Inflammation is part of the immune response that occurs in reaction to harmful stimuli like pathogens, injury or damaged cells. It takes place along three main steps:

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1. Introduction

1.1 Inflammation

Inflammation is part of the immune response that occurs in reaction to harmful stimuli like pathogens, injury or damaged cells. It takes place along three main steps:

the recruitment of innate immune cells and molecules to the site of infection to destroy the invading pathogen; the induction of local blood clotting to form a physical barrier to contain the spread of infection; the injured tissue repair. The main features that characterize an inflammatory reaction are explained by the Latin words calor, dolor, rubor and tumor, meaning heat, pain, redness and swelling [1]. These indeed are the effects of different changes in the local blood vessels: heat and redness are due to an increase in vascular diameter that results in the increased local blood flow and its reduction in the velocity. Moreover, the cell-adhesion molecules expressed by the epithelial cells of the blood vessels promote the attachment of circulating cells and their migration into the tissue. Increased permeability of blood vessels accounts for the swelling and pain, characterized by the flow of fluid and proteins from the blood to the tissue. The inflammatory reaction is initiated by immune cells resident in the tissue, able to recognize the harmful stimulus and release molecules responsible for the typical inflammation signs. Among the cells involved in the initiation of inflammation, monocytes/macrophages have a main role in this process and their behaviour is the object of our interest in the present work.

1.2 Innate immunity and monocytes

The immune system consists of specialized cells and molecules that protect the

body from a wide variety of pathogens and other harmful stimuli, and distinguish

them from the organism’s own molecules and tissues. Independently from the nature

of the dangerous agent, the host defence system in mammals is able to readily react

against the microorganisms with an innate immune response. This is genetically

determined and provides a first line of defence against pathogens, playing a crucial

part in the initiation of a rapid and unspecific immune reaction. When the innate

immune response is eluded or is not able to completely control an infection, an

adaptive immune response is triggered. The adaptive immunity develops later,

amplifying the innate immune effects, and is based on gene rearrangement resulting

in the assembly of antigen-specific receptors and antibodies. A key feature of the

adaptive immune response is the immunological memory, which confers lifelong

protective immunity to re-infection with the same pathogen, contributing to a more

rapid and effective response. Different subsets of specialized cells play different

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roles in the innate and adaptive immune systems. The cells of the innate immunity include monocytes/macrophages, granulocytes, mast cells, natural killer (NK) cells, and dendritic cells (DC), the latter forming a link between the innate and adaptive response. The key components of adaptive immunity are the B and T lymphocytes.

The present work is focused on monocytes/macrophages, which are among the first cells of the immune system that are recruited to the site of inflammation. In the blood there are different population of monocytes that are classified based on the expression of cell surface molecules. The classical monocytes are strongly positive for CD14, so they are called CD14

⁺

CD16

^-

monocytes (85% of blood monocytes), while another subtype of monocytes is the CD14

⁺

CD16

⁺

(5%). The two monocyte subtypes are responsible for IL-6, IL-8, and CCL2 and of TNF-" and IL-1!

production, respectively, in response to bacterial lipopolysaccharides (LPS). These CD14

⁺

cells are considered inflammatory monocytes and the expression of CD16 may correspond to an activation/differentiation state of CD14

⁺

monocytes. In addition, there is a third class of monocytes expressing low amounts of CD14 (~7%), the CD14

^dim

CD16

⁺

[2, 3]. This is considered a monocyte subset that patrols blood vessels and has a particular role in the inflammatory response against viruses and nucleic acids.

Macrophages are the mature form of monocytes: the latter circulate in the blood and continually migrate into tissues where they differentiate into mature resident tissue macrophages. Macrophages have different functions in the innate immune response, and also in maintaining homeostasis. They constantly patrol their surroundings for pathogens or signs of tissue damage. When a macrophage recognizes a pathogen, it phagocytises the microorganism and efficiently destroys it and, in addition, it stimulates other immune cells to respond against the danger signal and contribute to its removal. In fact, macrophages are able to orchestrate immune responses by inducing inflammation and secreting signalling proteins that activate and recruit other immune cells. In addition to fighting infections, tissue macrophages are also involved in tissue homeostasis by removing dying cells, cellular debris and harmful substances.

