Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy
1Correspondence: Department of Biotechnology and Bioscience, University of Milano–Bicocca, Piazza della Scienza, 2, 20126 Milano, Italy. E-mail: Paola.Castagnoli{at}unimib.it
ABSTRACT
At the 38th Annual Meeting of the Society for Leukocyte Biology held in Oxford this year, the biology of dendritic cells (DCs) and macrophages was discussed. In particular, functional genomics approaches were presented to investigate transcriptional changes during microbe and phagocytes interactions. Here, we report functional genomics studies likely to be of interest to the Journal of Leukocyte Biology readers with a particular emphasis on DC biology. DCs are professional antigen-presenting cells, which are essential for the initiation and regulation of natural killer, T, and T regulatory cell responses. Immature DCs, resident in peripheral sites, are specialized in antigen capture and continually sample soluble and particulate antigens in their local environment. DCs express receptors for cytokines, chemokines, endogenous danger signals, and microbial structures. The interactions between DCs and microorganism are complex, but progress in the past few years has shed light on several aspects of these processes. Infectious disease is the result of an intimate relationship between pathogens and hosts. Thus, understanding the cross-talk between host and pathogen is essential to improve our knowledge of infectious disease. Functional genomics and proteomics applied to DCs and macrophage biology are now providing powerful tools to dissect, at the molecular level, host-pathogen interactions.
Key Words: dendritic cells • functional genomics • host-pathogen interaction
INTRODUCTION
The immune system has evolved to protect individuals from invading pathogens. The generation of an appropriate response to fight the infections is essential, and higher animals have evolved complex recognition systems to ensure that relevant responses are induced [1 , 2 ]. The cross-talk between innate and adaptive immunity is regulated by a variety of cells, their secretion products, and their interactions with endogenous mediators. Antigen-presenting cells (APCs), in particular, dendritic cells (DCs) and macrophages, have a key role in regulating and modulating the immune response. Macrophages act as sentinel cells by engulfing foreign bodies by active phagocytosis and endocytosis and play a scavenger function. However, macrophages are poor activators of naive T cells. In contrast, DCs are professional APCs able to modulate adaptive responses through recognition of pathogens, directly or indirectly, by sensing perturbations in the microenvironment such as infectious agents, cell damage, or inflammation [3 , 4 ]. DCs represent the most potent APCs in the immune system, with the unique ability to induce not only primary immune responses against invading pathogens but also immunological tolerance. Following activation by appropriate signals, peripheral DCs begin to mature and migrate from the periphery to draining lymph nodes, where maturation is completed following interaction with the appropriate T cells [5 , 6 ]. DCs and macrophages are continuously produced from common hematopoietic stem cells within the bone marrow [7 , 8 ]. Developing DC precursors are thought to migrate from bone marrow to blood [9 ], from where they supply the interstitial DCs, which can be observed throughout the nonlymphoid peripheral organs of the body [10 ]. DCs have been found in heart, liver, thyroid, pancreas, bladder, kidney, ureter, and skin, where they are named Langerhans cells [11 , 12 ]. Fully developed DCs have also been observed in the circulatory networks of the body, including blood [13 ] and afferent lymphatics, where they are called veiled cells [14 ]. These cells represent DCs emigrating from peripheral organs into lymphoid tissues [14 ].
DCs AS SENSORS OF INFECTION
DCs have characteristics that make them ideally suited to monitor their environment for the presence of pathogens and to facilitate their uptake [10 ]. DCs represent a first line of defense against invading pathogen and are localized in close proximity with mucosal tissues of the respiratory tract, the gut, or the genito-urinary tract. Recently, it has been shown that DCs have molecular mechanisms to open the epithelial tight junctions and sample the gut lumen preserving the epithelial barrier [15 , 16 ].
Tissue resident immature DCs can identify pathogens directly by recognizing microbial-associated molecular patterns using highly conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) [17
]. Different TLR family members specific for various microbial molecules have been identified in mice and humans. Binding of TLRs to their microbial ligands leads to activation of phagocytes and direct killing of pathogens, as well as to the release of proinflammatory cytokines and antimicrobial peptides [18
]. In addition, these molecules activate DCs and are therefore important in the initiation of adaptive immune responses. Binding of ligands to TLRs triggers activation of the nuclear factor-
B (NF-B) signaling pathway [18
] required for the induction of inflammation. NF-B signaling induces the expression of chemokines, cytokines, adhesion molecules, matrix metalloproteases, nitric oxide synthase, and enzymes, which regulate prostanoid synthesis, leading to the initiation of an inflammatory response [17
].
