NF-jB as a potential therapeutic target in microbial diseases
The failure of conventional vaccines or antimicrobials to combat newly emerging pathogens such as new influenza viruses or antibiotic-resistant bacteria provides significant challenges in the identification of innovative therapeutic approaches and targets for microbial infections. Such therapies, directed towards host-cell molecules, may represent alternative options where conventional approaches face difficulties. We will largely focus on those strategies that directly target the host inflammatory response, specifically those that result in the activation of the nuclear transcription factor (NF)-kB. NF-kB plays a central role in the cellular stress response and in inflammation by controlling the expression of a network of inducers and effectors that define responses to pathogens. Therefore, the modulation of NF-kB activation and its signaling pathway offer an exceptional therapeutical strategy that could benefit from targeting a single host regulatory pathway. The use of NF-kB inhibitors or enhancers will be possible only if modulation between the host’s and pathogen’s advantage can be reached. Since different pathogens have developed various mechanisms to alter the activation of NF-kB, the present review will mainly focus on the role of NF-kB in microbial infections, highlighting its importance as a therapeutic target and reviewing the current understanding of how NF-kB inhibition can be considered a potential paradigm for the development of novel antimicrobial therapies.
Introduction
The immune cellular responses to bacterial or viral infections require the activation of specific signaling pathways that transduce extracellular signals from the cell surface to the nucleus1 where binding or modification of the activity of transcription factors play a critical role in the regulation of gene expression. Among these transcription factors, the nuclear factor-kB (NF-kB) family proteins are involved in many vital.
Dr Mariateresa Vitiello attended Biological Science under- graduate studies and obtained her Degree at the University of Naples ‘‘Federico II’’ in 1991. She became Consultant in Microbiology and Virology in 1995 and received her PhD in Microbiology in 1999. She held a Postdoctoral position in Micro- biology from 1999 to 2004 and from 2004 Dr Vitiello has been providing rigorous training in research and diagnostic skills to a wide range of students both graduate and undergraduate.
Marilena Galdiero
Dr Marilena Galdiero completed her medical undergraduate studies at the University of Naples ‘‘Federico II’’ in 1990. She had a PhD in Microbio- logy and research experience abroad before obtaining a faculty position at the Second University of Naples. She was appointed associate professor of Microbiology at the Faculty of Medicine of the Second Uni- versity of Naples in 2007. She has published in the area of microbiology focusing her studies on the biological activities She has published more than 50 peer-reviewed articles in the field of microbial pathogenesis and microbial-induced signal transmission.
Consequently, NF-kB, expressed in numerous cell types, controls the expression of several proinflammatory and immunoregulatory genes determining the outcome of the host innate immune response to microbial pathogens and activating almost every cell type involved in the immune response: neutrophils, macrophages, lymphocytes, endothelial and epithelial cells. Thus, while transient NF-kB activation represents a part of the normal physiological regulation, excessive and persistent activation of NF-kB leads to proinflammatory cytokine, chemokine overproduction and chronic inflammation. Several recent studies suggest that the hyperactivation of the NF-kB pathway is frequently associated with the development and progression of many human diseases including autoimmune disorders, cancer, cardiovascular and neurodegenerative disorders.3–6
Therefore, NF-kB appears to be an extremely attractive target for therapeutic intervention and a considerable amount of effort has been put into devising strategies that block NF-kB signaling.7 Recently, the study of the complex relationship between microbial pathogens and their host has led to a more extensive interpretation of how this liaison alters the host metabolism through signaling proteins, but also how pathogens hijack cellular signaling pathways and transcriptional factors, controlling them for their own advantage. The equilibrium between the advantage of the host and that of the pathogen from NF-kB activation is different depending on the kind of infection, in fact, micro- organisms have evolved strategies to exploit this cellular transcription factor to optimize their intracellular stage. Under- standing the molecular mechanisms utilized by the pathogen to interfere with the NF-kB pathway may enable us to develop novel antimicrobial therapies and more effective vaccines. The future challenge will be to conceive the most effective and selective inhibitor for a given physiological process. The under- standing of how specific modifications can regulate NF-kB activity in response to distinct stimuli could improve our knowledge in designing more selective inhibitors of NF-kB.
Since both the host and the pathogen are able to employ and gain an advantage from NF-kB activity, a therapeutical use of NF-kB inhibitors or enhancers will be possible only if modula- tion between the host’s and pathogen’s advantage can be reached. Therefore, we need to take into account the adverse effects that each treatment could exert on the normal cell functions. Inhibition of NF-kB to combat microbial infections can offer great clinical potential, but may be detrimental at the same time. Here, we will focus on the involvement of NF-kB in the pathogenesis of microbial infection, and discuss the possibility of therapeutic approaches based on the specific modulation of the NF-kB pathway.
NF-jB signaling pathway
The NF-kB signaling pathway has been the focus of extensive research over the last 20 years for its essential beneficial role in vertebrate physiology and for its deregulation in several human diseases.NF-kB belongs to the family of Rel proteins (Fig. 1). In mammalian cells, five members of the NF-kB family have been identified and cloned including NF-kB1 (p50 and its precursor member of the Biostructures and Bioimages Institute of the National Research Council of Italy. Recent research activities focus on the biological activity of bacterial porins and mechanisms of drug intracellular delivery.
The was appointed associate professor and then full professor of Microbiology in 2004. His more recent research interests are focused on the study of host–microbial interactions and antimicrobial development.
