LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P_BADcrp) and replacement of the msbB gene with the Escherichia colimsbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P_msbBmsbB_(EC) ΔP_crp21::TT araC P_BADcrp] for use with a balanced-lethal Asd-positive (Asd⁺) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE_Nt138-LcrV) was synthesized in χ10057 harboring an Asd⁺ plasmid (pYA5199, yopE_Nt138-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ∼240 median lethal doses (LD₅₀) (2.4 × 10⁴ CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.     

The N Terminus of Type III Secretion Needle Protein YscF from Yersinia pestis Functions To Modulate Innate Immune Responses

The type III secretion system is employed by many pathogens, including the genera Yersinia, Shigella, Pseudomonas, and Salmonella, to deliver effector proteins into eukaryotic cells. The injectisome needle is formed by the polymerization of a single protein, e.g., YscF (Yersinia pestis), PscF (Pseudomonas aeruginosa), PrgI (Salmonella enterica SPI-1), SsaG (Salmonella enterica SPI-2), or MxiH (Shigella flexneri). In this study, we demonstrated that the N termini of some needle proteins, particularly the N terminus of YscF from Yersinia pestis, influences host immune responses. The N termini of several needle proteins were truncated and tested for the ability to induce inflammatory responses in a human monocytic cell line (THP-1 cells). Truncated needle proteins induced proinflammatory cytokines to different magnitudes than the corresponding wild-type proteins, except SsaG. Notably, N-terminally truncated YscF induced significantly higher activation of NF-κB and/or AP-1 and higher induction of proinflammatory cytokines, suggesting that a function of the N terminus of YscF is interference with host sensing of YscF, consistent with Y. pestis pathogenesis. To directly test the ability of the N terminus of YscF to suppress cytokine induction, a YscF-SsaG chimera with 15 N-terminal amino acids from YscF added to SsaG was constructed. The chimeric YscF-SsaG induced lower levels of cytokines than wild-type SsaG. However, the addition of 15 random amino acids to SsaG had no effect on NF-κB/AP-1 activation. These results suggest that the N terminus of YscF can function to decrease cytokine induction, perhaps contributing to a favorable immune environment leading to survival of Y. pestis within the eukaryotic host.     

DNA Adenine Methylase Overproduction in Yersinia pseudotuberculosis Alters YopE Expression and Secretion and Host Immune Responses to Infection

Yersinia pseudotuberculosis mutants that overproduce the DNA adenine methylase (Dam) are highly attenuated, confer fully protective immune responses, and secrete several Yersinia virulence proteins (Yersinia outer proteins [Yops]) under conditions that are nonpermissive for secretion in wild-type strains. We examined here the effects of Dam overproduction on Yersinia virulence determinant expression and secretion, as well as the host immune response to Yersinia antigens. Western blot analysis with convalescent antisera identified several low-calcium-responsive antigens whose synthesis was affected by Dam overproduction. One of these antigens was shown to be the type III secretion effector protein, YopE, a cytotoxin involved in antiphagocytosis. Dam overproduction disrupted both the thermal and calcium regulation of YopE synthesis and relaxed the thermal but not the calcium dependence of YopE secretion. Altered expression and/or secretion of Yersinia proteins in Dam-overproducing strains may contribute to the decreased virulence and heightened immunity observed in vaccinated hosts and may provide a means by which to deliver heterologous antigens and/or immune modulators of the inflammatory response.     

Type III secretion decreases bacterial and host survival following phagocytosis of Yersinia pseudotuberculosis by macrophages

Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV(+) wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV(+) Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV(+) but not pYV-cured or DeltayopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV(+) bacteria internalized replicated by 24 h. In addition, approximately 20% of macrophages infected with wild-type pYV(+) Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV(+) Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.     

Type III secretion system-dependent translocation of ectopically expressed Yop effectors into macrophages by intracellular Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.     

