. P-Tyrstands for phospho-tyrosine. (C) BMDC from WT mice, ptpn22-/- mice or mice expressing a gain-of-function variant in Ptpn22 (619W)
were treated as in (A) and immunostained for NLRP3 (green) and LC3B (red). (D) BMDC from WT or ptpn22-/- mice
were treated as in (A) and NLRP3 precipitated from either cell lysates or autophagosome-enriched fractions. The
pictures show representative western blots for phospho-tyrosine (p-Tyr) and NLRP3 run in conventional SDS-PAGE.
(E) control-transfected WT BMDC, control-transfected ptpn22-/- BMDC, or ptpn22-/- BMDC transfected with a vector
expressing a loss-of-function variant in ptpn22 (263Q) were treated as in (A) and lysates run on Phos-tag gels, which
allow the separation of phosphorylated from nonphosphorylated proteins due to slower migration of the phosphorylated
forms. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n=3), except for confocal
microscopy, where the experiment has been performed only twice with 3 replicates. Images are representative for at
least 5 scanned areas for each depicted condition. Scale bars: 10 mm. P-Tyr stands for phospho-tyrosine. Numbers below
the western blot pictures show results of densitometric measurements.
图3. 磷酸化的NLRP3在自噬体中发现。 （A）在用MSU处理6小时之前，用upLPS将BMDC引发16小时以诱导NLRP3和IL1B表达的诱导。如图所示，图片显示来自细胞裂解物和自噬体富集级分中所示蛋白质的代表性蛋白质印迹。 （B）用LPS或LPS和MSU处理的THP-1细胞的共聚焦显微镜检查免疫染色NLRP3（绿色）和LC3B（红色;图左侧）或免疫染色NLRP3（绿色）和溶酶体标记物LAMP1（红色;图的右侧）;在图像的下方和旁边：在箭头位置的z堆叠。比例尺棒：10毫米。 P-Tyrstands用于磷酸酪氨酸。 （C）来自WT小鼠的BMDC，ptpn22-/-小鼠或在Ptpn22中表达功能获得性变异体的小鼠（619W）如（A）中处理并免疫染色NLRP3（绿色）和LC3B（红色）。 （D）如（A）处理来自WT或ptpn22 - / - 小鼠的BMDC，并从细胞裂解物或自噬体富集级分中沉淀NLRP3。图片显示代表性蛋白质印迹在常规SDS-PAGE中运行的磷酸酪氨酸（p-
Tyr）和NLRP3。 （A）处理（E）对照转染的WT BMDC，对照转染的ptpn22 - / - BMDC或用表达ptpn22中功能丧失变体的载体转染的ptpn22 - / - BMDC（263Q）运行于Phos-标签凝胶上，由于磷酸化形式的迁移较慢，所述凝胶允许从非磷酸化蛋白质中分离磷酸化蛋白质。数据代表3个独立实验中的1个，每个3个重复（n =
3），除了共焦显微镜，其中实验仅进行两次3次重复。对于每个描述的条件，图像代表至少5个扫描区域。比例尺：10毫米。 P-Tyr代表磷酸酪氨酸。低于蛋白质印迹图片的数字显示密度测量结果。
Only phosphorylated NLRP3 is detected in autophagosomes
在自噬体中只检测到磷酸化的NLRP3
We next precipitated NLRP3 from autophagosome-enriched fractions or whole cell lysates and probed for phospho-tyrosine
(p-Tyr).
我们接下来从富含自噬体的部分或全细胞裂解物中沉淀NLRP3并检测磷酸酪氨酸（p-Tyr）。
Interestingly, in autophagosomes, p-Tyr was increased upon MSU-treatment, whereas in whole cell lysates it was decreased
(Fig. 3D).
有趣的是，在自噬体中，MSU处理后p-Tyr增加，而全细胞裂解物中p-Tyr降低（图3D）。
In ptpn22-/- cells, we found no decrease in NLRP3 tyrosine phosphorylation upon inflammasome activation, and clearly more
NLRP3 was present in autophagosomes (Fig. 3A).
在ptpn22-/-细胞中，我们发现炎性小体激活后NLRP3酪氨酸磷酸化没有减少，并且自噬体中存在更多的NLRP3（图3A）。
Since similar findings were observed in cells expressing a loss-of-function PTPN22 variant (Fig. S4C), this effect seems to
be due to a loss of PTPN22 function, rather than absence of the protein per se.
