When a pathogenic microorganism first infects its host, there is usually a dramatic activation of the innate and adaptive immune responses, which can result in disease symptoms. If the pathogen and the host survive this initial interaction, the adaptive immune system usually clears the invading offender. However, some pathogenic bacteria are capable of maintaining infections in mammalian hosts even in the presence of inflammation, specific antimicrobial mechanisms, and a robust adaptive immune response, and can therefore be described as giving rise to persistent infection (Young et al., 2002; Rhen et al., 2003; Monack et al., 2004a, b). The persistence of the bacteria after a normal infection implies that an ‘immune status-quo' has been established after the acute response.
The host as well as the bacteria adopts various mechanisms, which bring about an ‘immune equilibrium'. Pathogens have acquired many mechanisms such as antigenic variation (Saunders, 1990; Reeves, 1993) and antigenic imitation, inhibition of synthesis of host proteins, inactivation of humoral immune components, and hiding in sites inaccessible to the immune system to avoid immune recognition and ensure their multiplication, survival, and persistence in the host even in the presence of an active immune response (Young et al., 2002). These responses can therefore be described as giving rise to persistent infections (Balaram et al., 2009).
Role of macrophages in S. Typhimurium persistence
For Salmonella, as for many other facultative intracellular pathogens, the key to a successful infection lies in the outcome of their encounter with the host's macrophages. Salmonella can escape killing by these phagocytes and survive and multiply within them, giving rise to chronic infections.
It has been well established that macrophages serve as host cells for Salmonella in vitro or in acute infection models (Fields et al., 1986; Groisman & Saier, 1990; Gulig et al., 1998). Similarly, persistent Salmonella reside within MOMA-2+ macrophages of chronically infected mice (Monack, 2004). The fate of macrophages that are persistently infected with Salmonella is not known, nor is it clear how the bacteria infect new host cells over time. It is possible that bacteria persist within macrophages for the lifetime of the host cell and then infect a new macrophage. However, S. Typhimurium is able to induce host cell death in vivo (Richter-Dahlfors et al., 1997; Monack et al., 2000), providing a potential mechanism by which Salmonella can escape from an infected cell to infect neighboring cells.
In a similar model of chronic infection, Salmonella was found within hemophagocytic macrophages, cells that have engulfed white and red blood cells. In vitro assays showed that these cells might represent a survival niche for Salmonella (Nix et al., 2007). However, it is not clear whether these cells are actively targeted by bacteria or whether the presence of Salmonella is owing to the engulfment of previously infected cells. Interestingly, studies of the intracellular replication of Salmonella revealed that the vast majority of infected cells contain very few intracellular organisms (Sheppard et al., 2003; Monack et al., 2004a). It appears that active repression of growth rate by a bacteria-directed mechanism may be critical for Salmonella virulence (Tierrez & García-del Portillo, 2005). More recently, a study suggested that Salmonella enters a dormant-like stage upon entry into macrophages (Helaine et al., 2010). These studies are in line with the average number of bacterial cells found within macrophages (3–4) in a chronic infection model (Monack et al., 2004a). However, the replicative state of Salmonella in persistently infected cells remains to be elucidated.
Intracellular trafficking of the Salmonella phagosome and the interactions between the bacteria and host factors have been analyzed in epithelial cell models or in tissue culture macrophages from susceptible mice (Haraga et al., 2008). However, the trafficking and gene expression patterns of persistent intracellular Salmonella have not yet been investigated. About 80% of the bacteria present in persistently infected MLN are found within MOMA-2 expressing macrophages (Monack et al., 2004a). The remaining 20% could be extracellular or in unidentified cells. Other in vivo models using susceptible mouse strains have shown that Salmonella colonizes predominantly macrophages in the liver and CD18+ cells (macrophages and/or DCs) in the blood (Richter-Dahlfors et al., 1997; Vazquez-Torres & Fang, 2000). Phagocytes also account for the majority of cells containing intracellular bacteria in the spleen (Salcedo et al., 2001). Infection of neutrophils and DCs by Salmonella has also been documented in spleen and MLN (Dunlap et al., 1992; Yrlid et al., 2001; Cheminay et al., 2005) in acute models of infection.