Pathogen Recognition by the Innate Immune System

Microbial infection initiates complex interactions between the pathogen and the host. Pathogens express several signature molecules, known as pathogen-associated molecular patterns (PAMPs), which are essential for survival and pathogenicity. PAMPs are sensed by evolutionarily conserved, germline-encoded host sensors known as pathogen recognition receptors (PRRs). Recognition of PAMPs by PRRs rapidly triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines, chemokines and type I interferons. These responses also initiate the development of pathogen-specific, long-lasting adaptive immunity through B and T lymphocytes. Several families of PRRs, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and DNA receptors (cytosolic sensors for DNA), are known to play a crucial role in host defense. In this review, we comprehensively review the recent progress in the field of PAMP recognition by PRRs and the signaling pathways activated by PRRs.     

Host Innate Immune Response to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

This review focuses on recent progress in our understanding of Mycobacterium tuberculosis survival in macrophages, the interaction of M. tuberculosis with Toll-like receptors (TLRs) and the establishment of the link between innate and adaptive immunity, and TLRs and interferon-γ-mediated antimicrobial pathways in macrophages. We also propose a paradigm that TLR2 signaling regulates the magnitude of the host Th1 response leading to either M. tuberculosis persistence and latent infection or replication and disease.     

Susceptibility to Yersinia pseudotuberculosis Infection is Linked to the Pattern of Macrophage Activation

T helper 1 cells play a crucial role in the clearance of Yersinia pseudotuberculosis infection. By producing cytokines and presenting antigens to T cells, activated macrophages can orientate the adaptive immune response. The pathway used by macrophages to metabolize arginine has been employed as an important parameter to discriminate their activation state. In this study, the pattern of macrophage activation in Y. pseudotuberculosis- infected BALB/ c ( Yersinia -susceptible) and C57BL/6 ( Yersinia -resistant) mice and their immunostimulatory capacity were analysed. In the early phase of infection, macrophages obtained from C57BL/6 mice produced higher levels of NO, lower arginase activity, and larger amounts of IL-12 and TNF-α than macrophages from BALB/ c mice. On the other hand, macrophages derived from BALB/ c mice produced higher levels of IL-10 and TGF-β than C57BL/6 mice. The Y. pseudotuberculosis infection leads to a fall in the macrophage immunostimulatory capacity of both strains of mice, with T-cell proliferation significantly reduced 12 h after infection. Moreover, we observed in the supernatant of co-culture of macrophages from infected mice with T lymphocytes from heat-killed Yersinia -immunized mice lower IFN-γ production by cells from BALB/ c mice than by C57BL/6 mice, and IL-4 was produced only by BALB/ c mice on the first- and third-day post-infection. These results suggest that the pattern of macrophage activation is associated with susceptibility and resistance to Y. pseudotuberculosis infection in BALB/ c and C57BL/6 mice.     

Kinetics of Innate Immune Response to Yersinia pestis after Intradermal Infection in a Mouse Model

A hallmark of Yersinia pestis infection is a delayed inflammatory response early in infection. In this study, we use an intradermal model of infection to study early innate immune cell recruitment. Mice were injected intradermally in the ear with wild-type (WT) or attenuated Y. pestis lacking the pYV virulence plasmid (pYV⁻). The inflammatory responses in ear and draining lymph node samples were evaluated by flow cytometry and immunohistochemistry. As measured by flow cytometry, total neutrophil and macrophage recruitment to the ear in WT-infected mice did not differ from phosphate-buffered saline (PBS) controls or mice infected with pYV⁻, except for a transient increase in macrophages at 6 h compared to the PBS control. Limited inflammation was apparent even in animals with high bacterial loads (10⁵ to 10⁶ CFU). In addition, activation of inflammatory cells was significantly reduced in WT-infected mice as measured by CD11b and major histocompatibility complex class II (MHC-II) expression. When mice infected with WT were injected 12 h later at the same intradermal site with purified LPS, Y. pestis did not prevent recruitment of neutrophils. However, significant reduction in neutrophil activation remained compared to that of PBS and pYV⁻ controls. Immunohistochemistry revealed qualitative differences in neutrophil recruitment to the skin and draining lymph node, with WT-infected mice producing a diffuse inflammatory response. In contrast, focal sites of neutrophil recruitment were sustained through 48 h postinfection in pYV⁻-infected mice. Thus, an important feature of Y. pestis infection is reduced activation and organization of inflammatory cells that is at least partially dependent on the pYV virulence plasmid.     