由于在表达功能丧失PTPN22变体的细胞中观察到类似的发现（图S4C），这种效应似乎是由于PTPN22功能丧失所致，而不是由于缺乏蛋白本身。
This finding strongly indicates that upon MDP- or MSU-treatment, phosphorylated NLRP3 is sequestered into phagophores.
这一发现强烈表明，在MDP或MSU处理后，磷酸化的NLRP3被隔离进入吞噬泡。
To address whether nonphosphorylated NLRP3 is also present in autophagosomes, we next ran whole cell lysate and
autophagosome-enriched fractions on a Phos-tag gel (Fig. 3E).
为了解决非磷酸化NLRP3是否也存在于自噬体中，我们接下来在Phos-标签凝胶上运行全细胞裂解物和富集自噬体的级分（图3E）。
On these gels, phosphorylated proteins migrate at ab slower speed than their nonphosphorylated forms.39 In the lysate of
nontreated cells, NLRP3 appeared as a double band with approximately 50% appearing in the upper (phosphorylated) and
50% in the lower (nonphosphorylated) band.
This is consistent with our previous findings that roughly half of NLRP3 is phosphorylated in nonactivated cells.9 In lysates,
phosphorylation was decreased upon MDP treatment, where only a faint upper (phosphorylated) band was detectable. Of note,
in ptpn22-/- cells or cells expressing a loss-of-function variant in Ptpn22 (263Q), MSU treatment did not decrease
phosphorylation of NLRP3.
Furthermore, in autophagosomes, the lower, nonphosphorylated band was not visible (Fig. 3E), indicating that only
phosphorylated NLRP3 was sequestered into phagophores. 此外，在自噬体中，较低的非磷酸化条带不可见（图3E），表明只有磷酸化的NLRP3被隔离进入吞噬泡。
NLRP3 lacking the phosphorylation site is not recruited to phagophores
缺乏磷酸化位点的NLRP3不被募集到吞噬泡
To further test the hypothesis that phosphorylation of NLRP3 is required for its recruitment into phagophores, we transfected
BMDC from NLRP3-deficient mice with either wild-type NLRP3 or a mutated form of NLRP3 that lacks the phosphorylation
site at tyrosine 859 (Y859F NLRP3). When analyzing autophagosome-enriched fractions for the presence of NLRP3, we only
found NLRP3 in autophagosomes from BMDC transfected with WT NLRP3, but not in cells transfected with NLRP3Y859F,
which lacks the phosphorylation site (Fig. 4A, Fig. S5A). In cells transfected with NLRP3Y859F, no colocalization of LC3B
puncta with NLRP3 aggregates was observed (Fig. 4B). This further indicates that phosphorylated NLRP3— but not
nonphosphorylated NLRP3—is sequestered to the phagophore upon inflammasome induction. Of interest, presence of the
NLRP3Y859F construct resulted in increased CASP1 cleavage and enhanced IL1B secretion upon MSU and MDP treatment
(Fig. 4C, Fig. S5). This observation indicates that phosphorylation of NLRP3 and subsequent degradation via autophagy is a
regulatory mechanism of NLRP3 activation.
In summary, these data indicate that NLRP3 phosphorylation results in sequestering of NLRP3 into phagophores and that this
might be the mechanism for how phosphorylation of NLRP3 reduces its activation.
总之，这些数据表明NLRP3磷酸化导致NLRP3被隔离到吞噬泡中，这可能是NLRP3的磷酸化如何降低其活化的机制。
Figure 4. NLRP3 lacking the phosphorylation site is not recruited to phagophores. BMDC from nlrp3-/- mice were
transfected with a wild-type (WT) Nlrp3 expression vector, or a Nlrp3 construct where Tyr859 in NLRP3 is replaced
with a phenylalanine (Y859F; Y>F) to abolish NLRP3 phosphorylation. The cells were primed for 16 h with upLPS to
induce NLRP3 and IL1B expression, before activation with MSU (150 mg/ml) for 6 h. Shown is (A) representative
western blot pictures from cell lysates and autophagosome-enriched fractions, (B) confocal microscopy of LC3B (red)
and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI, scale bar: 10 mm; and (C) IL1B in the cell
culture supernatant. Data are representative for 1 out of 3 independent experiments with 3 replicates each (n=3), except
for confocal microscopy where the experiment has been performed only twice with 3 replicates. Images are
representative for at least 5 scanned areas for each depicted condition. Numbers below the western blot pictures show
results of densitometric measurements. *=p<0.05, one-way ANOVA with Bonferroni correction.
