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New insights into the crosstalk between Shigella and T lymphocytes
Review
New insights into the crosstalk between Shigella and T lymphocytes Wilmara Salgado-Pabo´n1*, Christoph Konradt1**, Philippe J. Sansonetti1,2, and Armelle Phalipon1 1 2
Unite´ de Pathoge´nie Microbienne Mole´culaire, Institut Pasteur, INSERM U786, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France Chaire de Microbiologie et Maladies Infectieuses, Colle`ge de France, Paris, France
Subversion of host immune responses is the key infection strategy employed by most, if not all, human pathogens. Modulation of the host innate response by pathogens has been vastly documented. Yet, especially for bacterial infections, it was only recently that cells of the adaptive immune response were recognized as targets of bacterial weapons such as the type III secretion system (T3SS) and its effector proteins. In this review, we focus on the recent advances made in the understanding of how the enteroinvasive bacterium Shigella flexneri interferes with the host adaptive response by targeting T lymphocytes, especially their migration capacities. Host immune response subversion Pathogens have evolved strategies to avoid or resist host defense mechanisms. Although there is ample information on the mechanisms used by pathogenic bacteria to subvert the innate immune defense system, very little is known about how they affect the adaptive immune response. The key role of dendritic cells (DCs) as antigen-presenting cells (APCs) bridging innate and adaptive immunity renders them particularly attractive for pathogen targeting, as recently reviewed [1]. Besides DCs, B and T lymphocytes are two other critical cell types of adaptive immunity, acting as cellular effectors ensuring protective immunity. Induction of long-term protective immunity to acute infection is a complex process, with T cell activation being a key element for eliciting both efficient antibody- and cellmediated immune responses. T cell activation relies on productive encounters between APCs bearing foreign antigens and cognate T lymphocytes, which are present at low frequencies. Motility of naı¨ve T cells within lymph nodes (LNs), secondary lymphoid organs where pathogen-specific immune response are orchestrated, is of Corresponding author: Phalipon, A. ([email protected]). Keywords: Shigella; type III secretion system; adaptive immunity; T lymphocyte; lymph node; two-photon microscopy. * Current address: University of Iowa, Carver College of Medicine, Department of Microbiology, 51 Newton Road, Iowa City, IA 52242, USA. ** Current address: Department of Pathobiology, School of Veterinary Medicine, 380 South University Avenue, University of Pennsylvania, PA, USA. 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.002
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utmost importance for these encounters to occur rapidly and efficiently [2,3], and high motility coefficients have been proposed to play a key role in optimizing the effector response to pathogens [4]. Chemoattractant-mediated T cell motility is highly dependent on the actin cytoskeleton. In the context of Shigella infection, it is critical to realize that Shigella has the capacity to trigger massive actin cytoskeleton rearrangements, as demonstrated in intestinal epithelial cells [5,6]. Hence, if Shigella were to target the actin cytoskeleton in T lymphocytes, it could significantly affect T cell motility and subsequently the priming of specific immune responses. This was the premise of the studies discussed in this review, where evidence is provided that upon infection Shigella targets T lymphocytes and alters their migratory properties. Host innate and adaptive immune responses elicited upon Shigella infection Shigella is an enteroinvasive pathovar of Escherichia coli and the causal agent of the acute recto-colitis shigellosis, otherwise known as bacillary dysentery. Children under the age of 5 years living in endemic areas of developing countries are the main targets of the disease. Although the mortality rate has significantly decreased in the last two decades, morbidity remains unacceptably high and hence problematic. Unfortunately despite unrelenting efforts, there is no vaccine available to protect children against shigellosis [7–10]. Therefore, deciphering the bacterial strategies that impair host immune responses and render humans continuously susceptible to Shigella infection throughout childhood is critical in designing new rational-based vaccine candidates. Potent and acute inflammation is the hallmark of the host innate immune response to Shigella infection, characterized by a rapid influx of polymorphonuclear cells (PMNs) leading to massive tissue destruction [11,12]. The pathogenesis of Shigella relies on the expression of a T3SS and its secreted effector proteins. A first wave of secreted effectors is required for cellular invasion and initiation of the inflammatory response upon bacterial targeting of resident macrophages and intestinal epithelial cells. A second wave of T3SS effectors targets mitogenactivated protein (MAP) kinases and the nuclear factor-kB (NF-kB) signaling pathway to control the inflammatory process and to promote bacterial survival [13–15]. Detailed molecular mechanisms involving T3SS virulence effectors
Review and their cellular protein targets have been recently reviewed [16–18]. The inflammatory response also includes the recruitment of innate T cells. The virulence protein ShiA encoded by a Shigella pathogenicity island located on the chromosome has been shown to control the level of inflammation induced upon infection, by limiting not only PMNs, but also innate T lymphocyte recruitment [19]. This is a relevant bacterial strategy considering that innate T lymphocytes, such as mucosal-associated invariant T (MAIT) cells, have been recently shown to be very effective in detecting and efficiently lysing Shigella-infected epithelial cells [20]. Whether T3SS effectors or other virulence proteins interfere with the priming of Shigella-specific immune responses is not known. However, indirect evidence suggests that effector-mediated targeting of adaptive immune cells is a possible mechanism of immune evasion. It has been known for decades that natural infection with Shigella fails to elicit long-lasting protective immunity, and several infection episodes are required to generate a shortterm, mainly serotype-specific, antibody-mediated protection [21,22]. Interestingly, a similar phenomenon has been reported upon Plasmodium falciparum infection leading to malaria [23]. These are suggestive of pathogen strategies utilized to dampen the acquired immune response. Impairing pathogen-specific protection, and thus providing the possibility of infecting the same individual multiple times, is obviously an advantage for human-restricted pathogens such as Shigella. Furthermore, upon Shigella infection it is well established that high levels of DC, B cell, and T cell death occurs during infection, as observed in rectal biopsies of Shigella-infected individuals. The factors that lead to cell death during infection are still under investigation, but evidence suggests that acute inflammation plays a critical role. In a mouse model of infection mimicking acute inflammation occurring upon natural infection, the proinflammatory cytokine environment elicited in response to the bacterium is prone for the predominant induction of Shigella-specific CD4+ T helper (Th) 17 cells (Th17 cells) [24]. In addition, even though Shigella is a facultative intracellular bacterium and despite its ability to actively secrete proteins into the host cytoplasm, another type of T lymphocytes, that is CD8+ T lymphocytes that play a prominent role in generating adaptive immune responses to cytosolic microbial pathogens and their products, fail to be primed upon infection [24,25]. Finally, the proinflammatory property of Shigella also affects DCs, especially their recruitment, by decreasing the production of chemokine (C–C motif) ligand 20 (CCL20), which is required for DC migration to infected tissues [26]. Besides the effects of Shigella-induced acute inflammation on B and T lymphocytes, whether Shigella directly targets T or B cell function and the consequence of this was examined, focusing up to now on T lymphocytes. Direct interaction between Shigella and lymphocytes are likely to occur: (i) in the lymphoid follicles associated with the colonic mucosa after bacterial crossing of the intestinal barrier via M cells located within the lymphoid follicleassociated epithelium, (ii) in the lamina propria, the connective tissue underlying the intestinal epithelium, and/or (iii) the draining mesenteric LNs, which constitute the last