Fig. 1 (A) NF-kB/Rel proteins have in common a 300-amino acid conserved N-terminus (known as the Rel homology domain (RHD)). The RHD contains important coded information which enables dimerization with other Rel members. The NF-kB/Rel proteins can be subdivided, according to differences in the sequences of their C-terminus: (a) RelA, RelB and c-Rel proteins, in which the C-terminal region contains the transactivating domain (TAD), which is essential for NF-kB-mediated gene transactivation. RelB contains a leucine zipper domain (LZ) in the N-terminal sequence; (b) p50 and p52 are produced as precursor proteins (p105 and p100, respectively) which are cleaved in the cytosol, by a ubiquitin-proteasome pathway, to produce the smaller mature peptides (p50 and p52). The C-terminal domains contain seven repeats of a conserved 33 amino acid sequence, called ankyrin (ANK) repeats (also present in IkB proteins). The N-terminus and C-terminus are linked by a flexible glycine-rich region (GRR). (B) IkB proteins (IkBa, IkBb, IkBg, IkBe, Bcl-3) have in common a conserved core of five to seven ankyrin repeat-motifs that are similar to the Rel/NF-kB protein. (C) IkB kinase proteins (IKKa, IKKb, IKKg). IKKa and IKKb contain protein-kinase domains at their N-terminals, and leucine-zippers (LZ) and helix-loop-helix motifs (HLH) in their C-terminal regions; IKKg (also known as NEMO) is an unrelated structural/regulatory subunit which receives and integrates signals from upstream signaling pathways and receptor- associated proteins.
In most resting cells NF-kB dimers are seized in an inactive form in the cytoplasm through physical association of their RHDs with NF-kB inhibitor proteins, called collectively IkBs (IkBa, IkBb, IkBg, IkBe, BCL3, p100, p105, and IkBL) which mask the DNA binding domains.10,11
NF-kB1 and NF-kB2 are first expressed as precursor proteins (p105 and p100, respectively) and are involved in the control of NF-kB activity. They present structural homology with IkBs in their C-terminal portions and thus act as IkB molecules when unprocessed, but provide the active subunits p50 and p52 upon processing.
Exposure of cells to a variety of stimuli leads to the rapid phosphorylation and proteolytic degradation of IkB, which allows NF-kB to translocate to the nucleus activating gene transcription.Although many stimuli including proinflammatory cytokines, bacteria, fungi, viruses and their products, physiological stress conditions, apoptotic mediators and mitogens have the potential to activate the NF-kB pathway, the responses elicited are both cell and stimulus specific, suggesting that not all activators utilize the same signaling components and cascades. Two major NF-kB activating signal transduction pathways have been described: the classical and alternative pathways (Fig. 2).12 The classical (or canonical) pathway depends on the IKK (IkB kinase) complex composed of two catalytically active subunits of IKKa and IKKb, and a regulatory scaffolding subunit, NEMO (NF-kB essential modulatory), which is also called IKKg, while the alternative (or noncanonical) pathway depends on IkKa homodimers and NF-kB inducing kinase (NIK).13,14 In the classical pathway, NF-kB activation is mediated by IkBa degradation.
In particular, upon stimulation by highly conserved pattern recognition receptors (PRRs) that recognize both endogenous and exogenous ‘‘danger signals’’,15 which include pathogen- associated molecular patterns (PAMPs) such as lipopoly- saccharide (LPS), lipoteichoic acid, and peptidoglycan as well as endogenous danger signals,16 signal transduction pathways induce the activation of IKK complex. This event leads to the phosphorylation and ubiquitination of IkBa at specific N-terminal serine residues and its consequent proteasomal degradation allowing the nuclear translocation of associated NF-kB subunits (p50–p65) DNA binding and gene transcrip- tion. Activation of the classical pathway usually occurs within minutes and triggers the induction of several immediate early genes involved in immune or inflammatory responses, apoptosis and cell proliferation.
Fig. 2 Schematic representation of NF-kB signaling pathway in microbial diseases. In the model illustrated, bacterial and viral antigens interact with different receptors at the cell surface that in turn activate intracellular signaling molecules via a set of adaptor proteins and kinases initiating a cascade of phosphorylations. The activation of classical signaling pathway (IKKb/IKKg-dependent) leads to proteasomal degradation of cytosolic IkB proteins and subsequent nuclear translocation of predominately RelA/p50 dimers promoting a rapid response that ultimately results in the transcription of genes involved in inflammation, immunity, cell growth and apoptosis. The alternative signaling pathway (IKKa-dependent) activates the cleavage of the p100 precursor bound to RelB; processing of p100 results in nuclear translocation of RelB/p52 dimers that activate target genes involved in development and differentiation.
The alternative NF-kB pathway is NEMO-independent and is initiated by a subset of tumor necrosis factor (TNF) receptor family members such as B-cell activating factor,17 lymphotoxin b,18 and CD40 ligand.19 In particular, NF-kB- inducing kinase (NIK) is a serine/threonine kinase that plays a pivotal role in the activation of alternative signaling.20 Inter- action of the stimuli with cell surface receptors leads to the activation of NIK that directly phosphorylates IKKa to trigger the phosphorylation and proteolytic cleavage of p100 to produce p52. The p52 subunit can dimerize with RelB, p65 or c-Rel. However, only the p52/RelB heterodimer is able to translocate to the nucleus, while p52/p65 and p52/c-Rel are activated through the classical pathway.14 In contrast to the classical pathway activation, p100 processing and p52/RelB activation are delayed for several hours.