图4.缺乏磷酸化位点的NLRP3不被募集到吞噬细胞。用野生型（WT）Nlrp3表达载体或Nlrp3构建体转染来自nlrp3 - / - 小鼠的BMDC，其中NLRP3中的Tyr859被苯丙氨酸（Y859F; Y> F）取代以消除NLRP3磷酸化。在用MSU（150mg / ml）活化6小时之前，用upLPS将细胞引发16小时以诱导NLRP3和IL1B表达。所示为（A）来自细胞裂解物和自噬体富集级分的代表性蛋白质印迹图片，（B）LC3B（红色）和NLRP3（绿色）免疫染色细胞的共聚焦显微镜;蓝色：DNA用DAPI染色，比例尺：10mm;和（C）细胞培养上清液中的IL1B。数据代表3个独立实验中的1个，每个3个重复（n = 3），除了共焦显微镜，其中实验仅进行两次3次重复。对于每个描述的条件，图像代表至少5个扫描区域。低于蛋白质印迹图片的数字显示密度测量结果。 * = p
<0.05，具有Bonferroni校正的单向ANOVA。
Phosphorylated NLRP3 interacts with SQSTM1 upon inflammasome activation
炎性体激活后，磷酸化的NLRP3可与SQSTM1互作
To understand how phosphorylated NLRP3 is recruited into phagophores we addressed whether NLRP3 interacts with
molecules involved in targeting substrates to the phagophore.
为了理解磷酸化的NLRP3如何募集到吞噬泡中，我们研究了NLRP3是否与将底物靶向吞噬泡的分子互作。
Indeed, we found that NLRP3 interacts with SQSTM1 upon inflammasome activation (Fig. 5A).
事实上，我们发现NLRP3在炎症小体活化后与SQSTM1互作（图5A）。
Of note, this interaction was strictly dependent on phosphorylation of NLRP3, because in cells lacking the phosphorylation
site no interaction between SQSTM1 and NLRP3 was observed.
值得注意的是，这种相互作用严格依赖于NLRP3的磷酸化，因为在缺乏磷酸化位点的细胞中没有观察到SQSTM1和NLRP3之间的相互作用。
On the other hand, a phospho-mimetic variant of NLRP3 (NLRP3Y859E; reported in Ref. 9) resulted in increased interaction
between SQSTM1 and NLRP3 (Fig. 5A).
另一方面，NLRP3的磷酸化拟似变体（NLRP3Y859E;报告于参考文献9）导致SQSTM1和NLRP3之间的相互作用增加（图5A）。
Further, loss of PTPN22 resulted in increased interaction of SQSTM1 and NLRP3 (Fig. 5B).
此外，缺乏PTPN22导致SQSTM1和NLRP3的相互作用增加（图5B）。
Figure 5. NLRP3 interacts with SQSTM1 upon its activation. (A) BMDC from nlrp3-/- mice were transfected with
a wild-type (WT) Nlrp3 expression vector, a Nlrp3 construct where Tyr859 in NLRP3 is replaced with a phenylalanine
(Y859F; Y>F) to abolish NLRP3 phosphorylation, or with a phsopho-mimetic NLRP3, where Tyr859 was replaced with
a glutamate (Y859E; Y>E). The cells were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before
activation with MSU (150 mg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and probed for the
indicated proteins. (B) BMDC from WT or ptpn22-/- cells were primed for 16 h with upLPS to induce NLRP3 and IL1B
expression, before activation with MSU (150 mg/ml) for 6 h. NLRP3 or SQSTM1 were precipitated from the lysate and
probed for the indicated proteins. Data is representative for 1 out of 3 independent experiments with 3 replicates each
(n=3). Numbers below the western blot pictures show results of densitometric measurements.