Cell-Contact-Stimulated Formation of Filamentous Appendages by Salmonella typhimurium Does Not Depend on the Type III Secretion System Encoded by Salmonella Pathogenicity Island 1

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717–724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer’s patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.     

Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1β secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.Legionella pneumophila is a Gram-negative, facultative intracellular bacterium that causes a severe form of pneumonia called Legionnaires disease in humans. Essential for L. pneumophila pathogenesis is its capacity to replicate within alveolar macrophages; thus, the bacteria utilize a type IVB secretion system called Dot/Icm to modulate phagosome biogenesis and create an intracellular niche that supports bacterial multiplication (reviewed in references 16, 26, and 53). The Dot/Icm is composed of about 26 genes that encode a diverse set of proteins: some are contained in the cytoplasm, such as IcmW (60), IcmQ, IcmR, IcmS (14), and the ATPase DotB (50); some are periplasmic, such as IcmX (36); and some are known to localize in the inner bacterial membrane, such as DotA (60), DotL (9), DotU, and IcmF (49, 54). Collectively, this system builds a complex that facilitates the secretion of a large repertoire of bacterial proteins directly into the host cell cytoplasm. The secreted proteins, so-called effectors, target different host cell processes and, whereas deletions of the Dot/Icm structural components abolish bacterial multiplication in bone marrow-derived macrophages (BMMs), deletions of single effector proteins have little impact on the capacity of L. pneumophila to multiply in BMMs (20, 44).In contrast to human cells, macrophages obtained from most inbred mice strains are nonpermissive for L. pneumophila replication (57). We have previously demonstrated that caspase-1 activation effectively contributes to the restriction of L. pneumophila multiplication by mechanisms not yet identified (58). In addition to caspase-1, Naip5 and Nlrc4/Ipaf, which belong to the Nod-like receptor (NLR) family of proteins, are also required for the effective restriction of L. pneumophila multiplication in murine BMMs (5, 24, 41, 46, 58). This restriction is also dependent on bacterial flagellin as bacterial mutants deficient in flaA fail to trigger caspase-1 activation and freely multiply in restrictive BMMs (5, 41, 46). The demonstration that both Nlrc4^−/− and Naip5^−/− BMMs fail to trigger caspase-1 activation in response to L. pneumophila flagellin (35) has led to the speculation that both proteins form an inflammasome responsible for flagellin recognition. Whereas the Nlrc4/Naip5 inflammasome is required for flagellin recognition in a process that culminates with the restriction of L. pneumophila multiplication, another inflammasome seems to be engaged in response to L. pneumophila infection (11). This second inflammasome is dependent on ASC (apoptosis-associated speck-like protein with a caspase recruitment domain) but independent of Nlrp3/Nalp3 and apparently plays no role in the restriction of Legionella multiplication in BMMs (11). Interestingly, ASC^−/− BMMs were severely impaired for caspase-1 activation and interleukin-1β (IL-1β) secretion in response to L. pneumophila; therefore, the ASC-dependent inflammasome may play a major role in the processing and secretion of IL-1β (11, 58). Although extensively investigated, the mechanism by which IL-1β is secreted from BMMs is still not clear; the protein lacks the signal for canonical endoplasmic reticulum (ER)-Golgi apparatus protein secretion. Possible mechanisms to explain IL-1β secretion include the following: (i) release upon host cell lysis, (ii) release via exocytosis of secretory lysosomes, (iii) release from shed plasma membrane microvesicles, (iv) release via fusion of multivesicular bodies with the plasma membrane and subsequent release of IL-1β-containing exosomes, or (v) release through a transporter or pores formed in host cell membranes (19).In this context, Fink and Cookson have recently demonstrated that Salmonella enterica serovar Typhimurium (S. Typhimurium) induced pore formation in the membranes of J774 macrophage-like cells in a process temporally associated with secretion of active IL-1β and IL-18 (22). This process was inhibited by caspase-1 inhibitor Z-YVAD-FMK and was dependent on the S. Typhimurium type III secretion system (22). Interestingly, previous studies have demonstrated that L. pneumophila triggers pore formation in BMMs by mechanisms dependent on the Dot/Icm secretion system (31, 60). However, it was not clear whether this pore was a host cell response or the result of membrane damage induced directly by the Dot/Icm secretion system. Because recent reports demonstrated that the cytotoxic effects of L. pneumophila were independent of ASC and Nlrp3 but dependent on flagellin, Naip5, and Nlrc4 (11, 35, 46), we used L. pneumophila as a model to assess the host and bacterial factors involved in pore formation in BMMs and their relation to IL-1β secretion. By using several isogenic mutants of L. pneumophila in combination with primary BMMs from different knockout mice, we obtained results that account for the determination of host and bacterial factors responsible for pore formation in response to L. pneumophila infection. Our data support the hypothesis that pore formation is not the result of membrane damage induced by Dot/Icm activity. Instead, pore formation is a highly coordinated host response that requires host Nlrc4 and caspase-1, and it is triggered in response to pathogenic bacteria expressing virulence factors such as flagellin and type IV secretion systems.     

