Here we report that zebrafish localized otic infection is suited for real-time observation of neutrophil infiltration and identification of host signaling pathways that sensitize neutrophils to bacterial infection. Neutrophils are recruited immediately to the site of infection (Movie S2
) and pathogen-leukocyte interactions are readily observed at the onset of the infection (). In contrast, neutrophils respond 90 min post infection with another strain of Pseudomonas
, PA14, injected into the hindbrain ventricle (HBV) in 2 dpf larvae (Phennicie et al., 2010
). The potential difference in the dynamics of neutrophil infiltration into the ear or HBV may be due to the developmental stage of the larvae, dose and type of bacterial strains used, or the immune-privileged nature of the brain, which is well known in humans and other higher vertebrates. Although direct comparison of neutrophil recruitment in the ear and HBV infection is technically difficult (Levraud et al., 2008
), our study indicates that neutrophil responses to infection can be readily observed with the ear infection model in 3 dpf larvae. The yolk sac, on the other hand, is not a good site to investigate pathogen-leukocyte interactions since neutrophils fail to infiltrate into the yolk sac, making the larvae more susceptible to infection, consistent with a previous report (van der Sar et al., 2003
). Nonetheless, neutrophils are able to sense the presence of bacteria inside the yolk, indicated by the increased numbers and motility of neutrophils patrolling on top of the yolk in response to infection ( and data not shown). Taken together, our findings identify the localized otic infection as a powerful model system to characterize mechanisms that mediate leukocyte recruitment to sites of localized bacterial infection using real-time imaging.
Since the identification of the requirement of H2
in mediating leukocyte detection of wounds and transformed cells, the enhanced production of H2
has been speculated to be a universal mechanism used by the host to sense the destruction of tissue homeostasis by various insults and to activate the appropriate innate immune response (Feng et al., 2010
; Yoo and Huttenlocher, 2009
). In animal cells, H2
is known to serveas a second messenger that regulates transcription, proliferation or enzyme activity (Bedard and Krause, 2007
). Surprisingly, we visualize minimal H2
generation upon localized Pseudomonas aeruginosa
infection (). Accordingly, neutrophil recruitment to localized infection with both Gram-negative and Gram-positive bacteria is independent of tissue generated H2
(, , , , ), suggesting a context dependent requirement of H2
signaling in sensitizing professional phagocytes. However, whether tissue generated H2
is required for efficient clearance of bacterial infection in zebrafish has not been determined (due to the limitation of current tools).
is considered an antiseptic and is routinely used for treating minor wounds, it remains questionable whether physiologically generated H2
could directly kill microbes since H2
is only microbicidal at high concentrations (Hampton et al., 1998
). Secondary oxidants with a higher destructive capacity, such as HOCl generated by neutrophil-derived myeloperoxidase, are more likely to conduct direct microbicidal activities (Klebanoff, 1974
; Rosen et al., 2002
). Therefore, tissue generated H2
may primarily modulate the local environment, which can mediate leukocyte infiltration and inflammation. Leukocytes, on the other hand, are the major effectors that clear microbes by phagocytosis, releasing ROS and neutrophil extracellular traps.
The identities of the signals that mediate neutrophil recognition of bacterial infection and recruitment in vivo still remain elusive. The neutrophil chemoattractant could be bacterially-derived peptides with formylated N-terminal methionine groups, which are recognized by the host N-formyl peptide receptor (FPR) and mediate neutrophil chemotaxis (Migeotte et al., 2006
). However, fMLP, a synthetic peptide that mimics the activity of bacterially-derived peptides, fails to attract neutrophils when injected into the ear (Figure S2A, B
). In addition, treatment with cyclosporin H, an FPR inhibitor, does not affect neutrophil recruitment to infection (Figure S2C, D
). Collectively, these results suggest that N-formyl peptides are not likely to be involved in rapid neutrophil detection of infection in zebrafish. Other candidate signaling molecules include those in the complement system, such as C5a. The complement system has been extensively studied in zebrafish (Sun et al., 2010
) and multiple mannose binding lectin
loci have been identified (Jackson et al., 2007
). However, it is not clear whether the complement system mediates rapid neutrophil detection of pathogens.
The responses of neutrophils can also be driven by a diverse array of pattern recognition receptors (PRRs) that bind pathogen-associated molecular patterns (PAMPs). The toll-like receptor (TLR) family of cell surface receptors recognizes different foreign biomolecules including those exposed on the cell surface of both Gram-negative and Gram-positive bacteria, such as lipopeptides, lipopolysaccharide (LPS) and flagellin. TLRs signal through several intracellular adaptor molecules, among which, MyD88 plays a pivotal role. TLRs and MyD88 are well conserved in the zebrafish (van der Sar et al., 2006
). However, depleting endogenous MyD88 with two separate morpholino oligonucleotides indicates a dispensable role for MyD88 in mediating neutrophil recruitment to PAK infection (Figure S3
). It is possible that MyD88 plays roles in later sustained neutrophil recruitment or activation, but not in the initial rapid neutrophil response. It is also possible that another adaptor protein, such as TRAM can function in place of MyD88 and mediate functional PRR signaling (Yamamoto et al., 2003
). In contrast, we find that MyD88 is involved in neutrophil wound detection (Figure S3
), which is not entirely surprising since TLRs are implicated in recognition ofendogenous ligands that are derived from necrotic cells, such as HMGB1 (Apetoh et al., 2007
; Scaffidi et al., 2002
) or extracellular matrix components that are generated as a result of tissue injury (Jiang et al., 2006
). A challenge for future investigation will be to identify the specific signaling mechanisms that mediate leukocyte attraction to infected tissues in live animals.
In summary, we have identified a differential requirement for tissue generated H2O2 in mediating neutrophil recruitment to tissue injury and bacterial infection. Our results indicate that signals mediating leukocyte response to infection or acute injury are indeed unique and suggest the potential of identifying novel signaling events that are differentially involved in sensitizing immune cells to non-pathogen driven versus pathogen driven inflammation.