La diferenciación entre las FFLL de verbo soporte y las realizativas

The initial recognition by resident immune cells and respiratory epithelial cells represents an important step in the successful induction of specific immunity. DCs and macrophages are specialized inflammatory cells that are strategically localized throughout the body near potential pathogen entry portals, including the respiratory tract. These cells act as sentinels for the immune system by sensing the presence of potential pathogens through PRRs. Their main aim is to recognize and process antigens and present these to the adaptive immune system. PRRs recognize distinct evolutionary conserved microbial components called pathogen-associated molecular patterns (PAMPs) such as lipids, lipoproteins, proteins, and nucleic acids, derived from a wide range of bacteria, viruses, parasites, and fungi (reviewed by (192, 193)). There are many different classes of PRRs, including surface-expressed Toll-like receptors (TLRs), cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors, C-type lectin receptors, and retinoic acid-inducible gene-I-like receptors. Prevention of an effective immune response, either through directly preventing recognition or by modulating the downstream pathways, is an effective survival mechanism for bacteria. The biological mechanisms by which B. pertussis achieves this will be discussed here.

Most efforts aimed at elucidating the interaction between B. pertussis and innate immune cells have looked at human monocyte-derived DCs (MDDCs), as these cells are relatively easy to culture in vitro. Immature MDDCs express a repertoire of PRRs, such as TLR2, TLR4, TLR5, but not TLR9, as well as a range of intracellular PRRs, that are able to recognize several

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bacterial PAMPs.

TLR4 is involved in the recognition of LPS (192, 194). Typically, LPS structures are comprised of three distinct structural domains, the lipid A, the core oligosaccharide, and O-antigenic repeats. LPS is bound by the LPS-binding protein, which forms a complex with CD14, followed by the transfer of LPS to the surface-exposed TLR4-MD-2 complex. Following recruitment of MyD88 and TRIF to the cytoplasmic domain of TLR4, NF-κB and interferon regulatory factor 3 (IRF3) signaling pathways are induced, leading to the production of pro- inflammatory cytokines including type I interferons and IL-6 (193).

In contrast to most Gram-negative bacteria, B. pertussis produces a LOS that has a branched core structure with a non-repetitive trisaccharide, rather than a long repeating O-side chain (195). The lipid A moiety of B. pertussis LOS also activates TLR4 signaling pathways in MDDCs, albeit significantly less efficient compared to the typical lipid A domain present on the LPS structure of enteric Gram-negative pathogens (196, 197).

Activation of TLR4 signaling pathways by LOS is dependent on recognition of the relatively conserved lipid A structure moiety (193). In recent years, it has become apparent that variation in bacterial LPS is not restricted to the O-antigen repeats but also occurs in the lipid A region, leading to differential inflammatory responses (198). Initially, Preston et al. showed that the lipid A moiety of B. bronchiseptica LPS is modified through the palmitoyl transferase activity of the Bvg+ _{phase-specific PagP enzyme (199). By expressing B. bronchiseptica PagP}

and its antagonist PagL in B. pertussis, Geurtsen et al. revealed that these enzymes can modify B. pertussis LOS in a similar fashion. Importantly, they showed that the PagP-mediated palmitoylation is crucial for the endotoxic activity of LOS (200). However, it should be mentioned that although the genome of B. pertussis contains both genes, they are both pseudogenes and therefore cannot explain the high degree of LPS variation that was observed by the authors (200). This implies that other enzymes with similar activity may be expressed in B. pertussis, such as the outer membrane phospholipase A.