However, also pathogenic stimulation by LPS, Helicobacter pylori, human T-cell leukaemia virus and Epstein–Barr virus21–24 has been shown to induce alternative signaling.Focusing on LPS, recent studies have demonstrated that this structural component of the outer envelope of all gram- negative bacteria induces activation of both canonical and non-canonical pathways of NF-kB in human colonic epithelial cells. The non-canonical pathway requires phosphorylation of BCL10 (serine 138) and NIK, opening new avenues for therapeutic interventions.25
Several reports have suggested complex feedback between the canonical and non-canonical pathways and the potential of the non-canonical pathway to provide a more sustained activation of specific genes.14,25,26
Recently published data demonstrate that alternative NF-kB signaling down-regulates proinflammatory cytokine production in dendritic cells, suggesting an important role of the alternative NF-kB pathway in the regulation of immunity.27 Therefore, cross-regulation between the classical and alternative signaling pathways appears to be crucial in promoting an optimally protective response characterized by the balance between inflammation and self-tolerance.28
Role of NF-jB in inflammation
Currently, the NF-kB pathway is one of the most important proinflammatory gene expression regulators activated at sites of inflammation in several disease processes such as inflammatory, neoplastic and infectious diseases.4,5,29 Also, there is a better understanding of the precise pathophysiological mechanism of NF-kB that leads to inflammation.
Inflammation is a response induced by noxious stimuli and conditions, such as infections involving the delivery of blood cells at the infection site. This response has been characterized best for microbial infections such as bacterial infections, in which it is triggered by receptors of the innate immune system, the Toll-like receptors.
Among the potential initiating stimuli (infection, tissue injury, tissue stress and malfunction), the activation of an immune response is associated only with infection-induced inflammation. Furthermore, a successful acute inflammatory response results in the elimination of the infectious agents followed by a resolution and repair phase, which is mediated mainly by tissue-resident and recruited macrophages.30
If the acute inflammatory response is unable to eliminate the pathogen, the inflammatory process persists and acquires new characteristics. The neutrophil infiltrate is replaced with macrophages followed by T and B lymphocytes, and if the combined effect of these cells is still insufficient, the onset of a chronic inflammatory state can be evidenced.
Several studies have demonstrated that inflammatory response represents the ‘‘common soil’’ of multifactorial diseases, including a wide variety of conditions such as chronic inflam- matory disorders, cardiovascular, neurodegenerative and autoimmune diseases, cancer and various symptoms of aging.31 One of the most intriguing aspects of studying inflammation is the plurality of the inflammatory mediators continuously discovered. In this context, NF-kB is well characterized as a primary mediator of inflammatory responses during infection and immune reactions.
It is well established that one of the major functions of NF-kB is its key involvement in creating an effective immune and inflammatory response against bacterial and viral infections, not only inducing the transcription of proinflammatory cyto- kines (TNF-a, IL-1b, IL-6, IL-8), but also regulating the expression of adhesion molecules (ICAM-1, V-CAM-1, E-selectin).32–34 This indicates an important role of leukocyte adhesion and transmigration resulting in an accumulation of immune cells at inflammation sites. NF-kB also induces the expression of enzymes whose proteins have a connection to the pathogenesis of the inflammatory process, such as inducible cyclooxygenase, inducible nitric oxide synthase and nitric oxide. All of these molecules play critical roles in key biological events involving cell recruitment, attachment, differentiation, proliferation and activation which contribute to an active and specific inflammatory response.However, one unresolved question is whether blocking a single proinflammatory cytokine will suffice for an adequate treatment. In this regard, anti-NF-kB therapy, which is focused on inhibiting the expression of some inflammatory mediators, including TNF-a and IL-1, might represent the future in development of anti-microbial treatments.
NF-jB and bacterial infection
NF-kB modulates the expression of many proteins with important functions in innate and adaptive immunity regulating numerous aspects of immune function required for resistance to infection. Bacterial infections are a major cause of mortality and morbidity.35 It is known that an adequate immune response against bacterial pathogens depends on appropriate recognition of the invading organism by the host immune cells. The recognition of PAMPs by PRRs leads to the activation of several mitogen-activated protein kinase (MAPK) pathways36 and subsequently specific gene expression by activating several transcription factors, including NF-kB and activator protein-1 (AP-1).
Understanding the cellular and molecular events that occur during the interaction of individual pathogenic components with host cells is critical for preventing both infection and the resulting tissue damage. In this regard, bacterial surfaces are of utmost importance when considering the interaction with host cells in the context of pathogenesis and immunity to infections.37 Indeed, several bacterial components, including peptidoglycan,38 outer membrane proteins (OMPs),39,40 and LPS,41 are involved in the modulation of pathogen–host cell interactions.
In gram-negative bacteria, the MAPKs activation initiated by components present on their outer membrane such as LPS or OMPs plays an important role in transducing inflammatory signals associated with bacterial infection.40 Among the OMPs, porins are the most represented and are usually present in the form of homotrimers allowing the diffusion through the outer membrane of small hydrophilic solutes.42 In addition to their pore function, bacterial porins also appear to be targets of the immunological system. Currently, there are several reports that highlight their ability to stimulate a protective role.43,44 Porins induce many cellular responses including cellular activa- tion, cytokine release, and immunological effects.40,43,45 Several reports have demonstrated that porin-stimulated cells are activated with an evident phosphorylation of many cellular proteins, such as the MAPKs and nuclear transcription factors such as NF-kB, AP-1, and STAT1/STAT3.46,47
Synthetic peptides corresponding to loops 5, 6, and 7 can mimic the role of intact porin P2 from Haemophilus influenzae type b (Hib) by inducing the activation of the MAPK cascade, essentially c-Jun amino-terminal kinase (JNK) and p38,48 and the release of proinflammatory cytokines TNF-a and IL-6.49 Results from MacLeod et al.50 have shown that PorB from Neisseria meningitidis is able to induce protein tyrosine kinase (PTK) activity, the phosphorylation of ERK1/2 and IkBa, leading to NF-kB nuclear translocation in murine B cells.
