Activation of NOD receptors by Neisseria gonorrhoeae modulates the innate immune response

Abstract

NOD1 and NOD2 are members of the NOD-like receptor family of cytosolic pattern recognition receptors that recognize specific fragments of the bacterial cell wall component peptidoglycan. Neisseria species are unique amongst Gram-negative bacteria in that they turn over large amounts of peptidoglycan during growth. We examined the ability of NOD1 and NOD2 to recognize Neisseria gonorrhoeae, and determined the role of NOD-dependent signaling in regulating the immune response to gonococcal infection. Gonococci, as well as conditioned medium from mid-logarithmic phase grown bacteria, were capable of activating both human NOD1 and NOD2, as well as mouse NOD2, leading to the activation of the transcription factor NF-κB and polyubiquitination of the adaptor receptor-interacting serine-threonine kinase 2. We identified a number of cytokines and chemokines that were differentially expressed in wild type versus NOD2-deficient macrophages in response to gonococcal infection. Moreover, NOD2 signaling up-regulated complement pathway components and cytosolic nucleic acid sensors, suggesting a broad impact of NOD activation on innate immunity. Thus, NOD1 and NOD2 are important intracellular regulators of the immune response to infection with N. gonorrhoeae. Given the intracellular lifestyle of this pathogen, we believe these cytosolic receptors may provide a key innate immune defense mechanism for the host during gonococcal infection.

Keywords: Innate immunity; NOD-like receptors; Neisseria gonorrhoeae.

Figure 1. N. gonorrhoeae activates human NOD1 and NOD2

HEK293 cells, transiently transfected with human NOD1 (hNOD1) or NOD2 (hNOD2) and an NF-κB driven luciferase reporter, were incubated for 24 hrs in the presence of N. gonorrhoeae supernatant (GC sup) or N. gonorrhoeae crude lysate (GC lys), prepared as described in the Methods; or in the presence of the NOD1 ligand iEDAP (10 μg/ml), the NOD2 ligand MDP (10 μg/ml), human TNF-α (50ng/ml), LPS (100 ng/ml), P₃CSK₄ (100 ng/ml), or medium only (M). All conditions were tested in triplicate. Cell lysates were prepared and assessed for luciferase activity as described in the Methods. Data is presented as fold increase over the baseline luciferase activity in HEK 293 cells transfected with an empty vector. *=p<0.05; **=p<0.005; NS=not statistically significant. Each experiment was repeated at least 3 times with similar findings.

Figure 2. N. gonorrhoeae activates the NOD1/2 adapter RIPK2 in human cervical epithelial cells and human macrophages

(A) HeLa cells or (B) human macrophages were incubated with the indicated Neisseria gonorrhoeae preparations, or NOD1 ligand C12-iEDAP (10 μg/ml; iEDAP), the NOD2 ligand MDP (10 μg/ml), or medium alone (M) for 24 hr, unless otherwise indicated. Protein lysates were made and subjected to immunoprecipitation with an anti-RIPK2 antibody, followed by immunoblot with an anti-ubiquitin antibody, as described in the Methods. Activation of NOD1 or NOD2 results in polyubiquitination of the adaptor protein RIPK2, which appears as a smear in this assay. Immunoblot for RIPK2 is shown below as an indicator of protein loading. Each experiment was repeated at least 2 times with similar findings.

Figure 3. NOD activation by N. gonorrhoeae alters chemokine induction in cervical epithelial cells

HeLa cells were primed overnight with IFN-γ (100 U/ml), except as indicated in (A), followed by incubation with live Neisseria gonorrhoeae (GC) or heat killed Neisseria gonorrhoeae (HK GC) (both MOI 50:1), the NOD1 ligand C12iEDAP (10 μg/ml), human TNF-α (100 ng/ml), LPS (100 ng/ml), P3CSK4 (100 ng/ml), or medium only (M). Where indicated, the RIPK2 inhibitor SB 203580 (B) or the Nod1 inhibitor ML130 (Fig. 3C) was added 1 hour prior to treatment at the indicated concentrations. Cell supernatants were collected after 22 hrs and analyzed for the presence of IL-8 by ELISA. *=p<0.05; **=p<0.005; ***=p<0.0005; NS=not statistically significant; ND=not detected. All conditions were tested in triplicates. Each experiment was repeated 2 times with similar findings.

