A Spaetzle-like role for nerve growth factor β in vertebrate immunity to Staphylococcus aureus

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Science  31 Oct 2014:
Vol. 346, Issue 6209, pp. 641-646
DOI: 10.1126/science.1258705

Overcoming staph infections is hardwired

Several evolutionarily conserved components of antistaphylococcal immunity have been identified, using Drosophila as a model organism. However, no vertebrate ortholog has been identified for the Toll ligand Spaetzle, which plays a key role in controlling gram-positive infection in flies. Hepburn et al. have now identified NGF-β as a functional equivalent to Spaetzle in vertebrates. NGF-β acts as a paracrine “alarmin” orchestrating macrophage and neutrophil responses to S. aureus infection. People with deleterious mutations in genes encoding NGF-β or its high-affinity receptor TRKA are predisposed to recurrent and severe staph infections. S. aureus proteins selectively trigger macrophage production of NGF-β, which enhances uptake and superoxide-dependent killing of S. aureus, stimulates proinflammatory cytokine production, and promotes neutrophil recruitment. Moreover, TrkA silencing in vivo increases susceptibility to S. aureus. Thus, the NGF-β–TRKA pathway is a critical, evolutionarily conserved component of vertebrate immunity to S. aureus infection.

Science, this issue p. 641


Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity.

Staphylococcus aureus causes a range of serious infections, including skin ulceration, osteomyelitis, pneumonia, and septicaemia (1, 2). Several evolutionarily conserved components of antistaphylococcal immunity have been identified using Drosophila as a model organism (3, 4). One of the key mediators of immunity to Gram-positive bacteria in Drosophila is the soluble protein Spaetzle which, when activated by Spaetzle processing enzyme (SPE) upon infection, triggers effector immunity in an autocrine and paracrine manner through Toll receptor activation (3, 58). To detect potential vertebrate equivalents of Spaetzle, we searched the human proteome using a relatively tolerant PROSITE pattern [C-X(35,45)-C-X(10,15)-C-X(25,32)-C-X-C; modified from (9)] to identify 166 soluble proteins potentially containing a >10-membered cystine knot domain (see the supplementary materials). We identified the neurotrophin nerve growth factor β (NGFβ) as a possible vertebrate ortholog of Spaetzle (Fig. 1, A and B). NGFβ regulates the survival, differentiation, and function of central and peripheral neurons (10, 11), predominantly through activation of its high-affinity receptor, tropomyosin-related kinase receptor A (TRKA). Like Spaetzle, NGFβ is generated by enzymatic cleavage of a precursor proprotein to form a biologically active cystine-knot dimer (11) (Fig. 1C). Because NGFβ is implicated in the modulation of inflammation in non-neuronal cells (1214), we asked whether NGFβ could play a Spaetzle-like role in coordinating vertebrate immunity to S. aureus.

Fig. 1 NGFβ is implicated in antistaphylococcal immunity and is released from macrophages after S. aureus infection.

(A) Bioinformatic identification of potential human orthologs of Spaetzle. The human proteome was searched using a PROSITE pattern to find soluble proteins potentially containing a >10-membered cystine-knot domain, which were then subjected to multifactorial analysis, incorporating structural prediction of disulphide bond formation with other structural and sequence parameters (see supplementary materials for details) to identify NGF (red) as the closest human ortholog to Spaetzle (Spz, blue). The other human neurotrophins [BDNF, neurotrophic factor 3 (NTF3), and NTF4 (black)] are also highlighted. (B) Phylogenetic alignment of vertebrate neurotrophic factors (NGF, BDNF, and NTF 3 and 4), Drosophila neurotrophin (NT) 1 and 2, and the Drosophila immune regulator Spaetzle, with bootstrap values. (C) Dimeric protein structures (from the Protein Data Bank) of Spaetzle and human NGFβ. (D) Intracellular staining of NGFβ (green) in primary human macrophages uninfected (left) or infected (right) with S. aureus (SH1000) (red) and then treated with monensin for 14 hours to prevent secretion. Scale bar, 5 μm. (E) Time course of NGFβ release from primary human macrophages after infection with S. aureus. NGFβ secretion requires live bacteria because heat-killed (HK) or paraformaldehyde (PFA)–killed S. aureus do not trigger NGFβ release. (F) Release of pro-NGF and NGFβ from differentiated THP-1 cells upon infection with S. aureus for 12 hours. *P ≤ 0.05; **P ≤ 0.005. All experiments were carried out in at least triplicate and are representative of at least three independent repeats.