All this is possible because macrophages are characterized by a high plasticity that allows them to adapt their phenotype and their function to the changing environmental conditions, that activate them in different ways. A recent classification suggests three different macrophage activation programmes: the classical inflammatory activation, the wound-healing or alternative activation, and the regulatory activation [4]. Classically activated macrophages are induced by IFN-

# produced by NK cells and T helper 1 (Th1) cells, in addition to TNF-" or microbial

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products. These cells secrete high levels of pro-inflammatory cytokines like interleukin-12 (IL-12), IL-1, IL-6, TNF-" and IL-23, and have high capacity of host defence [5]. Conversely, alternative activation requires IL-4 as inducing stimulus, produced by granulocytes and T helper 2 (Th2) cells, or IL-13 produced by Th2 cells [6]. These macrophages are stimulated to produce high levels of IL-10, the type II IL-1 receptor (IL-1RII) and IL-1 receptor antagonist (IL-1Ra), and low levels of IL- 12 [7, 8]. The function of these cells is to dampen inflammation and secrete components of the extracellular matrix promoting tissue remodelling; they also contribute to the clearance of helminths and nematodes [7-10]. Regulatory macrophages can be generated by stress or during the last stage of adaptive immune responses, induced by different stimuli such as immune complexes, glucocorticoids, prostaglandins, apoptotic cells, transforming growth factors-! (TGF-!) and IL-10 [11-15]. As consequence, the cells start to produce IL-10 and down-regulate IL-12 to suppress immune responses [16]. Thus, monocytes are a heterogeneous population of cells and their polarization/activation state is influenced by local environmental conditions.

1.3 Viral inflammatory response

Many different kinds of bacteria and viruses exist in nature, each with a particular way of interacting with the human host. For this reason, it is not easy to describe a unique immune response against these infectious agents. The immune response against pathogens is mediated by different types of effector cells and by the production of inflammatory mediators. Among the effector cells, monocytes/macrophages have a key role in limiting the infection and in recruiting other immune cells to develop an adaptive immune response. The immediate outcome of the interaction between pathogens and monocytes/macrophages is the production and secretion of cytokines and chemokines that contribute to inducing and maintaining inflammation.

Activated macrophages can secrete a range of cytokines in response to bacterial

LPS, notably IL-1!, TNF-", IL-6, CXCL8 [17]. Conversely, in the response against

viruses CD14

⁺

monocytes produce high levels of IL-6, IL-8 and CCL2 when

stimulated with measles and HSV-1, while CD14

^dim

monocytes produce high amount

of TNF-" and IL-1! [3]. A recent study show that adenovirus vectors can activate the

inflammasome, resulting in increased production of mature IL-1! [18]. In other

studies it has been observed that in HIV/HPV positive tissues the expression and

protein production of IL-6, IL-1! and TNF-" was higher than in HIV/HPV negative

controls [19]. Moreover, monocytes stimulated with viruses respond by releasing

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chemokines such as CXCL8 (IL-8), CXCL10 (IP-10), RANTES (CCL5) and others [20, 21]. NK cells are also involved in containing virus infections, after activation by interferons or by macrophage-produced cytokines. NK cells can kill virus-infected cells by releasing cytotoxic granules inducing cell death. They can also secrete IFN-#

to activate macrophages. Although NK cells are not able to directly eliminate the virus, they can counteract viral replication, while T cells and antibodies come to clear the infection [22].