图5. NLRP3活化后可与SQSTM1互作。 （A）用野生型（WT）Nlrp3表达载体，Nlrp3构建体转染来自nlrp3-/-小鼠的BMDC，其中NLRP3中的Tyr859被替换为苯丙氨酸（Y859F; Y> F）以消除NLRP3磷酸化，或与
phsopho-模拟NLRP3，其中Tyr859被谷氨酸替代（Y859E; Y> E）。 在用MSU（150mg / ml）活化6小时之前，用upLPS将细胞引发16小时以诱导NLRP3和IL1B表达。 从裂解物中沉淀NLRP3或SQSTM1并探测指定的蛋白质。 （B）在用MSU（150mg / ml）活化6小时之前，来自WT或ptpn22 - / - 细胞的BMDC用upLPS引发16h以诱导NLRP3和IL1B表达。 从裂解物中沉淀NLRP3或SQSTM1并探测指定的蛋白质。 数据代表3次独立实验中的1次，每次3次重复（n = 3）。 低于蛋白质印迹图片的数字显示密度测量结果。
NLRP3 does not interact with SQSTM1 in PYCARD-deficient cells
NLRP3不与PYCARD缺陷型细胞中的SQSTM1互作
So far we showed that phosphorylated NLRP3 interacts with SQSTM1; however, this is only the case when NLRP3 is
activated.
到目前为止，我们结果表明磷酸化的NLRP3与SQSTM1相互作用;然而，只有当NLRP3被激活时才存在这种情况。
Because a portion of NLRP3 is phosphorylated in the steady-state, we wondered why phosphorylated NLRP3 is not recruited
to phagophores in resting cells.
因为NLRP3的一部分在稳定状态下被磷酸化，所以我们想知道为什么磷酸化的NLRP3不被募集到静止细胞中的吞噬泡。
Because NLRP3 activation results in its interaction with PYCARD, and we also found PYCARD in autophagosomes, we next
addressed whether the interaction of NLRP3 with SQSTM1 is PYCARD dependent.
因为NLRP3活化导致其与PYCARD互作，且我们还在自噬体中发现了PYCARD，我们接下来讨论NLRP3与SQSTM1的互作是否依赖性于PYCARD。
When we treated PYCARD-deficient BMDC with MSU, we did not detect NLRP3 in autophagosomes (Fig. 6A, B). 当我们用MSU处理PYCARD缺陷型BMDC细胞时，我们没有在自噬体中检测到NLRP3（图6A，B）。
Immunoprecipitation of NLRP3 revealed that NLRP3 was phosphorylated in pycard-/- cells, but it did not interact with
SQSTM1 (Fig. 6C).
NLRP3的免疫共沉淀显示NLRP3在pycard-/-细胞中被磷酸化，但它不与SQSTM1相互作用（图6C）。
This result indicates that the interaction of NLRP3 with SQSTM1 was dependent on NLRP3-PYCARD interaction.
这个结果表明NLRP3与SQSTM1的互作依赖于NLRP3-PYCARD的互作。
In sum, our results indicate that phosphorylated NLRP3 binds SQSTM1 and is subsequently recruited into phagophores, once
it interacts with PYCARD.
总之，我们的结果表明，磷酸化的NLRP3与PYCARD互作后，才能与SQSTM1结合，随后被募集到吞噬泡中，。
Upon NLRP3 activation, PTPN22 dephosphorylates NLRP3 and thereby protects it from degradation, allowing robust
inflammasome activity (summarized in Fig. S6).
在NLRP3激活后，PTPN22 使NLRP3去磷酸化，从而保护其免受降解，从而产生强大的炎性体活性（总结在图S6中）。
Figure 6. PYCARD is required for recruitment of NLRP3 into phagophores. BMDC from WT or pycard-/- cells
were primed for 16 h with upLPS to induce NLRP3 and IL1B expression, before activation with MSU (150 mg/ml) for
6 h. (A) confocal microscopy of LC3B (red) and NLRP3 (green) immunostained cells; blue: DNA stained with DAPI,
scale bar: 10 mm. (B) Western blots from autophagosome-enriched fractions and lysates were probed for the indicated
proteins. (C) NLRP3 or SQSTM1 were precipitated from the lysate or from autophagosome-enriched fractions and
probed for the indicated proteins. P-Tyr stands for phospho-tyrosine. Data are representative for 1 out of 3 independent
experiments with 3 replicates each (n=3), except for confocal microscopy where the experiment has been performed
only once with 3 replicates. Images are representative for at least 5 scanned areas for each depicted condition. Numbers
below the western blot pictures show results of densitometric measurements.
