Getting DAMP(s) Wets the Whistle for Neutrophil Recruitment
Authors: Emily R. Watts, Sarah R. Walmsley
Journal: Immunity (2018)

Neutrophil recruitment in response to pathogen invasion is mediated through "self" tissue damage signals (DAMPs) and pathogen associated signals (PAMPs). In this issue of Immunity, Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar demonstrate that DAMP signaling is a prerequisite for neutrophil recruitment. Neutrophil recruitment in response to pathogen invasion is mediated through "self" tissue damage signals (DAMPs) and pathogen associated signals (PAMPs). In this issue of Immunity, Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar demonstrate that DAMP signaling is a prerequisite for neutrophil recruitment. Neutrophilic inflammation represents a double-edged sword: effective inflammation is essential for pathogen clearance, but excessive or inappropriate inflammation is associated with a wide range of human diseases including chronic obstructive pulmonary disease (COPD) (Hoenderdos and Condliffe, 2013Hoenderdos K. Condliffe A. The neutrophil in chronic obstructive pulmonary disease.Am. J. Respir. Cell Mol. Biol. 2013; 48: 531-539Crossref PubMed Scopus (253) Google Scholar) and inflammatory arthritis (Németh and Mócsai, 2012Németh T. Mócsai A. The role of neutrophils in autoimmune diseases.Immunol. Lett. 2012; 143: 9-19Crossref PubMed Scopus (146) Google Scholar). Understanding the factors which regulate neutrophilic inflammation is therefore an important area of research. Neutrophil recruitment to the site of injury is an essential early step in initiating the immune response. Innate immune cells express a battery of receptors (for both pathogen and self-derived ligands) to enable this recruitment including Toll-like receptors (TLRs), formylated peptide receptors (FPRs), integrins and receptors for lipid mediators such as leukotriene B4 (LTB4). Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar address the important question of what if any physiological advantage there is to damage-associated molecular pattern (DAMP) signaling. It is clear that loss of integrity in epithelial barriers increases the risk of infection due to ingress of pathogens, thus there is a need to generate a reparative immune response. Meanwhile, pathogen-associated molecular pattern (PAMP) signaling has a clear role in immunity against invading pathogens but might be disadvantageous in the context of harmless pathogens such as commensal bacteria in the gut or on the skin. Indeed, we are exposed daily to such organisms and a rigorous immune response in this context is likely to be deleterious. How the innate immune system delineates a damaging from a harmless signal is not fully understood. The capacity of neutrophils to respond to "self" damage signals (DAMPs), as well as pathogenic signals (PAMPs), alone or in combination might be critical to drive an appropriate immune response. The interactions between these two types of signals are complex, allowing fine tuning of the innate response. This complexity is reflected in common signaling through pattern-recognition receptors (PRRs) exemplified by LPS and HMGB1 both signal through TLR4 and self- and pathogen-derived formylated peptides signaling through G-protein coupled FPRs (Piccinini and Midwood, 2010Piccinini A.M. Midwood K.S. DAMPening inflammation by modulating TLR signalling.Mediators Inflamm. 2010; 2010: 1-21Crossref Scopus (708) Google Scholar). Thus, while disentangling the relative contribution of DAMP and PAMP signaling is challenging, it provides crucial insights into recruitment and resolution of inflammatory responses. In this article (Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, the authors utilize an elegant model to measure the effect of tissue damage in the recruitment of neutrophils in the context of infection. The innate immune system in zebrafish larvae is closely related to that of more complex organisms with evidence of a fully active innate response but, at this stage of development, without confounding adaptive immune function. In addition, zebrafish larvae are almost fully transparent allowing real-time in vivo imaging. This, coupled with the relative ease of genetic manipulation through morpholino and other techniques allows effective interrogation of signaling pathways, making zebrafish an excellent model of innate immunity (Henry et al., 2013Henry K.M. Loynes C.A. Whyte M.K. Renshaw S.A. Zebrafish as a model for the study of neutrophil biology.J. Leukoc. Biol. 2013; 94: 633-642Crossref PubMed Scopus (103) Google Scholar). Through the use of both genetic manipulation and chemical inhibition, Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar identify the key molecular players in regulation of neutrophil recruitment. Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar take advantage of live imaging in LysC neutrophil reporter zebrafish larvae to model neutrophil responses to DAMP and PAMP signaling. Zebrafish are freshwater vertebrates and thus standard bathing solution is hypotonic. The introduction of freshwater during injury or infection leads to osmotic swelling, cell damage, and necrosis and the release of DAMPs. The authors control this DAMP signaling through the use of isotonic bathing media rather than freshwater which does not cause the described osmotic swelling and does not result in release of DAMPs. Infection with Pseudomonas Aeruginosa (PA) or Escherichia coli (E. Coli) generates a pathogen associated danger signal in both bathing conditions and so by simply altering the bathing media, Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar effectively tease apart PAMP signaling from DAMP signaling allowing assessment of the contribution of each to neutrophil recruitment. Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar demonstrated that in standard hypotonic bathing solution with PA injection, approximately one third of fish showed strong neutrophil recruitment with the remaining two thirds showing sporadic unsynchronized recruitment. When the bathing solution was switched to isotonic, the number of high responders was reduced to less than 10%. Injection with E. Coli in hypotonic conditions also resulted in a strong neutrophil response in approximately one third of larvae but in isotonic solution this was reduced to zero. The authors suggest that the residual number of high-responders observed with PA infection alone relates to the inherent pathogenicity of the pseudomonas strain and go on to demonstrate that loss of the virulence factor ExoU (which causes cellular necrosis) abrogated the residual neutrophil recruitment in isotonic solution. Furthermore, neutrophil recruitment could be restored in isotonic conditions by the addition of alternative (and importantly non-microbial) membrane disruptors such as melittin and digitonin. The protective role of DAMP signaling was highlighted by survival data which revealed a correlation between the rates of high-responders and rates of survival. Interestingly, isotonic bathing for the first 2 hr following infection was sufficient to determine survival outcome. Taken together, these data indicate that not only is DAMP signaling (through either osmotic or non-osmotic necrosis) crucial for effective immunity in this model but that the signaling must be early in the inflammatory response to provide protection. In order to understand the mechanisms determining neutrophil recruitment Huang and Niethammer, 2018Huang C. Niethammer P. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.Immunity. 2018; 48 (this issue): 1006-1013Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar undertook mRNA-seq analysis of larvae in three key conditions: uninfected, infected in hypotonic and infected in isotonic solution. They demonstrated that only the infected in hypotonic group exhibit a maximal transcriptional response, however, transcription of a subset of key inflammatory genes was induced in the infection plus isotonic group, i.e., by pathogen signals alone. The authors highlighted interleukin-1β(IL-1β) as an important example of a gene which was upregulated upon bacterial infection, irrespective of hypotonic damage cues and concluded that bacterial cues can therefore still be recognized and responded to in isotonic conditions and in the absence of DAMPs. Previous work by Philipp Niethammer's group (Enyedi et al., 2013Enyedi B. Kala S. Nikolich-Zugich T. Niethammer P. Tissue damage detection by osmotic surveillance.Nat. Cell Biol. 2013; 15: 1123-1130Crossref PubMed Scopus (75) Google Scholar, Enyedi et al., 2016Enyedi B. Jelcic M. Niethammer P. The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.Cell. 2016; 165: 1160-1170Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) has highlighted the role of calcium-dependent cytoplasmic phospholipase A2 (cPLA2) activity in leucocyte recruitment in a tail fin sterile injury model. Enyedi et al., 2013Enyedi B. Kala S. Nikolich-Zugich T. Niethammer P. Tissue damage detection by osmotic surveillance.Nat. Cell Biol. 2013; 15: 1123-1130Crossref PubMed Scopus (75) Google Scholar have demonstrated that this is reduced in tail fin injuries performed in isotonic bathing solution and that recruitment in this context can be restored by addition of exogenous arachidonic acid metabolites. In their current paper, they further interrogated the mechanisms underlying this phenotype. Through morpholino manipulation of TLR signaling and inflammasome signaling they identified the TLR-LPS recognition pathway as essential for PA recognition while the inflammasome might have a degree of redundancy in this context. Consistent with their previously published data, they demonstrated that cPLA2 silencing attenuated neutrophil recruitment and overexpression increased recruitment. Using a number of chemical inhibitors of arachidonic acid metabolism, they demonstrated that 5-lipoxygenase-mediated arachidonic acid metabolism (which generates neutrophil chemoattractants such as LTB4) was essential for osmotic damage sensing. Previous work has shown that Ca2+ flux and membrane stretch are required for translocation of cPLA2 to the nuclear membrane but in this work Huang et al. show that phosphorylation of cPLA2 was required for neutrophil recruitment in PA infection. They therefore identify cPLA2 as an important downstream integrator of mechanical and biochemical signals whose activation is required for robust neutrophil recruitment to sites of infection. In conclusion, Huang et al. show that neutrophil recruitment requires DAMP signaling in addition to PAMPs to enable the host to mount an effective immune response in the context of a pathogenic challenge (Figure 1). Conversely, this requirement for dual host and pathogen signals may also provide a mechanism for innate immune tolerance of non-pathogenic or commensal bacteria. Understanding the processes critical for defining the magnitude and duration of the innate immune response at its onset will enable the development of novel therapeutic strategies to target the dysfunctional neutrophilic inflammation observed in human disease states. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in ZebrafishHuang et al.ImmunityMay 15, 2018In BriefTissue damage signaling in the absence of pathogens causes harmful neutrophil infiltration into damaged organs with no obvious benefit for the host. By intravital imaging of zebrafish larvae, Huang and Niethammer reveal a beneficial role for tissue damage signaling as prerequisite for rapid neutrophil recruitment to microbial infection sites. Full-Text PDF Open Archive