SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides to Enhance the Nucleotide-Binding Oligomerizatio

SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides To Enhance the Nucleotide-Binding Oligomerization Domain This information is current as −Dependent Immune Response in Mouse of September 26, 2021. Bone Marrow−Derived Macrophages Yongjun Hu, Feifeng Song, Huidi Jiang, Gabriel Nuñez and David E. Smith Downloaded from J Immunol published online 21 May 2018 http://www.jimmunol.org/content/early/2018/05/18/jimmun ol.1800210 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/05/18/jimmunol.180021 Material 0.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 21, 2018, doi:10.4049/jimmunol.1800210 The Journal of Immunology

SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides To Enhance the Nucleotide-Binding Oligomerization Domain–Dependent Immune Response in Mouse Bone Marrow–Derived Macrophages

Yongjun Hu,* Feifeng Song,*,† Huidi Jiang,† Gabriel Nun˜ez,‡ and David E. Smith*

There is increasing evidence that proton-coupled oligopeptide transporters (POTs) can transport bacterially derived chemotactic peptides and therefore reside at the critical interface of innate immune responses and regulation. However, there is substantial contention regarding how these bacterial peptides access the cytosol to exert their effects and which POTs are involved in facilitating this process. Thus, the current study proposed to determine the (sub)cellular expression and functional activity of POTs in macrophages derived from mouse bone marrow and to evaluate the effect of specific POT deletion on the production of inflammatory Downloaded from cytokines in wild-type, Pept2 knockout and Pht1 knockout mice. We found that PEPT2 and PHT1 were highly expressed and functionally active in mouse macrophages, but PEPT1 was absent. The fluorescent imaging of muramyl dipeptide–rhodamine clearly demonstrated that PEPT2 was expressed on the plasma membrane of macrophages, whereas PHT1 was expressed on endosomal membranes. Moreover, both transporters could significantly influence the effect of bacterially derived peptide ligands on cytokine stimulation, as shown by the reduced responses in Pept2 knockout and Pht1 knockout mice as compared with wild- http://www.jimmunol.org/ type animals. Taken as a whole, our results point to PEPT2 (at plasma membranes) and PHT1 (at endosomal membranes) working in concert to optimize the uptake of bacterial ligands into the cytosol of macrophages, thereby enhancing the production of proinflammatory cytokines. This new paradigm offers significant insight into potential drug development strategies along with transporter-targeted therapies for endocrine, inflammatory, and autoimmune diseases. The Journal of Immunology, 2018, 201: 000–000.

he proton-coupled oligopeptide transporter (POT) family expressed in the proximal tubule of kidney for nutritional reabsorptive (also known as solute carrier family 15 [SLC15]) consists purposes (3) and in choroid plexus for the removal of neuropeptide T of four mammalian members: PEPT1 (SLC15A1), PEPT2 degradation products from cerebrospinal fluid (4). PHT1 and PHT2, by guest on September 26, 2021 (SLC15A2), PHT1 (SLC15A4), and PHT2 (SLC15A3). All POTs cloned initially from brain, are expressed ubiquitously and localized mediate the transport of di/tripeptides using an inwardly directed intracellularly, especially on the membrane of endosomes (5). They are proton gradient and membrane potential as the driving force (1). believed to help maintain acid–base homeostasis in endosomes via However, PHT1 and PHT2 can also transport the amino acid the cotransport of L-histidine and protons from within the endo- L-histidine and, as a result, are referred to as peptide/histidine trans- somes into the cytosolic compartment (6–8). POTs can transport up porters. Being localized at the apical surface of epithelial cells, PEPT1 to 400 dipeptides, 8000 tripeptides, and many peptide mimics, in- is predominantly expressed in the enterocytes of small intestine for cluding antibiotic, anticancer, and antiviral drugs. Thus, POT- nutritional absorptive purposes (2), whereas PEPT2 is predominantly mediated substrates have physiological and pharmacological relevance. They may also have significant value as targets in modu- lating gene expression in the innate immune system and *Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109; †Laboratory of Pharmaceutical Analysis and Drug inflammatory response (9–12). Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Innate immunity, the first line of defense against pathogenic ‡ Zhejiang, China 310058; and Department of Pathology, School of Medicine, invasion, is phylogenetically the oldest part of the immune system. University of Michigan, Ann Arbor, MI 48109 Major components of the innate immune system include epithelial ORCIDs: 0000-0002-1883-0868 (Y.H.); 0000-0002-1720-6115 (F.S.); 0000-0002- 2180-5153 (H.J.); 0000-0002-6301-7737 (D.E.S.). cells, which can prevent the entry of microbes, and leukocytes (e.g., Received for publication February 14, 2018. Accepted for publication May 2, 2018. neutrophils and macrophages), which can detect as well as elim- inate microbes that have penetrated through the epithelial barriers. This work was supported by National Institutes of Health/National Institute of Gen- eral Medical Sciences Grant R01-GM115481 (to D.E.S.) and by China Scholarship Microbe detection leads to a cascade of events in which the Council Grant CSC-201506320103 (to F.S.). sensing of pathogen-associated molecular patterns by specific Address correspondence and reprint requests to Dr. David E. Smith, University of pattern-recognition receptors results in the activation of immune Michigan College of Pharmacy, 428 Church Street, Room 4008, Ann Arbor, MI responses (13). One such class of pattern-recognition receptors is the 48109-1065. E-mail address: [email protected] nucleotide-binding oligomerization domain (NOD) proteins NOD1 The online version of this article contains supplemental material. and NOD2 (14–17). These intracellular proteins can respond to Abbreviations used in this article: BAF, bafilomycin A1; BMDM, bone marrow– derived macrophage; IBD, inflammatory bowel disease; MDP, muramyl dipeptide; bacterial cell wall fragments in which NOD1 detects peptidogly- NOD, nucleotide-binding oligomerization domain; POT, proton-coupled oligopeptide can motifs (e.g., L-alanyl-g-D-glutamyl-meso-diaminopimelic acid transporter; qRT-PCR, quantitative real-time PCR; SLC15, solute carrier family 15; [Tri-DAP]) that are found primarily in Gram2 bacteria and select Tri-DAP, L-alanyl-g-D-glutamyl-meso-diaminopimelic acid. + Gram bacteria, and NOD2 detects peptidoglycan motifs (e.g., L,D- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 muramyl dipeptide [MDP]; fMLF) that are widely found among both

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800210 2 ROLE OF SLC15A IN NOD-MEDIATED IMMUNE RESPONSE IN BMDMs

2 Gram and Gram+ bacteria. Once activated, the NOD signaling and water ad libitum provided by the Unit for Laboratory Animal Medi- pathway can induce transcription factors NF-kB and AP-1, thereby cine, University of Michigan, Ann Arbor, MI. Studies on gender-matched enhancing the production of proinflammatory cytokines (e.g., IL-6 mice, 6–8 wk of age, were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the and TNF-a). Moreover, LPS can interact with TLR4 to induce NF-kB National Institutes of Health. and AP-1 as well as transcription factors IRF3 and IRF7, leading Chemicals and materials to the stimulation of proinflammatory cytokines (e.g., IL-6, TNF-a, and IFN-g) (15, 17–19). Several mechanism(s) of how host cells may The Lineage Cell Depletion Kit and CD11b MicroBeads were from internalize pathogen-associated molecular patterns have been pro- Miltenyi Biotec (Auburn, CA); recombinant mouse M-CSF (1.5 mg/ml) and Quantikine ELISA mouse IL-6 and TNF-a kits were from R&D posed, such as phagocytosis of bacteria, bacterial-derived outer Systems (Minneapolis, MN); MDP, MDP control (L,L-isomer), iE-DAP, membrane vesicles, channels, pores, bacterial secretion systems, and Tri-DAP, MDP-rhodamine, and LPS-EB Ultrapure were from Invivo- endocytosis (17, 20, 21). So-called “transmembrane channels” have Gen (San Diego, CA); DAPI was from Molecular Probes (Eugene, OR); 3 3 14 also been implicated because MDP (dipeptide), Tri-DAP (tripeptide), [ H]GlySar (2.8 Ci/mmol), [ H]L-histidine (30 Ci/mmol), and [ C]mannitol and fMLF (tripeptide) were reported as substrates of POT family (53 mCi/mmol) were from Moravek Chemical (Brea, CA); GlySar, Gly- Pro, glycine, and L-histidine were from Sigma-Aldrich (St. Louis, MO); members (5, 22–27). However, the (sub)cellular location and func- rabbit anti-mouse PEPT1 and PEPT2 antisera were from Lampire Bio- tional relevance of POTs in the innate immune response and in- logical Laboratories (Pipersville, PA) (37); mouse anti-GFP, mouse b-actin flammatory bowel disease (IBD) are controversial. monoclonal, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP Abs It has been reported that PEPT1 was expressed aberrantly in the were from Bio-Rad (Hercules, CA); Oregon Green 488, goat anti-rabbit IgG, ProLong Diamond Antifade Mountant with DAPI, RMPI 1640 media, colon of patients with IBD but not in normal human colon (28). In and FBS were from Thermo Fisher Scientific (Waltham, MA); mouse studies using a DSS-induced mouse model of IBD, the same au- Pept1, Pept2, Pht1, Pht2, and Gapdh primers for quantitative real-time Downloaded from thors observed that the severity of inflammation was greater in PCR (qRT-PCR) were from Integrated DNA Technologies (Coralville, hPEPT1 transgenic mice than wild-type mice, in which hPEPT1 Iowa). All other chemicals and reagents were from standard sources. was overexpressed in the intestinal epithelial cells (29). In con- Preparation of mouse bone marrow cell subtypes trast, studies by a different group demonstrated that PEPT1 ex- Following euthanasia