RICERCA ITALIANA     

Analysis of the ability of Toll-like receptors to modulate effector and regulatory functions of innate immunity components

Università degli Studi di Roma "La Sapienza"

Abstract

The different cell populations of the innate immunity (namely monocyte/macrophages, and Natural Killer (NK) and gamma/delta T lymphocytes) coordinately concur to the early phase of the response against pathogens. Beside the direct antimicrobial activity, which is crucial for the limitation of microbial dissemination, these cell populations also modify the environment of the inflammatory site, and induce the recruitment and the activation of other cell populations, through the rapid secretion of cytokines and chemokines. In fact, multiple immunoregulatory circuits are put into action in the early phase of infection; they lead to the induction, potentiation, and support of the coordinate activities of the different cell populations belonging to the innate response, and set up the cytokine environment which instructs the adaptive response.
In this context, a family of recently identified membrane receptors, the Toll-like receptors (TLR), plays a central role, as they mediate the recognition of highly conserved microbial molecular structures (the pathogen-associated molecular patterns, PAMP), and determine the interaction of innate immunity cell populations with molecular components of pathogens. TLR-triggered cell functions depend on still partially unknown intracellular signalling pathways, and result in the production of proinflammatory cytokines, dendritic cell maturation, and T helper 1-type response polarization; moreover, TLR ligands can also directly modify antimicrobial capabilities. In particular, TLR3 and TLR9 intracellular receptors are specific for double stranded RNA (dsRNA) molecules, and unmethylated CpG-rich oligodeoxynucleotides CpG ODN), respectively, which are common products of microbial methabolism. The mechanisms responsible for TLR3 and TLR9-dependent modulation of innate immunity have been inadequately elucidated yet. Their ability to powerfully activate cells of the innate response may lead to the exploitment of their ligands as adjuvants, in novel immunostimulatory and/or vaccinal strategies. The cooperation among different effectors of the natural response (eventually potentiated by immunostimulatory therapeutical use of TLR ligands) could also overcome evasion strategies of pathogens. The tight and inextricable link between innate and adaptive immunity has important implications for a more rational vaccine design and immunotherapy. Given the therapeutic and prophylactic implications, direct evaluation of the regulatory circuits in the innate response and its impact on acquired immunity is required.
This project is aimed at the characterization of TLR3 and TLR9 ability to modulate the functions (microbicidal activity, cytoxicity, cytokine secretion, survival) of various cell populations of the innate response (macrophages, gamma/delta T lymphocytes, NK cells), with particular attention devoted to the regulation of cell survival and of the crosstalk between the different effector cells. The results obtained through this research program will lead to a better definition of the molecular basis responsible for the effects of microbial constituents, available either in the course of natural infections, or under an adjuvant immunotherapeutic approach, on the activation, the proliferation, and the persistence of different cell populations at the infection site. The novel information obtained through this study will constitute a rational basis for the design of immunomodulatory strategies aimed at the potentiation or the reconstitution of natural immunity functions in a variety of infectious pathological conditions. <<<

