Innate recognition of bacteria

Bacterial infections are a leading cause of morbidity and mortality worldwide. The first line of defense against pathogenic microorganisms is provided by the innate immune system. In order to recognize the presence of bacteria, innate effector cells, such as macrophages, neutrophils and dendritic cells, express an array of so-called pattern recognition receptors (PRRs) that sense conserved bacterial molecules, including lipids, nucleic acids or proteins.

The most well-studied PRRs are Toll-like receptors (TLRs), of which ten have been identified in humans. While TLR3, 7 and 8 are linked to antiviral immune responses, TLR1, 2, 4, 5, 6 and 9 predominantly signal the presence of bacteria [1]. Upon encounter of bacterial molecules by TLRs, signaling pathways, including MAPKs and nuclear translocation of nuclear factor (NF)-κB, result in the release of inflammatory mediators and activation of immune effector cells. TLRs are classical transmembrane proteins, with their ligand-binding domains pointing towards the extracellular milieu or corresponding membrane-enclosed intracellular compartments, such as the endosome. Most TLRs have been studied extensively on the basis of their ability to recognize distinct bacterial ligands in vitro. Consecutive in vivo studies using knockout mice do not always reveal such a strong phenotype and susceptibility to infection, as anticipated based on in vitro studies. While TLR4, which recognizes the Gram-negative bacterial component lipopolysaccharide, a well-known inducer of inflammation, is definitely required for any inflammatory response during endotoxemia, it also contributes to protective immune response mechanisms during Gram-negative bacterial infections [1].

By contrast, TLR2, which recognizes many Gram-positive cell wall components, such as peptidoglycan, lipopetides and lipoteichoic acid, was considered a key receptor required for host defense against Gram-positive infections; however, this assumption could not be unequivocally confirmed by studies that investigated infections using viable bacteria. In our hands, the presence of TLR2 on alveolar macrophages in vitro was absolutely required for an inflammatory response to the Gram-positive pathogen Streptococcus pneumoniae [2]. However, when TLR2^-/- mice were infected with S. pneumoniae, we could not discern an impact on survival nor aon ntibacterial defense [2]. Nevertheless, a partial effect was observed since the absence of TLR2 was associated with less pronounced pulmonary infiltrates. One might ask how much importance TLR2 contributes to this classical Gram-positive infection (pneumococcal pneumonia), that is to say, as long as bacteria are eliminated effectively, more pronounced pulmonary infiltrates will only harm a patient.

The requirement for single TLRs definitely depends on the type of pathogen, and the site and route of infection. In contrast to classical Gram-negative infections, in which lipopolysaccharide is the major immunogenic stimulus, with TLR4 being a prime receptor, host defense against Gram-positive infections apparently benefits from the simultaneous involvement of multiple PRRs. To stick with the pneumococcal pneumonia example, it has been demonstrated that additional PRRs, such as TLR4 and TLR9, contribute significantly to the inflammatory response [3,4]. Distinct from S. pneumoniae pneumonia, infection models utilizing the Gram-positive pathogen Staphylococcus aureus revealed a crucial role for TLR2, as demonstrated by the reduced survival rates in animals lacking this receptor [5,6].

What is the evidence for TLRs as important PRRs in humans? According to studies that investigated human TLR polymorphisms, varying degrees of altered susceptibility to particular bacterial infections were described [7]. While some TLR4 polymorphisms increased the risk of Gram-negative infections, the most common TLR2 polymorphisms were associated with mycobacterial infections [8,9]. The strongest indication that human TLRs indeed play an essential role in antibacterial host defense comes from the observation that an autosomal recessive mutation in the IL-1 receptor (IL-1R)-associated kinase (IRAK)4 is associated with recurrent bacterial infections [10–12]. IRAK4 is a crucial component of both TLR- and IL-1R-induced signaling pathways. Of note, almost all children that have been reported to carry an IRAK4 mutation suffered from recurrent S. pneumoniae infections. Although this finding is definitely of great importance, we do not know if this ‘IRAK4-mutation phenotype’ reflects the impaired transmission of TLR or IL-1R-mediated signals, or both. It would be interesting to know if these people are in turn compensated for by a decreased incidence of inflammatory diseases, such as rheumatoid arthritis, where IL-1 signaling is of importance.