and sterilization, the femurs and tibias of three mice, pression was consistently reduced during acute or chronic 6–8 wk of age, were removed accordingly (38). Bone marrow cells were experimental inflammation in mice (i.e., DSS-induced) and that flushed out with 0.5 ml RPMI 1640 media using a syringe with 26-gauge http://www.jimmunol.org/ MDP-induced cytokine expression was PEPT1-independent (30). needle and an aliquot obtained for Western blot analysis. The remaining These authors further reported that PEPT1 expression levels were, cells were resuspended by gentle pipetting and filtered with 30 mm nylon mesh for removing the cellular debris. The cells were centrifuged at in fact, decreased in the ileal and colonic epithelia of patients with 300 3 g for 10 min at 4˚C and washed with 5 ml RPMI 1640. Resident IBD during acute inflammation as compared with controls. Fi- macrophages were then purified by adherence to a Petri dish for 24 h, and nally, it was reported that immune cells, including macrophages, the protein lysate was harvested for Western blot analysis. Alternatively, 2 could express PEPT1, thereby interacting with bacterially derived Lin+ and Lin cells were prepared following the protocol provided in the products and regulating the production of proinflammatory cyto- Lineage Cell Depletion Kit (http://www.miltenyibiotec.com/en/products- and-services/macs-cell-separation/cell-separation-reagents/hematopoietic- kines (31, 32). However, our previous study demonstrated that stem-cells/direct-lineage-cell-depletion-kit-mouse.aspx). In brief, the cells PEPT1 was not required for fluorescent-labeled MDP uptake or were counted and then incubated with 10 ml of biotin-Ab mixture per 107 by guest on September 26, 2021 MDP-induced signaling in bone marrow–derived macrophages total cells for 10 min at 4˚C. A 30 ml aliquot of buffer and 20 ml of anti- 7 (BMDMs) from wild-type and Pept1 knockout mice (33). biotin MicroBeads were added per 10 cells and incubated for an additional 15 min at 4˚C. The cells were washed with 2 ml of buffer and These discrepancies might reflect the fact that previous studies centrifuged at 300 3 g for 10 min, after which the cells were resuspended only evaluated the expression of PEPT1 protein in human PBMCs and applied onto magnetic separation LS columns. The column was then and a human monocytic KG-1 cell line (31) and Pept1 transcripts but washed three times with 3 ml buffer, and the cells passed through the 2 not PEPT1 protein in immune cells obtained from mouse colon column were collected as the enriched lineage negative (Lin ) cell frac- (32). It is also possible that other POTs, such as PEPT2, PHT1, and tion with unlabeled cells. The retained cells were subsequently eluted as the enriched lineage positive (Lin+) cell fraction. Both enriched fractions PHT2 may be critical for MDP (or other bacterial products) to were centrifuged at 300 3 g for 10 min at 4˚C, and the protein lysates access intracellular compartments or organelles. For example, were prepared for Western blot analysis by adding NP-40 lysis buffer PEPT2 protein was reported to have high expression in a human (150 mM NaCl, 1.0% NP-40, 50 mM Tris-Cl, and proteinase inhibitor acute monocytic leukemia THP-1 cell line and to be phagosome- mixture, pH 8). associated for MDP transport into cytosol (23). Still, others have Preparation of mouse BMDMs reported that PEPT2 does not have to be internalized to mediate the Bone marrow cells were obtained from mice and prepared as described in uptake of MDP in mouse- and human-derived macrophages (34). the previous section. BMDMs were then isolated and differentiated by With this in mind, this study addresses two primary objectives: 1) standard methods (38, 39) and, in the presence of M-CSF, hematopoietic to determine the (sub)cellular expression and functional activity of stem/progenitor cells from the bone marrow were directed into a homog- ∼ 7 POTs in macrophages derived from mouse bone marrow and 2) to enous population of mature BMDMs. In brief, 10 cells were cultured in a 10-cm Ultra-Low Attachment Dish (Corning, Corning, NY) containing evaluate the effect of specific POT deletion on the production of 25 ml of RMPI 1640 media (plus 10% FBS+ 15 ng/ml M-CSF) and in- inflammatory cytokines in wild-type, Pept2 knockout, and Pht1 cubated at 37˚C in 5% CO2 for 3 d. The cells were washed three times with knockout mice. Our findings demonstrate for the first time, to our PBS (pH 7.4), detached by incubating in a refrigerator (4˚C) for 5 min, 5 knowledge, that PEPT2 and PHT1 work together in facilitating the reseeded (1 3 10 /well) in a 24-well plate containing RPMI 1640 media (plus 10% FBS+ 15 ng/ml M-CSF), and then cultured for another 3 d. It uptake of bacterially derived peptides into the cytosol of macro- should be noted that macrophage progenitors adhered to the cell culture phages, thereby playing a critical role in NOD ligand–triggered dish and were not washed away. Moreover, macrophages were fully dif- immune