Similarly, porins of Salmonella enterica serovar Typhimurium were shown to stimulate protein kinase A (PKA), PKC and PTK.51 Several studies illustrate that Bartonella henselae as well as B. henselae-derived OMPs induce an NF-kB-dependent upregula- tion of E-selectin and ICAM-1 in endothelial cells, which in turn results in enhanced polymorhonuclear rolling and adhesion.52 Similarly, the outer membrane ‘‘blebs’’ that are actively released by N. gonorrhoeae trigger an NF-kB activation which up-regulates the expression of the carcinoembryonic antigen-related cellular adhesion molecule to allow neisserial colonization and facilitate phagocytosis and cellular invasion.53,54
Hence, NF-kB modulation is able to shift the equilibrium between the advantage of the host or of the pathogen in consideration of the nature of the infection itself. Although during bacterial infection, microbial pathogens activate cellular signal transduction pathways that induce NF-kB activation, it has been shown that in some cases, the pathogens may interfere with the development of immune responses associated with antimicrobial immunity through active manipulation of the NF-kB system for their advantage. Enteroinvasive bacterial pathogens such as Salmonella, Shigella, Listeria and Escherichia species induce NF-kB DNA-binding and a wide range of cytokines.55–58 Increased activation of NF-kB can also contribute to the development of tissue damage providing a strategy for the pathogen to contaminate the environment. Indeed, continuous NF-kB activation and release of proinflammatory mediators may lead to disruption of the epithelial layer making the bacterial invasion easier and inducing a severe diarrhea in the host, an environmental contamination, and a consequent spread of the infection.59
However, for Salmonella, as well as for many other facultative intracellular pathogens, the disease is mediated by a dynamic interplay between host responses to bacterial components and bacterial virulence mechanisms that are triggered upon entry into the host environment. Interplay between host resistance factors and bacterial virulence factors is critical in determining the outcome of the infection. Given the role of NF-kB in both inflammation and apoptosis, it is not surprising that certain gram-negative bacteria have also evolved mechanisms to modulate NF-kB activity during infection. In this regard, gram-negative bacteria like Salmonella,60 Shigella,61 Yersinia62 and Pseudomonas63 have evolved powerful secretion machinery called type III secretion system that is used to optimize their virulence effect by delivering bacterial effectors to the membrane or into the host cell cytoplasm.64 For example, Yersinia pestis, Y. enterocolitica and Y. pseudotuberculosis produce effector proteins which can inhibit kinase activation within the target cells.62,65 The Yersinia outer protein YopJ targets the superfamily of MAPK kinases (MKKs), blocking both phosphorylation and subsequent activation of the MKKs inhibiting the extracellular signal-regulated kinase, JNK, p38, and NF-kB signaling path- ways, preventing cytokine synthesis and inducing apoptosis.66,67 YopJ-related proteins in S. enterica serovar Typhimurium and in plant pathogens suggest that this family of proteins could play a fundamental role in the modulation of host signaling responses.68,69
Other pathogens are able to inhibit NF-kB activation by interfering with the degradation of IkB. For example, uro- pathogenic E. coli (UPEC) was able to abrogate urothelial responses by blocking NF-kB translocation to the nucleus and by inhibiting NF-kB-dependent transcription in response to either LPS or TNF-a stimulation.70 As a consequence of the block of NF-kB, UPEC may also alter the expression of proinflammatory cytokines, thus leading to delayed or reduced neutrophil infiltration and associated phagocytosis of the pathogen. Furthermore, the ability of UPEC to invade bladder epithelial cells suggests that the NF-kB inhibition decreases inflammation and may allow the pathogen more time to be internalized by urothelial cells where they escape from the immune system and create a source of recurrent infection.71
As previously described, the pathogenesis of a number of chronic inflammatory diseases has been linked to dysregulated functioning of nuclear transcription factor NF-kB.Porphyromonas gingivalis is one of the suspected periodonto- pathic bacteria and is frequently isolated from the periodontal pockets of patients with chronic periodontal (CP) disease.72 Jotwani et al.73 demonstrated that P. gingivalis LPS, a key factor in the development of periodontitis, unlike E. coli LPS, induces an increase of the p50 : p65 ratio in monocyte-derived dendritic cells suggesting that increased levels of transcriptionally repressive p50 may be characteristic of CP disease and might be a result of suboptimal NF-kB activation and dendritic cell maturation by P. gingivalis. These findings suggest the possibility of selectively targeting the NF-kB p50 to break immunosuppres- sion and resolve chronic inflammation in CP disease.
Actually, although the treatment of bacterial infections with antibiotics is one of the key concepts of human medicine, it is evident that the effectiveness of antibiotics has become quite limited owing to an increase in bacterial antibiotic resistance, which represents a global health problem with a strong social and economic impact; for this reason, there is an urgent need for the development of molecules with a novel mechanism of action. Therefore, understanding the mechanisms used by pathogens to interact with the NF-kB system provides a broader picture of the complex interactions between the host and the pathogen and may represent an alternative therapeutic approach for a wide variety of inflammatory bacterial infections.
NF-jB and viral infection
In the past decade, significant advances in our understanding have been made regarding the mechanisms used by viruses to interfere with, and manipulate, the diverse host protective responses. It is well known that the survival of viruses strongly depends on their ability to evade or subvert the cellular innate protective antiviral responses.74–76 Host cell responses to patho- genic viruses are commonly mediated by phosphorylation- regulated signaling pathways affecting, for example, changes in gene expression patterns. These signaling processes can be initiated by the cell as a defense against a viral pathogen, but can also be used by the virus to support its replication.