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host barrier for Shigella, thus preventing systemic dissemination [15]. Here, we present the in vitro and in vivo approaches used to assess the Shigella–T cell interactions, and discuss the evidence for a role of the T3SS in modulating T cell motility and dynamics. Strategies evolved by bacterial pathogens to dampen T cell functions in general are also mentioned. Outcomes of Shigella–T lymphocyte crosstalk: inhibition of CD4+ T cell migration in vitro First, to simplify the complexity of the in vivo settings, whether there are direct interactions of Shigella with T cells was addressed in vitro. By using a classical in vitro model of cell migration towards a chemoattractant, T cell migration was severely impaired in activated human CD4+ T cells infected with invasive Shigella but not with a Shigella T3SS mutant. Surprisingly, injection of the T3SS effectors of Shigella into activated human CD4+ T cells with no subsequent bacterial invasion also caused impaired migration. Interestingly, Shigella only impairs the migration of activated T cells. Indeed, unactivated cells infected with invasive Shigella migrate at similar levels as uninfected cells. Hence, Shigella preferentially targets activated T lymphocytes [27]. Directional migration of T lymphocytes towards a chemoattractant relies at an early stage on a protein family of membrane cytoskeleton crosslinkers called ERM (ezrin, radixin, and meosin). ERMs are implicated in cell cortex organization and provide a conformationally regulated connection from the cortical actin cytoskeleton to the plasma membrane. The rapid conversion of activated ERMs (phosphorylated) to the inactivated (dephosphorylated) conformation is critical for its function and depends on the concentration of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane. Interestingly, PIP2 is a target for the Shigella T3SS effector invasion plasmid gene for Ipg (Ipg)D, a phosphoinositide 4-phosphatase that hydrolyses PIP2 into phosphatidylinositol 5-monophosphate (PI5P), decreasing the PIP2 pool at the plasma membrane and provoking a massive and sustained dephosphorylation of ERMs. In vitro studies with an IpgD deletion strain and its complemented counterpart provided evidence that IpgD is responsible for the CD4+ T cell migration deficiency observed in Shigella-infected cells [27]. It is therefore proposed that the enzymatic activity of IpgD on PIP2 affects posteriorization of the plasma membrane, known as polar-cap formation, essential for directional migration of T lymphocytes upon chemokine stimulation, with possible catastrophic consequences for the development of Shigella-specific adaptive immune responses (Figure 1). Phosphatidylinositol (PI) metabolism plays a key role in the regulation of receptor-mediated signal transduction, actin remodeling, and membrane trafficking in eukaryotic cells [28–30]. Thus, it is not surprising that several intracellular bacterial pathogens modulate and exploit PI levels, directly or indirectly, to ensure their survival and efficient intracellular replication. In addition to several previous reports, a new recent example is the HIV Tat protein that binds with a high affinity to PIP2, resulting in the perturbation of the PIP2-mediated recruitment of 193
Review
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Shigella IpgD
Transmembrane protein
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ERM acvaon through PIP2 binding and phosphorylaon
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IpgD reduces PIP2 levels and therefore impacts ERM acvaon
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Figure 1. Inhibition of T cell migration by Shigella. Shigella invasion of T lymphocytes requires a functional type III secretion system (T3SS) and results in inhibition of chemokine-induced T lymphocyte migration, an effect mediated by the T3SS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Left panel: ERMs, a protein family of membrane cytoskeleton crosslinkers (ezrin, radixin, and meosin), provide a conformationally regulated connection from the cortical actin cytoskeleton to the plasma membrane. Rapid conversion of ERMs from their active to inactive conformations, and vice versa, plays a key role in T cell polarization upon chemoattractant stimulus. Phosphatidylinositol 4,5-bisphosphate (PIP2) is key in the balance between the two forms of the ERMs. PIP2 hydrolysis in itself is in fact sufficient to induce ERM dephosphorylation [30]. Right panel: upon IpgD-mediated hydrolysis of PIP2, the pool of PIP2 at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus. Subsequently, the switch upon chemokine stimulation from unpolarized to polarized cell morphology that further allows T cell chemotaxis does not occur. Transmembrane proteins interacting with active, phosphorylated ERMs include CD43, CD44, intercellular adhesion molecule-1 (ICAM-1), and ICAM-2. Cytoplasmic proteins interacting with active, phosphorylated ERMs include Rho GDP dissociation inhibitor (RhoGDI), phosphatidylinositol 3-kinase (PI3K), synapse-associated protein 97 (SAP97), and focal adhesion kinase (FAK).
cellular proteins to the plasma membrane [31]. However, Shigella is the only pathogen documented to date that produces an effector protein (IpgD) that hydrolyses PIP2 and provokes a massive and sustained dephosphorylation of phospho-ERMs. PIP2 hydrolysis in itself is in fact sufficient to induce ERMs dephosphorylation, indicating a key role of PIP2 in ERM protein biology, namely hydrolysismediated ERM inactivation [32]. In addition, these data reveal that interfering with PI metabolism does not only help pathogens to improve their ability to enter or survive into host cells, but also offers the opportunity, depending on the targeted host cell type, to modulate the host immune response by affecting cell migration. Outcomes of Shigella–T lymphocyte crosstalks: impairment of CD4+ T cell dynamics in vivo Based on these in vitro findings, this phenomenon upon Shigella infection was examined in vivo. A mouse model was developed to accurately assess Shigella–T cell interactions within the LN, where specific immunity is initiated [33]. Mice were thus injected with Shigella within the foodpad and interactions with T cells were tracked in the draining popliteal LN [34]. Eradication of the infection foci and induction of longterm protection against re-infection rely on swift mobilization of activated, effector T lymphocytes to the infected sites. There, effector T lymphocytes activate macrophages to help kill engulfed or intracellular pathogens, or directly kill invaded cells, and secrete cytokines to sustain a pathogen-specific response. This process starts within the draining LNs, which receive macrophages and DCs carrying antigens or microorganisms from the infection foci, extracellular organisms that drain directly into the LN, and 194
inflammatory molecules produced at sites of infection. It is within this setting that pathogen-specific immune responses are generated. T lymphocyte migration and motility within LNs is critical because APCs displaying cognate antigen are scarce. Yet, this encounter is an absolute requirement for T cell activation, proliferation, differentiation into antigen-specific effector cells, and transmigration into infected tissues. In LNs, naı¨ve T cells migrate at an average velocity of 9–12 mm/min and can achieve peak velocities higher than 25 mm/min. These velocities (more than one cell diameter per minute) allow an individual naı¨ve T cell to scan an estimated 100 DCs per hour [35]. Consistent with in vitro findings, in vivo data from twophoton microscopy of Shigella-infected LNs revealed that Shigella indeed impacts on T cell dynamics. Two parameters reflecting T cell motility, mean velocity and arrest coefficient (representing the percentage of time a tracked individual T cell moves at a velocity less than 4 mm/min, considering that a T cell is arrested if the velocity is below 2 mm/min), were measured. Upon Shigella infection, the mean velocity decreased in parallel to the increase of the arrest coefficient as compared to uninfected LN or LN infected with a T3SS Shigella mutant [34]. Shigella induced CD4+ T cell migration paralysis very early (4 h) after arrival into the LN. At this time point, Shigella was predominantly found in the LN subcapsular sinus, either extracellularly or within subcapsular sinus macrophages and DCs. The Shigella-induced inhibition of CD4+ T cell migration is not dependent on antigen specificity because both polyclonal T cells and monoclonal cells expressing a T cell receptor of irrelevant specificity suffer the same fate. Still, Shigella’s interference with T cell migration is dependent on the expression of a functional T3SS.
