Diagnostic significance of the histopathology of bone marrow macrophages in forensic autopsies

Forensic pathologists often encounter autopsies that require an assessment of antemortem general conditions (e.g., infection, metabolic disorders). To establish evaluation clues for such cases, we quantitatively examined macrophages and the general pathology of bone marrow in samples from 180 forensic autopsy cases of decedents with various conditions. Hematoxylin-eosin staining, Berlin blue staining, and immunostainings for CD163, CD138, and CD61 were performed. We determined the numbers per field (density) of total macrophages, swollen macrophages, macrophages with hemophagocytosis, and hemosiderin-laden macrophages. Each density was standardized by identifying its ratio to the total number of macrophages. The decedents' background data (cause of death, other pathological findings, postmortem interval, antemortem symptoms, and presence of resuscitation) were extracted. No correlations were found between the postmortem interval and the other decedent data, indicating that these data are not affected by postmortem changes. In the group in which inflammatory disease was the cause of death, there were significant elevations in the ratio of the swollen macrophage density to total macrophages. Significantly higher ratios of the density of swollen and hemophagocytic macrophages were observed in the group in which conditions with a prolonged agonal period were the cause of death. The group with a return of spontaneous circulation to resuscitation showed a significantly higher ratio of macrophage density with hemophagocytosis. This study provides the first statistical analysis focused on bone marrow histopathology in forensic autopsies. The results will be useful for elucidating causes of death and agonal-period conditions.     

ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.     

芫根调节免疫功能的小肠转录组学研究

利用生物信息学技术初步探索芫根调控肠道免疫功能的分子生物学机制。方法 选择雄性SPF级BALB/c小鼠18只，随机分为3组，每组6只。SC1组小鼠每只每次灌胃芫根提取液150 μL，SC2组小鼠每只每次灌胃绞碎芫根原浆悬液150 μL，NC组小鼠每只每次灌胃生理盐水150 μL，连续7 d每日灌胃一次。分别取每组小鼠小肠组织样品提取RNA，总RNA的质检合格后，进行转录组测序。按GO功能和KEGG信号通路对差异表达基因聚类分析揭示差异表达高度富集的免疫相关通路。从免疫角度分析芫根灌胃小鼠小肠组织的基因表达变化情况。结果 转录组测序共检测到27733条 mRNA的表达，SC1组与NC组比较，显著差异表达基因1635个，其中上调差异表达基因1236个，下调差异表达基因399个；SC2组与NC组比较，显著差异表达基因2872个，其中上调差异表达基因2233个，下调差异表达基因639个（P＜0.05）。按GO功能和KEGG信号通路聚类分析显示差异表达基因在白介素分泌、干扰素反应、T细胞受体信号通路及维生素和脂肪消化吸收等途径的富集度在两个芫根组中均居于前20位（P＜0.01），肠黏膜趋化因子CCL20和巨噬细胞极化标志物CD274等免疫蛋白编码基因差异表达显著且同属于上述多个免疫通路。结论 芫根灌胃小鼠小肠的差异表达基因在白介素分泌、干扰素反应、T细胞受体信号通路、营养物质消化吸收等途径高度富集，提示芫根对肠道免疫系统及肠黏膜功能的调节，其中CD274的表达上调和CCL20的表达下调提示诱导M1型巨噬细胞极化和T细胞活化可能是芫根调控免疫和维护肠道正常结构功能的重要途径。     

Interleukin-34 drives macrophage polarization to the M2 phenotype in autoimmune hepatitis

BackgroundAutoimmune hepatitis is a chronic inflammatory disease, the abnormal immunological function is the main pathogenesis. Interleukin-34 is a newly identified cytokine that shares the same receptor as colony stimulating factor-1.MethodsWe used interleukin-34 knockout and wild-type mice in a Con A-induced hepatitis model and cocultured RAW264.7 macrophage cells with interleukin-34. We then detected associated inflammatory cytokine and chemokine levels to elucidate the role of interleukin-34.ResultsIn this study, we found that the loss of interleukin-34 resulted in higher sensitivity to Con A-induced hepatitis. RAW264.7 macrophage cells were able to differentiate to the M2 phenotype upon interleukin-34 stimulation.ConclusionsWe conclude that interleukin-34 may protect the liver from Con A-mediated hepatitis by driving M2 macrophage polarization and suppressing inflammation.     

The key role of macrophage depolarization in the treatment of COPD with ergosterol both in vitro and in vivo

Macrophages are the most abundant immune cells in the lung, which play an important role in COPD. The anti-inflammatory and anti-oxidation of ergosterol are well documented. However, the effect of ergosterol on macrophage polarization has not been studied. The objective of this work was to investigate the effect of ergosterol on macrophage polarization in CSE-induced RAW264.7 cells and Sprague-Dawley (SD) rats COPD model. Our results demonstrate that CSE-induced macrophages tend to the M1 polarization via increasing ROS, IL-6 and TNF-α, as well as increasing MMP-9 to destroy the lung construction in both RAW264.7 cells and SD rats. However, treatment of RAW264.7 cells and SD rats with ergosterol inhibited CSE-induced inflammatory by decreasing ROS, IL-6 and TNF-α, and increasing IL-10 and TGF-β, shuffling the dynamic polarization of macrophages from M1 to M2 both in vitro and in vivo. Ergosterol also decreased the expression of M1 marker CD40, while increased that of M2 marker CD163. Moreover, ergosterol improved the lung characters in rats by decreasing MMP-9. Furthermore, ergosterol elevated HDAC3 activation and suppressed P300/CBP and PCAF activation as well as acetyl NF-κB/p65 and IKKβ, demonstrating that HDAC3 deacetylation was involved in the effect of ergosterol on macrophage polarization. These results also provide a proof in immunoregulation of ergosterol for therapeutic effects of cultured C. sinensis on COPD patients.     