Deleterious biallelic mutations in the genes encoding NGFβ (NGF) (15, 16) or TRKA (NTRK1) (17) lead to a profound congenital sensory and autonomic neuropathy [termed hereditary sensory and autonomic neuropathy (HSAN) 4 and 5]. We found that these individuals also had frequent severe S. aureus infections of skin, teeth, joints, and bone (fig. S1), suggesting a pathogen-specific immune defect. To further explore the role of NGFβ in staphylococcal immunity, we measured its release from primary human macrophages obtained from healthy individuals. Infection of cells with live, but not killed, S. aureus stimulated de novo synthesis and secretion of both pro-NGF and mature NGFβ (Fig. 1, D to F). We found considerable variation in NGFβ stimulation by clinical isolates of S. aureus. Clones triggering lower levels of NGFβ were associated with increased all-cause patient mortality (fig. S1), again suggesting a protective role for NGFβ during S. aureus infection. The exact mechanisms generating mature NGFβ remain unclear, but it is likely that endogenous and exogenous host proteases [such as furins (18), matrix metalloproteinase (MMP) 7, and plasmin (19)], as well as bacterial proteases (fig. S1), combine to cleave pro-NGF during S. aureus infection, suggesting similarities with the regulation of Spaetzle processing (20).

We next examined whether other bacterial species were also able to stimulate NGFβ release from macrophages. Although a low-level response was seen with some other bacteria (such as Enterococcus faecalis), only S. aureus effectively triggered NGFβ release (fig. S1). Indeed, the closely related skin commensal Staphylococcus epidermidis was unable to stimulate NGFβ production effectively, suggesting that macrophages can discriminate between pathogenic and nonpathogenic staphylococcal species. Furthermore, macrophages only secreted NGFβ and not other neurotrophins [brain-derived growth factor (BDNF), NT3, and NT4] in response to infection (fig. S1). Thus, NGFβ may act as a specific and sensitive signal for S. aureus infection in man, potentially explaining the clinical phenotype of patients with HSAN 4 and 5 and suggesting a nonredundant and pathogen-specific role for NGFβ in innate immunity.

We then explored the cellular pathways triggering NGFβ generation. Rather than involving conventional surface pattern recognition receptors, S. aureus elicits NGFβ production through activation of nucleotide–binding and oligomerization domain (NOD)–like receptors (NLRs) (fig. S2), a well-recognized consequence of infection with this bacteria (21), and suggests an additional potential role for NGFβ during tissue damage.

To define the bacterial components responsible for NGFβ release from macrophages, we screened the Nebraska library of S. aureus transposon mutants (22) for their ability to stimulate NGFβ release from THP-1 cells. This identified a number of genes involved in bacterial cell wall synthesis, macromolecular transport, metabolism, and cellular regulation (fig. S3 and table S1), including the saeR/saeS 2 component gene system and autolysin, which regulate exoprotein and peptidoglycan release, respectively (23, 24). As expected, a number of purified S. aureus–derived exoproducts (protein A, peptidoglycan, and α-haemolysin) were able to stimulate NGFβ release in a proteinase K–dependent manner (fig. S3). Because most single exoprotein deletion mutants were still capable of stimulating NGFβ release, suggesting redundancy (fig. S4), we turned to comparative mass spectroscopy of conditioned media from wild-type and saeS-mutant S. aureus to define further bacterial components mediating NGFβ release (fig. S4) and identified alpha phenol–soluble modulins (α-PSMs), a recently described family of secreted peptides capable of membrane rupture (25), as putative factors (fig. S4). Thus, multiple S. aureus exoproteins can stimulate NGFβ release from macrophages. We asked whether this regulatory mechanism might be evolutionarily conserved to control Spaetzle production in Drosophila. Intriguingly, although the regulation of Spaetzle activity has focused on its SPE-mediated activation (26), pro-Spaetzle levels in Drosophila phagocytes (S2 cells) were stimulated by wild-type but not saeR-mutant S. aureus, by conditioned media, and by peptidoglycan (fig. S4), mirroring our results with NGFβ.

We then evaluated the effects of NGFβ on macrophage function. Primary human macrophages, which have constitutively high surface expression of TRKA but not the low-affinity NGF receptor p75, responded to NGFβ with sustained calcium signaling (Fig. 2A), which could be reconstituted in HeLa cells expressing wild-type TRKA but not the HSAN5-associated mutation G517E (Fig. 2B). TRKA signaling in macrophages also triggered rapid activation of calcium-dependent protein kinase C (PKC) isoforms (Fig. 2C), as well as other recognized components of TRKA signaling observed in neuronal cells (table S2). Because TRKA is thought to continue signaling after internalization, thereby permitting signal transmission along axons (27), we examined whether phagosomal TRKA activation might occur and found persistent tyrosine phosphorylation of TRKA within S. aureus–containing phagosomes (Fig. 2D). Functionally, TRKA activation led to enhanced phagocytosis (Fig. 2E), proinflammatory cytokine release from uninfected cells (Fig. 2F), and increased S. aureus–induced phagosomal superoxide generation (Fig. 2G). TRKA activation also enhanced intracellular killing of S. aureus in human and mouse macrophages (Fig. 2H) and in TRKA-transfected, but not control, THP-1 cells (Fig. 2I). This increased killing was dependent on intact receptor signaling (because it was not observed in cells from HSAN4 patients) and was principally mediated through enhanced superoxide generation (fig. S5) and autophagy (fig. S6). TRKA-dependent effector responses also depended on intact TLR signaling, because intracellular killing in S. aureus–infected cells and cytokine production in uninfected cells were abrogated in Myd88−/− and Trif−/− macrophages (fig. S7), suggesting an evolutionarily conserved interaction between cystine knot proteins and Toll family receptors.