Figure 4. N. gonorrhoeae activates NOD2 but not NOD1 in murine macrophages

(A) Primary murine BMDM or (B) immortalized murine BMDM derived from wild type C57BL/6 mice (WT), NOD1 knockout mice (NOD1 KO) or NOD2 knockout mice (NOD2 KO) were incubated for 2 hrs with either live Neisseria gonorrhoeae (Live GC) at the indicated MOIs, gonococcal lysates (GC Lys), supernatant from mid-log phase grown Neisseria gonorrhoeae (GC sup), the NOD2 ligand MDP (10 μg/ml), the TLR4 ligand LPS (100 ng/ml) or medium only (M). Protein lysates were made and subjected to immunoprecipitation with an anti-RIPK2 antibody, followed by immunoblot with an anti-ubiquitin antibody. Polyubiquitination of the adaptor protein RIPK2 appears as a smear in this assay. Each experiment was repeated at least 2 times with similar findings. (C) HEK293 cells transiently transfected with murine NOD1 (mNOD1) or NOD2 (mNOD2) and a NF-κB driven luciferase reporter, were incubated for 24 hrs in the presence of Neisseria gonorrhoeae supernatant (GC sup) or Neisseria gonorrhoeae crude lysate (GC lys), both diluted 1:10 or in the presence of the NOD1 ligand iEDAP (10 μg/ml), the NOD2 ligand MDP (10 μg/ml), human TNF-α (50 ng/ml), or medium only (M). All conditions were tested in triplicate. Cell lysates were prepared and assessed for luciferase activity as described in the Methods. Data is presented as fold increase over the baseline luciferase activity in HEK 293 cells transfected with an empty vector. *=p<0.05; **=p<0.005; NS=not statistically significant. Each experiment was repeated at least 3 times with similar findings.

Figure 5. NOD2 activation by N. gonorrhoeae alters levels of TNF-α, IL-6, IL-10, and RANTES in macrophages

(A) Immortalized murine BMDM derived from wild type C57BL/6 mice (WT) or NOD2 knockout mice (NOD2 KO) incubated with the following: live N. gonorrhoeae (MOI = 1 or 10); heat killed N. gonorrhoeae (MOI = 1 or 10), MDP (10 μg/ml), LPS (100 ng/ml), or a combination of the two; or medium alone (M). Cell supernatants were collected at 24 hours and assayed for TNF-α by ELISA. (B-E) Primary murine BMDM derived from wild type C57BL/6 mice (WT) or NOD2 knockout mice (NOD2 KO) were incubated with the indicated treatments. Cell supernatants were collected at 6 hours post infection, and assessed for presence of (B) TNF-α, (C) IL-6, (D) IL-10, and (E) RANTES by ELISA. *= p<0.05; **=p<0.005; ***=p<0.0005; NS=non statistically significant; ND=not detected. All conditions were tested in triplicate. Each experiment was repeated at least 3 times with similar findings.

Figure 6. Differential gene expression in response to N. gonorrhoeae infection in vitro

Representative genes out of the 127 genes found to be differentially expressed between wild type (WT) and NOD2 knockout (NOD2 KO) primary murine Bone BMDM infected with N. gonorrhoeae (GC) or medium only (M). Each condition was tested in triplicate. Highlighted in yellow are noted genes of particular interest, such as cytokines, chemokines, cytokine, chemokine receptors and growth factors (CSF1, Lif, IL23a, Ccl5, Tnfsf9, Cxcr4, Ccl24, Ccl22, Ccl17, Tnfsf4, Cx3cl1, Cxcl1), complement cascade related genes (Cfb, C3) and cytosolic DNA sensing pathway related genes (Zbp1=DAI, Dhx58=RIG-I).

Figure 7. NOD activation by N. gonorrhoeae affects multiple signaling pathways in macrophages

Primary murine BMDM from C57BL/6 mice (WT) or NOD2 knock out (NOD2−/−) mice were prepared as described in the Methods and incubated with live N. gonorrhoeae at an MOI 10:1 or medium only for 3 hrs. Each condition was tested in triplicate. Total RNA was extracted from the cell lysates and subjected to real time PCR as described in the Methods. Shown is the relative expression for Cfb, C3, CSF1 and IL23a normalized for GAPDH expression. *= p<0.05; **=p<0.005; ***=p<0.0005.