Review The question remains: how is polyclonal CD4+ T cell motility so broadly affected during infection? Direct inhibition of T cell migration, as demonstrated with invasion of CD4+ T cells in vivo, leads to migratory arrest. However, so far only activated T cells have been shown to be susceptible to this inhibitory mechanism. Another mechanism implicates the global effect of Shigella-induced inflammation on the tissue environment. This global effect may be mediated by chemokines and cytokines produced during infection. It could be also due to the impact of infiltrating neutrophils on the LN architecture. The dynamic behavior of T cells at inflammation sites appears to be largely driven by chemokines and cytokines produced by stimulated or invaded cells. Two-photon microscopy of S. flexneri-infected LNs labeled in vivo with CD169+-specific antibodies demonstrated that S. flexneri associates with and invades macrophages and DCs in the LN subcapsular area. One of the major proinflammatory mediators in S. flexneri-infected LNs 24 h post infection is indeed interleukin-1b (IL-1b), which uniquely results from caspase 1-mediated pyropoptosis, suggesting that Shigella interactions with LN-resident macrophages and DCs leads to cell death and cytokine production by known Shigella-specific mechanisms. Another proinflammatory mediator detected in the LN is the neutrophil chemotactic protein KC, which coincides with massive neutrophil infiltration into the LN subcapsular sinus [34]. The excessive recruitment of neutrophils and granule release within the subcapsular sinus may degrade the T cell reticular network that facilitates T cell motility or exacerbate the acute phase response. A careful analysis of the migration pattern of the T cells within different depths of the LN has provided evidence that invasive Shigella infection causes T cells to migrate at low speeds even deep within the LN where bacteria and neutrophils are undetectable [34]. Therefore, the T cell migration impairment observed deeper in the LN cannot be accounted for solely by the possible effect of neutrophils on the LN architecture in the subcapsular sinus. We propose that T cell migration inhibition could be the result of: (i) direct inhibition of T cell migration by Shigellaspecific effectors, (ii) effector-mediated induction of a variety of adhesion or recognition molecules by activated, invaded, dead, or dying cells, causing nonspecific interactions with lymphocytes, (iii) neutrophil reshaping of the LN architecture within the subcapsular sinus, and/or (iv) the effect of the massive inflammatory response that induces changes throughout the LN, leading to nonspecific interactions with APCs or cell death. Further cellular and molecular analysis to carefully dissect the contribution of the infiltrating and resident cell subtypes and their products on the observed decreased motility of polyclonal CD4+ T cells during Shigella infection is warranted and desired. Nevertheless, these data constitute the first report of the manipulation of CD4+ T cell dynamics within the LN upon bacterial infection. Indeed, the dynamic imaging of host–pathogen interactions in vivo including T cell behavior in LNs has been only reported so far for parasites and viruses [36–40], whereas dynamic imaging of T cell behavior upon bacterial infections has been studied in the spleen and in the liver upon Listeria monocytogenes [41] and Bacille Calmette-Gue´rin
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(BCG) infection [42], respectively. Briefly, interactions between T cells and DCs in the context of malaria have been visualized within the LN and infection has been shown to prevent priming of immune responses by antagonizing these cell–cell contacts, mainly by reducing T cell migration and downregulating activation markers on both T cells and DCs [43]. Similar to Shigella, Toxoplasma gondii infection also results in modulation of the behavior of non-parasite specific T cells. This T cell receptor (TCR)independent process correlates with the remodeling of the LN micro-architecture and changes in expression of CCL21 and CCL3, two chemokines stimulating T cell migration [39]. Viruses such as vaccinia virus or vesicular stomatitis virus were used to study the site of priming for T cell response within the LN. Naı¨ve CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region, just beneath the subcapsular sinus, the region reported as being the site of interactions between Shigella and T cells. Then, tight interactions with DCs were observed leading to complete T cell activation. Thus, antigen presentation at the LN periphery, not at lymphocyte exit sites in deeper LN venules as dogma dictates, has a dominant function in antiviral CD8+ T cell activation [40]. For bacterial infection, using the intracellular Gram-positive bacterium Listeria monocytogenes as a model for triggering an efficient priming of CD8+ T cells, the analysis of the migratory behavior and priming capacity of monocyte and DCs after infection of mice has highlighted the fact that the migratory route DCs take to drive T cell activation is independent of the architecture of lymphoid tissues [44]. Finally, by visualizing BCG granuloma formation in the mouse liver, the movement and behavior of T lymphocytes in granulomas were found to be very intriguing. These cells, which arrive at the granulomas within days, are in constant motion throughout the lesion. Their motion is such that each lymphocyte appears to wander through the entire granuloma, probably making direct contact with most of its macrophages. This report adds to the argument that the granuloma cannot be thought of as simply a barricade to contain mycobacteria, even after adaptive immunity is established [42]. Impairment of T lymphocyte functions by bacterial pathogens: beyond T3SS effectors and T cell migration These results emphasize the extraordinary power of the Shigella T3SS to deliver a given effector with a particular enzymatic activity, such as IpgD, into different cell types, thereby triggering a diversity of outcomes to modulate the host immune response, including dampening of the host inflammatory response [45]. T3SS host cell selectivity was previously illustrated with the Yersinia T3SS, where effector proteins were found to preferentially target phagocytic cells with high specificity for neutrophils in the LN and follicular B cells in the spleen [46,47]. The only other example of a T3SS effector impairing cell migration is SseI, the bacterial effector translocated into host cells by the Salmonella enterica pathogenicity island 2 T3SS. SseI inhibits normal cell migration of primary macrophages and DCs in vitro. Such an inhibition requires the host factor IQ motif containing GTPase activating protein, an important regulator of cell migration. DC and CD4+ T lymphocyte 195