DCs are involved in the dissemination of the bacteria from the intestine to the systemic organs. Salmonella-infected DCs are able to migrate to lymphoid tissue, as shown in an adoptive transfer mouse model (Zhao et al., 2006). CD103+ CD11b+ DCs are the main DC population that transports Salmonella from the intestinal tract to the MLN early after infection, and the migration of infected DCs is partially dependent on Toll-like receptor 5 (TLR5) and the chemokine receptor CCR7 (Bogunovic et al., 2009; Uematsu & Akira, 2009; Voedisch et al., 2009). DC migration does not appear to be necessary for Salmonella dissemination from the MLN to systemic organs (Voedisch et al., 2009). However, these observations have been made only 2 days after infection in a susceptible mouse model, which limits the conclusions one can make regarding DC-dependent Salmonella dissemination during chronic infections, especially because Salmonella is usually not detected in MLN from resistant mice until 4–5 days postinfection (unpublished data from our group). In fact, our group showed that DC migration was regulated by the Salmonella SPI2 effector SseI. SseI plays a role in blocking the migration of host immune cells and consequently attenuates the host's ability to clear systemic bacteria (McLaughlin et al., 2009).
DCs are also involved in antigen presentation to T cells. Uptake of Salmonella by DC and the activation of DC immune functions, including antigen presentation, are regulated by Salmonella LPS and bacterial effectors from both SPI1 and 2 T3SS (Halici et al., 2008; Zenk et al., 2009; Bueno et al., 2010). Moreover, the reduced intracellular proliferation of Salmonella within DCs limits antigen presentation and thus the development of a rapid T-cell response (Albaghdadi et al., 2009). The limited intracellular replication and the control of T-cell responses, through antigen presentation, are likely to be important factors for the establishment of a persistent infection, as they allow survival of both the host and the bacteria. Interestingly, a subset of infected DCs can migrate from the lamina propria into the intestinal lumen (Arques et al., 2009). This finding could be important regarding the chronic infection model, as this migration pattern could be involved in the continuous presence of Salmonella in the gut lumen. This hallmark of the chronic infection model is related to the shedding of bacteria in stools of infected mice, which is thought to be responsible for the transmission of Salmonella to other animals (Lawley et al., 2008).
Adaptive immune response to Salmonella chronic infection
The initial invasion of gut-associated lymphoid tissues by Salmonella induces a massive inflammatory response, characterized by the recruitment of neutrophils, DCs, inflammatory monocytes, and macrophages (Halle et al., 2007; Rydstrom & Wick, 2007). All of these cell types are important for containing the initial bacterial invasion and thus play a role in the establishment of a persistent infection (Griffin & McSorley, 2011). These early events, as well as the cells and molecules involved have been extensively studied with regard to the acute phase of infection; however, the potential role of these cell types in the immune status of bacterial carriers is scarce (Balaram et al., 2009).
The magnitude and duration of an immune response are modulated by the production of cytokines. CD4 T-helper (Th) cells play a central role in the production of cytokines during Salmonella infection. The most important signal for T-cell helper differentiation is the cytokine profile present at the time of T-cell stimulation. For example, a Th1 profile is induced by the IL-12 family and interferon, whereas a Th2 profile is induced by IL-10 and IL-4. Th1 is typically seen as pro-inflammatory and as inducing intracellular antimicrobial killing mechanisms, while Th2 is regarded as anti-inflammatory and responsible for the response to helminthes, allergies, and low dose antigens. The potential role of these immune profiles during persistent Salmonella infections has been studied in several mouse models of chronic infection.
The establishment of a persistent infection can be divided into two phases. First, an early resistance phase during which the immune system works to reduce the number of invading bacteria and has been characterized by a high Th1 and a low Th2 responses. Indeed, TNF-α, IL-12, IFN-g, and nitric oxide (NO) derivatives are all required for the control of Salmonella growth by the infected host in the acute stage of the infection (Butler & Girard, 1993; Trinchieri, 1995; Mastroeni, 2002; Mizuno et al., 2003; Netea et al., 2003; Stoycheva & Murdjeva, 2005; Sashinami et al., 2006). In addition, Blackwell et al. (1999) demonstrate that there is an in vivo bias toward the development of a Th1 response in mice bearing the wild-type Nramp1 allele, while a Th2 response is elicited in Nramp1 mutant mice. Because Nramp1 mutant mice are susceptible to Salmonella infection, this finding underscores the importance of a Th1 response in the resistance to the acute phase of Salmonella infection. This resistance phase is crucial in setting the equilibrium between the immune response and the bacteria that will allow for the establishment of the long-term infection state.
The second step is the maintenance of this equilibrium. Although this stage may require lower Th1 response than in the early phase, IFN-γ still plays an important role in maintaining the equilibrium, as injection of an anti-IFN-γ antibody into mice persistently infected with S. Typhimurium led to the reactivation of an acute infection (Monack, 2004). In addition, persistence of Salmonella Clp-XP-deficient bacteria in Balb/c mice required IFN-γ and TNF-α (Yamamoto et al., 2001). Furthermore, T-bet-deficient mice that did not generate IFN-γ-producing CD4 T cells had higher bacterial loads in spleens compared to WT mice and eventually succumbed to infection with attenuated Salmonella (Ravindran et al., 2005). Interestingly, the T-bet-deficient mice survived the first 3 weeks of infection, which correlated with significantly higher bacterial levels compared to WT mice at 3 weeks after infection. This would suggest a role for T-bet-dependent IFN-γ production in the persistence phase of an infection, rather than in the early response to the bacterial invasion.
