Christina Weisheit

CCR2 Mediates Homeostatic and Inflammatory Release of Gr1 high Monocytes from the Bone Marrow, but Is Dispensable for Bladder Infiltration in Bacterial Urinary Tract Infection

CCR2 is thought to recruit monocytes to sites of infection. Two subpopulations of murine blood monocytes differing in Gr1 and CCR2 expression have been described. The exact role of CCR2 in migration of CCR2lowGr1low and CCR2highGr1high monocytes into nonlymphoid tissue is controversial. In this study, we have addressed this question in a murine model of bacterial urinary tract infection. Only Gr1high monocytes were recruited into the infected bladder. CCR2 deficiency reduced their frequency in this organ, indicating a requirement of this chemokine receptor. Importantly, CCR2-deficient mice also showed reduced Gr1high monocyte numbers in the blood, but not in the bone marrow (BM), indicating that CCR2 acted at the step of monocyte release into the circulation. The same was found also in noninfected mice, indicating a further involvement of CCR2 in steady-state BM egress. An additional requirement of CCR2 in monocyte recruitment from the blood into the bladder was excluded by tracking particle-labeled endogenous monocytes and by adoptive transfer of BM-derived monocyte subsets. These findings demonstrate that CCR2 governs homeostatic and infection-triggered release of Gr1high monocytes from the BM into the blood but is dispensable for recruitment into a nonlymphoid tissue.

Exclusive CX3CR1 dependence of kidney DCs impacts glomerulonephritis progression

DCs and macrophages both express the chemokine receptor CX3CR1. Here we demonstrate that its ligand, CX3CL1, is highly expressed in the murine kidney and intestine. CX3CR1 deficiency markedly reduced DC numbers in the healthy and inflamed kidney cortex, and to a lesser degree in the kidney medulla and intestine, but not in other organs. CX3CR1 also promoted influx of DC precursors in crescentic glomerulonephritis, a DC-dependent aggressive type of nephritis. Disease severity was strongly attenuated in CX3CR1-deficient mice. Primarily CX3CR1-dependent DCs in the kidney cortex processed antigen for the intrarenal stimulation of T helper cells, a function important for glomerulonephritis progression. In contrast, medullary DCs played a specialized role in inducing innate immunity against bacterial pyelonephritis by recruiting neutrophils through rapid chemokine production. CX3CR1 deficiency had little effect on the immune defense against pyelonephritis, as medullary DCs were less CX3CR1 dependent than cortical DCs and because recruited neutrophils produced chemokines to compensate for the DC paucity. These findings demonstrate that cortical and medullary DCs play specialized roles in their respective kidney compartments. We identify CX3CR1 as a potential therapeutic target in glomerulonephritis that may involve fewer adverse side effects, such as impaired anti-infectious defense or compromised DC functions in other organs.

Ly6C(low) and not Ly6C(high) macrophages accumulate first in the heart in a model of murine pressure-overload.

Cardiac tissue remodeling in the course of chronic left ventricular hypertrophy requires phagocytes which degrade cellular debris, initiate and maintain tissue inflammation and reorganization. The dynamics of phagocytes in left ventricular hypertrophy have not been systematically studied. Here, we characterized the temporal accumulation of leukocytes in the cardiac immune response by flow cytometry and fluorescence microscopy at day 3, 6 and 21 following transverse aortic constriction (TAC). Cardiac hypertrophy due to chronic pressure overload causes cardiac immune response and inflammation represented by an increase of immune cells at all three time points among which neutrophils reached their maximum at day 3 and macrophages at day 6. The cardiac macrophage population consisted of both Ly6Clow and Ly6Chigh macrophages. Ly6Clow macrophages were more abundant peaking at day 6 in response to pressure overload. During the development of cardiac hypertrophy the expression pattern of adhesion molecules was investigated by qRT-PCR and flow cytometry. CD11b, CX3CR1 and ICAM-1 determined by qRT-PCR in whole cardiac tissue were up-regulated in response to pressure overload at day 3 and 6. CD11b and CX3CR1 were significantly increased by TAC on the surface of Ly6Clow but not on Ly6Chigh macrophages. Furthermore, ICAM-1 was up-regulated on cardiac endothelial cells. In fluorescence microscopy Ly6Clow macrophages could be observed attached to the intra- and extra-vascular vessel-wall. Taken together, TAC induced the expression of adhesion molecules, which may explain the accumulation of Ly6Clow macrophages in the cardiac tissue, where these cells might contribute to cardiac inflammation and remodeling in response to pressure overload.

