Nucleic Acid-Sensing Toll-Like Receptors Play a Dominant Role in Innate Immune Recognition of Pneumococci

Abstract

Streptococcus pneumoniae (or pneumococcus) is a highly prevalent human pathogen. Toll-like receptors (TLRs) function as immune sensors that can trigger host defenses against this bacterium. Defects in TLR-activated signaling pathways, including deficiency in the adaptor protein myeloid differentiation factor 88 (MyD88), are associated with markedly increased susceptibility to infection. However, the individual MyD88-dependent TLRs predominantly involved in antipneumococcal defenses have not been identified yet. Here we find that triple knockout mice simultaneously lacking TLR7, TLR9, and TLR13, which sense the presence of bacterial DNA (TLR9) and RNA (TLR7 and TLR13) in the phagolysosomes of phagocytic cells, display a phenotype that largely resembles that of MyD88-deficient mice and rapidly succumb to pneumococcal pneumonitis due to defective neutrophil influx into the lung. Accordingly, TLR7/9/13 triple knockout resident alveolar macrophages were largely unable to respond to pneumococci with the production of neutrophil-attracting chemokines and cytokines. Mice with single deficiencies of TLR7, TLR9, or TLR13 showed unaltered ability to control lung infection but were moderately more susceptible to encephalitis, in association with a decreased ability of microglia to mount cytokine responses in vitro. Our data point to a dominant, tissue-specific role of nucleic acid-sensing pathways in innate immune recognition of S. pneumoniae and also show that endosomal TLRs are largely capable of compensating for the absence of each other, which seems crucial to prevent pneumococci from escaping immune recognition. These results may be useful to develop novel strategies to treat infections by antibiotic-resistant pneumococci based on stimulation of the innate immune system. IMPORTANCE The pneumococcus is a bacterium that frequently causes infections in the lungs, ears, sinus cavities, and meninges. During these infections, body defenses are triggered by tissue-resident cells that use specialized receptors, such as Toll-like receptors (TLRs), to sense the presence of bacteria. We show here that pneumococci are predominantly detected by TLRs that are located inside intracellular vacuoles, including endosomes, where these receptors can sense the presence of nucleic acids released from ingested bacteria. Mice that simultaneously lacked three of these receptors (specifically, TLR7, TLR9, and TLR13) were extremely susceptible to lung infection and rapidly died after inhalation of pneumococci. Moreover, tissue-resident macrophages from these mice were impaired in their ability to respond to the presence of pneumococci by producing inflammatory mediators capable of recruiting polymorphonuclear leucocytes to infection sites. This information may be useful to develop drugs to treat pneumococcal infections, particularly those caused by antibiotic-resistant strains.

An in vivo atlas of host–pathogen transcriptomes during Streptococcus pneumoniae colonization and disease

“…Signaling from well-known bacterial recognition receptors, such as Toll-like receptors (TLRs), also lead to activation of both NF-κB and IFN regulatory factors that mediate the IFN response. Famà et al ( 48 ) demonstrated the effect of nucleic acid-sensing TLRs upon pneumococcal infection in mice. TLR7, TLR9, TLR13, and chaperone protein UNC93B1, responsible for TLR trafficking out of the endoplasmic reticulum, were shown to control pneumococcal infection as knockout mice lacking these genes were moderately to highly susceptible.…”

Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs

“…In view of the crucial role of neutrophil-derived Cxcl2 in host defenses (Girbl et al, 2018;Lentini et al, 2020), we sought to identify the bacterial products responsible for induction of this chemokine in bone marrow-derived murine neutrophils. We initially used mixtures of prokaryotic DNA + RNA, since it was previously found that innate antibacterial responses require the endosomal receptors TLR7, TLR9, and TLR13 (Signorino et al, 2014;Mohammadi et al, 2016;Fama et al, 2020;Lentini et al, 2020), which are known to recognize bacterial RNA (TLR7 and 13) and DNA (TLR9). Accordingly, a 1:1 DNA + RNA mixture potently induced TNF-a and Cxcl1 production in neutrophils at levels that were similar or slightly lower than those induced by whole bacteria, such as group B streptococcus (gram-positive) or Escherichia coli (gram-negative; Fig 1A and C).…”

“…To obtain bone marrow-derived macrophages, marrow cells were cultured for 6-7 days in RPMI 1640 supplemented with 10% FCS, penicillin (50 IU/ml), and streptomycin (50 lg/ml). Medium was supplemented with either 100 ng/ml macrophage colony-stimulating factor (M-CSF) or 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from PeproTech) to obtain bone marrowderived, M-CSF-polarized (M-CSF-MΦ) or GM-CSF-polarized (GM-CSF-MΦ) macrophages, respectively, as previously described (Fama et al, 2020). Isolated bone marrow-derived cells (5 × 10 5 per well in 0.2 ml of RPMI supplemented with 10% FCS) were seeded in microtiter plates and stimulated with HK or live bacteria grown to the midlog phase at the indicated concentrations or multiplicities of infection (MOI), respectively.…”

CodY Is a Global Transcriptional Regulator Required for Virulence in Group B Streptococcus

“…Mice showing signs of irreversible disease, such as prolonged hunching, inactivity, or neurological symptoms were humanely euthanized. In further experiments, mice were euthanized at 16 h after challenge and bacterial burden was determined in organ homogenates, as previously described ( Famà et al, 2020 ).…”