Review migration to the spleen is also suppressed in an SseIdependent manner in vivo [48]. Shigella is unique in that IpgD injection inhibits the capacity of T lymphocytes to reorganize their cytoskeleton to undergo directional migration, irrespective of whether or not injection is followed by bacterial invasion [27]. A few recent reports have highlighted the capacity of human-specific pathogens to target T cell migration via other types of virulence effectors. Bordetella pertussis produces adenylate cyclase toxin A (CyaA), an adenylate cyclase toxin that triggers the synthesis of cyclic AMP (cAMP) in peripheral blood lymphocytes. Accumulation of cAMP within T lymphocytes inhibits chemokine receptor signaling, and hence T cell chemotaxis, by a mechanism dependent on cAMP and the protein kinase A signaling pathway [49]. The Mycobacterium tuberculosis cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) desensitizes naı¨ve T cells to the LN egress signal sphingosine-1-phosphate (S1P), causing the preferential accumulation of C–C chemokine receptor type 5 (CCR5)+ CD4+ T cells in lung-draining LNs and impairing effector T cell migration to the infected lungs [50]. One example for viral infection is the HIV-1 protein Nef (negative regulator factor), which inhibits T cell chemotaxis by at least two mechanisms: down-modulation of LFA-1 (lymphocyte/leukocyte function-associated antigen-1), an adhesion molecule critical for generation and maintenance of the T cell receptor synapse, and phosphorylation (deactivation) of cofilin, an actin-depolymerizing factor that promotes T cell motility when dephosphorylated. These mechanisms lead to decreased adhesion and polarization of T cells and dysregulation of the assembly and disassembly of actin filaments, respectively [51,52]. Not only T lymphocyte trafficking, but also other T cell functions are targeted by bacterial pathogens. As far as T3SS effectors are concerned, the Yersinia outer membrane protein (YopH) virulence protein specifically targets adaptor molecules of the TCR signaling pathway, thus inhibiting T cell activation, whereas YopP inhibits CD8+ T cell priming in a mouse infection model [53,54]. This suggests that T3SS effectors have the potential to interfere with a diversity of T cell functions. Other virulence effectors have been shown to target other T cell functions. Bordetella pertussis CyaA triggers accumulation of cAMP within T lymphocytes resulting in impairment of T cell activation [49]. The Helicobacter pylori vacuolating cytotoxin A (VacA) virulence factor blocks T cell proliferation by inducing a G1/S cell arrest [55] and uses CD18 as a VacA receptor on human T lymphocytes [56]. Very recently, evidence was provided that the polysaccharide antigen Vi released from Salmonella typhi targets the T cell membrane prohibitin (belonging to a family of evolutionarily conserved proteins implicated in a large number of cellular functions such as cell proliferation, apoptosis, and cell signaling), thus interfering with T cell activation [57]. Concluding remarks Dampening the adaptive immune response by interfering with the key cellular actors required to mount a protective immunity is definitely part of the strategies used by pathogens for their own benefit. Undoubtedly, further in vivo 196
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investigations will reveal how much does the targeting of such cells, including T and B lymphocytes, impact on the specific cellular and humoral immune responses, and subsequently on protection against re-infection. Despite many pending questions (the most important ones being outlined in Box 1), the Shigella studies have unraveled a new mechanism that pathogens use to interfere with T lymphocytes and in particular with their dynamics. These findings suggest that direct manipulation can occur via bacterial invasion of T cells or injection of effectors into T cells without invasion. As for the latter mechanism, one may easily imagine the efficiency of a strategy consisting of injecting effectors as soon as bacterium–cell contacts occur without the need for invasion, a surprising observation in view of the intracellular lifestyle of Shigella. The importance of an ‘injection-only’ pathway to ‘freeze’ the host immune response has been largely underestimated and deserves further investigation. In addition, the molecular mechanisms underlying the susceptibility of activated T lymphocytes to Shigella invasion as compared to unactivated T cells that are refractory to invasion, but apparently not to injection of T3SS effectors, deserves to be deciphered. If such an ability to deal with T cells independently of their activation state occurs when Shigella reaches the intestinal mucosa, this would certainly confer a serious advantage to the bacterium for largely counteracting T cell functions. Developing the appropriate tools to address in vivo questions regarding the dynamics of Shigella–T cell interactions, whether it is in terms of bacterial invasion versus injection-only of T3SS effectors, is definitely a great challenge. The global impact on T cell migration induced by Shigella could certainly affect key steps required for the initiation of Shigella-specific responses, such as the cognate DC–T cell encounter, priming, and mobilization to infection sites. We speculate that the impairment of T cell dynamics discussed here is a mechanism that contributes to the development of poor Shigella-specific immune responses upon natural infection. However, we are aware that it will be difficult to demonstrate this assumption due to the lack of an experimental model of infection that closely mimics human infection upon oral inoculation.
Box 1. Outstanding questions What are the molecular mechanisms underlying Shigella invasion of activated but not unactivated T lymphocytes? Are the molecules involved in the entry process into epithelial cells those required of T cell invasion? Are there new targets specific for T lymphocytes that are upregulated upon activation? Are T lymphocyte subpopulations differentially impacted upon Shigella infection? What are the molecular mechanisms leading to the impairment of migration of non-invaded T lymphocytes in the LN? Does Shigella infection disrupt the reticular network that facilitates T cell migration? Does Shigella interfere with the chemokine gradient or alter T cell sensitivity to chemokines? Does neutrophil recruitment impact on T cell migration? Does Shigella target other T cell functions besides T cell movement? In the near future, will the ongoing development of human artificial LNs in vitro solve the issue of addressing these questions in the human context?
Review Altogether, these studies contribute to a better understanding of the complex interactions between bacteria and cells of the adaptive immune system. It also emphasizes the role of T3SS and its secreted effectors in the manipulation of the adaptive response in vivo, a so far very poorly studied topic [58]. It would certainly be worthwhile to take into account these findings for revisiting the design of live, attenuated Shigella vaccine candidates. Eventually, tissue imaging that allows one to more coherently analyze an infected tissue for a much better understanding of bacterial pathogenesis within the live host promises to reveal relevant interactions and angles for disease intervention [59]. Acknowledgments W.S.-P. was funded by the Pasteur Foundation and the Philips Foundation. C.K. was supported by fellowships from the European Consortium PATHOGENOMICS, the Institut Pasteur Transversal Research (PTR) Program no. 251 led by A.P., and the Howard Hughes Medical Institute (HHMI). The research leading to these results has received funding from PTR no. 251, the European Community’s PEOPLE Seventh Framework Program under grant agreement European Institute of Microbiology and Infectious Diseases (EIMID) IAPP–PIAP-GA-2008217768, the Domaine d’Inte´reˆt Majeur Malinf-Re´gion Iˆle de France, l’Agence Nationale de la Recherche under the project ‘PATHIMMUN’, and the Ministe`re des Affaires Etrange`res Projet P2R. P.J.S. is supported by the European Research Council (ERC) and is a HHMI Foreign Scholar.