responses. ferentiated at day 6. BMDM studies were performed on day 7. GlySar uptake and inhibition studies in BMDMs Materials and Methods 5 Animals BMDMs (1 3 10 /well) were prepared as described above and, at d 7 of culture, each well was washed with 1 ml PBS (pH 7.4) three times prior to Wild-type, Pept2 knockout (35), and Pht1 knockout (36) mice (all on uptake. A 0.5 ml volume of PBS (pH 7.4) containing 0.1 mCi [3H]GlySar C57BL/6 background) were bred and housed in a temperature-controlled (1 mM) and 0.2 mCi [14C]mannitol (1 mM) 6 potential inhibitors environment with 12 h light and dark cycles and received a standard diet (i.e., 5 mM GlyPro, L-histidine or glycine; or 1 mM MDP, MDP control, The Journal of Immunology 3 iE-DAP, or Tri-DAP) was then added to each well. After 5 min of incu- which 2 mg of total RNA was reverse transcribed into cDNA using the bation at 37˚C, uptake was terminated, and the cells were washed by Omniscript Reverse Transcription Kit (Qiagen) and 16-mer random pri- adding 1 ml ice-cold buffer three times to each well. A 0.3-ml aliquot of mers. The desired target gene was quantified on a 7300 Real-Time PCR Hyamine hydroxide was added to each well, the cells digested for 5 min at System (Applied Biosystems, Waltham, MA) where 50 ng of cDNA was room temperature, and the well was then placed in a scintillation vial. reacted with 25 ml of Power SYBR Green Master Mix (Applied Bio- Scintillation mixture (6 ml) was added, and the sample radioactivity systems). The primers were designed using Primer 3.0 (Applied Bio- was measured on a dual-channel liquid scintillation counter (LS 6000; systems) and synthesized by Integrated DNA Technologies. The forward Beckman Coulter, Brea, CA). Substrate (GlySar) uptake was calculated and reverse primers were: 59-GAGACAGCCGCATCTTCTTGT-39 and 59- according to the following equation (40): CACACCGACCTTCACCATTTT-39 for mGapdh; 59-CCACGGCCATT- h i TACCATACG-39 and 59-TGCGATCAGAGCTCCAAGAA-39 for mPept1; Mc 59-TGCAGAGGCACGGACTAGATAC-39 and 59-GGGTGTGATGAAC- Sc 2 Ss Ms GTAGAAATCAA-39 for mPept2; 59-GGCCATTGGGTGGATGAG-39 and GlySar Uptake ¼ Cs Ss 59-GCAGGTGGCAGCTGTTGA-39 for mPht1; and 59-CCTGTGATGG- TGACCCTTGTG-39 and 59-GGAGGACATAGGTGGACTGCAT-39 for where Sc is the dpm of GlySar in cells; Mc, the dpm of mannitol in cells; mPht2. The qRT-PCR thermal conditions were one cycle at 50˚C for 2 min, Ms, the dpm of mannitol in stock solution; Ss, the dpm of GlySar in stock 14 one cycle at 95˚C for 10 min, 40 cycles at 95˚C for 15 s, and 60˚C for solution; and Cs, the concentration of GlySar in stock solution (mM). [ C] 1 min. The ΔCT method was used to calculate the relative levels of target Mannitol was used to correct for nonspecific binding, and GlySar uptake gene transcripts in mice in which the ratio of target gene to mGapdh was was expressed as percent relative to control studies (i.e., no inhibitor equal to 22ΔCT, ΔCT = CT (gene) – CT (mGapdh). present) in wild-type mice. Western blot analysis LPS and MDP stimulation of BMDMs Protein levels were determined by immunoblotting (i.e., Western blot), as At day 7 of culture, the BMDMs were washed three times with PBS (pH 7.4) reported previously (41). In brief, membrane proteins were resolved by Downloaded from after which 1 ml of RMPI 1640 with M-CSF and LPS (0–10 ng/ml), 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane (Bio-Rad), containing either 10 mg/ml MDP, MDP control, or Tri-DAP, was added and the membrane was preincubated in 5.0% milk TBS buffer for 1 h. The to each well. For some studies, 1 mM bafilomycin A1 (BAF) was used to membrane was then incubated with mouse PEPT2 antisera (1:5000), 6 treat BMDMs for 30 min prior to 5 ng/ml LPS being added ( 10 mg/ml mouse PEPT1 antisera (1:3000), mouse GFP antisera (Thermo Fisher MDP). The cells were then incubated for 24 h at 37˚C, and the culture Scientific) (1:1000), or mouse GAPDH mAb (Bio-Rad) (1:1000) for media were collected. Cytokines TNF-a and IL-6 were quantified using 90 min at room temperature. The membrane was washed with 10 ml TBST the Quantikine ELISA Kits, according to the protocol supplied in the three times, and then the secondary Ab IgG-HRP (1:3000) was added and http://www.jimmunol.org/ manufacturer’s manual. incubated for 45 min. Finally, the membrane was washed with 10 ml TBST five times, the ECL substrate (Millipore, Billerica, MA) was applied, and Isolation of endosomes and L-histidine uptake studies the x-ray film was exposed and developed. All immunoblots had 25–30 mg The construction of mPht1 plasmid DNA and transfection was performed of protein loaded into each lane. as described below. Full-length Pht1 cDNA was amplified from the total RNA of mouse liver with forward primer 59-CGTCGCATGGAGGGC- Data and statistical analysis TCTG-39 and reverse primer 59-TGGCCCTCCTGCTGGTGG-39 using Data were reported as mean 6 SE unless otherwise noted. Statistical high fidelity PCR Taq DNA polymerase (Invitrogen). PCR conditions were differences between two groups were determined by an unpaired (two- as follows: one cycle at 95˚C for 2 min, 30 cycles at 95˚C for 45 s, 52˚C for sample) t test. Multiple group comparisons were performed by ANOVA, 45 s, and 68˚C for 2 min. The PCR product was then inserted into followed by Dunnett’s or Tukey’s test, using Prism v7.0 (GraphPad by guest on September 26, 2021 pcDNA3.1/CT-GFP-TOPO vector (Invitrogen). After the open reading Software, La Jolla, CA). A p value # 0.05 was considered significant. frame was confirmed by DNA sequencing (DNA Core Service, University of Michigan), the plasmid DNA was cut by Sal I and stably transfected into HEK293 cells. The positive colonies were selected with G418 and vali- Results dated by the signal of GFP (Supplemental Fig. 1). The cells were then POT expression in bone marrow and cell subtypes incubated with DMEM plus 10% FBS for preparation of endosomes. Endosomes from mouse liver, HEK293 mock cells, and mPhT1- Pept1 transcripts were not observed in the bone marrow of either transfected HEK293 cells were enriched using the Lysosome Enrichment wild-type or Pept2 knockout mice (Fig. 1A). This finding was Kit (Thermo Fisher Scientific) following the protocol for tissues provided by the manufacturer’s manual (https://www.thermofisher.com/order/catalog/ confirmed at the protein level where no band was found for PEPT1 product/89839). In brief, after washing the liver with PBS (pH 7.4), during immunoblot analysis of bone marrow from wild-type, Pht1 ∼200 mg of fresh tissue was minced into small pieces, 800 ml of Lysosome knockout, and Pept2 knockout mice (Fig. 1B). In contrast, tran- Enrichment Reagent A added, and 50 strokes applied to the mixture, on scripts were observed for Pept2 (Fig. 1C), Pht1 (Fig. 1D), and ice, with a Dounce Homogenizer (Thomas Scientific, Swedesboro, NJ). Pht2 (Fig. 1E) in wild-type mice, with the peptide/histidine For cells, three 10-cm plates (confluent) were washed with PBS (pH 7.4). An 800-ml volume of Lysosome Enrichment Reagent B was added and transporters showing greater expression than Pept2. Immunoblot mixed well. The tissue lysate was collected and centrifuged at 500 3 g for analysis confirmed that PEPT2 protein was present in the bone 10 min at 4˚C, followed by gradient ultracentrifugation at 145,000 3 g for marrow of wild-type and Pht1 knockout mice, but not in Pept2 2 h at 4˚C on an Optima L-90K Ultracentrifuge (Beckman Coulter) with knockout mice (Fig. 1F). Protein expression studies were not swing rotor. After centrifugation, the desired lysosome band was collected and diluted with two to three volumes of PBS (pH 7.4). The sample was reported for PHT1 and PHT2 because of the lack of specific Abs. mixed well, transferred into a new microcentrifuge tube, and then centri- When bone marrow cell subtypes were examined from wild-type fuged at 18,000 3 g for 30 min at 4˚C. The lysosomal pellet was stored on mice (Fig. 2A), PEPT2 protein was highly expressed in Lin+ cells ice until further processing. (consisting of T and B cells, macrophages, monocytes, gran- A 50-ml volume of the reaction mixture, which contained 10 mgof 3 ulocytes, and erythrocytes and their committed precursors) but not endosomes in PBS (pH 7.4) plus 0.01 mCi [ H]histidine (1 mM) and 0.02 2 mCi [14C]mannitol (1 mM) 6 potential inhibitors (1 mM MDP or 1 mM in Lin cells (consisting of hematopoietic and mesenchymal stem BAF), was incubated for 5 min. Ice-cold buffer (1 ml) was added to stop cells and their progenitors). As shown in Fig. 2B, PEPT2 protein the reaction, after which the mixture was centrifuged at 18,000 3 g for was also highly expressed in residential macrophages. 30 min at 4˚C. The pellet was then washed three times with 1 ml ice-cold buffer, the radioactivity was measured, and histidine uptake was calcu- PEPT2 protein expression in BMDMs and the uptake of GlySar lated, as described previously (40). in BMDMs from wild-type and Pept2 knockout mice Quantitation of mRNA with qRT-PCR PEPT2 is a plasma membrane–bound transporter that is widely distributed throughout the body. Because PEPT2 protein was mRNA levels were determined by qRT-PCR, as reported previously (37). + Briefly, total RNA was isolated from the small intestine and kidney and found in bone marrow–derived cells, Lin cells, and residential from BMDMs using the RNeasy Plus Kit (Qiagen, Hilden, Germany) in macrophages of this study, its expression was further evaluated in 4 ROLE OF SLC15A IN NOD-MEDIATED IMMUNE RESPONSE IN BMDMs Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. POT transporter expression in mouse bone marrow. Gene expression of Pept1 (A), protein expression of PEPT1 (B), gene expression of Pept2 (C), gene expression of Pht1 (D), gene expression of Pht2 (E), and protein expression of PEPT2 (F) in wild-type, Pht1 knockout (KO), and Pept2 knockout (KO) mice. The positive control was small intestine for PEPT1 and kidney for PEPT2. Quantiﬁcation of protein (i.e., transporter/b-actin ratio) is shown below the ﬁgure. One representative example is shown of three individual immunoblots.