Interaction of viral surface proteins with host cellular surfaces has been shown to initiate a cellular reaction that leads to the activation of host defense pathways that results in the production of cytokines such as interleukins, TNF-a and interferons (IFNs).77,78 In particular, induction of IFN-b gene expression is a tightly regulated process, in fact, the signal transduction pathway TANK-binding kinase 1/IFN regula- tory factor-3 (IRF-3) has been identified as an essential element for the activation of IFN-b gene expression.79 In addition to IRF-3 activation, efficient induction of IFN-b usually requires the activation of the NF-kB,80 suggesting that it is an essential component in the innate protective response to virus infection.
Among the host transcription factors, NF-kB is involved in the regulation of host innate antimicrobial and inflammatory responses. In this context, viruses, which obviously have established an intimate relationship with eukaryotic cells, are in many cases known to both depend on and to interfere with NF-kB.81,82
The NF-kB pathway is a prime target for viral evasion, therefore, several viruses have developed strategies to manipu- late this signaling through the use of multifunctional viral proteins that target either its activation or its direct signaling cascade members.83 The expression of a single viral protein is sufficient to NF-kB activation as seen with Tax from human T-cell leukemia virus (HTLV-1),84 E3/19K from adenovirus,85 and HBx from hepatitis B virus (HBV).86
The study of viral immunomodulatory proteins might help us to uncover new human genes that control immunity, and their characterization will increase our understanding not only of viral pathogenesis, but also of normal protective mechanisms. In addition, viral proteins indicate strategies of protective modulation that might have a therapeutic potential.
Work from our group recently revealed that hydrophobic domains of viral proteins, used by viruses to gain access into susceptible cells, are able to induce several transduction path- ways that lead to cytokine (IFN-b and IL-10) production, an event that appears to be dependent on early activation of AP-1 and NF-kB.34
However, it is now clear that viruses can directly activate NF-kB and utilize it in different ways.76 In particular, several studies indicate that NF-kB activation could be a strategy evolved by different viruses to block apoptosis and prolong survival of the host cell in order to gain time for replication and increase viral progeny production.87 This has been documented for some human viruses, including human immunodeficiency virus type 1 (HIV-1),88 HTLV-1,89 influenza virus,90 hepatitis B91 and C92 viruses (HCV), as well as poxviruses and herpesviruses93 that utilize different strategies to modulate the NF-kB pathway in order to facilitate and enhance viral replication avoiding host protective responses.
The ability of HIV virus to interfere with NF-kB signaling is associated with an inhibition of the immune response, even if several studies demonstrated that HIV replication is NF-kB- dependent.94 In particular, multiple mechanisms appear to be involved in the stimulation of the IKK complex mediated by HIV. The envelope glycoprotein gp120 can signal NF-kB by engaging the CD4 receptor in two different but related path- ways, one involving activation of the T-cell-specific tyrosine kinase p56lck 95 and the other utilizing phosphatidylinositol 3-kinase.96 Therefore, IKK rapidly phosphorylates IkBa and its two NH2-terminal serine residues. The phosphorylation and degradation of IkBa induce NF-kB release that can activate transcription of viral genes. In particular, the activa- tion and subsequent binding of NF-kB to the enhancer region in the long terminal repeat (LTR) of HIV-1 are essential in the regulation of the HIV-1 gene expression and transcription.97
Several reports indicate that deletions and/or mutations in NF-kB elements on the LTR abolished the transcription of viral genes and effective HIV replication.98,99 Moreover, HIV LTR- driven transactivation and HIV-1 replication can be blocked by IkBa overexpression.100 This observation suggests that the balance between NF-kB and IkBa at the nuclear level would be a key mechanism involved in both the maintenance of HIV latency and the induction of low level HIV replication.101
Among HIV-1-encoded proteins, Tat also inhibits IkBa degradation and nuclear translocation of RelA, participating in the regulation of NF-kB activity through the stimulation of IKK complex, PKR and PKC.Two examples of how pathogens can either inhibit NF-kB activation or, alternatively, promote induction of these transcrip- tion factors, depending on the cell types, are represented by two members of the gammaherpesvirinae subfamily, Epstein–Barr virus (EBV) and Kaposi Sarcoma herpesvirus (KSHV). While the ability of these viruses to inhibit NF-kB activity promotes apoptosis of activated T cells,103 the viral transformation of B cells and the development of lymphoproliferative disease are associated with NF-kB activation.104
Therefore, in the case of EBV and KSHV, there is a critical interdependence between the virus and host cell to modulate NF-kB activity, both positively and negatively, which has allowed survival and propagation of these viruses throughout evolution.EBV and KSHV, as other herpesviruses, have the tendency to develop a latent mode of infection in the host cell with a very restricted pattern of viral gene expression. Our current understanding supports that both EBV and KSHV latent infections persistently activate the NF-kB cascade and this activation is associated with the viruses ability to induce cellular transformation and tumor formation.105 Moreover, constitutive NF-kB activation is an effective strategy developed by EBV and KSHV to sustain latent infection, favoring viral persistence in vivo.
It is well known that EBV and KSHV encode some latent viral proteins each with a specific role in the virus-induced NF-kB activation and maintenance of latency.106 One of the best-characterized examples are represented by the EBV Latent Membrane Protein (LMP)-1 and KSHV FADD-like interleukin- 1-beta-converting enzyme (FLICE)/caspase-8-inhibitory protein (vFLIP), a latent viral protein that can prevent apoptotic cell death.
LMP-1 activates NF-kB preferentially through the NF-kB subunit c-Rel. This activation mechanism is essential for the survival of EBV-transformed cells and constitutes the most powerful transforming pathway used by EBV.107 KSHV-encoded vFLIP also can activate the IKK complex inducing activation of both the classical (IKKa/b—IkBa—p50/RelA) and alternative (NIK—IKKa—p52/RelB) NF-kB pathways108–110 and uses these pathways to promote cellular proliferation, survival, cytokine secretion and virus latency.
