Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1β, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1β were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.     

Mechanisms of NLRP3 priming in inflammaging and age related diseases

The NLRP3 inflammasome is a vital part of the innate immune response, whilst its aberrant activation drives the progression of a number of non-communicable diseases. Thus, NLRP3 inflammasome assembly must be tightly controlled at several checkpoints. The priming step of NLRP3 inflammasome activation is associated with increased NLRP3 gene expression, as well as post-translational modifications that control NLRP3 levels and licence the NLRP3 protein for inflammasome assembly. Increasing life expectancy in modern society is accompanied by a growing percentage of elderly individuals. The process of aging is associated with chronic inflammation that drives and/or worsens a range of age related non-communicable conditions. The NLRP3 inflammasome is known to contribute to pathological inflammation in many settings, but the mechanisms that prime NLRP3 for activation throughout aging and related co-morbidities have not been extensively reviewed. Here we dissect the biochemical changes that occur during aging and the pathogenesis of age related diseases and analyse the mechanisms by which they prime the NLRP3 inflammasome, thus exacerbating inflammation.     

Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation

Inflammation is not only a defensive mechanism against microbial invasion, but also frequently represents a critical response to tissue injury under sterile conditions. It is now well established that tissue injury leads to the release of endogenous molecules of intra- and extracellular origin acting as damage-associated molecular patterns (DAMPs). The small leucine-rich proteoglycans (SLRPs) can act as powerful DAMPs following their proteolytical release from the extracellular matrix. Recent investigations of SLRP signaling networks revealed new levels of complexity, showing that SLRPs can cluster different types of receptors and orchestrate a host of downstream signaling events. This review will summarize the evidence for the multifunctional proinflammatory signaling properties of the two archetypal SLRPs, biglycan and decorin. These secreted proteoglycans link the innate to the adaptive immune response and operate in a broad biological context, encompassing microbial defense, tumor growth and autoimmunity.     

Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog

Caspases-1, 4, 5, and 12 and other proteins containing caspase recruitment domains (CARDs) play crucial roles in the induction of inflammatory processes. Recently, hybrid caspase-1/4 mRNAs encoding proteases with two CARDs were identified in cat and dog, indicating that the molecular machinery of caspase-dependent inflammation has an unconventional composition in members of the order Carnivora. Here we extended these studies and identified, both in cat and dog, splice variants of caspase-12, which also contained two CARDs. Comparative genomics analysis of the repertoire of canine CARD proteins revealed that the gene encoding NLRC4/IPAF, which is implicated in the inflammatory response to cytosolic flagellin, was inactivated by deleterious mutations in the dog. Our results demonstrate that the repertoires of CARD proteins in cat and dog differ significantly from that of humans and suggest the existence of uncharacterized pathways of inflammasome-mediated signaling in Carnivora.     

Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.     

Cross reactivity of S. aureus to murine cytokine assays: A source of discrepancy

Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1β and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1β and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.     

炎性小体激活与细胞焦亡的研究进展

细胞焦亡是一种依赖天冬氨酸特异性半胱氨酸蛋白酶1(cysteinyl aspartate specific proteinase 1,caspase-1)/caspase-11的程序性细胞死亡方式。炎性小体的激活在细胞焦亡过程中扮演重要角色。当病原体入侵时,核苷酸结合寡聚化结构域样受体(nucleotide-binding oligomerization domain-like receptor,NLR)和黑色素瘤缺乏因子2(absent in melanoma 2,AIM2)等胞内模式识别受体(pattern recognition receptor,PRR)与相应配体结合,导致炎性小体多蛋白复合物组装和caspase-1/caspase-11激活,进而诱导细胞焦亡发生。深入研究炎性小体激活和细胞焦亡的相关机制,对认识炎症性疾病的发生发展非常重要。本文就炎性小体激活与细胞焦亡的研究进展进行综述。     

NLRP3炎症小体表达与慢性阻塞性肺疾病合并肺癌的相关性研究

目的:分析核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体表达与慢性阻塞性肺疾病(COPD)合并肺癌的相关性。方法:选取2015年1月-2018年2月我院收治的COPD合并肺癌患者62例作为实验组及同期88例COPD患者作为对照组。酶联免疫吸附法(ELISA)检测两组患者外周血IL-1β、IL-18浓度,免疫组化法检测两组患者术后肺病理组织中Caspase-1、ASC、NLRP3、IL-1β、IL-18蛋白相对表达量,并比较不同病理特征下患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量的差异,并分析其与COPD合并肺癌的相关性。结果:实验组患者外周血IL-1β、IL-18水平均明显高于对照组,差异具有统计学意义(P0.05);实验组患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量均明显高于对照组,差异均具有统计学意义(P0.05);中低分化、临床分期Ⅲ期、淋巴结有转移的急性加重期COPD合并肺癌患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量高于高分化、临床分期Ⅰ-Ⅱ期、淋巴结无转移的稳定期COPD合并肺癌患者,差异具有统计学意义(P0.05)。经Spearman秩相关性分析发现,患者术后肺病理组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18蛋白相对表达量与COPD合并肺癌患者病情严重程度、淋巴结转移情况、分化程度以及病情所处时期均呈正相关(r0,P0.05)。结论:NLRP3炎症小体通路可能参与了COPD合并肺癌的发展过程,其释放的细胞因子IL-1β、IL-18水平升高可能与患者持续炎症有关,并进一步导致机体免疫病理损伤,促进疾病进展。