In addition to TLRs, immature DCs also express several C-type lectins, such as the mannose receptor, DEC205, and DC-specific intercellular adhesion molecule (ICAM)-grabbing nonintegrin, which recognizes carbohydrate structures on pathogens [19
]. DCs also express multifunctional, soluble PRRs involved in innate immunity and inflammation, such as short and long pentraxins. The prototype pentraxin 3 (PTX)3 is highly expressed by DC and macrophages. PTX3 is able to recognize microbes, activate complement, and facilitate pathogen recognition by phagocytes [20
]. DCs sample the environment via multiple mechanisms of antigen uptake. These include receptor-mediated endocytosis through C-type lectins and Fc receptors [10
]. They also have a high capacity to endocytose particulates and solutes nonspecifically through phagocytosis and macropinocytosis [21
]. Although many of these pathways appear to be used for uptake of pathogen-related molecules, they may also be relevant for uptake of self-antigens [22
]. Immature DCs also express 
vß3-integrins, vß5-integrins, and CD36, which facilitate continuous uptake of apoptotic cells [23
]. This mechanism may be important in DC-mediated maintenance of peripheral self-tolerance [22
]. Indeed, internalized apoptotic cells are processed, and antigens are presented in association with major histocompatibility complex molecules.
THE PLASTICITY OF DC FUNCTION
In mice and humans, an unresolved paradigm in understanding DC biology is the existence of multiple DC functions, with a distinct role in natural killer (NK) cell or T and T regulatory activation [24 25 26 27 28 ]. An example of DC plasticity is provided by the study of intestinal DCs. One of the key questions in mucosal immunity is how tolerance to commensal bacteria is induced. We have shown that mouse DCs are widely distributed in the gut lamina propria and are involved in direct bacterial uptake across mucosal surfaces, preserving the integrity of the epithelial barrier [15 ]. In this environment, DCs sample pathogens and commensal bacteria through the formation of transepithelial dendrites. The extension of dendrites is regulated by the expression of the fractalkine receptor (CX3CR1 [16 ]). The decision between the induction of active immunity or tolerance will depend on the local microenvironment, which will likely play an important role in determining DC activation. Indeed, it is still not clear whether different DC subsets have different predetermined functions or whether the nature of the microenvironment and the maturation signals instruct the functionality of each individual DC during the immune response. It has been reported recently that human intestinal macrophages are characterized by a strong anergy, which provides unresponsiveness to immunostimulatory bacteria [29 ]. It remains to be established if this also applies to DCs. Conversely, it has also been shown that human DCs stimulated with different bacteria induce a distinct pattern of cytokines, sustaining the notion that human DCs have functional plasticity in their response to microbial stimuli [30 ]. Therefore, the factors that determine a given DC function probably depend on the state of DC maturation as well as on the local microenvironment.
MICROBIAL-INDUCED DC TRANSCRIPTOMES
The interactions between DCs and microorganisms are complex, but progress in the past few years has shed light on several aspects of these interactions. Pathogen-induced phenotypic changes in host cells are often associated with marked changes in gene expression. The advent of DNA microarray technology has greatly expanded our ability to monitor changes in the abundance of transcripts. Over the past 10 years, many studies have been published, which document changes in gene expression that occur during infections. These data have shown that host cells undergo marked reprogramming of their transcriptome during infection involving the differential expression of thousands of genes. Of particular interest are those studies investigating cell responses to live pathogens; a number of studies have investigated the effect of bacterial component, particularly, lipopolysaccharide (LPS) [31 ], which was able to induce a similar gene expression profile as whole bacteria, illustrating the major role played by LPS in stimulating the early DC response to bacteria infections [32 ]. In contrast, lipoteichoic acid elicited differential expression in only a small set of genes then using live Staphylococcus aureus [33 ]. Moreover, the response of peripheral blood mononuclear cell to dead Bordetella pertussis differed greatly from that induced by live Bordetella pertussis [34 ], indicating the relevance of using live pathogens to fully understand host-pathogen interactions.
We have applied functional genomics studies to dissect the kinetics of DC responses to pathogens [35
36
37
]. Mouse DCs have been treated with phylogenetically different organisms, such as bacteria, helmints, and parasites. A common core of the host-transcriptional program, which is shared among different organisms, has been generated. The group of genes always induced were those mediating inflammatory responses such as the chemokines CXC chemokine ligand 9 [CXCL9; monokine induced by interferon-
(IFN-)], CXCL10 (IFN-inducible protein 10), CC chemokine ligand 3 [CCL3; macrophage-inflammatory protein-1 (MIP-1)], CCL4 (MIP-1ß), CXCL1 [growth-related oncogene 1 (GRO1)], CCL1 (I-309), CXCL5 (granulocyte chemotactic protein-2), CXCL2 (GRO2), CCL17 (thymus and activation-regulated chemokine), CCL5 (regulated on activation, normal T expressed and secreted), CCL22 (macrophage-derived chemokine), and the proinflammatory mediators tumor necrosis factor (TNF-), interleukin (IL)-1, and IL-1ß. Another distinct group of genes was the IFN-induced genes; among these, we observed the expression of glucocorticoid-attenuated response gene (GARG)-16, GARG-39, GARG-49, IFN-inducible gene (IFI)-1, IFI-47, and IFN-stimulated gene (ISG) factor-3.