Pathogenesis of Necrotizing Enterocolitis: Modeling the Innate Immune Response

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant''s intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice.Necrotizing enterocolitis (NEC) is a disorder characterized by intestinal necrosis in premature infants that results in significant morbidity and mortality.¹ Approximately 7% of infants with a birth weight between 500 and 1500 g develop NEC.¹ The pathogenesis is characterized by intestinal inflammation that can progress to systemic infection/inflammation, multiorgan failure, and death. The bowel is distended and hemorrhagic on gross inspection. On microscopic examination, signs of inflammation, mucosal edema, epithelial regeneration, bacterial overgrowth, submucosal gas bubbles, and ischemic transmural necrosis are seen (Figure 1, A–E).²Open in a separate windowFigure 1Examples of the various grades of morphological damage in hematoxylin and eosin–stained specimens. A–E: Representative samples of premature infants with necrotizing enterocolitis. A: Age-matched control from patient with jejunal atresia. B: Mild injury with hemorrhagic necrosis of mucosa and loss of villus tip architecture. C: Progressive injury with inflammatory infiltration of muscularis with complete villus destruction. D: Severe muscular and epithelial damage with complete loss of mucosa. E: Perforation with transmural necrosis with complete loss of epithelial and muscular architecture. F–J: Representative samples from intestinal injury secondary to gavage feeding in the setting of hypothermia and hypoxia in neonatal rats. F: Intact morphology, grade 0. G: Sloughing of villus tips, grade 1. H: Mid-villus necrosis, grade 2. I: Loss of villi, grade 3. J: Complete destruction of the mucosa, grade 4. Insets in F–J show higher magnified portions of the same sections, corresponding to the boxed regions. K–O: Representative images of tissue injury secondary to 60 minutes of intestinal ischemia and 90 minutes of reperfusion in 2-week-old mice. K: Sham-operated mice (no ischemia). L: Villus tip necrosis. M: Mid-villus necrosis. N: Loss of villus architecture. O: Complete loss of mucosal architecture. F–J, reprinted with permission from Nature Publishing Group.²⁸ Scale bars = 50 μm (A–E, K–O). Original magnification, ×20 (A–O, main images, and F–J, insets).Currently the pathogenesis of NEC is believed to have multifactorial causes, including intestinal immaturity and microbial dysbiosis. Intestinal immaturity leads to a compromised intestinal epithelial barrier, an underdeveloped immune defense, and altered vascular development and tone. The compromised epithelial barrier and underdeveloped immune system, when exposed to luminal microbiota that have been shaped by formula feedings, antibiotic exposure, and Cesarean delivery, can lead to intestinal inflammation and sepsis. Despite therapeutic success in animal model systems, there are relatively few therapeutic strategies that have allowed for significantly improved outcomes in infants with NEC. Two hurdles that persist are our incomplete understanding of the developing immune system in premature infants and our inability to adequately replicate these complex factors in animal models.^3,4 This review summarizes the complex intestinal immune system in premature infants and details what is known about the involvement of innate immune factors in NEC, both in animal models and in human disease.     

Edwardsiella tarda-Induced Cytotoxicity Depends on Its Type III Secretion System and Flagellin

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection. Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS of E. tarda facilitates its survival and replication in murine bone marrow-derived macrophages, and E. tarda infection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1β in a T3SS-dependent manner. Deletion of the flagellin gene fliC of E. tarda results in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in the fliC mutant strain reduces its virulence. We propose that the host controls E. tarda infection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity.     