BMDMs. As shown in Fig. 2B, PEPT2 protein was highly uptake (35–75%) when studied in the presence of bacterially de- expressed in BMDMs even after 3 and 7 d of cell culture. Thus, rived peptides (i.e., MDP, MDP control, iE-DAP, and Tri-DAP). In the functional activity of PEPT2 was examined in this system by contrast, no signiﬁcant differences were observed for GlySar up- studying the uptake of a model dipeptide substrate (GlySar) in the take (6inhibitors) in BMDMs of Pept2 knockout mice. absence and presence of potential transport inhibitors. As shown in Fig. 3A, in the absence of inhibitors, GlySar uptake was re- MDP-rhodamine uptake in BMDMs from wild-type and Pept2 duced by ∼80% during PEPT2 ablation. A similar extent of in- knockout mice hibition was observed in wild-type mice when GlySar uptake was The previous inhibition study suggested that MDP might be a studied in the presence of GlyPro. Whereas the amino acid glycine substrate of PEPT2 and enter macrophages via this pathway. In this was without effect, signiﬁcant reductions were found in GlySar study, we used rhodamine-labeled MDP as a molecular probe to The Journal of Immunology 5

knockout mice, and the expression of these two cytokines was measured. As shown in Fig. 4A and 4B, the protein expression of IL-6 and TNF-a in BMDMs from Pept2 knockout mice was about one-half the values of BMDMs from wild-type animals, regardless of LPS concentration (except for 0 LPS). Similar results were observed when mouse BMDMs were stimulated with 1–10 ng/ml LPS plus 10 mg/ml Tri-DAP (Fig. 4C, 4D). Thus, PEPT2 ablation limited the entry of MDP (and Tri-DAP) into BMDMs and decreased the production of cytokines. In contrast, 10 ng/ml LPS plus 10 mg/ml MDP control (biologi- cally inactive L,L-isomer)hadnoeffectonIL-6andTNF-a production in wild-type BMDMs, as compared with Pept2 knockout mice (Fig. 4E, 4F, respectively). Thus, the observed effects in the current study were speciﬁc for NOD2 agonists and not for peptides in general. Cytokine expression in BMDMs from wild-type and Pht1 knockout mice after stimulation with LPS plus MDP

PHT1 is a transporter that is localized to the membrane of Downloaded from endosomes (and/or lysosomes), where it efﬂuxes substrates from the inside of endosomes into the cytosol. Thus, the dipeptide MDP was used as a model substrate to probe the functional activity of PHT1 in mouse BMDMs. As shown in Fig. 5A, we found that in BMDMs treated with 5 ng/ml LPS plus 10 mg/ml MDP, cytokine

production was reduced by 72 and 65%, respectively, for IL-6 and http://www.jimmunol.org/ TNF-a during PHT1 ablation. Additional studies were then performed with BAF, a specific inhibitor of vacuolar H+-ATPase (V-ATPase), to test whether a perturbation in the inside-acidic environment of endolysosomes would affect cytokine production. As shown in Fig. 5B, the production of IL-6 was substan- tially reduced in wild-type BMDMs when stimulated by LPS + MDPpretreatedwithBAF.ThisfindingsuggeststhatPHT1is expressed in the endolysosomes of macrophages, where a reduced proton gradient can attenuate the efflux of MDP from by guest on September 26, 2021 within the organelle into the cytosol and, thereby, reduce the production of cytokines. This aspect was further investigated in the subsequent two studies. FIGURE 2. Protein expression of PEPT2 in cell subtypes of mouse bone MDP-rhodamine uptake in BMDMs from wild-type and Pht1 A 2 marrow. PEPT2 protein was examined in ( ) Lin (includes hematopoietic knockout mice and mesenchymal stem cells and their progenitors) and Lin+ (includes T and B cells, macrophages, monocytes, granulocytes, erythrocytes, and their The previous inhibition study with BAF suggested that MDP might committed precursors) preparations and (B) residential macrophages and be effluxed from the endosomes of macrophages via PHT1. As a BMDMs isolated from wild-type mice. The positive control was wild-type result, MDP-rhodamine was used as a molecular probe to image the mouse kidney for (A) and bone marrow for (B) (which was validated in uptake of MDP in BMDMs from wild-type and Pht1 knockout Fig. 1F). Quantification of protein (i.e., transporter/b-actin ratio) is shown mice. As shown in Fig. 5C, an intense red fluorescent signal was below each figure. One representative example is shown of three individual observed in both BMDMs from wild-type and Pht1 knockout immunoblots. mice. However, more intense fluorescent granular particles were observed in the BMDMs of Pht1 knockout mice (i.e., 3-fold image the uptake of MDP in BMDMs from wild-type and Pept2 difference, as displayed in right-hand panel). This punctate knockout mice. As shown in Fig. 3B, a strong and extensive red pattern indicates that MDP-rhodamine is being accumulated in fluorescence was observed in BMDMs from wild-type mice, but some particular vesicle and even more so when the efflux of barely in BMDMs from Pept2 knockout mice (i.e., 4-fold differ- MDP-rhodamine from endosomes is being abrogated by PHT1 ence, as observed in right-hand panel). This result suggests that ablation. PEPT2 facilitates the uptake of MDP (and probably other bacterial L-Histidine uptake in endosomal preparations from wild-type peptide products) across the plasma membrane of macrophages. and Pht1 knockout mice and HEK293 studies Cytokine expression in BMDMs from