Principal Investigator

Gabriella PALMIERI Universita' degli Studi di ROMA

Research Objectives

Natural immunity components (including macrophages, gamma/delta T lymphocytes, and Natural Killer (NK) cells) play a pivotal role in the innate responses against infections, and in the instruction and polarization of the adaptive response. Their ability to perform pathogen killing, or the lysis of infected cells, is crucial for the limitation of microbial dissemination; their immunoregulatory activity relies on the capacity to rapidly secrete cytokines and chemokines, which induce the recruitment and activation of other cell populations at the inflammatory site, and to establish multiple cell-cell interactions, through distinct membrane receptors endowed with either activating, inhibitory, or costimulatory function. The functions of innate immunity cell populations are finely tuned by the interaction with molecular components of pathogens. In this context, a family of recently identified membrane receptors, the Toll-like receptors (TLR), plays a central role, as they mediate the recognition of highly conserved microbial molecular structures (the pathogen-associated molecular patterns, PAMP), and determine the functional activation, differentiation, and cell survival. In particular, TLR3 and TLR9 intracellular receptors, are specific for double stranded RNA (dsRNA) molecules, and unmethylated CpG-rich oligodeoxynucleotides CpG ODN), respectively. Their ability to powerfully activate cells of the innate response may lead to the exploitment of their ligands as adjuvants, in imunostimulatory therapeutical strategies, although most evidence on their biological role have been obtained in heterologous expression systems, and thus await to be validated in the physiological host cell.
This project is aimed at the characterization of TLR3 and TLR9 ability to modulate effector and regulatory functions of various cell populations of the innate response (macrophages, gamma/delta T lymphocytes, NK cells), with particular attention devoted to the regulation of cell survival and of the crosstalk between the different cell population. The results obtained through this research program will lead to a better definition of the molecular basis responsible for the effects of dsRNA and CpG ODN microbial constituents, available either in the course of natural infections, or under an adjuvant immunotherapeutic approach, on the activation, the proliferation, the functions, and the persistence of different cell populations at the infection site. The novel information obtained through this study will constitute a rational basis for the design of immunomodulatory strategies aimed at the potentiation or the reconstitution of natural immunity functions in a variety of infectious pathological conditions.
This proposal, which is articulated over two years, has the following objectives:
1. To study the expression, modulation, and functions initiated by TLR3 and TLR9 on different cell populations belonging to the innate response.
The groups afferent to the proposal are aimed to identify the mechanisms regulating macrophage, NK cell, and gamma/delta T lymphocytes responsivity to pathogen-derived nucleic acids, and to elucidate the role of TLR3 and TLR9 ligands in the regulation of their effector functions. Within this objective:
Unit Dieli will analyze the expression and modulation of TLR3 and TLR9 on different subsets of Vgamma9/Vdelta2 T lymphocytes, and will also investigate the intracellular distribution of the receptors (in collaboration with Unit Palmieri); this Unit will also evaluate the ability of TLR3 and TLR9 specific ligands to modulate proliferation, cytokine and chemokine production, cytotoxic activity and the progression from memory to effector stage in Vgamma9/Vdelta2 T cells;
Unit Fraziano will assess the capability of TLR3 and TLR9 expressed on human macrophages to augment natural defense mechanisms against tubercular infection; in particular, they will evaluate the possibility that dsRNA TLR3 ligand induces microbicidal activity of human macrophages, as already demonstrated in the case of other TLR;
Unit Palmieri will analyze the expression, modulation, and compartmentalization of TLR3 and TLR9 in human NK cells; they will also study the effect of TLR specific ligands on NK cell functions (natural cytotoxicity and ADCC, cytokine and chemokine production), and on the modulation of surface receptors involved in cell-cell interaction.
2. To analyze TLR3- and TLR9-dependent signalling pathways in different components of the innate response.
The evidence regarding the signalling pathways initiated by distinct TLR are still partial, and have chiefly been obtained in heterologous experimental systems. The involved Units are aimed at obtaining a better comprehension of the molecular basis that regulate TLR3 and TLR9 signalling ability in different cells of the innate system. To this purpose:
Unit Fraziano, that has recently demonstrated the ability of CpG ODN to activate phospholipase D in macrophages, will assess the activation of phospholipases D, C, and A2, and of NADPH oxidase induced by dsRNA and CpG;
Unit Palmieri will analyze the ability of TLR3 and TLR9 ligands to induce the activation of MAPK family members, and of NFkappaB and IRF3 transcription factors, and will identify the receptor-proximal events involved. In collaboration with Unit Fraziano, the activation of MAPK family members, of NFkappaB and IRF3 transcription factors, and of phospholipases will be extended to human macrophages.
3. To analyze the role of different TLR-triggered signal transduction pathways in the modulation of effector, antimicrobial and regulatory functions of innate immunity cells.
The proponents will identify, on macrophages, gamma/delta T lymphocytes, and NK cells, the modalities underlying the ability of TLR ligands to enhance the killing of the pathogen and/or of the infected cell, to affect apoptosis or cell survival, and to modulate cell-cell interactions among the different participants to the innate response, with particular attention to the role of NKG2D activating receptor and its ligands. In the context of this objective:
Unit Dieli will assess the ability of TLR9 ligands to induce the expression of NKG2D ligands on macrophages and dendritic cells, and thus activate Vgamma9/Vdelta2 T lymphocytes; this Unit will also analyze the role of this receptor:ligand couple in the different effector functions of Vgamma9/Vdelta2 cells and in their interaction with macrophages and NK cells (in collaboration with Units Fraziano and Palmieri);
Unit Fraziano will investigate the involvement of different TLR-activated phospholipases and their products in the antimicrobial activity of Mycobacterium tuberculosis-infected macrophages, as well as in regulating the biogenesis and maturation of phagolysosome. In collaboration with Units Dieli and Palmieri, they will assay the antimicrobial activity of gamma/delta T and NK cell lines, activated by TLR3 and TLR9 ligands, against Mycobacterium tuberculosis-infected macrophages;
Unit Palmieri will study the role of different signalling events in the TLR-mediated functions of human NK cells. In collaboration with Unit Fraziano, they will analyze the role of NKG2D ligands in the interaction between NK cells and Mycobacterium tuberculosis-infected or TLR3- and TLR9-stimulated macrophages. They will also ascertain whether TLR-mediated stimulation can affect the activation-induced apoptosis of NK cells. <<<

