Autophagy-associated Production of Antimicrobial Peptides hBD1 and LL37 Exhibits Anti-Bacillus Calmette-Guérin Effects in Lung Epithelial Cells

Antimicrobial peptides (AMPs) constitute important groups of bactericidal polypeptides against various microorganisms that exhibit their anti-bacteria activity through cleavage of precursor peptides into the active form of 50–100 amino acids in length. Various AMP cleavage mechanisms have been reported in different cell types; however, those in Mycobacterium tuberculosis (MTB)-infected lung epithelial cells remain unknown. In the present study, we found that MTB-infected lung epithelial cells expressed high level of the AMPs hBD1 and LL37 to kill intracellular MTB as the first-line immune barrier against MTB infection. Notably, their production in the lung epithelial cells was closely related to the function of autophagosomes and lysosomes. Experimental induction of autophagy in lung epithelial cells could enhance the expression of active hBD1 and LL37 at the post-transcriptional level, whereas silencing of these two active AMPs could decrease the bactericidal effect of autophagy. These findings indicated that cleavage of peptide precursors to form active AMPs might constitute a previously unrecognized antibacterial mechanism of autophagy. Author summary LM and RW conceived and designed the experiments; RW performed the experiments and analyzed the data; QW analyzed the data and contributed reagents/materials/analysis tools; HL performed the experiments; XZ, JY, YL and ZH analyzed the data. LM and RW drafted the manuscript.

remain unknown. In the present study, we found that MTB-infected lung epithelial  The human cathelicidin gene (CAMP) encodes an 18 kDa inactive precursor protein 71 (hCAP18). In human neutrophils, the hCAP18 C-terminus is released by proteolytic 72 hydrolysis, forming an active antimicrobial peptide consisting of 37 amino acids, 73 termed LL37 [19]. hCAP18 can also be cleaved by elastase from azurophilic granules 74 during the exocytosis and phagocytosis process in neutrophils [20]   Antibodies and reagents 125 The following reagents were used in this study: 3-methyladenine (3-MA;    Table 1.   214 Protein lysates used for Co-IP were prepared from A549 cells. Cells were 215 harvested, lysed in IP buffer containing 0.3% CHAPS Detergent (10810118001;

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The supernatant was collected and incubated with p62 primary antibodies and protein 219 G-agarose beads overnight at 4 C. The beads were washed five times with IP buffer 220 before processing for western blotting analysis.   BCG-infected A549 cells (Fig 1A). Therefore, A549 cells were infected with BCG 276 and LL37 and active hBD1 production was detected by qPCR or western blotting.

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BCG could effectively stimulate LL37 and hBD1 production at both mRNA and 278 protein levels in A549 cells (Fig 1B, 1C&1D). To confirm these results, we detected rapamycin treatment increased autophagy level in A549 cells (Fig 2A). Rapamycin Earle's balanced salt solution (EBSS) also increased AMP protein but not mRNA 318 levels ( Fig 2C&2D). Application of 3-MA to disturb the autophagic process in A549 319 and BEAS-2B cells decreased the active peptides of both AMPs whereas the mRNA 320 levels remained unchanged (Fig 2A&2B and S2D, S2E&S2F Fig). Moreover, we used 321 siRNA to silence autophagy-related protein 5 and 7 (ATG 5 and ATG 7) to generate 322 autophagy-defective A549 cells. Notably, BCG could not stimulate active LL37 and 323 hBD1 production in ATG 5-and ATG 7-deficient cells, despite increased mRNA 324 levels ( Fig 2E&2F). To further study the relationship between AMPs production and 325 autophagy, we overexpressed hBD1 and LL37 precursor-mCherry and LC3-GFP in 326 A549 cells. Upon BCG or rapamycin stimulation, hBD1 and LL37 precursor 327 co-localization with LC3 puncta could be observed (Fig 3&4), supporting that AMP 328 precursor was recruited into the autophagosomes. Conversely, under 3-MA treatment, 329 hBD1/LL37 precursor and LC3 puncta co-localization was decreased (Fig 2G). These and LL37 production ( Fig 4A) whereas it did not affect their mRNA levels ( Fig 4B). 345 We next applied NH 4 Cl to disturb lysosomal and autophagosomal function, which 346 obviously reduced active AMPs production ( Fig 4C) but did not affect their mRNA 347 levels ( Fig 4D).

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Furthermore, immunofluorescence to reflect the co-location of hBD1/LL37 349 precursor with autophagosomes and lysosomes showed that in A549 cells, the 350 precursors of hCAP18 exhibited colocalization with autophagosomes and lysosomes, 351 suggesting that the AMP precursors entered in autophagosomes (Fig 6&7). However,

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BCG infection of A549 cells treated with Baf A1 or NH 4 Cl did not alter the 18 353 colocalization among hCAP18, autophagosomes, and lysosomes, even upon 354 rapamycin stimulation (Fig 5E&5F). This indicated that autolysosome formation and 355 function were not involved in AMP precursor recruitment. Thus, whereas the fusion 356 of autophagosomes with lysosomes and lysosomal function are vital for active AMP 357 production, these exert no effect on AMP precursor recruitment. ( Fig 8C, Fig 9&10). Furthermore, we performed Co-IP to directly measure and further 376 confirm the interaction of p62 with AMP precursors. Under BCG stimulation, p62 377 interacted with hCAP18 (Fig 8D), suggesting that p62 is required for the autophagic 378 production of active AMPs.