It is tempting to speculate that B. pertussis is able to modulate its LOS in response to changing conditions within the host, as for example has been observed in B. bronchiseptica in which LPS biosynthesis is Bvg-regulated (201). In fact, B. pertussis has been found to substitute the lipid A phosphate groups of its LOS with glucosamine (GlcN), which also occurs in a BvgAS-dependent manner under Bvg+ _{conditions (202, 203). Importantly, GlcN}

substituents have been shown to be more potent inducers of the release of proinflammatory cytokines by macrophages (204). Although the in vivo significance of this observation has not yet been determined, it could be hypothesized that GlcN-modifications of LOS are important for skewing the host immune response, as they induce a different cytokine response. The mucosal surfaces of the upper respiratory tract are colonized with a plethora of other species and consequentially, the innate immune system is stimulated with a complex mixture of PAMPs, including B. pertussis LOS. In contrast, B. pertussis LOS may be able to interact in a more direct manner with the innate immune system in the lungs, which are normally sterile. It would be interesting to investigate if GlcN-modification of LOS is dependent on the location and differs between the upper and lower respiratory tract. One interesting alternative hypothesis for

the biological effect of the modified LOS is that it may also play a role in altering the outcome of bacterial competition by directing the innate immune response to competing flora. There is precedent for this, as Lysenko et al. have shown that species-specific stimulation of the innate immune response can be an effective strategy to outcompete nasal flora (205).

The lipid A and the oligosaccharide core domain of LPS can also be recognized by the surfactant proteins A and D (SP-A and SP-D, respectively), hydrophilic lipid-binding lectins that are ubiquitously expressed in the lower respiratory tract of humans (reviewed in (206)). Binding of SP-A to LPS induces agglutination, destabilizes the bacterial membrane, and facilitates phagocytosis (reviewed in (207)). Further, SP-A-mediated recognition of LPS can also modify LBP-CD14 complexes, thus contributing to an altered recognition of LPS by TLR4 (208). Interestingly, wild-type B. pertussis LOS is not recognized and bound by SP-A or SP-D (209, 210). However, after removal of one or more sugars from the terminal trisaccharide, LOS is recognized by SP-A and SP-D, leading to effective bacterial opsonization and phagocytosis (209). Thus, it appears that the terminal trisaccharide of B. pertussis LOS prevents access of SP-A and SP-D to the lipid A domain through steric hindrance, and thereby protects the bacteria from surfactant-mediated clearance.

Because LPS is such a characteristic and dominant feature of Gram-negative bacteria, the host has evolved multiple recognition receptors to sense the presence of this molecule, e.g. through surfactants. By expressing LOS, B. pertussis is able to effectively prevent surfactant-mediated recognition and clearance. Furthermore, by changing the composition of the lipid A moiety, it is able to modulate the LOS-mediated immune response. It remains to be investigated whether or not the stronger inflammatory response initiated by the Bvg+_-phase-

specific modification favors within-host survival.

Besides LPS, TLR4 has also been shown to recognize other factors expressed by pathogens, e.g. the pneumolysin that is released by Streptococcus pneumoniae or the fusion (F) protein on the surface of respiratory syncytial virus (211, 212). B. pertussis, Ptx also activates TLR4 signaling pathways in MDDCs (213). In addition to TLR4, stimulation of MDDCs with intact B. pertussis also induces TLR2 signaling, and Ptx has recently been established to be the agonist triggering these TLR2 signaling pathways by utilizing TLR2/TLR4 engagement (214, 215).

The intracellular cytosolic sensor Nod1, which recognizes peptidoglycans (216), has been shown to be activated by TCT, which essentially is a diaminopimelic (DAP)-containing tetrapeptide muramylpeptide (M-Tetra_DAP) (217). Surprisingly, this recognition was found to be mouse-specific. Whilst murine Nod1–mediated detection of TCT led to an efficient cytokine response and the production of NO (the mediators of cytotoxicity), human Nod1 was unresponsive to TCT (217). Differential recognition of TCT was shown to be dependent on a distinct preference of murine Nod1 for M-Tetra_DAP, whereas human Nod1 preferentially recognized DAP-containing tripeptide muramylpeptides (M-Tri_DAP). Importantly, this observation implies that the initially observed TCT-induced cytotoxicity in tracheal epithelial cells may not be due to the effect of TCT itself, but may predominately be caused by M-Tri_DAP, or alternatively by an unknown TCT-receptor.

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To conclude, host innate immune cells sense the presence of B. pertussis predominantly by TLR4-mediated recognition of LOS and to a minor extent by TLR2-mediated recognition of Ptx.