The Capsule of Acinetobacter baumannii Protects against the Innate Immune Response

Acinetobacter baumannii is an opportunistic pathogen that has recently emerged as a global threat associated with high morbidity, mortality, and antibiotic resistance. We determined the role of type I interferon (IFN) signaling in A. baumannii infection. We report that A. baumannii can induce a type I IFN response that is dependent upon TLR4-TRIF-IRF3 and phagocytosis of the bacterium. Phase variants of A. baumannii that have a reduced capsule, lead to enhanced TLR4-dependent type I IFN induction. This was also observed in a capsule-deficient strain. However, we did not observe a role for this pathway in vivo. The enhanced signaling could be accounted for by increased phagocytosis in capsule-deficient strains that also lead to enhanced host cell-mediated killing. The increased cytokine response in the absence of the capsule was not exclusive to type I IFN signaling. Several cytokines, including the proinflammatory IL-6, were increased in cells stimulated with the capsule-deficient strain, also observed in vivo. After 4 h in our acute pneumonia model, the burden of a capsule-null strain was significantly reduced, yet we observed increases in innate immune cells and inflammatory markers compared to wild-type A. baumannii. This study underscores the role of phase variation in the modulation of host immune responses and indicates that the capsule of A. baumannii plays an important role in protection against host cell killing and evasion from activation of the innate immune response.     

The Impact of Exogenous Factors on Respiratory Pathogen-Induced Innate Alveolar Macrophage Responses in COPD

Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have fundamentally impaired innate immune responses to toll-like receptor (TLR) ligands of nontypeable Haemophilus influenzae (NTHI). However, whether dysfunctional inflammatory responses in COPD extend to macrophage interactions with intact respiratory pathogens beyond NTHI has not been explored. Furthermore, the influences of exogenous factors, including active smoking and medications, on pathogen-induced innate immune responses have only begun to be investigated. We hypothesized that distinct alveolar macrophage impairments in COPD are not limited to NTHI TLR ligands and that active smoking and select COPD medications modulate innate responses.

Alveolar macrophages, obtained from COPD ex-smokers (n = 32) and active smokers (n = 64) by bronchoalveolar lavage (BAL), were incubated with NTHI, Moraxella catarrhalis, and Streptococcus pneumoniae, and with TLR2 and TLR4 ligands. Elicited IL-8 and TNF-α were measured by multianalyte microsphere flow cytometry to determine proinflammatory responsiveness.

Induced IL-8, but not TNF-α, was greater from alveolar macrophages of active smokers compared with ex-smokers, in response to NTHI (p = 0.04), M. catarrhalis (p = 0.003), and S. pneumoniae (p = 0.03). Both IL-8 and TNF-α induction by TLR2 and TLR4 ligands were greater in active smokers. While intergroup NTHI- and M. catarrhalis-induced TNF-α levels were no different, they were notably lower among ex-smokers taking anticholinergic medications (p < 0.04 for each), but not with any other bronchoactive medications. Our results support a paradigm of distinct immunologic responses of COPD alveolar macrophages of ex- and active smokers to diverse respiratory pathogens and highlight a subset of ex-smokers whose diminished alveolar macrophage responsiveness may be associated with anticholinergic agents.     

Interaction of Mucosal Microbiota with the Innate Immune System

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m², and the skin covers approximately 1.8 m² surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.     

Aging of the Innate Immune System: An Update

The relationship between advanced age and immunologic deficits is becoming an area of rapidly advancing research. Many of the clinical hurdles in the elderly population result from dysregulation of the immune system leading to the inability of the elderly to swiftly combat infection and to the increased incidence of chronic disease states and autoimmune conditions. Herein, we address the crucial alterations in the innate immune system that occur with advancing age. Specifically, we discuss how the effects of advanced age may lead to functional changes in the neutrophil, macrophage, dendritic cell, natural killer cell, and natural killer T cell populations in human and murine models that translate into aberrant innate immune responses. Furthermore, we elucidate how these changes may contribute to documented deficits in adaptive immunity as well as the pathological conditions and the increased morbidity and mortality seen in the elderly population.