Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self-propagating inflammatory program

Abstract

An overwhelming neutrophil-driven response causes both acute symptoms and the lasting sequelae that result from infection with Neisseria gonorrhoeae. Neutrophils undergo an aggressive opsonin-independent response to N. gonorrhoeae, driven by the innate decoy receptor CEACAM3. CEACAM3 is exclusively expressed by human neutrophils, and drives a potent binding, phagocytic engulfment and oxidative killing of Opa-expressing bacteria. In this study, we sought to explore the contribution of neutrophils to the pathogenic inflammatory process that typifies gonorrhea. Genome-wide microarray and biochemical profiling of gonococcal-infected neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ- and Tak1-dependent signaling cascade that results in the activation of an NF-κB-dependent transcriptional response, with consequent production of pro-inflammatory cytokines. Using an in vivo model of N. gonorrhoeae infection, we show that human CEACAM-expressing neutrophils have heightened migration toward the site of the infection where they may be further activated upon Opa-dependent binding. Together, this study establishes that the role of CEACAM3 is not restricted to the direct opsonin-independent killing by neutrophils, since it also drives the vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 thereby represents the tipping point between protective and pathogenic outcomes of N. gonorrhoeae infection.

Figure 1. Human CEACAM expression by mouse neutrophils results in neisserial capture and internalization.

(A) Human CEACAMs are expressed in CEABAC neutrophils in a manner reflecting that in human neutrophils. Human neutrophils (top), or wild type (WT) and CEABAC mouse neutrophils (bottom) were fixed and stained with antibodies specific for CEACAM1, CEACAM3, CEACAM6, or a mouse IgG isotype control, and analyzed by flow cytometry. Isotype histograms are shaded. (B) Immunoblot showing CEACAM expression in WT and CEABAC neutrophils. (C) Mouse neutrophils do not bind N. gonorrhoeae, while human neutrophils bind N. gonorrhoeae in an Opa-dependent manner. Mouse (top) or human (bottom) neutrophils were infected with non-opaque (Opa⁻) or Opa-expressing (Opa⁺) N. gonorrhoeae. Cells were visualized by staining filamentous actin with phalloidin , and bacteria are shown in green. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy (D). (E) WT and CEABAC PMNs kill Opa⁻ and Opa⁺ bacteria with similar kinetics. Adherent WT and CEABAC PMNs were infected with either Opa⁻ or Opa⁺ N. gonorrhoeae at an MOI = 1. Bacterial survival over time was evaluated as CFUs present in PMN lysates at each time point relative to bacterial CFUs present at time 0. N = 2. (F–G) N. gonorrhoeae infected CEABAC neutrophils respond analogously to human PMNs. Human (F) or mouse (WT and CEABAC) (G) neutrophils were infected with Opa⁻ or Opa⁺ N. gonorrhoeae and oxidative burst and degranulation responses were analyzed as described in Materials and Methods.

Figure 2. N. gonorrhoeae drives an acute inflammatory transcriptional program.

A gene array of WT and CEABAC PMNs infected with Opa⁺ N. gonorrhoeae was performed, and genes showing a significant increase in transcription over uninfected controls are illustrated. Genes with ≥2 fold induction over uninfected controls in both WT and CEABAC neutrophils 1 h post-infection are shown. (A) List of genes up-regulated to a similar level in both WT and CEABAC neutrophils relative to uninfected neutrophils. (B) List of genes differentially expressed between CEABAC and WT PMNs. The fold difference by which WT and CEABAC differ is indicated next to each bar.

Figure 3. Opa-expressing N. gonorrhoeae drive CEACAM-dependent production of pro-inflammatory cytokines in neutrophils.

(A–B) CEABAC PMNs secret pro-inflammatory cytokines in response Opa⁺ N. gonorrhoeae. Mouse (WT and CEABAC) neutrophils were infected with either Opa⁻ or Opa⁺ N. gonorrhoeae (MOI 10). MIP-1α, MIP-2, KC and TNFα production was measured 3 h post infection at (A) protein and (B) mRNA level. N≥3, error bars represent SEM. (C) Human neutrophils infected with Opa⁺ N. gonorrhoeae show increased levels of MIP-1α, MIP-2, TNFα, and IL-1α mRNA relative to PMNs infected with Opa⁻ bacteria. Each symbol represents an individual donor. Cytokine mRNA levels shown as relative to levels of GAPDH mRNA. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. For (A) and (B) stars indicate significance against all other conditions.

Figure 4. N. gonorrhoeae infection elicits an NF-κB and p38 MAPK signaling-dependent cytokine response in CEABAC neutrophils.