This observation provides a rationale for the use of NF-kB inhibitors as therapeutic agents for the future treatment of herpesvirus-associated malignancies. Although our knowledge of several compounds that inhibit NF-kB has increased substan- tially in the past few years, several studies speculate that for successful treatment of viral malignancies, NF-kB inhibitors will have to be used in conjunction with other drugs, perhaps those inducing apoptosis or inhibiting proliferation.111
Therapeutic developments to affect NF-jB signaling
NF-kB activation during microbial infections has been generally interpreted as a protective response of the host against the invading pathogen, but viruses and bacteria may well subvert NF-kB activity to their advantage to enhance replication. In fact, many pathogens can directly activate NF-kB and use the pathway to block apoptosis and, therefore, prolong survival of the host cell to increase their own chances to persist in the host and produce a sustained infection. This has, for example, been shown for HIV where Vpr was able to efficiently arrest the cell cycle at the G2/M stage and also to control apoptosis.112 In this view, NF-kB seems to be a good candidate as a potential target for inhibition of HIV-1 replication. Targeting NF-kB reduces problems of resistance because NF-kB is normally present in T-cells and is not subject to mutations. Recent reports have focused on the effect of a new NF-kB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) for HIV-1 infection and indicated that NF-kB is a potential molecular target for controlling active virus replication by regulating early and late phases of the HIV-1 life cycle.113,114 These observations support the importance of the develop- ment of novel antiretroviral agents having different targets for inhibition of HIV-1 replication that could be effective in the case of highly active antiretroviral therapy failure due to the cross-resistance of HIV-1.
A further example is given by the encephalomyocarditis virus (EMCV) where NF-kB-mediated suppression of apoptosis is a clear expression of EMCV virulence.115 Schwarz et al.115 showed that p50 knock-out mice (defective in NF-kB signaling) survive an EMCV infection that readily kills normal mice. The attenuation of the virus infection in mice lacking p50 could be explained by rapid apoptosis of infected cells which allows host cell phagocytes to clear infected cells before the viral sustained replication leading to a reduction of the viral burden and, as a consequence, survival of the mice.
The information on the role of NF-kB in microbial infections have reached a considerable amount and are still rapidly increasing, turning our attention to the potential therapeutic opportunities that NF-kB interference could offer. In brief, we can assert that the host utilizes NF-kB to trigger a protective response against the invading pathogen which, on the other hand, manipulates NF-kB-mediated cellular functions to gain a benefit for its survival. Since both the host and the pathogen are able to employ and gain an advantage from NF-kB activity, it is imperative to identify gaps for the therapeutical use of NF-kB inhibitors or enhancers in order to modulate the equilibrium between the host’s and pathogen’s advantage. Hence, in viral infections it should be possible to interfere with microbial replication either by enhancing antiviral signaling or by inhibiting proviral signaling, taking into account the adverse effects of each treatment on normal cell functions. Therefore, inhibition of NF-kB to combat microbial infections can offer great clinical potential, but may be detrimental at the same time. In fact, the balance between therapeutic benefit and potential damage that could derive from an impaired immune reaction has to be carefully pondered in order to design therapeutic strategies able to modulate NF-kB expression in consideration of the timing and modulation of each microbial infection. Understanding the molecular mechanisms utilized by different pathogens to interfere with the NF-kB pathway will enable us to exploit NF-kB as a new drug target for increasing our arsenal against microbial diseases.
Although therapeutic strategies targeting cellular signaling pathways, such as NF-kB, are still in a very early phase of pre-clinical development, it has been proven that it is indeed feasible without harmful side-effects or the emergence of resistance.Blocking the cellular mechanisms required for microbial replication may be an alternative approach to inhibit the growth and replication of pathogens. Inhibitors of microbial-induced intracellular signaling cascades came into focus, since the respective signaling processes are central regulators of many cellular responses that may support virus replication. The great advantage is that the virus cannot replace the missing cellular function and, thus, emergence of resistance should not easily occur. Focusing on the influenza viruses, several antiviral drugs have been developed to interfere with specific events in the replication cycle. Among them, the inhibitors of viral uncoating (amantadine), nucleoside inhibitors (ribavirin), viral transcription and neuraminidase inhibitors (zanamivir and oseltamivir) are reported as examples of traditional virus-based antiviral strategies.116 However, for most of them the efficacy is often limited by toxicity and the almost inevitable selection of drug-resistant viral mutants. Thus, the discovery of novel anti- influenza drugs that target general cell signaling pathways essential for viral replication, irrespective to the specific origin of the virus, would decrease the emergence of drug resistance and increase the effectiveness towards different strains of influenza virus.
The replication cycle of influenza viruses has been intensively studied and is receiving increased attention. In particular, two signaling pathways required for efficient influenza virus pro- pagation have attracted some attention as suitable targets for an antiviral approach, namely the Raf/MEK/ERK mitogenic kinase cascade117 and the IKK/NF-kB cascade.The most noteworthy example has been provided by Ludwig and his collaborators81 which demonstrated that inhibition of NF-kB has a broad activity against influenza viruses and may also have additional beneficial effects, such as the suppression of the overabundant cytokine expression that leads to the detrimental cytokine burst in patients infected with highly pathogenic influenza viruses.