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52 Stolp, B. et al. (2009) HIV-1 Nef interferes with host cell motility by deregulation of Cofilin. Cell Host Microbe 6, 174–186 53 Gerke, C. et al. (2005) The adaptor molecules LAT and SLP-76 are specifically targeted by Yersinia to inhibit T cell activation. J. Exp. Med. 201, 361–371 54 Trulzsch, K. et al. (2005) Yersinia outer protein P inhibits CD8 T cell priming in the mouse infection model. J. Immunol. 174, 4244–4251 55 Gebert, B. et al. (2003) Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 301, 1099–1102 56 Sewald, X. et al. (2008) Sticky socks: Helicobacter pylori VacA takes shape. Trends Microbiol. 16, 89–92 57 Santhanam, S.K. et al. (2014) The virulence polysaccharide Vi released by Salmonella typhi targets membrane prohibitin to inhibit T cell activation. J. Infect. Dis. http://dx.doi.org/10.1093/infdis/jiu064 58 Marketon, M.M. et al. (2005) Plague bacteria target immune cells during infection. Science 309, 1739–1741 59 Richter-Dahlfors, A. et al. (2012) Tissue microbiology provides a coherent picture of infection. Curr. Opin. Microbiol. 15, 15–22
New insights into the crosstalk between Shigella and T lymphocytes Wilmara Salgado-Pabo´n1*, Christoph Konradt1**, Philippe J. Sansonetti1,2, and Armelle Phalipon1 1 2
Unite´ de Pathoge´nie Microbienne Mole´culaire, Institut Pasteur, INSERM U786, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France Chaire de Microbiologie et Maladies Infectieuses, Colle`ge de France, Paris, France
Subversion of host immune responses is the key infection strategy employed by most, if not all, human pathogens. Modulation of the host innate response by pathogens has been vastly documented. Yet, especially for bacterial infections, it was only recently that cells of the adaptive immune response were recognized as targets of bacterial weapons such as the type III secretion system (T3SS) and its effector proteins. In this review, we focus on the recent advances made in the understanding of how the enteroinvasive bacterium Shigella flexneri interferes with the host adaptive response by targeting T lymphocytes, especially their migration capacities. Host immune response subversion Pathogens have evolved strategies to avoid or resist host defense mechanisms. Although there is ample information on the mechanisms used by pathogenic bacteria to subvert the innate immune defense system, very little is known about how they affect the adaptive immune response. The key role of dendritic cells (DCs) as antigen-presenting cells (APCs) bridging innate and adaptive immunity renders them particularly attractive for pathogen targeting, as recently reviewed [1]. Besides DCs, B and T lymphocytes are two other critical cell types of adaptive immunity, acting as cellular effectors ensuring protective immunity. Induction of long-term protective immunity to acute infection is a complex process, with T cell activation being a key element for eliciting both efficient antibody- and cellmediated immune responses. T cell activation relies on productive encounters between APCs bearing foreign antigens and cognate T lymphocytes, which are present at low frequencies. Motility of naı¨ve T cells within lymph nodes (LNs), secondary lymphoid organs where pathogen-specific immune response are orchestrated, is of Corresponding author: Phalipon, A. ([email protected]). Keywords: Shigella; type III secretion system; adaptive immunity; T lymphocyte; lymph node; two-photon microscopy. * Current address: University of Iowa, Carver College of Medicine, Department of Microbiology, 51 Newton Road, Iowa City, IA 52242, USA. ** Current address: Department of Pathobiology, School of Veterinary Medicine, 380 South University Avenue, University of Pennsylvania, PA, USA. 0966-842X/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.02.002
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utmost importance for these encounters to occur rapidly and efficiently [2,3], and high motility coefficients have been proposed to play a key role in optimizing the effector response to pathogens [4]. Chemoattractant-mediated T cell motility is highly dependent on the actin cytoskeleton. In the context of Shigella infection, it is critical to realize that Shigella has the capacity to trigger massive actin cytoskeleton rearrangements, as demonstrated in intestinal epithelial cells [5,6]. Hence, if Shigella were to target the actin cytoskeleton in T lymphocytes, it could significantly affect T cell motility and subsequently the priming of specific immune responses. This was the premise of the studies discussed in this review, where evidence is provided that upon infection Shigella targets T lymphocytes and alters their migratory properties. Host innate and adaptive immune responses elicited upon Shigella infection Shigella is an enteroinvasive pathovar of Escherichia coli and the causal agent of the acute recto-colitis shigellosis, otherwise known as bacillary dysentery. Children under the age of 5 years living in endemic areas of developing countries are the main targets of the disease. Although the mortality rate has significantly decreased in the last two decades, morbidity remains unacceptably high and hence problematic. Unfortunately despite unrelenting efforts, there is no vaccine available to protect children against shigellosis [7–10]. Therefore, deciphering the bacterial strategies that impair host immune responses and render humans continuously susceptible to Shigella infection throughout childhood is critical in designing new rational-based vaccine candidates. Potent and acute inflammation is the hallmark of the host innate immune response to Shigella infection, characterized by a rapid influx of polymorphonuclear cells (PMNs) leading to massive tissue destruction [11,12]. The pathogenesis of Shigella relies on the expression of a T3SS and its secreted effector proteins. A first wave of secreted effectors is required for cellular invasion and initiation of the inflammatory response upon bacterial targeting of resident macrophages and intestinal epithelial cells. A second wave of T3SS effectors targets mitogenactivated protein (MAP) kinases and the nuclear factor-kB (NF-kB) signaling pathway to control the inflammatory process and to promote bacterial survival [13–15]. Detailed molecular mechanisms involving T3SS virulence effectors
Review and their cellular protein targets have been recently reviewed [16–18]. The inflammatory response also includes the recruitment of innate T cells. The virulence protein ShiA encoded by a Shigella pathogenicity island located on the chromosome has been shown to control the level of inflammation induced upon infection, by limiting not only PMNs, but also innate T lymphocyte recruitment [19]. This is a relevant bacterial strategy considering that innate T lymphocytes, such as mucosal-associated invariant T (MAIT) cells, have been recently shown to be very effective in detecting and efficiently lysing Shigella-infected epithelial cells [20]. Whether T3SS effectors or other virulence proteins interfere with the priming of Shigella-specific immune responses is not known. However, indirect evidence suggests that effector-mediated targeting of adaptive immune cells is a possible mechanism of immune evasion. It has been known for decades that natural infection with Shigella fails to elicit long-lasting protective immunity, and several infection episodes are required to generate a shortterm, mainly serotype-specific, antibody-mediated protection [21,22]. Interestingly, a similar phenomenon has been reported upon Plasmodium falciparum infection leading to malaria [23]. These are suggestive of pathogen strategies utilized to dampen the acquired immune response. Impairing pathogen-specific protection, and thus providing the possibility of infecting the same individual multiple times, is obviously an advantage for human-restricted pathogens such as Shigella. Furthermore, upon Shigella infection it is well established that high levels of DC, B cell, and T cell death occurs during infection, as observed in rectal biopsies of Shigella-infected individuals. The factors that lead to cell death during infection are still under investigation, but evidence suggests that acute inflammation plays a critical role. In a mouse model of infection mimicking acute inflammation occurring upon natural infection, the proinflammatory cytokine environment elicited in response to the bacterium is prone for the predominant induction of Shigella-specific CD4+ T helper (Th) 17 cells (Th17 cells) [24]. In addition, even though Shigella is a facultative intracellular bacterium and despite its ability to actively secrete proteins into the host cytoplasm, another type of T lymphocytes, that is CD8+ T lymphocytes that play a prominent role in generating adaptive immune responses to cytosolic microbial pathogens and their products, fail to be primed upon infection [24,25]. Finally, the proinflammatory property of Shigella also affects DCs, especially their recruitment, by decreasing the production of chemokine (C–C motif) ligand 20 (CCL20), which is required for DC migration to infected tissues [26]. Besides the effects of Shigella-induced acute inflammation on B and T lymphocytes, whether Shigella directly targets T or B cell function and the consequence of this was examined, focusing up to now on T lymphocytes. Direct interaction between Shigella and lymphocytes are likely to occur: (i) in the lymphoid follicles associated with the colonic mucosa after bacterial crossing of the intestinal barrier via M cells located within the lymphoid follicleassociated epithelium, (ii) in the lamina propria, the connective tissue underlying the intestinal epithelium, and/or (iii) the draining mesenteric LNs, which constitute the last
