| The epidermis constantly encounters invasions that disrupt its architecture, yet whether the epidermal immune system utilizes damaged structures as danger signals to activate self-defense is unclear. C.elegans as a model organism, the main body of its epidermis is a monolayer and multinucleated giant cell, and compared to the complex structure of mammalian epidermis, without the various kinds of immune cells interference make C.elegans the perfect model to study the immune function of epidermis. Here, we used the C. elegans epidermis model in which skin penetrating infection or injury activates immune defense and antimicrobial peptide(AMP) NLP-29 and CNC-2 family production. By systemically disrupting each architectural component, we found that only disturbance of the apical hemidesmosomes triggered an immune response and robust AMP expression. The epidermis recognized structural damage through apical hemidesmosomes transmembrane receptor MUP-4 and its associated STAT-like protein STA-2, whose disruption led to detachment of STA-2 molecules from apical hemidesmosomes and transcription of AMPs. This machinery enabled the epidermis to bypass certain signaling amplification and directly trigger AMP production when subjected to extensive architectural damage. We also extended our findings in the C.elegans epidermis towards mammalian systems in the primary human keratinocytes. Together, our findings uncover an evolutionarily conserved mechanism for the epithelial barriers to detect danger and activate immune defense. Here we briefly summarize our study from the aspects of objectives, methods, results, conclusions and keywords.Objectives:1, Identify immune-activating epidermal structural components.2, Elucidate molecular mechanisms underlying damage-induced immune response in the C. elegans epidermis.3, Explore physiological significance of damage-induced immune response in the C. elegans epidermis.4, Extend findings in C. elegans epidermis towards mammalian systems.Methods:1, a) we first mapped out the relative positions of major supporting structures comprising the epidermal architecture of the C. elegans by immunostaining. b) We next disrupted individual epidermal supporting structure by RNAi or drug treatment. Their effects on innate immune response were then analyzed by examining the expression of nlp-29 and cnc-2.2, a) We utilized RNAi or loss-of-function mutations to inactivate most C.elegans hemidesmosomes(Ce HDs)-related genes to investigate which Ce HDs components contribute to the induction of AMP expression. b) We examined most players known to be required for epidermal AMP induction upon external insults to find out through which signaling pathway the apical Ce HDs drive AMP production. c) We utilized immunostaining against STAT-like protein STA-2 and Ce HD marker MH4 to examine the subcellular of endogenous STA-2 and check out the spatial relationship between STA-2 and Ce HDs. We performed co-IP approach in C. elegans to test if STA-2 form a protein complex with the apical Ce HDs transmembrane protein MUP-4 which is the main player responsible for structural damage induced innate immune activation.3, a) We employed a severe epidermal injury approach using micrometer-scale fine glass shards, which allows us to introduce multiple wounds in the epidermis of a single worm. b) We injured sek-1(km4)( p38MAP2K) and sta-2(ok1860) mutants by using two different kinds of injury methods to check out whether there is a difference of the involved signaling pathway mediating the epidermal immune response caused by the mild injury and severe injury.4, a) We disrupted the supporting structural components of primary adult human epidermal keratinocytes(HEKa) individually.We next quantified the expression levels of several key innate immune effectors produced by skin keratinocytes following disruption of each structure. b) We further investigated the involvement of all seven human STATs, p38-MAPK and NF-k B signaling pathways in HD-mediated immune activation in HEKa cells.Results:1, a) In general, most epidermal supporting structures are organized into parallel stripe patterns and display minimum entanglement. This highly ordered 3D architecture allow us to perform targeted destruction of one structure at a time without causing extensive collateral damage. b) We disrupted each epidermal supporting structure and checked the immune response using the antimicrobial peptide Pnlp-29 ::GFP reporter. The results show that only damage of the apical Ce HDs, and their neighboring structures could induce an epidermal immune response. Specifically, inactivation of apical Ce HDs receptor mup-4 or its extracellular partner bli-1 triggers robust AMP production. Knockdown of ifb-1 results in moderate nlp-29 up-regulation. Loss of VAB-10A/Plakin also mildly induces nlp-29 transcription.2, a) Compared to mup-4, loss of most other Ce HDs components does not dramatically increase AMP expression. b) The QPCR results show that the functional loss of most known pathways or players does not block nlp-29 or cnc-2 up-regulation caused by mup-4 inactivation, but with the sole exception of STA-2/STAT5 B. c) Immunostaining results show that most endogenous STA-2 is distributed in a characteristic Ce HD-like pattern in the epidermis of the healthy worms and the stripes of STA-2 colocalize with those of the Ce HDs. The co-IP results showed that STA-2 and MUP-4 belong to the same protein complex in C. elegans epidermis.3, a) From the results of phalloidin staining of worms subjected to needle wounding or extensive wounding, we can see that the severe epidermal injury approach indeed create multiple wounds compared with the needle wounding. b) Mild injury in the epidermis caused by microinjection needle puncture(1 wound per animal) trigger nlp-29 up-regulation in a p38 and STA-2-dependent manner. However, when multiple wounds are produced, the induction of nlp-29 no longer goes through p38-MAPK pathway, but it still requires STA-2 function.4, a) Consistent with findings using C. elegans epidermis model, collapse of actin or microtubule cytoskeletons in HEKa cells does not significantly affect any immune effectors tested. Only disassembly of cell-matrix junctions HPCs triggers robust and specific transcription of beta-defensins. b) The induction of beta-defensin by HPC disassembly was analyzed by QPCR after inactivation of STATs, p38 or NF-k B. The results show that among the seven STATs, inactivation of STAT3 or STAT5 B significantly attenuate AMP up-regulation after HPC disassembly in HEKa cells. Neither p38-MAPK nor NF-k B appears to be required for the induction of beta-defensin.Conclusions:1. C. elegans epidermal structures display highly organized spatial distribution patterns.2. Epidermal architectural damage induces AMP transcription in a spatially restricted manner.3. The apical Ce HD transmembrane protein MUP-4 is a key component of immune activation.4. Ce HD-induced immune response requires a STAT protein, but not other known AMP-regulating molecules.5. STA-2 is localized to Ce HD attachment structures at the apical membrane.6. Extensive injury bypasses p38-MAPK signaling and directly activates STA-2 to drive AMP expression.7. Hemidesmosome disassembly induces AMP expression in primary human epidermal keratinocytes, and this immune response requires STAT3 or STAT 5B. |