图6. PYCARD是将NLRP3募集到吞噬细胞中所必需的。 来自WT或pycard - / - 细胞的BMDC在用MSU（150mg / ml）活化6小时之前用upLPS引发16h以诱导NLRP3和IL1B表达。 （A）LC3B（红色）和NLRP3（绿色）免疫染色细胞的共聚焦显微术; 蓝色：DNA用DAPI染色，比例尺：10mm。 （B）对来自自噬体富集级分和裂解物的蛋白质印迹进行所述蛋白质的探测。 （C）NLRP3或SQSTM1从裂解物或富含自噬体的级分中沉淀出来并探测指定的蛋白质。 P-Tyr代表磷酸酪氨酸。 数据代表3个独立实验中的1个，每个3个重复（n = 3），除了共焦显微镜，其中实验仅进行一次3次重复。 对于每个描述的条件，图像代表至少5个扫描区域。 低于蛋白质印迹图片的数字显示密度测量结果。
Discussion
We have previously demonstrated that loss of PTPN22 reduces IL1B secretion via enhanced phosphorylation of the
inflammasome receptor NLRP3.9 Here, we demonstrate that loss of functional autophagy abrogates the reduction of NLRP3
activation observed in PTPN22-deficient cells. This effect is explained by our observation that phosphorylation of NLRP3
mediates recruitment of NLRP3 into phagophores, which seems to be responsible for reduced NLRP3 activity. Our data
clearly show that in autophagy-deficient cells phosphorylated NLRP3 is still able to efficiently form inflammasome complexes,
most likely due to the lack of sequestration into phagophores. In turn, dephosphorylation of NLRP3—as observed upon robust
inflammasome induction—protects NLRP3 from being sequestered into phagophores, and allows full NLRP3 activation.
我们以前已经证实，缺乏PTPN22通过增强炎性小体受体NLRP3的磷酸化来降低IL1B的分泌。在这里，我们证明功能性自噬的丧失会消除在PTPN22缺陷细胞中观察到的NLRP3激活的减少。 我们的观察解释了这种效应，即NLRP3的磷酸化介导将NLRP3募集到吞噬泡中，这似乎负责降低NLRP3活性。 我们的数据清楚地表明，在自噬缺陷细胞中，磷酸化的NLRP3仍然能够有效地形成炎性复合体，最可能是由于缺乏对吞噬泡的隔绝作用。 反过来，NLRP3的去磷酸化（如在强烈炎性体诱导下观察到的）保护NLRP3免于被隔离进入吞噬泡，并且允许NLRP3完全活化。
Autophagy and inflammasome activation both play a crucial role in the development of several inflammatory disorders,
including neurodegenerative diseases, cancer, rheumatoid arthritis, multiple sclerosis, IBD, and many more (reviewed in refs.
40-42); hence, understanding the mechanisms for how these pathways influence each other is of great relevance for
understanding underlying pathomechanisms, and ultimately for the development of novel therapeutic strategies. In the
intestines, autophagy plays a crucial role for tissue homeostasis and defense against invading bacteria.27,30 This role is
highlighted by the fact that genetic variants resulting in defects in the autophagy machinery enhance the risk to develop
IBD.17,33,43 Although rather protective in many cells and diseases, in some disorders autophagy might also drive pathologies:
Enhanced autophagy promotes survival of inflammatory synovial fibroblasts in rheumatoid arthritis,44 and protects cancer
cells from cell death.45 For the NLRP3 inflammasome the situation is a little different: in classical inflammatory disorders,
such as arthritis, diabetes, or SLE, an increase of NLRP3 activation clearly promotes disease onset/progression.46-49 In the
intestine, however, the role of NLRP3 is still a matter of dispute, with some reports showing protective effects upon loss of
NLRP3, whereas others demonstrate that NLRP3-induced inflammasome activation is important for promoting wound
healing and tissue repair.50-52
自噬和炎性体激活在几种炎性疾病的发展中起着至关重要的作用，包括神经退行性疾病，癌症，类风湿性关节炎，多发性硬化症，IBD等等（参见参考文献40-42）。因此，理解这些途径如何相互影响的机制对于理解潜在的病理机制以及最终开发新的治疗策略具有重要意义。在肠道中，自噬对组织平衡和防御入侵细菌起着至关重要的作用。这一作用突出表明，遗传变异导致自噬机制的缺陷增加了发展IBD的风险.尽管在许多细胞和疾病中具有相当的保护作用，但在某些疾病中，自噬也可能会引发病理：增强自噬促进类风湿性关节炎中滑膜成纤维细胞的存活，并保护癌细胞免受细胞死亡。对于NLRP3炎症小体，情况有点不同：在典型的炎性疾病中，如关节炎，糖尿病或SLE，NLRP3激活的增加明显促进疾病的发生/发展。然而，在肠道中，NLRP3的作用仍然是一个争议问题，一些报道显示缺失NLRP3后才具有保护作用，而另一些表明NLRP3诱导的炎性体活化对于促进伤口愈合和组织修复是重要的.