wild-type and Pept2 Because PHT1 was considered to be expressed predominately on knockout mice after stimulation with LPS (plus MDP, MDP the membrane of endosomes (and as suggested by our previous control, or Tri-DAP) results), it was decided to enrich endosomes from the liver of IL-6 and TNF-a are two proinflammatory cytokines that can be wild-type and Pht1 knockout mice and to then study the PHT1- stimulated by LPS and be further enhanced by the entry of bac- mediated uptake of L-histidine (6potential inhibitors such as terially derived peptides in BMDMs. Thus, 10 mg/ml MDP, MDP and BAF). As shown in Fig. 5D, the uptake of L-histidine in MDP control, or Tri-DAP was added to the cell culture media of endosomes prepared from wild-type mice was only 15% of the 1–10 ng/ml LPS-stimulated BMDMs from wild-type and Pept2 value observed in Pht1 knockout mice. Moreover, the presence of 6 ROLE OF SLC15A IN NOD-MEDIATED IMMUNE RESPONSE IN BMDMs Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. GlySar and MDP-rhodamine uptake studies in BMDMs of wild-type and Pept2 knockout mouse. (A) Effect of potential inhibitors on 3 theuptakeof1mM[ H]GlySar in which GlyPro or glycine were present at 5 mM and MDP (L,D-isomer), MDP control (L,L-isomer), iE-DAP, or Tri- DAP were present at 1 mM. GlySar uptakes were corrected for nonspeciﬁc binding using [14C]mannitol and represented as percentage of control, relative to wild-type mice. Data are expressed as mean 6 SE (n = 6) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey test. (B) Uptake of MDP-rhodamine in wild-type and Pept2 knockout mice. MDP-rhodamine (red) was marked by arrows, and the nuclei (blue) were stained by DAPI (original magniﬁcation 340). Density measurements are shown in the right-hand panel (LabWorks Image Acquisition and Analysis Software v4.5; UVP, Upland, CA) and expressed as mean 6 SE (n = 10 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t test. ***p # 0.001.

MDP or BAF resulted in a 3-fold increase in L-histidine uptake in mock cells. In contrast, PEPT2 protein was not observed in mouse endosomes from wild-type mice. However, in Pht1 knockout liver endosomes (Fig. 5F). mice, no difference was observed in the uptake of L-histidine Thus, the concerted effort by PEPT2 and PHT1 optimizes the across all three treatments (i.e., L-histidine alone and in the presence of NOD ligands in the cytosol milieu and the activation of presence of MDP or BAF). This finding suggests that PHT1 is cytokine production, as schematically shown in Fig. 6. expressed and functionally active in the endosomes of native mouse tissue (in this case, the liver). Discussion This finding was supported using an anti-GFP Ab to probe There is increasing evidence that POTs play an important role in innate endosomes enriched from HEK293 cells transfected with pcDNA3.1- immunity and regulation by facilitating NOD-like receptor signaling mPhT1/CT-GFP plasmid DNA (or blank pcDNA-CT-GFP plasmid pathways and the downstream production of proinflammatory cyto- DNA). As shown in Fig. 5E, the immunoblot analysis showed a kines. However, the (sub)cellular expression, functional activity, and 90-kDa band (i.e., 27 kDa for GFP plus 63 kDa for mPHT1) in the relevance of POT family members are controversial, especially with endosomes of Pht1-transfected cells but not in the endosomes of respect to immune cells such as macrophages. This is unfortunate The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Effect of PEPT2 on the expression of IL-6 and TNF-a in BMDMs of wild-type and Pept2 knockout mice. BMDMs were treated for 24 h with 0–10 ng/ml LPS plus 10 mg/ml MDP [L,D-isomer (A and B)], Tri-DAP (C and D), or 10 ng/ml LPS plus 10 mg/ml MDP control [L,L-isomer (E and F)], after which cytokine concentrations in the cell culture media were measured. Data are expressed as mean 6 SE (n = 4) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t test. *p # 0.05, **p # 0.01, ***p # 0.001. because several POT family members have been associated with In particular, we found the following: 1) PEPT2 protein was diabetes (42), IBD (22, 29), and systemic lupus erythematosus (43). highly expressed in mouse bone marrow, Lin+, resident macro- With the use of novel animal models, along with biochemical, cel- phages, and BMDMs, whereas PEPT1 protein was absent from lular, and molecular approaches, several major findings were revealed mouse bone marrow; 2) GlySar uptake in wild-type BMDMs was in this article by studying macrophages derived from the bone mar- significantly reduced by MDP, iE-DAP, or Tri-DAP, as was the row of wild-type, Pept2 knockout, and Pht1 knockout mice. fluorescence imaging of MDP-rhodamine in Pept2 knockout mice 8 ROLE OF SLC15A IN NOD-MEDIATED IMMUNE RESPONSE IN BMDMs Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Effect of PHT1 on the LPS-MDP–stimulated immune response and uptake studies of MDP or L-histidine. (A) BMDMs from wild-type and Pht1 knockout mice were treated for 24 h with 5 ng/ml LPS plus 10 mg/ml MDP, after which IL-6 and TNF-a concentrations in the cell culture media were measured. Data are expressed as mean 6 SE (n = 6) in which each experiment was performed in triplicate. Statistical differences between the two genotypes were determined by an unpaired (two-sample) t test. (B) BMDMs from wild-type mice were treated for 24 h