Although NF-kB covers a pivotal role in the innate immune defence,118 two independent studies showed that replication of influenza viruses may be impaired rather than enhanced in cells where the pathway was blocked.119,120 In fact, influenza viruses replicate with high efficiency in cells with pre-activated NF-kB, whereas progeny virus production is greatly impaired when viruses are grown in host cells in which NF-kB signaling has been impaired by specific inhibitors such as BAY11-7085 or BAY11-7082.120 From these studies it could be concluded that influenza viruses have acquired the capability to turn the anti-viral activity of NF-kB into a virus-supportive action. The underlying mechanism for the enhancing effect of NF-kB on virus propagation was found to be the induction of proapoptotic factors such as the tumor necrosis factor apoptosis ligand (TRAIL) and FasL/CD95L, that in the context of an influenza virus infection mediate the activation of caspases,119 which are responsible for a nuclear retention of viral ribonucleo- protein (RNP) complexes.121,122 Moreover, the suppressor of cytokine signaling-3 (SOCS-3) gene is strongly up-regulated in a NF-kB dependent manner in cells infected with influenza viruses,123,124 and inhibitors of NF-kB also differentially regulate viral RNA synthesis.125 The dependence of influenza viruses on NF-kB activity prompted investigations on the use of NF-kB for anti-viral intervention. Acetylsalicylic acid (ASA), also known as aspirin, efficiently blocked replication of influenza viruses in cell culture. Application of the compound into the lethally infected mouse lung as an aerosol by tracheal intubation reduced virus titres and significantly increased survival.121 In summary, NF-kB inhibitors can serve as anti-influenza agents in vivo without toxic side effects or the tendency to induce viral resistance.
The NF-kB signaling pathway has also particular relevance in several liver diseases including viral hepatitis induced by HBV and HCV. Recently, published studies have illustrated that the NF-kB signaling pathway is a potential target for the development of hepatoprotective agents.126 Several types of drugs including: antioxidants, proteasome inhibitors, IKK inhibitors and nucleic acid-based decoys have been shown to interfere with NF-kB activity at different levels and may be useful for the treatment of liver diseases.127 In chronic hepatitis C the activation of NF-kB is markedly modulated by viral proteins such as core protein and nonstructural ones, particularly NS5A and NS3.128 In hepatitis B the major factor influencing NF-kB function appears to be the HBx protein. Effects of viral proteins on NF-kB function in relation to the course of hepatitis are complex. They participate in the perpetua- tion of the inflammatory state in the liver, inhibit apoptosis of hepatocytes, as well as differentiate antigen-presenting cells. The latter effect has a deleterious impact on the formation of a specific immune response to viral peptides. It seems that both viruses, C and B, acquired the ability to modify NF-kB function, contributing to the establishment of chronic persistence.129 The question remains as to whether NF-kB can really be exploited for the development of therapeutics for these pathologies in the diseased human liver.
Also HSV-1 promotes its own replication by inducing persistent activation which, in turn, activates NF-kB viral gene transcription.130 A good example of the effect of the block of NF-kB activation on viral replication is given by the inhibition of IKK function by cyclopentenone prostanoids (cyPGs) during HSV-1 infections.130 In fact, the molecular target of cyPGs has been found to be IKK.131 CyPGs have been demonstrated to possess a potent antiviral activity against a variety of DNA and RNA viruses in vitro and in vivo.132 Treatment with the cyPG prostaglandin A1 (PGA1) completely blocked HSV-1-induced IKK activity in infected cells, thus preventing degradation of IkBa and consequent NF-kB activa- tion.130 NF-kB inhibition following cyPGs treatment results in a block of HSV-1 gene expression and in a dramatic decrease in the production of HSV-1 infectious particles. The ability of cyPGs to block viral gene expression was also shown during acute HIV-1 infection.133
Given that the NF-kB pathway is a multi-component pathway, there are a variety of inhibitors and several different strategies to inhibit NF-kB signaling that have been the topic of excellent reviews in the recent past.There are three general types of inhibitors that have been used to target NF-kB: biological inhibitors, that include several plant, marine, and microbial compounds targeting various steps in the NF-kB signaling; synthetic inhibitors, such as proteasome inhibitors and glucocorticoids; and biomole- cular inhibitors that include decoy oligonucleotides, siRNA, ribozymes and interfering peptides. These NF-kB inhibitors may be classified into those that (a) block signaling upstream of IKK, (b) block signaling at the IKK step, (c) block signaling at the IkBa degradation step, and (d) prevent the nuclear activation of NF-kB (Fig. 3).
Molecules that act upstream of the IKK complex include inhibitors of receptor activation by the natural ligand, inhibitors of adaptor protein recruitment to the receptor and inhibitors of IKK-activating kinases. In particular, inhibitors of cytokine binding to its receptors are very relevant for certain inflammatory diseases. For example, anti-TNF antibodies (e.g. Remicade) and a soluble TNF receptor (e.g. Enbrel) have been shown to suppress NF-kB activation in patient with inflammatory bowel diseases.137 Among the IL-1 receptor antagonist molecules that inhibit IL-1 action, anakinra has been approved by FDA for use in rheumatoid arthritis patients that are refractory to more conventional forms of treatment.138
Another strategy for inhibiting NF-kB activation is to block IKK activation and function.139 Although several agents have been shown to inhibit NF-kB activation at the IKK step, few studies have investigated the mechanism by which a given agent can inhibit IKK or its activation. CHS-828, SC-514, BM-345541 and non-steroidal anti-inflammatory drugs (NSAIDs) are probably the most studied of the IKK inhibitors.140–143
It has been suggested for a long time that IKKs are the most specific and effective pharmacological targets in the NF-kB activation pathway, because it appeared very likely that all of their target proteins are exclusively involved in NF-kB activa- tion. In the meantime, this point of view has changed, and it is well known that IKKs are also able to phosphorylate proteins that are participants in distinct signal transduction path- ways.144 In the past few years, several compounds have been reported to suppress HIV-1 gene expression and replication through the inhibition of NF-kB activation using IKK as a molecular target.145 Most of the studied inhibitors against NF-kB can block the induction of HIV-1 gene expression in latently infected cells, using as a target the kinases upstream IkBa, accompanied by a diverse range of stimuli from cytokines such as TNF-a and mitogen such as phorbol ester.146,147
Fig. 3 Potential pharmacological inhibitors of NF-kB and their mole- cular targets in the classical activation pathway.