First Results

The expected results obtained in the first phase of the project will consist of:
- the comprehension of the mechanisms regulating TLR3 and TLR9 expression and modulation on innate immunity effectors;
- the characterization of the role of TLR3- and TLR9-specific ligands on the activation, functions and differentiation of macrophages, NK cells, and gamma/delta T lymphocytes;
- the identification of the main signal transduction pathways initiated by TLR3 and TLR9 in distinct cell populations of innate immunity.The expected results of this second phase of the project will be represented by:
- the establishment of the role of TLR3 and TLR9 ligands in directly or indirectly modulating the interactions and the functional crosstalk between the different components of natural immunity;
- the identification of TLR-triggered signalling events involved in the biogenesis and maturation of macrophage phagolysosome, and in the different functional activities of NK cells;
- the definition of the role of cellular environment in modulating the signalling ability of TLR3 and TLR9;
- the establishment of the role of TLR ligands in affecting NK cell survival. <<<

Timescale

24 months

National and international background

The different cell populations of the innate immunity concur to the early phase of the response against pathogens. Monocyte/macrophages, and Natural Killer (NK) and gamma/delta T lymphocytes, are important effectors of the natural resistance against infections by many intracellular pathogens. Although they have been initially characterized for the direct antimicrobial activities, these cell populations also modify the environment of the inflammatory site, induce the recruitment and activation of other cell populations, and thus contribute to the instruction of the adaptive response, through the rapid secretion of cytokines and chemokines.
Tissue macrophages and circulating monocytes, which rapidly infiltrate the inflammatory site, represent an early warning system for pathogens, by acting as both sentinels and the first line of defense against infections. Besides carrying out the intracellular killing of a variety of microbes, they also secrete a vast array of biological mediators, such as cytokines and chemokines, leading to the coordinated recruitment and activation of other cell populations responsible for microbial eradication (1); they also play a role in the instruction and polarization of adaptive response, by acting as antigen presenting cells to T lymphocytes. Antimicrobial effector functions and immunoregulatory activity of macrophages are finely tuned by soluble mediators and by membrane receptors involved in the interactions with pathogens or with the other participants to the immune response.
T lymphocytes bearing a gamma/delta receptor account for a small population (5%) of peripheral blood T cells, but are the predominant lymphocyte subset in the epithelia. Most gamma/delta T cells in the peripheral blood express a Vgamma9/Vdelta2-type TCR, and recognize non-proteic substances characterized by phosphate groups, thus named phosphoantigens. These low-molecular weight ligands may be of microbial origin, or ubiquitous metabolites, or still uncharacterized components of tumor cells (2). Differently from alpha/beta lymphocytes, phosphoantigen recognition by Vgamma9/Vdelta2 T cells requires neither processing nor MHC-dependent presentation (2). They show cytotoxic activity against a number of different tumor cell lines, and can also kill Mycobacterium tuberculosis-infected macrophages and intracellular pathogens, through mechanisms dependent on granule-associated perforin and granulysin (3, 4). Recent evidence have led to the hypothesis that Vgamma9/Vdelta2 lymphocytes are functionally heterogeneous; in particular, Unit Dieli has characterized the presence of four subsets in the peripheral blood, displaying different phenotype, functional properties and localization (5). Although the progression from naive to terminally differentiated is presumable, the factors responsible for this process are mostly unknown, and it is still possible that besides phosphoantigens, cytokines and polyclonal stimuli also contribute to the maintenance and/or the expansion of memory-type cells, and to their progression to effector and terminally differentiated cells.
