Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-α. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-α(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-α(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-α by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-α expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.     

Ly-6G+CCR2- myeloid cells rather than Ly-6ChighCCR2+ monocytes are required for the control of bacterial infection in the central nervous system

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b(+)Ly-6G(+)CCR2(-) granulocytes, also CD11b(+)Ly-6C(high)CCR2(+) but not Ly-6C(low)CCR2(-) monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b(+)Ly-6C(high)CCR2(+) inflammatory monocytes modulated local CXCL2 and IL-1beta production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6C(high)CCR2(+) inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b(+)Ly-6G(+)CCR2(-) myeloid cells are involved in the restriction of the extracellular bacteria.     

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.     

A unique hybrid renal mononuclear phagocyte activation phenotype in murine systemic lupus erythematosus nephritis

Renal infiltration with mononuclear cells is associated with poor prognosis in systemic lupus erythematosus. A renal macrophage/dendritic cell signature is associated with the onset of nephritis in NZB/W mice, and immune-modulating therapies can reverse this signature and the associated renal damage despite ongoing immune complex deposition. In nephritic NZB/W mice, renal F4/80(hi)/CD11c(int) macrophages are located throughout the interstitium, whereas F4/80(lo)/CD11c(hi) dendritic cells accumulate in perivascular lymphoid aggregates. We show here that F4/80(hi)/CD11c(int) renal macrophages have a Gr1(lo)/Ly6C(lo)/VLA4(lo)/MHCII(hi)/CD43(lo)/CD62L(lo) phenotype different from that described for inflammatory macrophages. At nephritis onset, F4/80(hi)/CD11c(int) cells upregulate cell surface CD11b, acquire cathepsin and matrix metalloproteinase activity, and accumulate large numbers of autophagocytic vacuoles; these changes reverse after the induction of remission. Latex bead labeling of peripheral blood Gr1(lo) monocytes indicates that these are the source of F4/80(hi)/CD11c(int) macrophages. CD11c(hi)/MHCII(lo) dendritic cells are found in the kidneys only after proteinuria onset, turnover rapidly, and disappear rapidly after remission induction. Gene expression profiling of the F4/80(hi)/CD11c(int) population displays increased expression of proinflammatory, regulatory, and tissue repair/degradation-associated genes at nephritis onset that reverses with remission induction. Our findings suggest that mononuclear phagocytes with an aberrant activation profile contribute to tissue damage in lupus nephritis by mediating both local inflammation and excessive tissue remodeling.     

Plasticity of Ly-6C(hi) myeloid cells in T cell regulation

CD11b(+)Ly-6C(hi) cells, including inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs), are important in infectious, autoimmune, and tumor models. However, their role in T cell regulation is controversial. In this article, we show that T cell regulation by IMCs and IDCs is determined by their activation state and is plastic during an immune response. Nonactivated IMCs and IDCs function as APCs, but activated IMCs and IDCs suppress T cells through NO production. Suppressive IMCs are induced by IFN-γ, GM-CSF, TNF-α, and CD154 derived from activated T cells during their interaction. In experimental autoimmune encephalomyelitis, CD11b(+)Ly-6C(hi) cells in the CNS are increasingly activated from disease onset to peak and switch their function from Ag presentation to T cell suppression. Furthermore, transfer of activated IMCs or IDCs enhances T cell apoptosis in the CNS and suppresses experimental autoimmune encephalomyelitis. These data highlight the interplay between innate and adaptive immunity: immunization leads to the expansion of Ly-6C(hi) myeloid cells initially promoting T cell function. As T cells become highly activated in the target tissue, they induce activation and NO production in Ly-6C(hi) myeloid cells, which in turn suppress T cells and lead to the contraction of local immune response.     

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16−, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62–3.87) and of CD14+/CD16+ (2.59, 1.28–4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16− (0.66, 0.47–1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16− monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.     

The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or DeltaactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset. In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes. Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C(high) monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.     

Identification of mouse serum miRNA endogenous references by global gene expression profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.     

Functional and phenotypic alterations in T cell subsets during the course of MAIDS, a murine retrovirus-induced immunodeficiency syndrome

The functional and phenotypic characteristics of Ly-4(CD4)+ and Ly-2(CD8)+ T cells were studied after induction of murine AIDS with LP-BM5 murine leukemia virus. Assays of spleen cells for their ability to generate in vitro CTL responses to TNP-modified autologous cells (self + x CTL) and to alloantigens (allo CTL) showed that self + x CTL responses were greatly impaired at 3 to 4 wk postinfection and were undetectable thereafter. Allo CTL responses were normal at 3 to 4 wk, but were reduced at 8 to 9 wk and absent at 14 wk postinfection. This sequential loss of self + x and allo CTL responses was related to a selective defect in Ly-4(CD4)+ Th cell function associated with impaired production of IL-2 and deficient proliferative responses to Con A or to soluble Ag. Changes in the functional characteristics of Ly-4(CD4)+ T cells were unrelated to changes in their frequency in spleen, but did correlate with marked alterations in their distribution among four subsets defined by mAb SM3C11 and SM6C10. Assays of CTL responses generated by mixtures of spleen cells from normal and infected mice suggested that active suppression of Ly-4(CD4)+ Th function may contribute to this defect. Studies of Ly-2(CD8)+ T cells showed that infection with LP-BM5 murine leukemia virus also induced a major phenotypic shift in subpopulations defined by their reactivity with mAb 6C10. However, this phenotypic change did not appear to correlate with major functional defects.     

MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)