The balance between Th1 and Th2 levels is likely to be critical for the maintenance of a persistent microbial infection. Various studies have suggested that the pathogenesis and persistence of various chronic bacterial, viral, or parasite infections involve a Th1 to Th2 cytokine switch (Fitzgerald, 1992; Becker, 2004). Mice persistently infected with Salmonella have high antibody titers, which is consistent with a Th2-biased immune response (Monack, 2004). In addition, a Th2 response is generally associated with a higher bacterial load. In support of this, mice exposed to lead (Pb), which leads to a Th2-skewed immune response, have significantly higher bacterial burdens compared to control mice (Fernandez-Cabezudo et al., 2007). In addition, co-infection by Salmonella and Schistosoma, which induces a Th2-skewed host immune response (Sacco et al., 2002; Wynn et al., 2004; Schramm & Haas, 2010), increased Salmonella growth and prolonged the duration of Salmonella infection (Rocha et al., 1971; Njunda & Oyerinde, 1996; Bouree et al., 2002; Muniz-Junqueira et al., 2009). However, surprisingly, a study of mice deficient for Nramp1 and infected with a sublethal dose of Salmonella showed a reduced bacterial load was associated with a higher Th2 response (Caron, 2006). Taken together, these findings suggest that the cytokine profiles differ in acute infection compared to persistent Salmonella infections. It appears that a Th1 response is beneficial for the clearance of a high load of bacteria and at the same time conducive for the persistence of low bacterial load. Thus, the Th1/Th2 balance appears to be critical in maintaining an exquisite equilibrium between pathogen survival and host clearance mechanisms.
In addition to Th1 and Th2 T cells, there has been a great deal of interest recently in the role of Th17-producing T cells in the control of Salmonella infections. Th17 cells are defined as a distinct lineage from Th1 and Th2 cells and are characterized by the expression of cytokines such as IL-17A and F, IL-22, and IL-26. IL-17A and IL-22 are induced during some Salmonella infections, and recent work in mice, calves, and the macaque has suggested that these cells are critical for controlling local invasion by Salmonella (Raffatellu et al., 2008; Godinez et al., 2009; Liu et al., 2009). However, a recent study suggested that IL-17 may not be as important for the inflammatory response to S. Typhimurium in the gut (Songhet et al., 2010). In addition, a recent study indicated that IL-17A-producing cells had a limited role in the maintenance of the protective immunity against Salmonella (Schulz et al., 2008). Thus, more studies are necessary to fully address the potential role of Th17 T cells during persistent Salmonella infection. Finally, the role of another subset of effector CD4 T cells, regulatory T cells (Tregs), has been studied in a chronic model of Salmonella infection. The Tregs suppressive potency modulates the effectiveness Th1 responses during the course of Salmonella infection and thus influences the progression of persistent infection (Johanns et al., 2010).
Resolution of a primary S. Typhimurium infection in mice is a combination of innate and T-cell-mediated effects with a less significant role for B cells and antibodies, because B cell-deficient mice can clear infection. However, the Th1 response these mice develop is transient, and they are not protected from re-infection with a virulent strain (Mastroeni et al., 2000a, b; Mastroeni, 2002). In a model of susceptible mice persistently infected with an attenuated Salmonella strain, B cells were shown to contribute to the early phase of T-cell programing via a MyD88-dependent mechanism and are required in a BCR-dependent process for the development of the memory T-cell response (Barr et al., 2010). Thus, TLR activation of B cells optimizes the generation of the primary Th1 response, a process that does not require Ag presentation, but relies on B cell cytokine secretion. Thus, BCR recognition and B cell Ag presentation are an absolute requirement for the development of Th1 memory cells and hence, protective immunity to Salmonella (Barr et al., 2010).
What is the role of the indigenous intestinal microbiota during Salmonella persistence? The interactions between the gut microbiota and the immune response are under intense study because of the effects of this relationship with the health of an individual. The microbiota has been shown to play an important role in the host susceptibility to acute Salmonella infection (Bohnhoff et al., 1954; Collins & Carter, 1978; Stecher et al., 2005; Sekirov et al., 2008). In chronically infected mice, the microbiota is required to lower the bacterial load in the gut and thus lower the fecal shedding of the bacteria. This effect seems to be because of the microbiota itself rather than by a microbiota-induced mucosal response (Endt et al., 2010).