Among these, ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6- hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile) has been devel- oped and evaluated as a potent and specific inhibitor for IKKa and IKKb with the ability to inhibit HIV-1 replication in cells latently infected with virus.Kwon et al.148 have examined the effect of tetracycline- inducible expression of transdominant repressors of IkBa (TD-IkBa) on HIV-1 multiplication using Jurkat T cells. TD-IkBa was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kB DNA binding activity and LTR-directed gene activity. Furthermore, TD-IkBa expression altered dramatically de novo HIV-1 infection of Jurkat cells and increased sensitivity of virus-infected cells to apoptosis. The most direct strategy for blocking NF-kB activation is to block NF-kB from binding to specific kB sites on DNA. Some sesquiterpene lactones have been reported to inhibit NF-kB by interacting NF-kB subunit DNA binding.149 Blocking specific NF-kB DNA binding can also be accomplished with decoy oligodeoxynucleotides that have kB sites and competes for NF-kB dimer binding to specific genomic promoters.150 These agents have been reported to have therapeutic potential in a number of animal models of inflammation.151
Collectively, these observations show that it is almost certain that there will be continued interest in the identification and characterization of compounds that can reduce NF-kB activity, and that IKK and NF-kB can be considered attractive targets for therapeutic intervention at least in viral diseases.
Conclusions and perspectives
The discovery of NF-kB transcription factors, that rapidly reprogram an exceptionally large number of genes in different situations as a result of their distinctive regulation, has modified the investigations and interpretation of the immunology of micro- bial diseases and introduced novel prospects for therapeutic intervention.7 The NF-kB pathway ability to react to several stimuli, the NF-kB members’ ability to form homo- and hetero- dimers, and the presence of canonical and non-canonical activa- tion can explain its complex and sometimes apparently conflicting effect in different systems and pleiotropic phenotypic traits in which it is involved.
Activation of the transcription factor NF-kB is one of the hallmark cell responses to infection with different pathogens. The central role of NF-kB in inflammation suggests that an adaptation of microbes to a host could involve an interference with the function of NF-kB, or a modulation of concomitant signal transduction events. The observation that several pathogens have evolved different strategies to evade or manip- ulate the NF-kB pathway likely reflects the selective pressure that this system places on the evolution of pathogens.152 It is also worth remembering that the relentless rise of antibiotic resistance, combined with a steady decline in the discovery of new antibiotics, emphasize the need for conceptually novel therapeutic strategies against microbial infections.
Greater comprehension of the mechanisms by which microbes induce or inhibit the activation of NF-kB may have a funda- mental role in understanding molecular aspects of microbial pathogenesis and improving strategies for the development of alternative antimicrobial agents. In the post-genomic era, improved knowledge and novel technologies have contributed to the discovery of several unknown cellular processes that modulate NF-kB. In particular, the identification of a number of cellular and microbial proteins that regulate this cascade has shed light on the molecular mechanisms and molecular targets in the NF-kB pathway for drug development.
The possibility to interfere with NF-kB activation provides an obvious selective advantage to invading microorganisms, but what is sometimes less clear is whether the activation of NF-kB in response to infection benefits the host or the pathogen.Several experimental evidence indicates that inhibition of NF-kB will be a valuable tool for treating many human diseases.153 Many different therapeutic NF-kB inhibitors have been reported in such inflammatory conditions including septic shock, autoimmune diseases, atherosclerosis and cancer, but very few have been translated to the clinical and approved for human use.134 One concern about the use of NF-kB inhibitors is the lack of specificity. NF-kB is a convergence point for cellular response to multiple stimuli and regulates genes involved in the development of inflammatory disease regulating apoptosis and cell proliferation. However, these same functions of NF-kB are required for normal cellular life. NF-kB unselective and complete inhibition can have serious adverse consequences such as immune suppression and tissue damage. It is the balance between therapeutic benefits and potential changes in normal cellular function and consequences of NF-kB inhibition that will dictate whether NF-kB inhibitors prove to be clinically successful.154
Our opinion regarding the future directions in NF-kB-based therapeutics is the possibility to design selective inhibitors that modulate NF-kB effects specifically in infected cell without altering its physiological functions in other contexts. A possible option could be to associate selective NF-kB inhibitors or activa- tors to a cell-type specific delivery system to direct NF-kB modulators to infected cells in order to avoid unnecessary and deleterious hyperactivation or suppression of the NF-kB physio- logical function. Even though no viable cell-specific delivery systems are currently in use, the emergence of new nanotech- nological tools may render this objective feasible in the future.155,156 In addition, improving the knowledge of the microbial protein sites which potently bind to host proteins, disrupt their function, and modulate the NF-kB signaling pathway will be helpful for designing peptides or peptidomimetics based on these sites may be also useful as therapeutic strategies.
There is no doubt that a more complete understanding of the NF-kB modulation mechanisms is likely to offer great potential for the development of novel therapeutics but must be approached with caution. A combinatorial therapy to block several key mediators of the immune pathways in infected cells in association with drugs targeting pathogen replication may be more effective in controlling diverse infectious diseases. Thus, it is a fertile area for future investigations.
We believe that the possibilities offered by a deeper understanding of the regulation of inflammatory signaling, including not just NF-kB but also other pathways, open up the promise of novel therapeutic approaches able to modulate the inflam- matory host-response at different steps of C25-140 infection.