Fig. 2 Effects of NGFβ-TRKA signaling in human macrophages.

(A) Addition of NGFβ (250 ng/ml; 9.25 μM) triggers sustained calcium oscillations in Fluo3-loaded primary human macrophages (detected by single-cell confocal imaging). Three representative recordings normalized for starting fluorescence (F/F0) are shown. (Inset) Surface expression of TRKA and p75R (black) compared with isotype control (dark gray) or unstained cells (gray fill) on primary human macrophages. (B) Single-cell calcium signaling in GCamp3-expressing HeLa cells transfected with wild-type TRKA (blue), HSAN4-associated TRKA mutation (G517E; green) or empty vector (black) in response to NGFβ (250 ng/ml; 9.25 μM). (Inset) Surface expression of TRKA in transfected HeLa cells. (C) TRKA signaling in macrophages triggered rapid activation of calcium-dependent PKC isoforms. (D) Colocalization of intracellular phospho-TRKA (green) with red fluorescent protein (RFP)–labeled S. aureus (SH1000; red) in primary human macrophages treated for 30 min with 100 ng/ml (3.7 μM) NGFβ. (E and F) Addition of NGFβ to primary human macrophages increased (E) phagocytosis of RFP-labeled S. aureus and (F) release of TNFα and IL-8 (measured after 24 hours). (G) (i) Luminol-based detection of superoxide in response to S. aureus, NGFβ, or phorbol 12-myristate 13-acetate (PMA). (ii) The generation of phagosomal superoxide, monitored by DHR123-labeled heat-killed S. aureus, is increased in cells treated with the TRKA-specific agonist gambogic amide (250 nM; red) compared with vehicle (white; P < 0.005) or bacteria without cells (black). Four representative fluorescence traces from individual cells are shown for each group. (H and I) TRKA activation (by gambogic amide; 250 nM) enhanced intracellular killing of S. aureus in (H) primary human macrophages (left) and the mouse macrophage cell line RAW 264.7 (right) and (I) TRKA-transfected (blue), but not control (black), THP-1 cells. (Inset) Surface TRKA expression in THP-1 cells transfected with TRKA (blue) or empty vector (black) compared with isotype control (gray) and unstained cells (gray fill). All experiments were carried out in at least triplicate and are representative of at least three independent repeats.

We next determined the role of NGFβ-TRKA in human neutrophils, which are critical components of the host response to S. aureus infection (28). Neutrophils constitutively expressed TRKA (Fig. 3A) and released NGFβ in response to live S. aureus and peptidoglycan (Fig. 3B). As seen in macrophages, NGFβ stimulated neutrophils to generate superoxide (Fig. 3C) and secrete proinflammatory cytokines (Fig. 3D) and enhanced intracellular killing of S. aureus (Fig. 3E). NGFβ also stimulated chemokinesis and chemotaxis in a TRKA- and calcium-dependent manner (Fig. 3, F and G, movie S1, and fig. S8), suggesting that NGFβ may be an important chemotactic signal for neutrophil recruitment to sites of S. aureus infection.

Fig. 3 NGFβ-TRKA signaling stimulates functional activation of neutrophils.

(A) TRKA expression on untreated, IFN-γ (10 ng/ml) or granulocyte-macrophage colony-stimulating factor (100 ng/ml)–primed primary human neutrophils. (B) Neutrophils secrete NGFβ in response to live S. aureus, peptidoglycan (PGN) but not lipopolysaccharide (LPS; 100 ng/ml) or lipoteichoic acid (LTA; 5 μg/ml). (C and D) Neutrophils generate superoxide (C) and release interleukin-8 (IL-8) (D) in response to S. aureus, PMA, and/or NGFβ (red). (E) Killing of S. aureus by human neutrophils is enhanced by treatment with NGFβ (100 ng/ml; red) compared with control (white). (F) Representative plots of x-y displacement and calcium levels in individual neutrophils after addition of vehicle (control) or NGFβ. (G) Chemokinesis of human neutrophils assessed using a transwell assay in response to increasing concentrations of NGFβ. *P ≤ 0.05; **P ≤ 0.005. All experiments were carried out in at least triplicate and are representative of at least three independent repeats.

