Efficacy of daptomycin in implant-associated infection due to methicillin-resistant Staphylococcus aureus: importance of combination with rifampin.

Limited treatment options are available for implant-associated infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). We compared the activity of daptomycin (alone and with rifampin [rifampicin]) with the activities of other antimicrobial regimens against MRSA ATCC 43300 in the guinea pig foreign-body infection model. The daptomycin MIC and the minimum bactericidal concentration in logarithmic phase and stationary growth phase of MRSA were 0.625, 0.625, and 20 microg/ml, respectively. In time-kill studies, daptomycin showed rapid and concentration-dependent killing of MRSA in stationary growth phase. At concentrations above 20 microg/ml, daptomycin reduced the counts by >3 log(10) CFU/ml in 2 to 4 h. In sterile cage fluid, daptomycin peak concentrations of 23.1, 46.3, and 53.7 microg/ml were reached 4 to 6 h after the administration of single intraperitoneal doses of 20, 30, and 40 mg/kg of body weight, respectively. In treatment studies, daptomycin alone reduced the planktonic MRSA counts by 0.3 log(10) CFU/ml, whereas in combination with rifampin, a reduction in the counts of >6 log(10) CFU/ml was observed. Vancomycin and daptomycin (at both doses) were unable to cure any cage-associated infection when they were given as monotherapy, whereas rifampin alone cured the infections in 33% of the cages. In combination with rifampin, daptomycin showed cure rates of 25% (at 20 mg/kg) and 67% (at 30 mg/kg), vancomycin showed a cure rate of 8%, linezolid showed a cure rate of 0%, and levofloxacin showed a cure rate of 58%. In addition, daptomycin at a high dose (30 mg/kg) completely prevented the emergence of rifampin resistance in planktonic and adherent MRSA cells. Daptomycin at a high dose, corresponding to 6 mg/kg in humans, in combination with rifampin showed the highest activity against planktonic and adherent MRSA. Daptomycin plus rifampin is a promising treatment option for implant-associated MRSA infections.

Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues.

Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.

Initiation of protein O glycosylation by the polypeptide GalNAcT-1 in vascular biology and humoral immunity.

Core-type protein O glycosylation is initiated by polypeptide N-acetylgalactosamine (GalNAc) transferase (ppGalNAcT) activity and produces the covalent linkage of serine and threonine residues of proteins. More than a dozen ppGalNAcTs operate within multicellular organisms, and they differ with respect to expression patterns and substrate selectivity. These distinctive features imply that each ppGalNAcT may differentially modulate regulatory processes in animal development, physiology, and perhaps disease. We found that ppGalNAcT-1 plays key roles in cell and glycoprotein selective functions that modulate the hematopoietic system. Loss of ppGalNAcT-1 activity in the mouse results in a bleeding disorder which tracks with reduced plasma levels of blood coagulation factors V, VII, VIII, IX, X, and XII. ppGalNAcT-1 further supports leukocyte trafficking and residency in normal homeostatic physiology as well as during inflammatory responses, in part by providing a scaffold for the synthesis of selectin ligands expressed by neutrophils and endothelial cells of peripheral lymph nodes. Animals lacking ppGalNAcT-1 are also markedly impaired in immunoglobulin G production, coincident with increased germinal center B-cell apoptosis and reduced levels of plasma B cells. These findings reveal that the initiation of protein O glycosylation by ppGalNAcT-1 provides a distinctive repertoire of advantageous functions that support vascular responses and humoral immunity.

Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment.

Lymphocyte homing is mediated by specific interaction between L-selectin on lymphocytes and the carbohydrate ligand 6-sulfo sialyl Lewis X on high endothelial venules. Here we generated mice lacking both core 1 extension and core 2 branching enzymes to assess the functions of O-glycan-borne L-selectin ligands in vivo. Mutant mice maintained robust lymphocyte homing, yet they lacked O-glycan L-selectin ligands. Biochemical analyses identified a class of N-glycans bearing the 6-sulfo sialyl Lewis X L-selectin ligand in high endothelial venules. These N-glycans supported the binding of L-selectin to high endothelial venules in vitro and contributed in vivo to O-glycan-independent lymphocyte homing in wild-type and mutant mice. Our results demonstrate the critical function of N-glycan-linked 6-sulfo sialyl Lewis X in L-selectin-dependent lymphocyte homing and recruitment.

Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues.