Dendritic Cells and Macrophages: Sentinels in the Kidney

The mononuclear phagocytes (dendritic cells and macrophages) are closely related immune cells with central roles in anti-infectious defense and maintenance of organ integrity. The canonical function of dendritic cells is the activation of T cells, whereas macrophages remove apoptotic cells and microbes by phagocytosis. In the kidney, these cell types form an intricate system of mononuclear phagocytes that surveys against injury and infection and contributes to organ homeostasis and tissue repair but may also promote progression of CKD. This review summarizes the general functions and classification of dendritic cells and macrophages in the immune system and recapitulates why overlapping definitions and historically separate research have created controversy about their tasks. Their roles in acute kidney disease, CKD, and renal transplantation are described, and therapeutic strategy to modify these cells for therapeutic purposes is discussed.

Antibiotics regulate the immune response in both presence and absence of lipopolysaccharide through modulation of Toll-like receptors, cytokine production and phagocytosis in vitro

The inflammatory response to pathogen-associated molecular patterns such as lipopolysaccharide (LPS) in sepsis is mediated via Toll-like receptors (TLRs). Since TLRs also trigger various immune functions, including phagocytosis, their modulation is a promising strategy in the treatment of sepsis. As antibiotics have immunomodulatory properties, this study examined the effect of commonly used classes of antibiotics on i) the expression of TLRs and cytokines and ii) the phagocytic activity under sepsis-like conditions in vitro. This was achieved by incubating THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) obtained from patients after open-heart surgery with the addition of LPS and six key antibiotics (piperacillin, doxycycline, erythromycin, moxifloxacin or gentamicin). After 24h, mRNA levels of both cytokines (IL-1β, IL-6) and TLRs (1, 2, 4, and 6) were monitored and phagocytosis was determined following coincubation with Escherichia coli. Each antibiotic differentially regulated the gene expression of the investigated TLRs and cytokines in monocytes. Erythromycin, moxifloxacin and doxycyclin displayed the strongest effects and changed mRNA-levels of the investigated genes up to 5.6-fold. Consistent with this, antibiotics and, in particular, moxifloxacin, regulated the TLR-and cytokine expression in activated PBMCs obtained from patients after open-heart surgery. Furthermore, piperacillin, doxycyclin and moxifloxacin inhibited the phagocytic activity of monocytes. Our results suggest that antibiotics regulate the immune response by modulating TLR- and cytokine expression as well as phagocytosis under septic conditions. Moxifloxacin, doxycycline and erythromycin were shown to possess the strongest immunomodulatory effects and these antibiotic classes should be considered for future immunomodulatory studies in sepsis.

Linezolid, vancomycin and daptomycin modulate cytokine production, Toll-like receptors and phagocytosis in a human in vitro model of sepsis