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host barrier for Shigella, thus preventing systemic dissemination [15]. Here, we present the in vitro and in vivo approaches used to assess the Shigella–T cell interactions, and discuss the evidence for a role of the T3SS in modulating T cell motility and dynamics. Strategies evolved by bacterial pathogens to dampen T cell functions in general are also mentioned. Outcomes of Shigella–T lymphocyte crosstalk: inhibition of CD4+ T cell migration in vitro First, to simplify the complexity of the in vivo settings, whether there are direct interactions of Shigella with T cells was addressed in vitro. By using a classical in vitro model of cell migration towards a chemoattractant, T cell migration was severely impaired in activated human CD4+ T cells infected with invasive Shigella but not with a Shigella T3SS mutant. Surprisingly, injection of the T3SS effectors of Shigella into activated human CD4+ T cells with no subsequent bacterial invasion also caused impaired migration. Interestingly, Shigella only impairs the migration of activated T cells. Indeed, unactivated cells infected with invasive Shigella migrate at similar levels as uninfected cells. Hence, Shigella preferentially targets activated T lymphocytes [27]. Directional migration of T lymphocytes towards a chemoattractant relies at an early stage on a protein family of membrane cytoskeleton crosslinkers called ERM (ezrin, radixin, and meosin). ERMs are implicated in cell cortex organization and provide a conformationally regulated connection from the cortical actin cytoskeleton to the plasma membrane. The rapid conversion of activated ERMs (phosphorylated) to the inactivated (dephosphorylated) conformation is critical for its function and depends on the concentration of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane. Interestingly, PIP2 is a target for the Shigella T3SS effector invasion plasmid gene for Ipg (Ipg)D, a phosphoinositide 4-phosphatase that hydrolyses PIP2 into phosphatidylinositol 5-monophosphate (PI5P), decreasing the PIP2 pool at the plasma membrane and provoking a massive and sustained dephosphorylation of ERMs. In vitro studies with an IpgD deletion strain and its complemented counterpart provided evidence that IpgD is responsible for the CD4+ T cell migration deficiency observed in Shigella-infected cells [27]. It is therefore proposed that the enzymatic activity of IpgD on PIP2 affects posteriorization of the plasma membrane, known as polar-cap formation, essential for directional migration of T lymphocytes upon chemokine stimulation, with possible catastrophic consequences for the development of Shigella-specific adaptive immune responses (Figure 1). Phosphatidylinositol (PI) metabolism plays a key role in the regulation of receptor-mediated signal transduction, actin remodeling, and membrane trafficking in eukaryotic cells [28–30]. Thus, it is not surprising that several intracellular bacterial pathogens modulate and exploit PI levels, directly or indirectly, to ensure their survival and efficient intracellular replication. In addition to several previous reports, a new recent example is the HIV Tat protein that binds with a high affinity to PIP2, resulting in the perturbation of the PIP2-mediated recruitment of 193
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Shigella IpgD
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Figure 1. Inhibition of T cell migration by Shigella. Shigella invasion of T lymphocytes requires a functional type III secretion system (T3SS) and results in inhibition of chemokine-induced T lymphocyte migration, an effect mediated by the T3SS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Left panel: ERMs, a protein family of membrane cytoskeleton crosslinkers (ezrin, radixin, and meosin), provide a conformationally regulated connection from the cortical actin cytoskeleton to the plasma membrane. Rapid conversion of ERMs from their active to inactive conformations, and vice versa, plays a key role in T cell polarization upon chemoattractant stimulus. Phosphatidylinositol 4,5-bisphosphate (PIP2) is key in the balance between the two forms of the ERMs. PIP2 hydrolysis in itself is in fact sufficient to induce ERM dephosphorylation [30]. Right panel: upon IpgD-mediated hydrolysis of PIP2, the pool of PIP2 at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus. Subsequently, the switch upon chemokine stimulation from unpolarized to polarized cell morphology that further allows T cell chemotaxis does not occur. Transmembrane proteins interacting with active, phosphorylated ERMs include CD43, CD44, intercellular adhesion molecule-1 (ICAM-1), and ICAM-2. Cytoplasmic proteins interacting with active, phosphorylated ERMs include Rho GDP dissociation inhibitor (RhoGDI), phosphatidylinositol 3-kinase (PI3K), synapse-associated protein 97 (SAP97), and focal adhesion kinase (FAK).
cellular proteins to the plasma membrane [31]. However, Shigella is the only pathogen documented to date that produces an effector protein (IpgD) that hydrolyses PIP2 and provokes a massive and sustained dephosphorylation of phospho-ERMs. PIP2 hydrolysis in itself is in fact sufficient to induce ERMs dephosphorylation, indicating a key role of PIP2 in ERM protein biology, namely hydrolysismediated ERM inactivation [32]. In addition, these data reveal that interfering with PI metabolism does not only help pathogens to improve their ability to enter or survive into host cells, but also offers the opportunity, depending on the targeted host cell type, to modulate the host immune response by affecting cell migration. Outcomes of Shigella–T lymphocyte crosstalks: impairment of CD4+ T cell dynamics in vivo Based on these in vitro findings, this phenomenon upon Shigella infection was examined in vivo. A mouse model was developed to accurately assess Shigella–T cell interactions within the LN, where specific immunity is initiated [33]. Mice were thus injected with Shigella within the foodpad and interactions with T cells were tracked in the draining popliteal LN [34]. Eradication of the infection foci and induction of longterm protection against re-infection rely on swift mobilization of activated, effector T lymphocytes to the infected sites. There, effector T lymphocytes activate macrophages to help kill engulfed or intracellular pathogens, or directly kill invaded cells, and secrete cytokines to sustain a pathogen-specific response. This process starts within the draining LNs, which receive macrophages and DCs carrying antigens or microorganisms from the infection foci, extracellular organisms that drain directly into the LN, and 194
inflammatory molecules produced at sites of infection. It is within this setting that pathogen-specific immune responses are generated. T lymphocyte migration and motility within LNs is critical because APCs displaying cognate antigen are scarce. Yet, this encounter is an absolute requirement for T cell activation, proliferation, differentiation into antigen-specific effector cells, and transmigration into infected tissues. In LNs, naı¨ve T cells migrate at an average velocity of 9–12 mm/min and can achieve peak velocities higher than 25 mm/min. These velocities (more than one cell diameter per minute) allow an individual naı¨ve T cell to scan an estimated 100 DCs per hour [35]. Consistent with in vitro findings, in vivo data from twophoton microscopy of Shigella-infected LNs revealed that Shigella indeed impacts on T cell dynamics. Two parameters reflecting T cell motility, mean velocity and arrest coefficient (representing the percentage of time a tracked individual T cell moves at a velocity less than 4 mm/min, considering that a T cell is arrested if the velocity is below 2 mm/min), were measured. Upon Shigella infection, the mean velocity decreased in parallel to the increase of the arrest coefficient as compared to uninfected LN or LN infected with a T3SS Shigella mutant [34]. Shigella induced CD4+ T cell migration paralysis very early (4 h) after arrival into the LN. At this time point, Shigella was predominantly found in the LN subcapsular sinus, either extracellularly or within subcapsular sinus macrophages and DCs. The Shigella-induced inhibition of CD4+ T cell migration is not dependent on antigen specificity because both polyclonal T cells and monoclonal cells expressing a T cell receptor of irrelevant specificity suffer the same fate. Still, Shigella’s interference with T cell migration is dependent on the expression of a functional T3SS.