Among the inflammatory mediators, the most important molecules produced against a viral infection are the type-I interferons (IFN-"/!), which are secreted mainly by the plasmacytoid dendritic cells (pDCs) among leucocytes, and by a wide range of other cell types. IFNs are a class of cytokines that “interfere” with viral replication. In addition to inducing resistance to viral replication, interferons have immunomodulatory effects such as increasing antigen presentation, inducing chemokines that recruit other lymphocytes, activating immune cells such as macrophages, DCs and NK cells. In addition they can inhibit cell growth or promote apoptosis [23, 24].

The first event that is essential for triggering the immune response against viral pathogens is the direct interaction of the invading agent with the host cells, followed by the pathogen entry into the cells mediated by endocytosis or other mechanisms.

Each virus preferentially infect a specific cell type, but it can anyway come in contact with immune cells and trigger an innate immune reaction. For some viruses, the entry in immune cells mediated by phagocytosis, is a sufficient step to induce innate responses [20].

1.4 Pattern recognition receptors

The first step that is necessary to start the fight against a pathogen it is the

recognition of specific nonself motifs. Indeed, the organism can distinguish its own

molecules from those of a microorganism by germline-encoded pattern-recognition

receptors (PRRs) that can recognize molecular structures known as pathogen-

associated molecular patterns (PAMPs). PRRs include the Toll-like receptors

(TLRs), RIG-like receptors (RLRs), Nod-like receptors (NLRs) and the cytosolic

DNA or AIM2-like receptors (ALRs) [25-27]. TLRs are pathogen receptors that are

conserved during evolution. Toll was first identified in Drosophila melanogaster as a

gene that controls the correct dorso-ventral patterning in the embryo, while in adult

insects its signal induces the expression of host-defence mechanisms [28]. TLRs are

type I transmembrane proteins, characterized by an extracellular leucine-rich repeat

(LRR) domain that recognizes and binds ligands, a transmembrane domain, and a

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cytoplasmic Toll-interleukin (IL)-1 receptor (TIR) domain, which interacts with other TIR-type domains and activates signalling pathways [29, 30]. So far, 10 different TLRs have been identified in humans, suggesting different ability in recognition of PAMPs and a sort of redundancy [29, 31-34]. TLRs are expressed by many cell types, such as macrophages, DCs, B cells, T cells, NK cells and some epithelial cells (Table 1), and they are localized in different cellular compartments:

TLR1, TLR2, TLR4, TLR5, TLR6 and probably TLR10 are located at the cell surface, whereas TLR3, TLR7, TLR8 and TLR9 (but also TLR4) are intracellular receptors, expressed on the membranes of endosomes or of the endoplasmic reticulum (ER) [25, 35].

Table 1

Cell distribution/localisation and selectivity of recognition of human TLRs TLR Cellular

localization PAMPs recognized Cytokines

induced Expression pattern TLR1 Plasma

membrane

Triacyl lipopeptides Inflammatory cytokines

B cells,

monocytes/macrophages, NK cells, pDCs, T cells TLR2 Plasma

membrane

Peptidoglycan, LAM, hemagglutinin, phospholipomannan, glycosylphosphophatidyl inositol mucin

Inflammatory cytokines, type I IFNs

Monocytes/macrophages, B cells, NK cells, T cells

TLR3 Endosome ssRNA, dsRNA Inflammatory cytokines, type I IFNs

NK cells, T cells

TLR4 Plasma membrane/

Endosome

LPS, mannan,

glycoinositolphospholipids, envelope proteins

Monocytes/macrophages, DCs, B cells, NK cells

TLR5 Plasma membrane

Flagellin Inflammatory

cytokines

Monocytes/macrophages, T cells, NK cells, DCs

TLR6 Plasma membrane

Diacyl lipopeptides, LTA, zymosan

Inflammatory cytokines

B cells,

monocytes/macrophages, NK cells, pDCs, T cells

TLR7 Endosome ssRNA Inflammatory

cytokines, type I IFNs

pDCs, B cells,

monocytes/macrophages

TLR8 Endosome ssRNA Inflammatory

cytokines, type I IFNs

Monocytes/macrophages

TLR9 Endosome dsDNA, CpG motifs, hemozoin

pDCs, B cells, T cells, monocytes/macrophages, NK cells

TLR10 unknown B cells, pDCs

TLRs on the cell surface recognized mainly molecules derived from microbial

membrane components, whereas the intracellular TLRs are more specific for nucleic

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acids, detecting primarily viruses. Their localization on the endosomal membranes make possible a discrimination between host and foreign nucleic acids, because host DNA is not usually within endosomes [36].