Dendritic cells (DCs) have long been recognized as key regulators of immune responses. However, the process of their recruitment to peripheral tissues and turnover during homeostasis remains largely unknown. The chemokine CXCL14 (BRAK) is constitutively expressed in skin and other epithelial tissues. Recently, the human chemokine was proposed to play a role in the homeostatic recruitment of macrophage and/or DC precursors toward the periphery, such as skin. Although so far no physiological function could be demonstrated for the murine CXCL14, it shows a remarkable homology to the human chemokine. In order to elucidate the in vivo role of CXCL14, we generated a mouse defective for this chemokine. We studied various components of the immune system with emphasis on monocytes/macrophages and DC/Langerhans cell (LC) populations in different tissues during steady state but did not find a significant difference between knockout (CXCL14(-)(/)(-)) and control mice. Functionally, LCs were able to become activated, to migrate out of skin, and to elicit a delayed type of hypersensitivity reaction. Overall, our data indicate that murine CXCL14 is dispensable for the homeostatic recruitment of antigen-presenting cells toward the periphery and for LC functionality.

Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus.

Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin-sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue-derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.

Compartment-specific expression and function of the chemokine IP-10/CXCL10 in a model of renal endothelial microvascular injury.

The recruitment of inflammatory cells into renal tissue, mainly T cells and monocytes, is a typical feature of various renal diseases such as glomerulonephritis, thrombotic angiopathies, allograft rejection, and vasculitis. T cells predominantly infiltrate the tubulointerstitium, whereas monocytes are present in the tubulointerstitial and glomerular compartment. Because chemokines play a pivotal role in leukocyte trafficking under inflammatory conditions, this study investigated whether a differential expression of chemokines contributes to the precise coordination of leukocyte subtype trafficking in a rat model of renal microvascular endothelial injury. Renal microvascular endothelial injury was induced in rats by selective renal artery perfusion with an anti-endothelial antibody. Induction of the disease led to severe glomerular and tubulointerstitial endothelial injury with subsequent upregulation of chemokines followed by inflammatory cell recruitment. Among the analyzed chemokine mRNA, IP-10/CXCL10 (119-fold), acting via CXCR3 on activated T cells, and MCP-1/CCL2 (65-fold), acting via CCR2 on monocytes, were by far the most strongly upregulated chemokines. In situ hybridization revealed that IP-10/CXCL10 mRNA was selectively expressed by endothelial cells in the tubulointerstitial area, co-localizing with infiltrating T cells. Despite extensive damage of glomerular vasculature, no IP-10/CXCL10 expression by glomerular endothelial cells was detected. MCP-1/CCL2 mRNA in contrast was detectable in the glomerulus and the tubulointerstitium. Treatment with a neutralizing anti-IP-10/CXCL10 antibody significantly reduced the number of infiltrating tubulointerstitial T cells without affecting monocyte migration and led to an improved renal function. Our study demonstrates a role of IP-10/CXCL10 on T cell recruitment in a rat model of renal endothelial microvascular injury. Furthermore, a differential chemokine expression profile by endothelial cells in different renal compartments was found. These findings are consistent with the hypothesis that functional heterogeneity of endothelial cells from different vascular sites exists and provide an insight into the molecular mechanisms that may mediate compartment-specific T cell and monocyte recruitment in inflammatory renal disease.

T cell-regulated neutrophilic inflammation in autoinflammatory diseases.

Previous studies of acute generalized exanthematous pustulosis, a peculiar drug hypersensitivity reaction, suggested that CXCL8-producing T cells regulate sterile, polymorphonuclear neutrophil-rich skin inflammations. In this study, we test the hypothesis of whether CXCL8-producing T cells are present in autoinflammatory diseases like pustular psoriasis and Behçet's disease. Immunohistochemistry of normal skin revealed few CD4+ and CD8+ T cells, few CXCL8+ cells, and no neutrophilic infiltration, whereas in acute exacerbations of atopic dermatitis, numerous CD4+ T cells but few CD8+ T cells, neutrophils, or CXCL8+ cells were detected. In contrast, a pronounced infiltration of neutrophils and of predominantly CD4+ T cells was observed in skin biopsies from pustular psoriasis, Behçet's disease, and acute generalized exanthematous pustulosis, with infiltrating T cells strongly positive for CXCL8 and the chemokine receptor CCR6. Skin-derived T cell clones from pustular skin reactions were positive for CCR6 but negative for CCR8 and secreted high amounts of CXCL8 and GM-CSF, often together with IFN-gamma and TNF-alpha after in vitro stimulation. Moreover, some skin-derived T cell clones from Behçet's disease and from pustular psoriasis predominantly produced CXCL8 and GM-CSF, but failed to secrete IL-5 and IFN-gamma. These cells might represent a particular subset as they differ from both Th1 as well as Th2 T cells and are associated with a unique, neutrophil-rich sterile inflammation. Our findings suggest that CXCL8/GM-CSF-producing T cells may orchestrate neutrophil-rich pathologies of chronic autoinflammatory diseases like pustular psoriasis and Behçet's disease.