Review The question remains: how is polyclonal CD4+ T cell motility so broadly affected during infection? Direct inhibition of T cell migration, as demonstrated with invasion of CD4+ T cells in vivo, leads to migratory arrest. However, so far only activated T cells have been shown to be susceptible to this inhibitory mechanism. Another mechanism implicates the global effect of Shigella-induced inflammation on the tissue environment. This global effect may be mediated by chemokines and cytokines produced during infection. It could be also due to the impact of infiltrating neutrophils on the LN architecture. The dynamic behavior of T cells at inflammation sites appears to be largely driven by chemokines and cytokines produced by stimulated or invaded cells. Two-photon microscopy of S. flexneri-infected LNs labeled in vivo with CD169+-specific antibodies demonstrated that S. flexneri associates with and invades macrophages and DCs in the LN subcapsular area. One of the major proinflammatory mediators in S. flexneri-infected LNs 24 h post infection is indeed interleukin-1b (IL-1b), which uniquely results from caspase 1-mediated pyropoptosis, suggesting that Shigella interactions with LN-resident macrophages and DCs leads to cell death and cytokine production by known Shigella-specific mechanisms. Another proinflammatory mediator detected in the LN is the neutrophil chemotactic protein KC, which coincides with massive neutrophil infiltration into the LN subcapsular sinus [34]. The excessive recruitment of neutrophils and granule release within the subcapsular sinus may degrade the T cell reticular network that facilitates T cell motility or exacerbate the acute phase response. A careful analysis of the migration pattern of the T cells within different depths of the LN has provided evidence that invasive Shigella infection causes T cells to migrate at low speeds even deep within the LN where bacteria and neutrophils are undetectable [34]. Therefore, the T cell migration impairment observed deeper in the LN cannot be accounted for solely by the possible effect of neutrophils on the LN architecture in the subcapsular sinus. We propose that T cell migration inhibition could be the result of: (i) direct inhibition of T cell migration by Shigellaspecific effectors, (ii) effector-mediated induction of a variety of adhesion or recognition molecules by activated, invaded, dead, or dying cells, causing nonspecific interactions with lymphocytes, (iii) neutrophil reshaping of the LN architecture within the subcapsular sinus, and/or (iv) the effect of the massive inflammatory response that induces changes throughout the LN, leading to nonspecific interactions with APCs or cell death. Further cellular and molecular analysis to carefully dissect the contribution of the infiltrating and resident cell subtypes and their products on the observed decreased motility of polyclonal CD4+ T cells during Shigella infection is warranted and desired. Nevertheless, these data constitute the first report of the manipulation of CD4+ T cell dynamics within the LN upon bacterial infection. Indeed, the dynamic imaging of host–pathogen interactions in vivo including T cell behavior in LNs has been only reported so far for parasites and viruses [36–40], whereas dynamic imaging of T cell behavior upon bacterial infections has been studied in the spleen and in the liver upon Listeria monocytogenes [41] and Bacille Calmette-Gue´rin
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(BCG) infection [42], respectively. Briefly, interactions between T cells and DCs in the context of malaria have been visualized within the LN and infection has been shown to prevent priming of immune responses by antagonizing these cell–cell contacts, mainly by reducing T cell migration and downregulating activation markers on both T cells and DCs [43]. Similar to Shigella, Toxoplasma gondii infection also results in modulation of the behavior of non-parasite specific T cells. This T cell receptor (TCR)independent process correlates with the remodeling of the LN micro-architecture and changes in expression of CCL21 and CCL3, two chemokines stimulating T cell migration [39]. Viruses such as vaccinia virus or vesicular stomatitis virus were used to study the site of priming for T cell response within the LN. Naı¨ve CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region, just beneath the subcapsular sinus, the region reported as being the site of interactions between Shigella and T cells. Then, tight interactions with DCs were observed leading to complete T cell activation. Thus, antigen presentation at the LN periphery, not at lymphocyte exit sites in deeper LN venules as dogma dictates, has a dominant function in antiviral CD8+ T cell activation [40]. For bacterial infection, using the intracellular Gram-positive bacterium Listeria monocytogenes as a model for triggering an efficient priming of CD8+ T cells, the analysis of the migratory behavior and priming capacity of monocyte and DCs after infection of mice has highlighted the fact that the migratory route DCs take to drive T cell activation is independent of the architecture of lymphoid tissues [44]. Finally, by visualizing BCG granuloma formation in the mouse liver, the movement and behavior of T lymphocytes in granulomas were found to be very intriguing. These cells, which arrive at the granulomas within days, are in constant motion throughout the lesion. Their motion is such that each lymphocyte appears to wander through the entire granuloma, probably making direct contact with most of its macrophages. This report adds to the argument that the granuloma cannot be thought of as simply a barricade to contain mycobacteria, even after adaptive immunity is established [42]. Impairment of T lymphocyte functions by bacterial pathogens: beyond T3SS effectors and T cell migration These results emphasize the extraordinary power of the Shigella T3SS to deliver a given effector with a particular enzymatic activity, such as IpgD, into different cell types, thereby triggering a diversity of outcomes to modulate the host immune response, including dampening of the host inflammatory response [45]. T3SS host cell selectivity was previously illustrated with the Yersinia T3SS, where effector proteins were found to preferentially target phagocytic cells with high specificity for neutrophils in the LN and follicular B cells in the spleen [46,47]. The only other example of a T3SS effector impairing cell migration is SseI, the bacterial effector translocated into host cells by the Salmonella enterica pathogenicity island 2 T3SS. SseI inhibits normal cell migration of primary macrophages and DCs in vitro. Such an inhibition requires the host factor IQ motif containing GTPase activating protein, an important regulator of cell migration. DC and CD4+ T lymphocyte 195