When TLRs recognize PAMPs, they can form homodimers or heterodimers, like TLR1/2 or TLR2/6, and activate a signalling pathway through their cytoplasmic TIR domains. After dimerization, signalling requires the recruitment of TIR-containing adaptor proteins (e.g. MyD88, TIRAP, TRIF, TRAM) that initiate a transduction signal cascade, resulting in activation and nuclear translocation of NF-$B, AP1 or IRF transcription factors. Ultimately, the outcome of TLRs activation is the production of inflammatory cytokines, chemokines and antiviral type I interferons (IFNs), depending on the stimulating agents and on the cell type [35-37].

RIG-like receptors are RNA helicases able to recognize viral RNA in the cytoplasm and comprise three proteins: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated 5) and LGP2 (laboratory of genetic and physiology 2) [38]. RIG-I has been identified as an inducer of IFNs in response to transfection with polyinosinic-polycytidylic acid (polyI:C), a synthetic dsRNA [39].

RIG-I and MDA5 structurally consist of two N-terminal caspase recruitment

domains (CARDs), responsible for activation of signalling pathways, and an internal

DExD/H box RNA helicase domain for RNA recognition and signalling activation

that requires ATP [40]. Moreover, RIG-I and LGP2 have a C-terminal repressor

domain (RD) that keeps the molecules in an inactive state in the absence of

activating RNA [41]. Unlike RIG-I and MDA5, LGP2 does not contain the N-

terminal CARD and previous studies demonstrated that it can inhibit signalling

mediated by RIG-I and MDA5 [40]. However, more studies are needed to better

understand the role of LGP2 in RLR signalling. RIG-I and MDA5 can recognize

different type of viruses and distinguish viral RNA from self-RNA. The 5’-

triphosphate terminal of host transcripts have a 5’ cap of 7-methylguanosine, as for

mRNA, or are removed by maturation processes as for tRNA and rRNA. RIG-I is

able to specifically sense the unmodified viral ssRNA, containing 5’-ppp ends, and

also short dsRNA (<25 bp) with at least a single phosphate at the 5’- or 3’ ends [42-

44]. On the contrary, MDA5 can recognize long dsRNA (>1kb), or particular

ssRNA, that are not sensed by RIG-I [45]. An example is the picornavirus RNA,

which has a viral peptide VPg attached to the 5’-terminal of the RNA that prevent

the recognition by RIG-I [43, 46]. To initiate signalling after binding of RNA, RIG-I

and MDA5 need an adapter protein, interferon-! promoter stimulator 1 (IPS-1), also

known by the names mitochondrial antiviral signalling (MAVS), virus-induced

signalling adapter (VISA), and CARD adapter inducing IFN-! (Cardif) [47-50]. IPS-

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1 interact with RLRs by the CARD domain and this interaction induces the recruitment of signalling molecules, such as tumor necrosis factor (TNF) receptor- associated factor (TRAF) family members [48, 51, 52]. In turn, these molecules activate inhibitor of NF-$B (I$B) kinase (IKK) family members, protein kinases that activate the transcription factors IRFs and NF-$B.

Cytosolic DNA receptors include AIM2, IFI16 (interferon-inducible protein 16), DAI (DNA-dependent activator of IFN-regulatory factors, IRFs) and RNA polymerase III. AIM2 is an IFN-inducible receptor that can bind dsDNA through a HIN domain, and can activate the inflammasome. DAI is a cytoplasmic DNA receptor induced by IFNs and able to bind dsDNA, inducing type I IFNs production.

