Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity

Abstract

Humans are colonized by a large and diverse bacterial flora (the microbiota) essential for the development of the gut immune system. A broader role for the microbiota as a major modulator of systemic immunity has been proposed; however, evidence and a mechanism for this role have remained elusive. We show that the microbiota are a source of peptidoglycan that systemically primes the innate immune system, enhancing killing by bone marrow-derived neutrophils of two major pathogens: Streptococcus pneumoniae and Staphylococcus aureus. This requires signaling via the pattern recognition receptor nucleotide-binding, oligomerization domain-containing protein-1 (Nod1, which recognizes meso-diaminopimelic acid (mesoDAP)-containing peptidoglycan found predominantly in Gram-negative bacteria), but not Nod2 (which detects peptidoglycan found in Gram-positive and Gram-negative bacteria) or Toll-like receptor 4 (Tlr4, which recognizes lipopolysaccharide). We show translocation of peptidoglycan from the gut to neutrophils in the bone marrow and show that peptidoglycan concentrations in sera correlate with neutrophil function. In vivo administration of Nod1 ligands is sufficient to restore neutrophil function after microbiota depletion. Nod1(-/-) mice are more susceptible than wild-type mice to early pneumococcal sepsis, demonstrating a role for Nod1 in priming innate defenses facilitating a rapid response to infection. These data establish a mechanism for systemic immunomodulation by the microbiota and highlight potential adverse consequences of microbiota disruption by broad-spectrum antibiotics on innate immune defense to infection.

Figures

Figure 1. The microbiota enhances neutrophil function via Nod1 signaling

(a,b) Killing of S. pneumoniae (a) and S. aureus (b) by neutrophil-enriched PECs obtained from WT and Nod1−/− mice stimulated with heat-killed H. influenzae (i.p.) and unstimulated control mice. (c,d) Killing of S. pneumoniae (c) and S. aureus (d) byneutrophils harvested from bone marrow of WT mice treated with broad-spectrum antibiotics and untreated control mice. (e,f) Killing of S. pneumoniae (e) and S. aureus (f) by neutrophils harvested from bone marrow of germ-free WT mice and previously germ-free conventionalized mice. (g,h) Killing of S. pneumoniae (g) and S. aureus (h) by neutrophils harvested from bone marrow of WT, Nod1−/−, Tlr4−/−, and Nod2−/− mice treated with broad-spectrum antibiotics and untreated control mice. For all assays bacterial viability is expressed relative to control assays without neutrophils. Values represent at least three independent experiments performed in triplicate ± SEM. *P< 0.05, **P<0.01 and ***P<0.001.

Figure 2. Peptidoglycan is present systemically in sera and bone marrow cells, and levels are decreased in the absence of the microbiota

(a,b) Detection of translocated peptidoglycan in blood, feces and bone marrow cell samples from mice inoculated with [3H]-DAP labeled E. coli via oral gavage, by liquid scintillation counting. CFU in feces (—) and CPM in feces (– –) (a); CPM in serum fraction (—) and CPM in bone marrow fraction (– –) (b). Data is relative to CPM of total inoculum, time is post-oral inoculation. Statistical comparison of peptidoglycan accumulation in bone marrow cells at 72 hour is relative to 24 hour time point. (c,d) A HEK293T cell bioassay to detect peptidoglycan recognized by Nod1 and Nod2 in the sera of antibiotic treated and non-antibiotic treated WT mice (c); and conventionalized and germ-free WT mice (d). HEK293T cells were co-transfected with a NF-κB-luciferase reporter and either a Nod1 or Nod2 construct; luciferase expression was measured 24 h post-transfection and considered a measure of NF-κB activation. The specificity of NF-κB activation was confirmed using sera from germ-free mice with MDP and MurNAcTriDAP added in vitro to control Nod1 and Nod2 bioassays. Values represent fold increase in luciferase expression above empty vector controls, and are based on three independent experiments performed in triplicate ± SEM. *P< 0.05, **P<0.01.

Figure 3. Peptidoglycan recognized by Nod1 restores neutrophil function after microbiota depletion and can activate neutrophils directly via NF-κB signaling, and Nod1 is required for early responses during pneumococcal sepsis

(a) Killing of S. pneumoniae P1121 by neutrophils from WT mice treated with broad-spectrum antibiotics and then administered (i.p., day 7) either MurNAcTriDAP, MDP, or vehicle controls. 24 hours after stimulation neutrophils were harvested from the bone marrow and their ability to kill S. pneumoniae compared. Bacterial viability is expressed relative to control assays without neutrophils. (b) Survival of Nod1−/− mice and WT mice challenged intranasally with 5 × 107 CFU of S. pneumoniae P1547. (c) Killing of S. pneumoniae by differentiated HL-60 cells treated with either MurNAcTriDAP, MDP, or vehicle controls for 4 hours. For NF-κB inhibition, cells were pretreated with 6-amino-4-(4-phenoxyphenylethylamino) quinazoline for 2 hours at 37 °C prior to stimulation with peptidoglycan. Bacterial viability is expressed relative to control assays without neutrophils. (d) Killing of S. pneumoniae by human neutrophils treated with MurNAcTriDAP for 2 hours, bacterial viability is shown relative to vehicle control treatment. Values represent at least three independent experiments performed in triplicate ± SEM. *P< 0.05, **P<0.01.