Cutaneous CXCL14 targets blood precursors to epidermal niches for Langerhans cell differentiation.

Dendritic cells (DCs) are major constituents of peripheral tissues, where they control immunity to foreign and self-antigens. The process of continuous DC renewal under homeostatic conditions is largely undefined. Here, we demonstrate that CD14+ DC precursors, either derived from CD34+ hematopoietic progenitor cells or isolated from blood, were attracted by the chemokine CXCL14, which is constitutively produced in healthy skin and other epithelial tissues. In a tissue model we show that human epidermal equivalents profoundly affected CD14+ DC precursors, including their suprabasal positioning and survival as well as their differentiation into Langerhans cell-like cells with potent antigen-presentation functions. Our model assigns unprecedented roles to CXCL14 and epidermal tissue as attractant and niche of differentiation, respectively, in the renewal of Langerhans cells under steady-state conditions.

Chemokine-mediated control of T cell traffic in lymphoid and peripheral tissues.

Antigen-driven T cell education and subsequent pathogen elimination present particular challenges for the immune system. Pathogens generally enter the body at peripheral sites such as the skin, gastrointestinal tract or lung, areas from which naïve T cells are largely excluded. Instead, naïve T cells constantly recirculate through secondary lymphoid organs, such as lymph nodes and Peyer's patches, in search for antigen brought to these locations by means of afferent lymphatic channels. Here, antigen-loaded dendritic cells present antigen-peptide-MHC complexes to clonotypic T cells and provide appropriate co-stimulatory signals for immune response initiation. As a result, short-lived effector T cells and long-lived memory T cells are generated that reach the peripheral tissue for participation in immune responses and immune surveillance. Effector and memory T cell relocation is non-random, due to tissue-specific "address codes" that allow proper tissue homing. This process involves adhesion molecules, including selectins, integrins, and corresponding vascular ligands as well as the large family of chemokines and their receptors. Here, we discuss the changes in chemokine receptor expression that occur during T cell activation and differentiation, and the ways in which these changes impact on the migration potential of naïve, effector, and memory T cells. We summarize our current understanding of T cell homing to the T zone and B cell follicles within secondary lymphoid tissues and highlight the two chemokine receptors CCR7 and CXCR5 that recognize chemokines constitutively present either in the T zone (CCR7 ligands CCL19/ELC and CCL21/SLC) or follicular compartment (CXCR5 ligand CXCL13/BCA-1). CCR7 is characteristic for naive and central memory T (T(CM)) cells whereas CXCR5 distinguishes follicular B helper T (T(FH)) cells. In addition, we further subdivide long-lived memory T cells into CCR7-negative effector memory T (T(EM)) cells and peripheral immune surveillance T (T(PS)) cells. The latter term designates the extraordinarily large subset of memory T cells with primary residence in normal (healthy) peripheral tissues. Our current understanding of T(PS) cell migration and function is highly fragmentary, but these cells are thought to provide immediate protection locally at the site of pathogen entry. Here, we propose that the tissue distribution of T(PS) cells is determined by a distinct set of chemokines and corresponding receptors that differs from those operating in secondary lymphoid tissues and inflammatory sites.

Chemokines: control of primary and memory T-cell traffic.