IFI16 is a newly discovered DNA sensor localized to the nucleus that directly induces type I IFNs and other inflammatory mediators, and is himself induced by IFN-". DAI is cell type-specific but his deficiency does not affect the DNA recognition because of the redundancy of DNA receptors. RNA polymerase III is indirectly involved in initiating signalling, as it transcribes AT-rich dsDNA in dsRNA containing a 5’-triphospate, which can in turn activate RIG-I [26, 53-56].

1.5 Cytokines and their receptors

One of the effects of activation of PRRs is the production by different cells of cytokines, small proteins involved in intercellular communication and modulation of the immune response. These molecules are grouped into different families on the basis of their structure. One of these is the IL-1 family that includes 11 members: IL- 1" (IL-1F1), IL-1! (IL-1F2), IL-1Ra (IL-1F3), IL-18 (IL-1F4), IL-36Ra (IL-1F5), IL-36" (IL-1F6), IL-37 (IL-1F7), IL-36! (IL-F8), IL-36# (IL-1F9), IL-38 (IL-1F10), IL-33 (IL-1F11) [57]. Cytokines are able to respond to stimuli by inducing responses through binding to specific receptors. Other important cytokines are IL-6 and TNF-", and a particular category of cytokines that are called chemokines for their ability to induce chemotaxis in responsive cells. Different studies have demonstrated the involvement of some of these molecules in the inflammatory response against viruses and bacteria, so they were chosen for analysis of our model [3, 19, 20, 21, 58].

IL-1!, IL-1Ra and IL-1RII

IL-1! is one of the main pro-inflammatory cytokines that act during immune

responses. It was considered an endogenous pyrogen because it is an endogenous

molecule that can induce fever [59]. It is produced mainly by activated monocytes,

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macrophages and DCs, but also by B lymphocytes, NK cells and epithelial cells [60].

The expression of the cytokine can be induced by different factors, such as complement components or hypoxia, but most of the mRNA is degraded because of an instability element in the coding region [61]. However, in the presence of TLR ligands (e.g. LPS) or IL-1! itself, IL-1! is translated and accumulates in the cytoplasm as an inactive precursor, pro-IL-1! [62]. The cleavage/activation process of the pro-protein is regulated by an intracellular enzymatic system, in which the cysteine protease capsase-1 cleaves the pro-IL-1!. Mature IL-1! is then secreted to the extracellular space [63]. This cytokine plays a key role in initiating the inflammatory response and is a link between innate and adaptive immunity. To exert its effector function IL-1! binds a membrane receptor complex, comprising the IL-1 receptor type I (IL-1RI), which binds the cytokine, and by IL-1R accessory protein (IL-1RAcP), which enhances the affinity of IL-1! for IL-1RI and is required for signal transduction [64]. IL-1! signalling goes mostly through the MyD88-dependent activation of NF-$B in target cells, and consists in T lymphocyte activation and cytokine production and B cell proliferation. IL-1!-induced activation can be inhibited by IL-1R antagonist (IL-1Ra), which competes with IL-1! to bind IL-1RI and does not recruit IL-1RAcP. Another inhibitor of IL-1! is the type II IL-1R (IL- 1RII) [65]. This receptor exists in a membrane and a soluble form that capture IL-1 respectively on the cell surface or in the microenvironment, but it does not contain the intracellular TIR domain for signal transduction. IL-1RII is considered a “decoy”

receptor because it can bind IL-1! with high affinity, but is unable to initiate signalling pathway. In addition, the membrane IL-1RII can recruit IL-1RAcP after binding to IL-1!, so that the accessory protein is not available to form the receptor complex with IL-1RI [64].

