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Paneth Cell Α-Defensins HD-5 and HD-6 Display Differential Degradation Into Active Antimicrobial Fragments

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Paneth Cell Α-Defensins HD-5 and HD-6 Display Differential Degradation Into Active Antimicrobial Fragments Paneth cell α-defensins HD-5 and HD-6 display differential degradation into active antimicrobial fragments D. Ehmanna, J. Wendlera, L. Koeningera, I. S. Larsenb,c, T. Klaga, J. Bergerd, A. Maretteb,c, M. Schallere, E. F. Stangea, N. P. Maleka, B. A. H. Jensenb,c,f, and J. Wehkampa,1 aInternal Medicine I, University Hospital Tübingen, 72076 Tübingen, Germany; bQuebec Heart and Lung Institute, Department of Medicine, Faculty of Medicine, Cardiology Axis, Laval University, G1V 4G5 Quebec, QC, Canada; cInstitute of Nutrition and Functional Foods, Laval University, G1V 4G5 Quebec, QC, Canada; dElectron Microscopy Unit, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany; eDepartment of Dermatology, University Hospital Tübingen, 72076 Tübingen, Germany; and fNovo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Genomics, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark Edited by Lora V. Hooper, The University of Texas Southwestern, Dallas, TX, and approved January 4, 2019 (received for review October 9, 2018) Antimicrobial peptides, in particular α-defensins expressed by Paneth variety of AMPs but most abundantly α-defensin 5 (HD-5) and -6 cells, control microbiota composition and play a key role in intestinal (HD-6) (2, 17, 18). These secretory Paneth cells are located at the barrier function and homeostasis. Dynamic conditions in the local bottom of the crypts of Lieberkühn and are highly controlled by microenvironment, such as pH and redox potential, significantly af- Wnt signaling due to their differentiation and their defensin regu- fect the antimicrobial spectrum. In contrast to oxidized peptides, lation and expression (19–21). Transgenic mice expressing HD-5 or some reduced defensins exhibit increased vulnerability to proteolytic HD-6 under control of their endogenous promoters were protected degradation. In this report, we investigated the susceptibility of against Salmonella typhimurium infections, and both peptides strongly Paneth-cell–specific human α-defensin 5 (HD-5) and -6 (HD-6) to in- influenced the microbiota composition (10, 22–24). A normal Paneth testinal proteases using natural human duodenal fluid. We systemat- cell function in humans is needed to allow a balanced host–microbe ically assessed proteolytic degradation using liquid chromatography– relationship and a functional intestinal barrier, while an abnormal mass spectrometry and identified several active defensin fragments Paneth cell with reduced expression or secretion of HD-5 or HD-6 MICROBIOLOGY capable of impacting bacterial growth of both commensal and path- is associated with inflammatory bowel disease (2, 9, 25, 26). ogenic origins. Of note, incubation of mucus with HD-5 resulted in Of note, HD-5 and HD-6 are functionally different. While the – 255 8,000 new antimicrobial combinations. In contrast, HD-6 remained strong antimicrobial activity of HD-5 has been known for years stable with consistent preserved nanonet formation. In vivo studies (27, 28), little is known about the antimicrobial activity of HD-6. demonstrated proof of concept that a HD-5 fragment shifted micro- It has, however, recently been demonstrated that HD-6 forms Akkermansia biota composition (e.g., increases of sp.) without decreas- self-assembled nanonets that trap bacteria and prevent infections ing diversity. Our data support the concept that secretion of host (23). Moreover, under reducing conditions, which is the natural peptides results in an environmentally dependent increase of an- state in the gut (29), a direct antimicrobial activity of HD-6 could timicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host’s ability to control pathogens and commensals. These findings broaden Significance our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus Paneth cells provide intestinal host defense against pathogens providing promising avenues to explore for drug development. and control the healthy microbiota by secreting antimicrobial peptides. We show that the most abundant secreted Paneth cell antimicrobial peptides | α-defensins | intestinal barrier | proteolytic products, human defensin HD-5 and HD-6, show a distinct sus- digestion | host–microbiota interaction ceptibility to proteolytic digestion by human duodenal fluid. WhileHD-5isdigestedinmanyfragments,HD-6isstableandstill able to form nanonets. The occurring fragments of HD-5 were uman body surfaces, especially the intestine, are host to antimicrobially active against microorganisms. We provide proof trillions of microbial