Although there is evidence that autophagy is involved in mediating unconventional secretion of inflammasome substrates,53
several reports show that loss of functional autophagy results in enhanced inflammasome activation.35-37 One mechanism
proposed to mediate this effect is the accumulation of aged mitochondria upon loss of autophagy,54 and subsequently enhanced
levels of reactive oxygen species in the cytosol, which induces inflammasome activity.55
虽然有证据表明自噬参与了非常规炎性基质分泌的调控[53]，但一些报道显示功能性自噬的丧失导致炎症小体激活增强。其中一种机制是调节这种效应的机制之一是老化线粒体在失去 自噬[54]，随后胞浆中活性氧的水平增加，从而诱导炎性体活性。
Autophagy also controls inflammasome activation directly: ubiquitinated inflammasomes37 as well as IL1B35 can be found
in autophagosomes. We demonstrate here an additional mechanism—how autophagy affects NLRP3-mediated IL1B secretion,
which is dependent on the recruitment of phosphorylated NLRP3 into phagophores. Thereby, phosphorylation of NLRP3
mediates its interaction with SQSTM1 and subsequent accumulation in autophagosomes upon inflammasome activation;
hence autophagy removes NLRP3 from the cytosol and thereby directly reduces NLRP3-mediated inflammasome assembly.
Conversely, PTPN22-mediated dephosphorylation protects NLRP3 from recruitment into phagophores and thereby promotes
inflammasome activation. Consistent with this, we only detected phosphorylated NLRP3 in autophagosomes, but not
nonphosphorylated NLRP3. These findings are well in line with our previous results, where we observed that an increase in
NLRP3 phosphorylation in PTPN22-deficient cells resulted in a reduction of inflammasome activation.9
自噬还直接控制炎症小体激活：泛素化炎性体以及IL1B35可以在自噬体中发现。我们在这里展示一个额外的机制
- 自噬如何影响NLRP3介导的IL1B分泌，这依赖于磷酸化NLRP3募集到吞噬泡中。因此，NLRP3的磷酸化介导其与SQSTM1的相互作用并随后在炎性体活化后在自噬体中积累;因此自噬将细胞质中的NLRP3清除，并由此直接减少NLRP3介导的炎性体组装。相反，PTPN22介导的去磷酸化保护NLRP3免于募集到吞噬泡中，从而促进炎症小体激活。与此一致，我们仅在自噬体中检测到磷酸化的NLRP3，但未检测到非磷酸化的NLRP3。这些发现与我们之前的结果一致，其中我们观察到PTPN22缺陷细胞中NLRP3磷酸化的增加导致炎性体活化的减少.
Of note, treatment with the NLRP3 activators MSU and MDP did not promote protein expression levels of PTPN22; hence it
might be surprising that PTPN22 has such a strong impact on downstream signaling events induced by these 2 molcules.
Nevertheless, we previously showed that signals, which induce robust NLRP3 activation, also promote the interaction between
NLRP3 and PTPN22,9 and increase the phosphatase activity of PTPN22.23
值得注意的是，用NLRP3激活剂MSU和MDP处理不能促进PTPN22的蛋白质表达水平; 因此PTPN22对由这两种分子诱导的下游信号传导事件具有如此强烈的影响可能是令人惊讶的。 然而，我们以前表明，诱导强大的NLRP3活化的信号也促进NLRP3和PTPN22之间的相互作用，并增加PTPN22的磷酸酶活性。
Further, we found that the recruitment of NLRP3 into phagophores was accompanied by the interaction of NLRP3 with
SQSTM1, a molecule that targets its binding partners to autophagic degradation. We therefore speculate that the recruitment
of NLRP3 into phagophores is a result of NLRP3-SQSTM1 interaction, which is induced upon inflammasome activation. We
observed an increase in autophagy in both MDP- and MSUtreated cells; hence, sequestration of NLRP3 might also be the
result of enhanced autophagy, and not the result of inflammasome activation. Of interest, however, neither the interaction of
NLRP3 with SQSTM1 nor recruitment of NLRP3 into phagophores was observed in cells lacking PYCARD. Since NLRP3
only interacts with PYCARD upon inflammasome induction, this strongly indicates that inflammasome activation is necessary
for the interaction of NLRP3 with SQSTM1, and subsequently also for NLRP3 sequestration into phagophores.