Natural Killer (NK) cells represent a small lymphocyte subpopulation in peripheral blood and in some lyumphoid and non lymphoid organs (6). They rapidly infiltrate infection sites, where they carry out their cytotoxic functions, either "natural", or antibody-dependent, thanks to the presence, on the vast majority of these cells, of CD16, the low-affinity receptor for IgG Fc fragment, FcgammaRIII (7). NK cell functions are strictly regulated by a balance between positive (provided by a vast array of activating receptors) and negative (initiated by MHC class I-specific inhibitory receptors) signals, following interaction with target cells and with the other participants to the immune response (8-12). NKG2D activating receptor, in particular, recognizes cellular ligands which are rapidly induced on different cell types upon infection (MIC-A and B, ULBPs) (8, 9). NK cell functions depend on multiple signalling pathways which, as studied by Unit Palmieri too, include tirosine and serin/threonine kinases, lipid kinases, small GTPases (belonging to rho/rac and ras families), phospholipases, and mitogen-activated kinases (MAPK); the ultimate effectors of the different signalling cascades organize and coordinate the functional response (13-17). The amplitude and dynamics of in vivo NK responses, and the persistence of these cells at the infection site, are tightly regulated by a variety of cytokines, both of the innate and adaptive immunity, by heterogeneous substances collectively known as "biological response modifiers" (BRM) (6, 18), and by multiple cell-cell interactions; in this context, it has been previously demonstrated that CD16 engagement induces the apoptotic program of IL-2-activated NK cells, mainly through Fas ligand (FASL)- and TNF-mediated pathways (19, 20).
It has been recently identified a family of evolutionarily conserved membrane receptors, the Toll-like receptors (TLR), which are involved in the recognition of molecular components of microbial origin (Pathogen-Associated Molecular Patterns, PAMP) (21, 22). They provide a rapid and efficient surveillance system of the innate immunity, capable of guiding and activating the subsequent antigen-specific responses of adaptive immunity (21-23). Ten different members have been currently identified, specific for distinct molecular patterns. Some TLR are specific for nucleic acids of pathogen origin: TLR3 has been demonstrated to recognize double-stranded RNA (dsRNA) molecules, a common intermediate in the replicative cycle of many viruses (24), and TLR9 binds to unmethylated, CpG-rich oligodeoxynucleotides (CpG ODN), abundantly represented in DNA of bacterial origin (25); very recently, TLR7 and TLR8 have been shown to recognize single-stranded RNA (26, 27). This TLR subgroup is endowed with a a distinct subcellular distribution: it has been reported that TLR9 localizes in a lysosome-like intracellular compartment, whose integrity is crucial for receptor function; this feature seems to be shared by TLR3 also, in a cell type-dependent fashion (28, 29). The ability of different TLR to recognize a repertoire of microbial molecular patterns, their surface or phagosomal localization, and their powerful signal transduction pathways, make the members of TLR family an excellent system for pathogen recognition.
The expression and function of TLR3 and TLR9 in the hemopoietic compartment have been initially characterized on dendritic cells and macrophages, where the specific nucleic acid ligands trigger the production of proinflammatory cytokines endowed with antimicrobial activity, such as type I IFN, IL-12, IL-6, TNF, promote dendritic cell maturation, and polarize a T helper 1 type response (21-23); the possibility that these ligands, similarly to what reported for other TLR (30), also modulate cell survival is still unknown.
Beside directing the development of adaptive immunity, TLR3- and TLR9-mediated activation of innate immunity populations can also be directly involved in the antimicrobial activity (23, 31). In particular, microbicidal activity of human and murine macrophages against different intracellular pathogens is upregulated by stimulation with CpG ODN, through still partially uncharacterized mechanisms, as demonstrated by Unit Fraziano and other authors (32-35). The ability of TLR ligands to induce macrophage anti-mycobacterium activity is particularly relevant, as it has been unequivocally ascribed only to few molecules, sofar (36-38). Finally, a key role in the development of these microbicidal mechanisms is played by macrophage-derived phospholipase D (35, 38).
TLR expression and function has been poorly investigated in lymphocytes of the innate response: evidence regarding TLR3 and TLR9 expression and modulation are scarce, and the ability of these cells to directly interact with dsRNA and CpG ODN is still controversial (28, 39-42); on the other hand, the capacity of in vivo stimulation with polyinosinic-polycytidylic acid (poly I:C), a synthetic mimic of dsRNA, to rapidly enhance NK responses has been shown by many authors (18, 43, 44). Recent results obtained by Unit Palmieri indicate that highly purified human NK cell populations, either fresh or short-term cultured, and a panel of NK cell tumors, express TLR3 mRNA; they also show that NK cell stimulation with poly I:C augments both natural and CD16-dependent cytotoxicity, and stimulates CXCL10 (IP10) chemokine production (Pisegna and Palmieri, manuscript in preparation). Although expression of TLR3 and TLR9 on human Vgamma9/Vdelta2 T lymphocytes has not been evaluated sofar, recent reports indicate that some murine gamma/delta lymphocyte subsets constitutively express various TLR (45) that, upon stimulation with the specific ligand, determine their activation.