Motility is a hallmark of leukocytes, and breakdown in the control of migration contributes to many inflammatory diseases. Chemotactic migration of leukocytes largely depends on adhesive interaction with the substratum and recognition of a chemoattractant gradient. Chemokines are secreted proteins and have emerged as key controllers of integrin function and cell locomotion. Numerous distinct chemokines exist that target all types of leukocytes, including hematopoietic precursors, leukocytes of the innate immune system, as well as naive memory, and effector lymphocytes. The combinatorial diversity in responsiveness to chemokines ensures the proper tissue distribution of distinct leukocyte subsets under normal and pathological conditions. Inflammatory chemokines are readily detected in lesional tissue and local cellular infiltrates carry corresponding chemokine receptors. Blocking of inflammatory chemokines represents a promising strategy for the development of novel anti-inflammatory therapeutics. This review focuses on a separate class of chemokines, termed homeostatic chemokines, with steady-state production at diverse sites, including primary and secondary lymphoid tissues as well as peripheral (extralymphoid) tissues. More precisely, we discuss the chemokines involved in T-cell traffic during the initiation of adaptive immunity and compare the distinct migration properties of short-lived effector T cells and long-lived memory T cells. Memory T cells are currently classified according to the presence of the lymph node-homing receptor CCR7 into CCR7+ central memory T (T(CM)) cells and CCR7- effector memory T (T(EM)) cells. For better understanding memory T-cell function, we propose the distinction of a third category, termed peripheral immune surveillance T (T(PS)) cells, which typically reside in healthy peripheral tissues, such as skin, lung, and gastrointestinal tract. Localization and relocation of memory T cells is strictly related to their function in recall responses. Therefore, detailed knowledge of their generation and tissue distribution may help to design better vaccination strategies.

Characterization of human T cells that regulate neutrophilic skin inflammation.

It is unknown whether neutrophilic inflammations can be regulated by T cells. This question was analyzed by studying acute generalized exanthematous pustulosis (AGEP), which is a severe drug hypersensitivity resulting in intraepidermal or subcorneal sterile pustules. Recently, we found that drug-specific blood and skin T cells from AGEP patients secrete high levels of the potent neutrophil-attracting chemokine IL-8/CXCL8. In this study, we characterize the phenotype and function of CXCL8-producing T cells. Supernatants from CXCL8(+) T cells were strongly chemotactic for neutrophils, CXCR1, and CXCR2 transfectants, but not for transfectants expressing CXCR4, CX3CR1, human chemokine receptor, and RDC1. Neutralization experiments indicated that chemotaxis was mainly mediated by CXCL8, but not by granulocyte chemotactic protein-2/CXCL6, epithelial cell-derived neutrophil attractant-78/CXCL5, or growth-related oncogene-alpha,beta,gamma/CXCL1,2,3. Interestingly, approximately 2.5% of CD4(+) T cells in normal peripheral blood also produced CXCL8. In addition to CXCL8, AGEP T cells produced large amounts of the monocyte/neutrophil-activating cytokine GM-CSF, and the majority released IFN-gamma and the proinflammatory cytokine TNF-alpha. Furthermore, apoptosis in neutrophils treated with conditioned medium from CXCL8(+) T cells could be reduced by 40%. In lesional skin, CXCL8(+) T cells consistently expressed the chemokine receptor CCR6, suggesting a prominent role for CCR6 in early inflammatory T cell recruitment. Finally, our data suggest that CXCL8-producing T cells facilitate skin inflammation by orchestrating neutrophilic infiltration and ensuring neutrophil survival, which leads to sterile pustular eruptions found in AGEP patients. This mechanism may be relevant for other T cell-mediated diseases with a neutrophilic inflammation such as Behçet's disease and pustular psoriasis.

A skin-selective homing mechanism for human immune surveillance T cells.

Effective immune surveillance is essential for maintaining protection and homeostasis of peripheral tissues. However, mechanisms controlling memory T cell migration to peripheral tissues such as the skin are poorly understood. Here, we show that the majority of human T cells in healthy skin express the chemokine receptor CCR8 and respond to its selective ligand I-309/CCL1. These CCR8(+) T cells are absent in small intestine and colon tissue, and are extremely rare in peripheral blood, suggesting healthy skin as their physiological target site. Cutaneous CCR8(+) T cells are preactivated and secrete proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma, but lack markers of cytolytic T cells. Secretion of interleukin (IL)-4, IL-10, and transforming growth factor-beta was low to undetectable, arguing against a strict association of CCR8 expression with either T helper cell 2 or regulatory T cell subsets. Potential precursors of skin surveillance T cells in peripheral blood may correspond to the minor subset of CCR8(+)CD25(-) T cells. Importantly, CCL1 is constitutively expressed at strategic cutaneous locations, including dermal microvessels and epidermal antigen-presenting cells. For the first time, these findings define a chemokine system for homeostatic T cell traffic in normal human skin.