To establish whether NGFβ-TRKA signaling represents a critical, evolutionarily conserved component of vertebrate immunity to S. aureus infection, we examined its role during in vivo infection of zebrafish. Effective morpholino knockdown of trkA was confirmed by immunohistochemistry, where we observed the expected loss of trkA protein in the forebrain and nose of zebrafish larvae (Fig. 4A). Knockdown of trkA had a major effect on the host response to S. aureus: trkA morphants were more susceptible to S. aureus infection than controls, a phenotype that could be rescued by concomitant injection of morpholino-resistant trkA RNA (Fig. 4B) and was only partially rescued in a transgenic line expressing trkA specifically in macrophages (fig. S9), suggesting the critical importance of trkA signaling in other cells (such as neutrophils). Bacterial counts in trkA-deficient fish rose faster and remained significantly higher than in controls (Fig. 4C). We then explored the relationship between the ability of bacteria to stimulate NGFβ release from macrophages and the in vivo effect of silencing trkA expression during infection (Fig. 4D). We observed a greater effect of trkA knockdown in fish infected with wild-type (SH1000) S. aureus compared to animals infected with bacteria less able to trigger NGFβ release from macrophages: the saeR–S. aureus mutant (causing a mild infection) and Enterococcus (causing a severe infection). Furthermore, trkA knockdown compromised neutrophil migration to sites of S. aureus infection (Fig. 4, E and F) as well as sterile inflammation (Fig. 4, G and H), supporting a role for NGFβ as an “alarmin” for both S. aureus infection and nonspecific tissue damage.

Fig. 4 Disruption of NGFβ-TrkA signaling compromises S. aureus immunity in vivo.

(A) Reduced TrkA protein expression (assessed by immunohistochemistry) in the forebrain and nose of 72 hours post-infection zebrafish larvae injected with trkA-targeted (bottom) but not control (top) morpholinos. (B) Kaplan-Meier survival curves of fish infected with S. aureus. TrkA morphants (red) were more susceptible to S. aureus infection than controls (black) and could be rescued by concomitant injection of morpholino-resistant trkA RNA (blue). N of at least 45 fish per group performed as three independent experiments. (C) Numbers of viable S. aureus were significantly greater in trkA morphant (red) than control (white) fish, assessed as colony-forming units (CFU) per embryo. (D) Morpholino trkA knockdown (red) caused a greater effect on mortality in fish infected with wild-type (SH1000) S. aureus (black) compared to animals infected with bacteria less able to trigger NGFβ release from macrophages: the saeR– S. aureus mutant (causing a mild infection; blue) and Enterococcus faecalis (causing a severe infection; green). (E to H) Reduced migration of green fluorescent protein–tagged neutrophils to sites of S. aureus infection [(E) and (F)] or sterile inflammation [(G) and (H)] in trkA morphants (red) compared with controls (white). Representative images at 4 hours after infection (E) (scale bar: brightfield, 200 μm; fluorescence, 100 μm) or tail injury (G) (scale bar, 100 μm). N of at least 32 fish per group performed as three independent experiments. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Unless otherwise stated, data shown are representative of at least three independent experiments.

In summary, our results indicate a critical role for NGFβ-TRKA signaling in controlling vertebrate innate immunity during S. aureus infection. It is also conceivable that other vertebrate cystine-knot proteins might play similar roles to NGFβ for other bacterial pathogens. The recent finding that Spaetzle also functions as a neurotrophin in Drosophila (29) suggests an evolutionarily conserved dual function for cystine-knot proteins in both nerve development and antistaphylococcal immunity and may explain stimulation of aberrant nerve growth by soft-tissue infection by S. aureus (30). Our findings reveal pleotropic effects of the NGFβ-TRKA pathway that may particularly influence innate immunity to S. aureus infection, suggesting that, potentially, person-to-person variability in phagocyte secretion of, or response to, NGFβ may influence vulnerability to S. aureus infection and may provide opportunities for therapeutic intervention, particularly in multidrug-resistant disease.

Supplementary Materials

Figs. S1 to S9

Movie S1

Databases S1 and S2

References (3164)

References and Notes

  1. Acknowledgments: We thank S. Clegg and E. Henderson for help with patient samples, R. Mifsud and D. Cusens for initial phylogenetic and functional analysis, A. Segal for provision of Nod2−/− mouse bone marrow, and the aquarium staff of the Bateson Centre, University of Sheffield for zebrafish husbandry. This work was supported by The Wellcome Trust [Senior Clinical Research Fellowship to R.A.F. (084953), project grant to S.J.F./S.A.R. (089981), The Medical Research Council, UK (Research center grant (G0700091), Senior Clinical Fellowship to S.A.R. (G0701932)], Papworth Hospital and the National Institute for Health Research Cambridge Biomedical Research Centre, and the Intramural Research Program of NIAID, NIH.
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