Review migration to the spleen is also suppressed in an SseIdependent manner in vivo [48]. Shigella is unique in that IpgD injection inhibits the capacity of T lymphocytes to reorganize their cytoskeleton to undergo directional migration, irrespective of whether or not injection is followed by bacterial invasion [27]. A few recent reports have highlighted the capacity of human-specific pathogens to target T cell migration via other types of virulence effectors. Bordetella pertussis produces adenylate cyclase toxin A (CyaA), an adenylate cyclase toxin that triggers the synthesis of cyclic AMP (cAMP) in peripheral blood lymphocytes. Accumulation of cAMP within T lymphocytes inhibits chemokine receptor signaling, and hence T cell chemotaxis, by a mechanism dependent on cAMP and the protein kinase A signaling pathway [49]. The Mycobacterium tuberculosis cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) desensitizes naı¨ve T cells to the LN egress signal sphingosine-1-phosphate (S1P), causing the preferential accumulation of C–C chemokine receptor type 5 (CCR5)+ CD4+ T cells in lung-draining LNs and impairing effector T cell migration to the infected lungs [50]. One example for viral infection is the HIV-1 protein Nef (negative regulator factor), which inhibits T cell chemotaxis by at least two mechanisms: down-modulation of LFA-1 (lymphocyte/leukocyte function-associated antigen-1), an adhesion molecule critical for generation and maintenance of the T cell receptor synapse, and phosphorylation (deactivation) of cofilin, an actin-depolymerizing factor that promotes T cell motility when dephosphorylated. These mechanisms lead to decreased adhesion and polarization of T cells and dysregulation of the assembly and disassembly of actin filaments, respectively [51,52]. Not only T lymphocyte trafficking, but also other T cell functions are targeted by bacterial pathogens. As far as T3SS effectors are concerned, the Yersinia outer membrane protein (YopH) virulence protein specifically targets adaptor molecules of the TCR signaling pathway, thus inhibiting T cell activation, whereas YopP inhibits CD8+ T cell priming in a mouse infection model [53,54]. This suggests that T3SS effectors have the potential to interfere with a diversity of T cell functions. Other virulence effectors have been shown to target other T cell functions. Bordetella pertussis CyaA triggers accumulation of cAMP within T lymphocytes resulting in impairment of T cell activation [49]. The Helicobacter pylori vacuolating cytotoxin A (VacA) virulence factor blocks T cell proliferation by inducing a G1/S cell arrest [55] and uses CD18 as a VacA receptor on human T lymphocytes [56]. Very recently, evidence was provided that the polysaccharide antigen Vi released from Salmonella typhi targets the T cell membrane prohibitin (belonging to a family of evolutionarily conserved proteins implicated in a large number of cellular functions such as cell proliferation, apoptosis, and cell signaling), thus interfering with T cell activation [57]. Concluding remarks Dampening the adaptive immune response by interfering with the key cellular actors required to mount a protective immunity is definitely part of the strategies used by pathogens for their own benefit. Undoubtedly, further in vivo 196
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investigations will reveal how much does the targeting of such cells, including T and B lymphocytes, impact on the specific cellular and humoral immune responses, and subsequently on protection against re-infection. Despite many pending questions (the most important ones being outlined in Box 1), the Shigella studies have unraveled a new mechanism that pathogens use to interfere with T lymphocytes and in particular with their dynamics. These findings suggest that direct manipulation can occur via bacterial invasion of T cells or injection of effectors into T cells without invasion. As for the latter mechanism, one may easily imagine the efficiency of a strategy consisting of injecting effectors as soon as bacterium–cell contacts occur without the need for invasion, a surprising observation in view of the intracellular lifestyle of Shigella. The importance of an ‘injection-only’ pathway to ‘freeze’ the host immune response has been largely underestimated and deserves further investigation. In addition, the molecular mechanisms underlying the susceptibility of activated T lymphocytes to Shigella invasion as compared to unactivated T cells that are refractory to invasion, but apparently not to injection of T3SS effectors, deserves to be deciphered. If such an ability to deal with T cells independently of their activation state occurs when Shigella reaches the intestinal mucosa, this would certainly confer a serious advantage to the bacterium for largely counteracting T cell functions. Developing the appropriate tools to address in vivo questions regarding the dynamics of Shigella–T cell interactions, whether it is in terms of bacterial invasion versus injection-only of T3SS effectors, is definitely a great challenge. The global impact on T cell migration induced by Shigella could certainly affect key steps required for the initiation of Shigella-specific responses, such as the cognate DC–T cell encounter, priming, and mobilization to infection sites. We speculate that the impairment of T cell dynamics discussed here is a mechanism that contributes to the development of poor Shigella-specific immune responses upon natural infection. However, we are aware that it will be difficult to demonstrate this assumption due to the lack of an experimental model of infection that closely mimics human infection upon oral inoculation.
Box 1. Outstanding questions What are the molecular mechanisms underlying Shigella invasion of activated but not unactivated T lymphocytes? Are the molecules involved in the entry process into epithelial cells those required of T cell invasion? Are there new targets specific for T lymphocytes that are upregulated upon activation? Are T lymphocyte subpopulations differentially impacted upon Shigella infection? What are the molecular mechanisms leading to the impairment of migration of non-invaded T lymphocytes in the LN? Does Shigella infection disrupt the reticular network that facilitates T cell migration? Does Shigella interfere with the chemokine gradient or alter T cell sensitivity to chemokines? Does neutrophil recruitment impact on T cell migration? Does Shigella target other T cell functions besides T cell movement? In the near future, will the ongoing development of human artificial LNs in vitro solve the issue of addressing these questions in the human context?
Review Altogether, these studies contribute to a better understanding of the complex interactions between bacteria and cells of the adaptive immune system. It also emphasizes the role of T3SS and its secreted effectors in the manipulation of the adaptive response in vivo, a so far very poorly studied topic [58]. It would certainly be worthwhile to take into account these findings for revisiting the design of live, attenuated Shigella vaccine candidates. Eventually, tissue imaging that allows one to more coherently analyze an infected tissue for a much better understanding of bacterial pathogenesis within the live host promises to reveal relevant interactions and angles for disease intervention [59]. Acknowledgments W.S.-P. was funded by the Pasteur Foundation and the Philips Foundation. C.K. was supported by fellowships from the European Consortium PATHOGENOMICS, the Institut Pasteur Transversal Research (PTR) Program no. 251 led by A.P., and the Howard Hughes Medical Institute (HHMI). The research leading to these results has received funding from PTR no. 251, the European Community’s PEOPLE Seventh Framework Program under grant agreement European Institute of Microbiology and Infectious Diseases (EIMID) IAPP–PIAP-GA-2008217768, the Domaine d’Inte´reˆt Majeur Malinf-Re´gion Iˆle de France, l’Agence Nationale de la Recherche under the project ‘PATHIMMUN’, and the Ministe`re des Affaires Etrange`res Projet P2R. P.J.S. is supported by the European Research Council (ERC) and is a HHMI Foreign Scholar.
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