IL-18 and IL-18BP

Another important cytokine of the IL-1 family is IL-18. It was originally identified as an IFN-# inducing factor (IGIF) in Th1 and NK cells, and as a Th1 activating factor in combination with IL-12 [66]. In addition, IL-18 induces cytokine and chemokine production, cell adhesion molecules and cytotoxicity. One report also showed that IL-18 alone could play a role in inducing Th2 cells response, stimulating IL-4 production. Thus, IL-18 can promote the maturation process of Th1 and Th2 cells depending on genetic and microenvironmental conditions. IL-18 is produced mainly by monocytes/macrophages and DCs, but is expressed also by epithelial cells;

keratinocytes can produce inactive IL-18, but they cannot express caspase-1 [67]. IL-

18, like IL-1!, is synthesized as a biological inactive precursor. This pro-protein

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requires a proteolytic processing to be activated to the mature form and this process is regulated mainly by the caspase-1, as for IL-1! [67]. The expression of IL-18 can be stimulated by microbial products like LPS, or by IFNs. The receptor complex for IL-18 binding is composed by the IL-18 receptor chain " (IL-18R") and chain ! (IL- 18R!). Both receptor chains are necessary for signalling, with IL-18R" functioning as binding receptor, while IL-18R! is the accessory protein that increase the binding affinity of IL-18 [67, 68]. The natural inhibitor of IL-18 activity is IL-18-binding protein (IL-18BP). This is a soluble molecule able to interact with mature IL-18, preventing its binding to IL-18R". IL-18BP mRNA is constitutively expressed in different tissues, particularly in immunologically active tissues like spleen and intestinal tract [69]. In humans there are 4 different isoforms generated by alternative splicing: IL-18BPa, b, c, d. Among these, only IL-18BPa and IL-18BPc are able to neutralize IL-18 [70]. IL-18BP expression is regulated by IFN-#, so it depends on IL- 18 in a auto-regulatory feedback mechanism [71].

IL-6

IL-6 is a cytokine produced mainly by monocytes, fibroblasts and endothelial

cells, but also by T and B lymphocytes [72]. The biological activities of IL-6 are

mediated by a receptor complex, which consists of the binding receptor IL-6R, and

by the membrane protein gp130 that transmits the signal. The signalling complex can

be a tetrameric structure, as for other receptors, or a hexameric complex, and the

kind of structure seems to be regulated by the low or the high concentration of IL-6

respectively [73]. IL-6 has also a soluble receptor, but in contrast to the soluble

receptors of other cytokines, the sIL-6R amplifies signal from IL-6, by binding to

and activating membrane-bound gp130. Recent studies demonstrate that the

signalling of IL-6 through sIL-6R promotes pro-inflammatory activities. Conversely,

anti-inflammatory and regenerative activities of IL-6 seems to depend on the

membrane receptor [74]. IL-6 has pleiotropic functions: together with various

chemokines is involved in attraction of neutrophils in the initial phase of an acute

inflammation. IL-6 can also induce recruitment of monocytes and T cells by

enhancing the production of specific chemokines (e.g. CCL2, CCL8, CXCL5) [75,

76]. IL-6 can also regulate monocyte differentiation to macrophages. Upregulation of

cell adhesion molecules on endothelial cells and lymphocytes by IL-6 results in

enhancement of leukocyte transmigration [75]. Eventually, IL-6 stimulates B cell

maturation into plasma cells; it can indirectly stimulate antibodies production; it

induces T cell growth and cytotoxic T cell differentiation [77].

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TNF-"

Tumor necrosis factor-alpha (TNF-") is a cytokine involved in the acute phase of systemic inflammation and it was first identified as a factor with antitumor activity.