Deoxyelephantopin decreases the release of inflammatory cytokines in macrophage associated with attenuation of aerobic glycolysis via modulation of PKM2

Growing evidence suggests that activated immune cells undergo metabolic reprogramming in the regulation of the innate inflammatory response. Remarkably, macrophages activated by lipopolysaccharide (LPS) induce a switch from oxidative phosphorylation to aerobic glycolysis, and consequently results in release of proinflammatory cytokines. Pyruvate Kinase M2 (PKM2) plays a vital role in the process of macrophage activation, promoting the inflammatory response in sepsis and septic shock. Deoxyelephantopin (DET), a naturally occurring sesquiterpene lactone from Elephantopus scaber, has been shown to counteracts inflammation during fulminant hepatitis progression, but the underlying mechanism remains unclear. Here, we studied the function of the DET on macrophage activation and investigated the anti-inflammatory effects of DET associated with interfering with glycolysis in macrophage. Our results first demonstrated that DET attenuates LPS-induced interleukin-1β (IL-1β) and high-mobility group box 1 (HMGB1) release in vitro and in vivo and protected mice against lethal endotoxemia. Furthermore, DET decreased the expression of pyruvate dehydrogenase kinase 1 (PDK1), glucose transporter 1(GLUT1), lactate dehydrogenase A (LDHA), and reduced lactate production dose-dependently in macrophages. Moreover, we further revealed that DET attenuates aerobic glycolysis in macrophages associated with regulating the nuclear localization of PKM2. Our results provided a novel mechanism for DET suppression of macrophages activation implicated in anti-inflammatory therapy.     

NLRP12 negatively regulates EtOH-induced liver macrophage activation via NF-κB pathway and mediates hepatocyte apoptosis in alcoholic liver injury

Alcohol-induced liver injury is characterized by abnormal liver dysfunction and excessive inflammation response. Recent years a wealth of data have been yielded indicating that EtOH (ethyl alcohol)-induced macrophage activation along with liver inflammation plays a dominating role in the progression of alcohol-induced liver injury. Here we found high expression of NLRP12 (Nucleotide-binding oligomerization domain protein 12, which is generally considered to be a negative regulator of inflammatory response) in EtOH-fed mouse liver tissue, primary Kupffer cells and EtOH-induced RAW264.7 cells. Additionally, overexpression of NLRP12 following Ad (adenovirus)-NLRP12-EGFP contributed to the attenuation of steatosis and inflammation in EtOH-fed mice model and EtOH-primed RAW264.7 cells. In parallel, Knockdown of NLRP12 aggravated the inflammatory response in RAW264.7 cells triggered by EtOH. Meanwhile, after administration of overexpression or inhibition of NLRP12 expression in vitro, the expression of phosphorylated protein of NF-kB signaling pathway was significantly affected. After increasing or decreasing the expression of NLRP12 in RAW264.7 cells, AML-12 cells were cultured with the supernatant of RAW264.7 cells stimulated by EtOH, and the percent of apoptosis ratio of AML-12 cells was remarkably altered. The study suggested that reduced inflammatory response induced by NLRP12-mediated inhibition of NF-kB pathway participated in the decrease of hepatocyte apoptosis in alcohol-induced liver injury. Collectively, these findings suggested the significance of NLRP12-mediated macrophage activation in alcohol-induced liver injury.     

血清趋化因子检测在肺癌早期诊断中的应用价值

目的探讨血清趋化因子检测在肺癌早期诊断中的应用价值。方法采用回顾性总结研究方法,选择肺癌患者72例(肺癌组)、肺炎患者72例(肺炎组)与健康人72例(对照组),检测3组人群的白细胞计数,检测血清趋化因子MIP-3α、IL-8、Fractalkine水平,随访记录患者的预后,判断诊断价值。结果肺癌组与肺炎组的外周白细胞计数均显著低于对照组(P<0.05),但肺癌组与肺炎组对比差异无统计学意义(P>0.05)。肺癌组与肺炎组的血清MIP-3α、IL-8、Fractalkine水平都高于对照组(P<0.05),肺癌组高于肺炎组(P<0.05)。随访至2018年9月,肺癌组中患者死亡6例,存活66例,死亡率为8.3%。多因素logistic回归分析淋巴结转移、临床分期、组织学分化、MIP-3α、IL-8、Fractalkine为影响患者预后死亡的主要独立危险因素(P<0.05)。结论MIP-3α、IL-8、Fractalkine等趋化因子联合现有的肿瘤标志物,可用于诊断早期肺癌与预测预后。     

Development and function of tissue resident macrophages in mice

Macrophages are important for tissue development, homeostasis as well as immune response upon injury or infection. For a long time they were only seen as one uniform group of phagocytes with a common origin and similar functions. However, this view has been challenged in the last decade and revealed a complex diversity of tissue resident macrophages. Here, we want to present the current view on macrophage development and tissue specification and we will discuss differences as well as common patterns between heterogeneous macrophage subpopulations.