While the principal importance of membrane-anchored TLRs during bacterial infections is apparent and confirmed by numerous studies, it soon became clear that some host defense mechanisms cannot be solely accounted for by TLRs. The observation that some pathogens only induce an inflammatory response when localized inside a cell soon led to the identification of two cytoplasmatic receptors called nucleotide-binding oligomerization domain (Nod)1 and Nod2 [13]. Nod1 is expressed ubiquitously and is able to sense peptidoglycan-containing mesodiaminopimelic acid, whereas Nod2 expression is restricted to intestinal epithelial cells, monocytes, macrophages and dendritic cells, and signals the presence of muramyldipeptide, another peptidoglycan motif [13]. Considering that the involvement of Nod1 or Nod2 induces an inflammatory response through the activation of transcription factors, including nuclear translocation of NF-κB, this led to the idea that Nods are the intracellular counterpart of membrane-bound TLRs.

Consecutive studies disclosed an entire family of related proteins, referred to as Nod-like receptors (NLRs). Just like Nod1 and 2, these proteins contain a C-terminal leucine-rich repeat (LRR) unit, a Nod domain (also termed NACHT) and signaling modules, such as a caspase activation and recruitment domain, pyrin or baculoviral inhibitor of apoptosis domain, respectively. The most well investigated members of this NLR family so far are NACHT, LRR and pyrin domain-containing protein (Nalp)1 and Nalp3, ICE protease-activating factor (Ipaf) and neuronal apoptosis inhibitory protein (Naip). Distinct from Nod1 and Nod2, the main function attributed to these members concentrates on the assembly of the inflammasome, which is a multiprotein complex responsible for the activation of caspase-1. Active caspase-1 in turn cleaves pro-IL-1β and pro-IL-18 to its active form, followed by the secretion of these important proinflammatory cytokines. In other words, while TLRs sense extracellular bacteria and are able to induce pro-IL-1β, a second stimulus is required to activate caspase-1, which will then induce the release of mature IL-1β. Ipaf and Naip have been reported to recognize cytosolic flagellin from bacteria, such as Salmonella typhimurium or Legionella pneumophila, irrespective of TLR5 (which is known as the sensor of extracellular flagellin) [14,15]. However, while the in vitro detection of L. pneumophila flagellin correlated with survival in Naip^-/- mice in vivo, the biological role of Ipaf during S. typhimurium infection is less clear [15–17].

The molecules activating the Nalp3 inflammasome are multiple and, intriguingly enough, involve microbial components, as well as endogenous danger signals [17,18]. The latter, that is the recognition of danger signals by Nalp3, is of great interest considering the idea that tissue damage during infection and inflammation might signal the presence of pathogenic bacteria. Adding the fact that activation and release of IL-1β requires two signals (TLR and NLR mediated), it is intriguing to speculate that this dual mechanism alerts the host to dangerous pathogens but protects us from detrimental inflammation when facing harmless microbes. In turn, this could explain, for example, why some bacteria colonize the respiratory tract without causing severe inflammation. Host cells might recognize the presence of these bacteria via TLRs, but only cell-damaging effects that lead to the release of danger molecules would provide the second signal required for full-blown inflammation. However, the definite ligands of Nalp3 are not entirely understood, but several reports have demonstrated activation of Nalp3 via ATP via potassium efflux-inducing bacterial toxins but also uric acid [18]. Together, these mechanisms indicate that the pathways that are commonly associated with cell damage result in the activation of the Nalp3 inflammasome and release of mature IL-1β.

What about the role of PPRs as targets for future therapies of bacterial infections or their most severe complication, namely sepsis? At this juncture, there are two Phase III trials under way that attempt to prevent the overwhelming inflammatory response during severe sepsis by means of TLR blockade [19]. Eritoran (E5564, Eisai Pharmaceutical, USA) is a TLR4 antagonist and TAK242 (Takeda Pharmaceutical, Japan) blocks the TLR4-induced signaling pathway. Both compounds showed favorable effects in earlier studies and patients are currently being recruited. At this point, there are no clinical trials under way that investigate drugs targeting the inflammasome during sepsis. Nevertheless, caspase-1 inhibitors are available and clinical studies in sterile inflammatory diseases, such as psoriasis and rheumatoid arthritis, are ongoing [20]. In the future, it will be exciting to see whether these new compounds might influence the tissue-damaging effects of the inflammatory response against bacteria.

In conclusion, PRRs, such as TLRs and NLRs, are essential receptors signaling the presence of bacterial infections and their interaction crucially contributes to an effective host defense system. How and to what extent distinct members of these PRRs are involved in systemic and overwhelming inflammation, as seen during sepsis, still remain to be clarified. However, current evidence supports the idea that the interplay of these PRRs is causally related to both beneficial and detrimental inflammation induced by bacteria. The finding that endogenous mediators are able to activate either of the two PRR families makes it tempting to speculate that blocking one particular pathway may assist in the prevention of overwhelming inflammation without interfering with beneficial host defense pathways.