Genes known to activate the immune response were also induced, such as the transcription factors NF-B, activated protein-1, JUN, FOS, factors that mediates the effect of IFN [signal transducer and activator of transcription (STAT)-1, STAT-4, and STAT-5A], and components of the signal-transduction cascade [myeloid differentiation primary-response protein 88, TNF receptor-associated factor (TRAF)1, and TRAF6]. All microorganisms analyzed, also induced genes important to limit the immune response, such as inhibitor of B- (IkB-) and IkB-
. Genes involved in lymphocyte activation (CD80, CD86, CD40), cell adhesion [CD44, CD34, CD36, ICAM1, integrin-5 (ITGA5), ITGA1, ITGA4], and interleukins are commonly produced; in particular, IL-15, IL-2, IL-1, IL-6, IL-12B, Ebi3, and lymphotoxin are known to induce proliferation, activation, or differentiation of immune cells.
It is interesting that macrophages and DC up-regulate similar sets of genes in response to pathogens [38 ], as they share similarity on more than 96% of basal gene expression. Conversely, their response to various types of pathogens differs dramatically. In particular, the ISG shows higher induction in pathogen-activated DCs than in macrophages similarly activated. Also, the cytokines produced are slightly different, and we have shown that mouse and human DCs are able to produce large amounts of the IL-2 cytokine early after pathogen encounter [35 ], suggesting a role for DC-derived IL-2 in the induction and control of innate immune responses. In fact, we have been able to show that DC-derived IL-2 is involved in the activation of NK cells during the initial phase of bacterial infections [35 ]. One of the differences, although not exhaustive, which we could measure between macrophages and DCs, resides in the inability of macrophage to produce IL-2. Therefore, it seems that DCs have a few specialized functions clearly distinct from other phagocytes.
At the 38th Annual Meeting of the Society for Leukocyte Biology, held in Oxford this year, David. A. Hume (University of Queensland, Australia) has also summarized our current understanding of macrophage biology using a genome-wide transcriptional profiling induced by LPS. An effective immune system requires rapid and appropriate activation of inflammatory mechanisms but equally rapid and effective resolution of the inflammatory state. Macrophage activation is a key determinant of pathology in a variety of inflammatory diseases. Hume and co-workers have reported that bacterial products such as LPS and CpG DNA down-modulated the expression of the colony-stimulating factor-1 receptor (CSF-1R) on primary murine macrophage cell membranes. The ability of bacterial products to down-modulate the CSF-1R rendered macrophages unresponsive to CSF-1 as assessed by Akt and extracellular signal-regulated kinase 1/2 phosphorylation. Using cDNA microarrays, they have identified in macrophages a cluster of similarly CSF-1-repressed genes, which were repressed by CSF-1 and were induced by LPS, only in the presence of CSF-1. LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors, suggesting that this mechanism leads to transcriptional reprogramming in macrophages. This new mechanism of macrophage activation, in which LPS as well as other TLR agonists regulate gene expression by switching off the CSF-1R signal, is particularly interesting: This finding indeed provides a biological relevance to the well-documented ability of macrophage activators to down-modulate the expression of the CSF-1R at the cell surface. The requirement for CSF-1 during DC homeostasis is still an open question. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced green fluorescent proteins in cells of the myeloid lineage, Hume and co-workers [39 , 40 ] have shown that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation, supporting a close relationship between DCs and macrophages.
CONCLUSIONS
Coevolution of microbes and the immune system has resulted in the selection of sophisticated mechanisms, which provide advantages to the host or to the microbe. We are now beginning to understand the complex nature of the receptors repertoire expressed on DCs, which recognize specific molecular patterns of infectious agents and transduce signals that condition the immune response.
Understanding host gene expression changes following microbial encounter represents one view of the host-pathogen interaction. The use of arrays that contains probes for human and pathogen genes will allow monitoring the activities of host and pathogen genomes simultaneously. The use of these arrays in time-course experiment will show how gene expression changes in the host correlate with those observed in the microorganism. A detailed understanding of the common responses is likely to give insight into the basic molecular mechanisms governing these interactions, whereas genes that are regulated in a cell-specific or pathogen-specific matter will provide information about specific gene expression programs that regulate specific cells upon pathogen encounter. These studies will ultimately allow the dissection of a regulatory network, which underlies the transcriptional response to infection.
Received October 3, 2005; revised January 31, 2006; accepted February 1, 2006.
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