CEABAC-Opa interaction leads to activation of NF-κB signaling. (A) WT and CEABAC PMNs were left untreated or pre-incubated with DPI (10 µM) or cytochalasin D (10 mg/ml). PMNs were then infected with either Opa⁻ or Opa⁺ N. gonorrhoeae and MIP-1α levels were measured 3 h post infection. N = 3, error bars represent SEM. One-Way ANOVA indicates no significant differences between corresponding samples. (B) WT and CEABAC PMNs were infected with Opa⁺ N. gonorrhoeae (MOI = 10 bacteria/PMN) for times indicated, and levels of IkBα were then detected by immunoblot with IκBα antibody. Assessment of tubulin levels confirmed equal protein loading. (C, D) CEABAC-Opa interaction promotes activation of p38 MAPK. WT and CEABAC PMNS were infected as in (B). Activation of MAP kinases p38, Erk1, and Erk2 was determined by immunoblot with phospho-specific antibodies. Total p38 and Erk1/2 levels were assessed to ensure equal protein loading. (D, F) WT and CEABAC PMNs were left untreated or preincubated with (D) p38 inhibitor (SB203580, 10 µM) or (E) TAK1 inhibitor ((5z)-7-oxozeaenol, 500 nM). PMNs were then infected with either Opa⁻ or Opa⁺ N. gonorrhoeae and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Stars indicate significance against all other conditions.

Figure 5. CEACAM3 signaling is required for the PMN cytokine response to N. gonorrhoeae.

Inhibition of Src-family kinases and Syk leads to decreased cytokine production by infected PMNs. (A, B) WT and CEABAC PMNs were left untreated or pre-incubated with (A) Src-family kinase inhibitor (PP2, 10 µM), or (B) Syk inhibitor (piceatannol, 50 µM). PMNs were then infected with either non-opaque (Opa⁻) or Opa-expressing (Opa⁺) N. gonorrhoeae, and MIP-1α and MIP-2 levels were measured 3 h post infection. N≥3, error bars represent SEM. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (C) Opa⁺ N. gonorrhoeae infection leads to phosphorylation of PKCδ. WT and CEABAC PMNs were infected with Opa⁺ N. gonorrhoeae (MOI = 10 bacteria/PMN) for times indicated times. PKCδ activation was measured by immunoblot using phospho-PKCδ antibody. Immunoblot for PKCδ indicates equal protein loading. (D) CEABAC PMNs were left untreated or pre-incubated with PKC inhibitor (BIS II, 10 µM). PMNs were then infected with Opa⁻ or Opa⁺ N. gonorrhoeae, and MIP-1α levels were measured 3 h post infection. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001. Unless otherwise indicated, stars indicate significance against all other conditions. (E, F) Inhibition of SFK, Syk, TAK1 or p38 does not affect bacterial binding (E) or phagocytosis (F). WT and CEABAC PMNs were left untreated or pre-incubated with indicated inhibitors. PMNs were then infected with Opa⁺ N. gonorrhoeae (MOI = 25) for 30 min. Intracellular and total bacteria were differentially stained, and quantified via immunofluorescence microscopy. (G) Schematic representation of proposed signaling pathway resulting in bacterial engulfment, activation of oxidative burst and degranulation, and cytokine production.

Figure 6. CEACAM binding stimulates the inflammatory response to N. gonorrhoeae in vivo.

(A) Neutrophil migration assay. PMN migration speed towards N. gonorrhoeae infected CEABAC or WT neutrophil-derived supernatants was measured using a Zigmond chamber. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, ***P<0.001 (B) Neutrophil infiltration is more pronounced in human CEACAM-expressing mice in an infected subcutaneous air pouch model. Manual neutrophil counts of wash fluids collected from air pouches. ‘PBS’ denotes mice injected with sterile PBS. One-Way ANOVA was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (C) Giemsa-Wright stain of wash fluid collected from CEABAC air pouch infected with Opa⁺ N. gonorrhoeae. The arrows point towards N. gonorrhoeae inside the neutrophil. (D) Neutrophil-expressed CEACAMs mediate N. gonorrhoeae binding within the air pouch. Cells from trypsinized air pouches were grown on coverslips in the presence of antibiotics, and infected in vitro with Opa⁺ N. gonorrhoeae. Cells were visualized by staining for filamentous actin , CEACAMs (green), DNA (blue) and bacteria (cyan). (E) Levels of MIP-1α, MIP-2, KC, and IL-1β were measured in wash fluids collected from air pouches. PMN⁻ refers to mice in which neutrophils were depleted by administration of the Gr1-specific clone RB6-8C5 antibody one day prior to infection with Opa⁺ N. gonorrhoeae. One-Way ANOVA (with Tukey's post-test) was performed for relevant samples, *P<0.05, **P<0.01, ***P<0.001 (F) Inhibition of pro-inflammatory signaling reduces neutrophil infiltration into the air pouch. Neutrophil counts of wash fluids collected from CEABAC mice infected with Opa⁺ N. gonorrhoeae in the presence or absence of TAK1 inhibitor.

Figure 7. CEACAM3-mediated inflammation promotes immunopathology associated with N. gonorrhoeae infection.

Upon in vitro infection, CEACAM3 allows efficient bacterial phagocytosis and clearance via activation of neutrophil antimicrobial responses including degranulation and oxidative burst. Concomitantly, CEACAM3 promotes de novo transcription of pro-inflammatory cytokines by the neutrophil (left panel). In vivo, this cytokine response recruits more neutrophils to the site of infection. As they arrive, these neutrophils will become activated via N. gonorrhoeae binding to CEACAM3, driving a self-propagating inflammatory process leading to the immunopathology associated with gonococcal disease (top right panel). By limiting the CEACAM3-dependent inflammatory cascade without affecting bacterial engulfment, such as via Tak1 inhibitor used in this study, bacterial clearance can continue while inflammation is reduced (bottom right panel).