Conventional antibiotics exhibit immunomodulatory properties beneficial in the treatment of sepsis. Antibiotic-resistant Gram-positive bacteria have become a problem in sepsis therapy, giving rise to increased use of last-resort antibiotics; for example, linezolid (LIN), vancomycin (VAN) and daptomycin (DAP). As the immunomodulatory properties of these antibiotics in treating sepsis are unknown, this study examined the effect of VAN, LIN and DAP on the immune response under sepsis-like conditions in vitro. Lipopolysaccharide (LPS)-activated THP-1 monocytes were incubated with LIN, VAN or DAP. Gene expression of cytokines (TNFα, IL-1β, IL-6, IL-10) and Toll-like receptors (TLR1, 2, 4, 6, 7 and 9) was monitored and phagocytosis was determined following coincubation with E. coli. The antibiotics differentially modulated the gene expression of the investigated cytokines. While LIN and VAN upregulated the expression of all TLRs, DAP downregulated mRNA levels of TLR1, TLR2 and TLR6, which recognize pathogen-associated molecular patterns from Gram-positive bacteria. In addition, LIN inhibited, whereas VAN promoted the phagocytic activity of monocytes. Our results suggest that LIN and VAN possess pro-inflammatory properties, whereas DAP might reduce the immune response to Gram-positive bacteria in sepsis. Furthermore, VAN might be beneficial in the prevention of Gram-negative infections by increasing the phagocytosis of E. coli.

Beta2-adrenoceptor stimulation suppresses TLR9-dependent IFNA1 secretion in human peripheral blood mononuclear cells.

Introduction IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs. Methods PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay. Results TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells. Conclusion We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice. Our results have implications for the future treatment of human patients, in which the endogenous immune response plays a pivotal role, such as during viral infections, inflammatory diseases and cancers.

Synergistic Stimulation with Different TLR7 Ligands Modulates Gene Expression Patterns in the Human Plasmacytoid Dendritic Cell Line CAL-1.

Objective. TLR7 ligation in plasmacytoid dendritic cells is promising for the treatment of cancer, allergy, and infectious diseases; however, high doses of ligands are required. We hypothesized that the combination of structurally different TLR7 ligands exponentiates the resulting immune response. Methods. CAL-1 (human pDC line) cells were incubated with the TLR7-specific adenine analog CL264 and single-stranded 9.2s RNA. Protein secretion was measured by ELISA. Microarray technique was used to detect modified gene expression patterns upon synergistic stimulation, revealing underlying functional groups and networks. Cell surface binding properties were studied using FACS analysis. Results. CL264 in combination with 9.2s RNA significantly enhanced cytokine and interferon secretion to supra-additive levels. This effect was due to a stronger stimulation of already regulated genes (by monostimulation) as well as to recruitment of thus far unregulated genes. Top scoring canonical pathways referred to immune-related processes. Network analysis revealed IL-1β, IL-6, TNF, and IFN-β as major regulatory nodes, while several minor regulatory nodes were also identified. Binding of CL264 to the cell surface was enhanced by 9.2s RNA. Conclusion. Structurally different TLR7 ligands act synergistically on gene expression patterns and on the resulting inflammatory response. These data could impact future strategies optimizing TLR7-targeted drug design.

Antifungal antibiotics modulate the pro-inflammatory cytokine production and phagocytic activity of human monocytes in an in vitro sepsis model

AIMS The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes. MAIN METHODS THP-1 monocytes were incubated with L-AMB, ITC or ANI and LPS. Gene expression levels of cytokines (TNF-, IL-1, IL-6, and IL-10) were measured after 2h, 6h, and 24h. Cytokine protein levels were evaluated after 24h and phagocytic activity was determined following co-incubation with Escherichia coli. KEY FINDINGS All antimycotics differentially modulated the gene and protein expression of cytokines in sepsis-like conditions. In the presence of LPS, we identified L-AMB as immunosuppressive, whereas ITC demonstrated pro-inflammatory properties. Both compounds induced remarkably less phagocytosis. SIGNIFICANCE Our study suggests that antimycotics routinely used in septic patients alter the immune response in sepsis-like conditions by modulating cytokine gene and protein expression levels and phagocytic activity. Future treatment strategies should consider the immune status of the host and apply antimycotics accordingly in bacterial septic patients with secondary fungal infections.

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation.

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4 -/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4 -/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4 -/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.