Macrophage Phenotype Is Associated with Disease Severity in Preterm Infants with Chronic Lung Disease

Background

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation.

Objectives

To investigate airway innate immune cellular phenotypes in preterm infants with respiratory distress syndrome (RDS) or CLD.

Methods

Bronchoalveolar lavage (BAL) fluid was obtained from term and preterm infants requiring mechanical ventilation. BAL cells were phenotyped by flow cytometry.

Results

Preterm birth was associated with an increase in the proportion of non-classical CD14+/CD16+ monocytes on the day of delivery (58.9±5.8% of total mononuclear cells in preterm vs 33.0±6.1% in term infants, p = 0.02). Infants with RDS were born with significantly more CD36⁺ macrophages compared with the CLD group (70.3±5.3% in RDS vs 37.6±8.9% in control, p = 0.02). At day 3, infants born at a low gestational age are more likely to have greater numbers of CD14⁺ mononuclear phagocytes in the airway (p = 0.03), but fewer of these cells are functionally polarized as assessed by HLA-DR (p = 0.05) or CD36 (p = 0.05) positivity, suggesting increased recruitment of monocytes or a failure to mature these cells in the lung.

Conclusions

These findings suggest that macrophage polarization may be affected by gestational maturity, that more immature macrophage phenotypes may be associated with the progression of RDS to CLD and that phenotyping mononuclear cells in BAL could predict disease outcome.     

Role of Dystrophin in Airway Smooth Muscle Phenotype,Contraction and Lung Function

Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca²⁺ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.     

How Mitochondrial Metabolism Contributes to Macrophage Phenotype and Functions

Metabolic reprogramming of cells from the innate immune system is one of the most noteworthy topics in immunological research nowadays. Upon infection or tissue damage, innate immune cells, such as macrophages, mobilize various immune and metabolic signals to mount a response best suited to eradicate the threat. Current data indicate that both the immune and metabolic responses are closely interconnected. On account of its peculiar position in regulating both of these processes, the mitochondrion has emerged as a critical organelle that orchestrates the coordinated metabolic and immune adaptations in macrophages. Significant effort is now underway to understand how metabolic features of differentiated macrophages regulate their immune specificities with the eventual goal to manipulate cellular metabolism to control immunity. In this review, we highlight some of the recent work that place cellular and mitochondrial metabolism in a central position in the macrophage differentiation program.     

Antigen Experience Shapes Phenotype and Function of Memory Th1 Cells

Primary and secondary (boosted) memory CD8 T cells exhibit differences in gene expression, phenotype and function. The impact of repeated antigen stimulations on memory CD4 T cells is largely unknown. To address this issue, we utilized LCMV and Listeria monocytogenes infection of mice to characterize primary and secondary antigen (Ag)-specific Th1 CD4 T cell responses. Ag-specific primary memory CD4 T cells display a CD62L^loCCR7^hi CD27^hi CD127^hi phenotype and are polyfunctional (most produce IFNγ, TNFα and IL-2). Following homologous prime-boost immunization we observed pathogen-specific differences in the rate of CD62L and CCR7 upregulation on memory CD4 T cells as well as in IL-2+IFNγco-production by secondary effectors. Phenotypic and functional plasticity of memory Th1 cells was observed following heterologous prime-boost immunization, wherein secondary memory CD4 T cells acquired phenotypic and functional characteristics dictated by the boosting agent rather than the primary immunizing agent. Our data also demonstrate that secondary memory Th1 cells accelerated neutralizing Ab formation in response to LCMV infection, suggesting enhanced capacity of this population to provide quality help for antibody production. Collectively these data have important implications for prime-boost vaccination strategies that seek to enhance protective immune responses mediated by Th1 CD4 T cell responses.     

核糖体合成调控蛋白RRS1的功能及其与疾病的关系

1999年,Tsuno等在酵母中发现了一个核糖体合成调控蛋白,命名为RRS1。在酵母中,RRS1是一个多功能的蛋白,与核糖体产物因子2相互作用,在25S r RNA成熟过程中起重要作用,并参与核糖体60S大亚基的生物合成。在哺乳动物细胞中,RRS1参与细胞周期中染色体在赤道板的聚集和端粒的聚集。RRS1在亨廷顿病等的发生中发挥着重要作用。我们简要概述RRS1的功能及其与疾病的关系。     

嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能分析

真核细胞中染色体浓缩调节因子（regulator of chromosome condensation 1, RCC1）是 RanGTPase 唯一的鸟嘌呤核苷酸交换因子. 染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成. 本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1（TTHERM_00530380）基因. 该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸. 实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高. 免疫荧光定位分析表明, HA RCC1在营养生长和饥饿时期,定位于大核和小核中|在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中. 过表达RCC1导致大核的无丝分裂异常, 细胞增殖变慢,最终产生无大核的后代细胞. 敲减RCC1导致了多小核的产生. 结果表明,RCC1参与调控了四膜虫细胞核的分裂, RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.     

Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background

Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.

Aim

To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.

Methodology and principal findings

Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4⁺ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.

Conclusions and significance

Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.     

人源细胞内物质转运调节分子ARFGAP1对细胞分泌功能的影响

 ARF GAP是重要的细胞内物质转运调节分子 .最近 ,在人胎肝 c DNA文库中发现一种新基因 ,其编码的氨基酸序列与大鼠的 ARF1 GAP有 32 %同源性 ,故将其命名为“ARFGAP1”.对ARFGAP1进行功能研究 ,利用分子克隆技术构建绿色荧光蛋白 (GFP) - ARFGAP1融合基因表达质粒 (p EGFP- C1 - ARFGAP1 ) ,经脂质体转染将其导入 COS- 7细胞瞬时表达 ,利用绿色荧光确定ARFGAP1的亚细胞定位 .结果显示 ,ARFGAP1位于细胞质部分 ,表达量高时 ,在核周高尔基体区聚集呈团块状或颗粒状 .构建真核表达质粒 pc DNA3.1 /myc- His- ARFGAP1 ,在 COS- 7细胞中表达 ,并用 ARFGAP1和分泌型碱性磷酸酶 (SEAP)真核表达质粒共同转染 COS- 7细胞 ,发现ARFGAP1在细胞中过表达能部分抑制 SEAP的分泌 .结果证明 ,ARFGAP1对细胞的物质转运和分泌功能有调节作用 .     

MicroRNA 21 Is a Homeostatic Regulator of Macrophage Polarization and Prevents Prostaglandin E2-Mediated M2 Generation

Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E₂ (PGE₂) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE₂ and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE₂. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE₂-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.     

The Ultrastructure of Mouse Lung: The Alveolar Macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1½ hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small (~6 mµ) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.