The molecular mechanisms responsible for TLR ability to modulate cell functions have been only partially identified. TLR signalling ability strictly depends on adaptor proteins, such as MyD88 (which associates with different TLR, including TLR9), and TRIF/TICAM-1 (specific for TLR3) (46), which confer selectivity to the functional capability of different TLR. These adaptors promote the association of still partially uncharacterized multimolecular complexes with the receptor intracellular domain, which are responsible for the initiation of enzymatic cascades leading to the activation of IRF3 and NFkappaB transcription factors, and of MAPK family members (ERK, JNK, and p38 kinases) (21, 22, 46), whose activity is ultimately responsible for the synthesis of cytokines, chemokines and costimulatory molecules. Related to this, Unit Palmieri has recently demonstrated that dsRNA stimulation induces p38 MAPK activation in human NK cells (Pisegna and Palmieri, manuscript in preparation), while results reported by Unit Fraziano indicate that CpG ODN treatment induces phospholipase D activation in M. tuberculosis infected macrophages, and underlie the role of its enzymatic products in the antimicrobial activity (35).
It is becoming increasingly clear that the functions of innate immunity cell populations may be significatively affected by microbial molecular components. The immunoregulatory circuits involving these cells, either through soluble mediators (cytokines and chemokines) or by membrane receptors, lead to both the potentiation of effector functions, and to the instruction of adaptive response. For example, Vgamma9/Vdelta2 and NK cells rapidly produce TNF alpha and IFN gamma, which play a crucial role in dendritic cell maturation, potentiation of macrophage microbicidal activity (6, 18, 43, 47), and in classical T cell or NK cell activation; moreover, they also secrete high levels of chemokines such as CXCL8, CCL3, CCL4, CCL5, XCL1 (6, 18, 48-50), which are extremely active to induce the recruitment of macrophages, NK cells and activated T cells to the inflammatory site (43, 51); activated macrophages, in turn, produce IL-12, potently able to stimulate IFN gamma secretion by NK cells. The prevalence of distinct cytokine and chemokine outlines may have a crucial role in determining the Th1/Th2 polarization of the adaptive response to infections.
Innate immunity functions are also finely regulated by cell-cell interactions; for example, phosphoantigen-dependent activation of Vgamma9/Vdelta2 is significatively augmented by the presence of macrophages or dendritic cells (52), suggesting the involvement of costimulatory molecules. NKG2D receptor, in particular, constitutively expressed on both NK cells (8, 9), and Vgamma9/Vdelta2 lymphocytes (53), may represent an important component in the interaction between different effectors at the infection site; in fact, besides being involved in the NK-mediated recognition of pathogen-infected target cells (8, 9, 50), it has been recently demonstrated that the interaction between NKG2D and MIC-A, which is expressed on stressed epithelial cells, and on macrophages and dendritic cells infected by Mycobacterium tuberculosis, strongly upregulates the phosphoantigen-induced activation of Vgamma9/Vdelta2 lymphocytes (53). Moreover, it has been recently reported that the TLR-mediated stimulation of murine macrophages upregulates the expression of NKG2D ligands (54).
Multiple immunoregulatory circuits are thus put into action in the early phase of infection; they lead to the induction, potentiation, and support of the coordinate activities of the different cell populations belonging to the innate response, and set up the cytokine environment which instructs the adaptive response. These cell-cell interactions may be profoundly modulated by the TLR-dependent recognition of microbial molecular constituents.
Starting from this rational basis, the possible exploitment of TLR as new formulation vaccine adjuvants has recently proposed (55). In this context, mucosal immunization with inactivated viruses, or with their components, in association with CpG ODN, has been shown to confer a strong protective immunity, of both systemic and mucosal type, in in vivo models of HSV-2 and HIV infections (56, 57). <<<