此外，我们发现将NLRP3募集到吞噬细胞中伴随着NLRP3与SQSTM1的相互作用，SQSTM1是一种将其结合伴侣靶向自噬降解的分子。 因此，我们推测将NLRP3募集到吞噬体中是NLRP3-SQSTM1相互作用的结果，其在炎性小体激活时被诱导。 我们观察到MDP和MSU处理的细胞中自噬的增加; 因此，NLRP3的隔离也可能是自噬增强的结果，而不是炎性体激活的结果。 然而，感兴趣的是，在缺乏PYCARD的细胞中，没有观察到NLRP3与SQSTM1的相互作用，也没有将NLRP3募集到吞噬泡中。 由于NLRP3仅在炎性体诱导后与PYCARD相互作用，这强烈表明炎性小体激活对于NLRP3与SQSTM1的互作是必要的，并且随后也是NLRP3募集进吞噬细胞中所必
需的。
Upon its activation, NLRP3 forms complexes that serve as a platform for the induction of PYCARD aggregates, which then
mediate CASP1 activation. We speculate that phosphorylated NLRP3 is less efficient in promoting the formation of PYCARD
specks, but promotes binding with SQSTM1 in a PYCARD-dependent manner. In a broader view, this would also be in line
with autophagy as a mechanism to remove damaged/nonfunctional proteins from the cytosol: if phosphorylated NLRP3 is
less efficient in promoting PYCARD specks, it might be regarded as a nonfunctional protein.
在其激活后，NLRP3形成复合物，作为诱导PYCARD聚集体的平台，然后介导CASP1激活。我们推测磷酸化NLRP3在促进PYCARD斑点形成方面效率较低，但促进与PYCARD中SQSTM1的结合 独立的方式。 从更广泛的角度来看，这也符合自噬作为从细胞质中去除受损/非功能蛋白的机制：如果磷酸化的NLRP3在促进PYCARD斑点方面效率较低，那么它可能被认为是非功能性蛋白。
Taken together, our results show that NLRP3 inflammasome activation and autophagy are closely interweaved pathways, and
that PTPN22 crucially interferes with the function of these 2 pathways. In conclusion, our results explain the mechanism for
how phosphorylation of NLRP3 reduces its activation and dampens secretion of IL1B, and why this effect is autophagy
dependent. Further, this explains how loss of PTPN22 and subsequent enhanced NLRP3 phosphorylation mediate a decrease
in NLRP3 inflammasome activation. Together with previous work from other groups, our findings in this and previous studies
suggest that PTPN22 might be an interesting therapeutic target for a broad number of inflammatory diseases. However, given
the important role of autophagy, it is obvious that understanding the molecular mechanism affected by PTPN22 in more detail
is of important relevance before the development of possible treatment options.
总之，我们的研究结果表明，NLRP3炎症小体激活和自噬是密切交错的途径，并且PTPN22严重干扰这两条途径的功能。 总之，我们的结果解释了NLRP3磷酸化如何降低其活化并抑制IL1B分泌的机制，以及为什么这种效应是自噬依赖性的。 此外，这解释了PTPN22的丧失和随后增强的NLRP3磷酸化如何介导NLRP3炎性体激活的减少。 结合其他研究小组以前的工作，我们在这项研究和以前的研究中的发现表明，PTPN22可能是广泛炎症性疾病的一个有趣的治疗靶点。 然而，考虑到自噬的重要作用，显而易见的是，在开发可能的治疗方案之前，更详细地理解受PTPN22影响的分子机制是重要的相关性。
Methods
Cell culture MDP, MSU, and ATP treatment Inhibitors, shRNA, and siRNA treatment Reagents and antibodies
NLRP3 vector transfections Western blot, immunoprecipitation, and Phos-tag gel electrophoresis
Autophagosome enrichment RNA isolation and real-time PCR Confocal microscopy Statistical analyses
Abbreviations
Disclosure of potential conflicts of interest
Funding
References
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