organisms, mostly bacteria (1), and H of concept about microbiome modulating capacities in vivo, collectively termed the “microbiota.” It is widely recognized that which includes an increase of Akkermansia sp. Our results in- the gut microbiota have multiple functions and that a disturbed dicate that fragmentation of defensins increases antimicrobial microbial consortium has been associated with multiple diseases diversity and further adds to the complexity of host microbial including multiple sclerosis, autism, obesity, and chronic in- – interaction at interfaces. Fragmentation could lead to new anti- flammatory bowel diseases (2 6). Most recently, the microbiome microbial peptides with possible therapeutic usage. composition has also been demonstrated to dramatically influ- ence human response rates and survival of anticancer treatments Author contributions: D.E., B.A.H.J., and J. Wehkamp designed research; D.E., J. Wendler, with checkpoint inhibitors (7, 8). L.K., I.S.L., J.B., and B.A.H.J. performed research; D.E., I.S.L., B.A.H.J., and J. Wehkamp It is indispensable for the host to preserve a functional intestinal analyzed data; and D.E., J. Wendler, L.K., I.S.L., T.K., A.M., M.S., E.F.S., N.P.M., B.A.H.J., barrier and to create a stable homeostasis with the microbiota (9– and J. Wehkamp wrote the paper. Conflict of interest statement: J. Wehkamp and D.E. are planning to apply for a patent on 12). One of the most important components of the intestinal barrier the HD-5 fragments. are antimicrobial peptides (AMPs), which are secreted from epi- This article is a PNAS Direct Submission. thelial cells throughout the gut. These peptides ensure a mostly This open access article is distributed under Creative Commons Attribution-NonCommercial- sterile inner mucus layer and protect against different microbes, NoDerivatives License 4.0 (CC BY-NC-ND). – fungi, and archaea viruses (13 15). Defensins are small cationic 1To whom correspondence should be addressed. Email: [email protected] molecules, characterized by three conserved disulphide bonds (16), tuebingen.de. and represent a main group of AMPs. In the small intestine, Paneth This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cellsplayakeyroleinbalancingthemicrobiotacompositionand 1073/pnas.1817376116/-/DCSupplemental. protecting the host from invading pathogens by secretion of a www.pnas.org/cgi/doi/10.1073/pnas.1817376116 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 be observed, while the bacteria-trapping nanonet formation was still present (30). The reduction—which is naturally present owing to the low redox potential in the gut or enzymatically generated by the thioredoxin system (30–33)—leads to an opening of the three disulphide bridges, followed by a change in the tertiary structure of these peptides (31, 34). This structural change leads to a post- translational modification of the antimicrobial spectrum (30, 31, 35). Importantly, these open, linear forms have been detected in the small intestine (31, 36). In contrast to the oxidized original peptide, we previously reported that the reduced linear peptide structures of hBD-1 were susceptible to proteases (37). It is commonly believed that the degradation process facilitates antimicrobial inactivation and/or loss of function (37). Here, we hypothesized that protein/ defensin degradation is rather a novel activation mechanism me- diated by the formation of new antimicrobial components. We thus investigated the susceptibility of HD-5 and HD-6 to proteolytic degradation from human duodenal fluid under reducing conditions to mimic the in vivo intestinal situation. We assessed the amino acid signature based on the exact masses and synthesized the newly identified fragments for further studies. We then systematically tested the antimicrobial spectrum of the resulting peptide frag- ments against a subset of commensal and pathogenic bacteria. Surprisingly, Paneth cell defensins were shown to be differentially affected by human mucus, but their overall antimicrobial spectrum was dramatically increased by the generation of new active antimi- Fig. 1. HD-6 nanonet formation is not affected by duodenal fluid. (A)Herewe crobial fragments, thus uncovering a key mucosal defense mecha- show the chromatograph of only exogenously added HD-6 incubated with du- nism for fine-tuning of host microbial interaction in the gut. odenal fluid after reduction with 2 mM TCEP. We detected only the oxidized and reduced full-length peptides due to their retention time with their m/z Results graphs and their two-, three-, four-, five- and sixfold protonated ions and neutral masses. (B) We incubated beads with 200 μg/mL reduced HD-6 or 0.01% Natural Human Duodenal Fluid Digests HD-5, While Nanonet-Forming HAc (control) and duodenal fluid
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