It is produced mainly by activated macrophages, but also by lymphocytes and NK cells. TNF-" can be expressed as a transmembrane protein that can also be cleaved in a soluble form, and both of them are biologically active. Its expression can be triggered by bacterial and viral pathogens, or by immunological stimuli. TNF-" acts by binding to its two receptors, TNF receptor 1 (TNFR1) and TNFR2 and has pleiotropic functions. It is involved in regulation of immune cells, inflammation, apoptosis, as well as cell proliferation, differentiation and migration [78, 79]. It also activates vascular endothelium and increases vascular permeability. As a pro- inflammatory molecule, TNF-" stimulates the production of a pro-inflammatory cytokine cascade, recruiting and activating immune cells such as macrophages, which can produce TNF-" that in turn can activates other macrophages and is responsible of their long-term survival [80, 81]. By inducing apoptotic cell death, especially in tumor cells, TNF-" can inhibit tumorigenesis. In contrast, it has also oncogenic activities by inducing cell proliferation and migration [82]. TNF-"

regulates immune cells, is able to induce fever and inflammation mediated by IL-6, IL-18, IL-8, and to inhibit viral replication [78].

CCL5 and CXCL8

Among cytokines there is a particular group of molecules named chemokines, produced by many different types of cells and acting as chemoattractants for immune cells, to recruit them into the site of infection or for promoting the homeostatic tissue replenishment and homing [83]. Chemokines are classified according to the presence of four conserved cysteine residues that play a key role to forming their structure. CC chemokines have two adjacent cysteine residues near the N-terminal; CXC chemokines have two cysteines separated by a single amino acid; C chemokines have only one cysteine near the N-terminal; CX3C chemokines have three amino acids between the two cysteines [84].

CCL5, also known as RANTES (Regulated And Normal T cell Expressed and

Secreted), is a chemokine produced by many cell types such as T cells, macrophages,

platelets, eosinophils, fibroblasts, endothelial and epithelial cells. CCL5 functions by

binding to its receptors CCR1, CCR3 and CCR5 and acting on monocytes, NK and T

cells, basophils, eosinophils, DCs [85]. High levels of CCL5 have been correlated

with inflammatory responses, and the chemokine seems to be involved not only in

chronic inflammation, but also in wound repair and inflammatory angiogenesis. The

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main role of CCL5 is to recruit immune cells to the site of infection and injury. It plays also a role in regulating the immune response against viruses, although this role it is poorly understood. In fact, some studies demonstrate the involvement of RANTES in HIV pathogenesis, while others suggest an antiviral function against HIV and other viruses [86-88].

CXCL8 is a chemokine produced by monocytes, macrophages, fibroblasts, epithelial and endothelial cells, and attracting monocytes, macrophages, neutrophils, basophils and T cells. CXCL8 also promote the activation of monocytes and neutrophils [89]. CXCL8 binds to two receptors, CXCR1 and CXCR2, and is induced by different stimuli, such as inflammatory signals (IL-1!, TNF-", IL-6), stress factors and steroid hormones [90]. It is produced early during an inflammatory response and remains active for a long time, because of its relative long half-life [91]. CXCL8 has also a role as mediator of angiogenesis in wound healing and neoplasia, stimulating endothelial proliferation and capillary tube formation [89].

1.6 Aim of work

Inflammation is an extremely complex reaction that involves many different actors, interacting with each other and with environmental factors. This complexity is an obstacle to the understating of the role and function of individual elements, so that a simplification of the system is necessary if we want to focus on the specific behaviour of a particular element, like monocyte during a viral inflammatory reaction. The aim of this work is to build an in vitro model based on human monocytes that simulates the key steps of a prototypical inflammatory reaction against viruses, from pathogen-induced triggering to eventual resolution of inflammation and re-establishment of homeostasis. This work could help in defining the profile of the physiological response against a viral challenge and in comparing it with a bacterial-induced immune reaction. The use of culture allows to highly controlling and easily manipulating the environment conditions. Our intent was trying to mimic the in vivo cell behaviour in a way as much realistic as possible. For this reason, freshly isolated monocytes were preferred to cell lines, as the latter acquired the ability to proliferate indefinitely through random or induced mutation, introducing an artificial condition to the culture. Moreover the in vitro model offers the opportunity to avoid the use of laboratory animals, according to the method of