with 5 ng/ml LPS alone and in the presence of 1 mM BAF, 10 mg/ml MDP, or 10 mg/ml MDP plus 1 mM BAF. BMDMs were pretreated with BAF for 30 min prior to being coincubated with MDP and/or LPS. IL-6 concentrations in the cell culture media were then measured. Data are expressed as mean 6 SE (n = 3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey test. (C) Uptake of MDP-rhodamine in BMDMs prepared from wild-type and Pht1 knockout mice. MDP-rhodamine (red) was marked by arrows and the nuclei (blue) were stained by DAPI (original magnification 3100). Scale bars, 10 mm. Particle density is shown in the right-hand panel and expressed as mean 6 SE (n = 6–8 cells). Statistical differences between the two genotypes were determined by an unpaired (two-sample) t test. (D) Effect of potential inhibitors on the uptake of 1 mM[3H]histidine into endosome-enriched liver preparations from wild-type and Pht1 knockout mice. Endosomes were pretreated with MDP (1 mM) or BAF (1 mM) for 30 min prior to experimentation. Data are expressed as mean 6 SE (n = 3) in which each experiment was performed in triplicate. Treatment groups with the same letter were not statistically different, as determined by ANOVA and Tukey test. (E) Fusion protein of GFP-mPHT1 (90 kDa) was detected with an anti-GFP mAb in the endosome of pcDNA3.1-mPht1/CT-GFP transfected HEK293 cells but not in pcDNA3.1-CT-GFP transfected HEK293 (mock) cells. (F) Expression of PEPT2 protein in mouse liver endosomes. One representative example is shown of three individual immunoblots. ***p # 0.001. as compared with wild-type animals; 3) the LPS plus MDP (or inhibition studies, along with MDP–rhodamine fluorescence im- Tri-DAP)–stimulated expression of IL-6 and TNF-a was signifi- aging of BMDMs from wild-type and Pht1 knockout mice, sug- cantly reduced in BMDM cell media during Pept2 ablation gested an endosomal expression of this transporter; and 5) (a similar finding was observed during Pht1 ablation); 4) BAF L-histidine uptake and MDP–rhodamine imaging studies, along The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ FIGURE 6. Schematic depicting the PEPT2- and PHT1-mediated uptake of bacterially derived products (e.g., MDP and Tri-DAP) into the cytosol of macrophages and their effect on NOD signaling and downstream production of proinflammatory cytokines. with immunoblot analyses, confirmed the functionality and pro- transporters of MDP in fly and human phagocytes (23). Based on tein expression of PHT1 in endosomes. Collectively, our results the high levels of PEPT2 expression in human THP-1 monocyte- point to PEPT2 being expressed on the plasma membrane of derived cells, the ability of PEPT2 to enhance NOD2-dependent macrophages, where it can facilitate the cellular uptake of bac- NF-kB activation, and the association of PEPT2 with phagosome terially derived chemotactic peptides. For engulfed bacteria (and membranes, these authors suggested PEPT2 as a phagosome- by guest on September 26, 2021 their degradation products), peptidoglycans can be effluxed from associated transporter of MDP in human phagocytes. However, endosomes into the cytosol of macrophages by PHT1 (see Fig. 6). the uptake of MDP was not tested directly but instead indirectly The expression and functional relevance of PEPT1 in immune by the MDP-stimulated enhanced production of NF-kBby cells are controversial. Previous studies reported that PEPT1 was PEPT2-transfected HEK293T cells. Because other peptide trans- expressed in immune cells, thereby regulating the secretion of porters were not evaluated, such as PHT1 and PHT2, it remains proinflammatory cytokines triggered by bacteria (and/or bacterial unclear as to the role of peptide/histidine transporters relative products) and the induction of colitis (31, 32). However, in one to PEPT2 in the release of MDP from phagolysosomes into the study (31), immunoblot analyses and immunofluorescence stain- cytosol. ing of PEPT1 expression were performed on human macrophages Our results with PEPT2 corroborate previous findings (23), isolated from peripheral blood and, in the other study (32), colonic suggesting that PEPT2 has an important role in immune cell immune cells from mice were analyzed for Pept1 gene expression regulation via NOD signaling pathways and triggered cytokine and not protein. Thus, it is uncertain as to whether PEPT1 protein production. In this regard, GlySar uptake studies demonstrate that would be found in the colon of native tissue, especially in colonic MDP and Tri-DAP can inhibit dipeptide uptake in the BMDMs of immune cells. Recently, we reported that PEPT1 protein was wild-type but not Pept2 knockout mice (Fig. 3A). Additional absent from the colonic tissue and immune cells of normal mice support is shown by the reduced fluorescence of MDP-rhodamine and DSS-treated mice (26). Our current study confirms this finding (Fig. 3B), along with cytokine production, in BMDMs during in that neither PEPT1 transcripts nor protein were observed in Pept2 ablation (Fig. 4). Based on these data alone, we are unable mouse bone marrow (Fig. 1). However, PEPT2 gene and protein to discern whether PEPT2 is operating as a transporter of MDP or expression were clearly demonstrated in mouse bone marrow, as a receptor for subsequent endocytic accumulation in the cell, Lin+, and residential macrophages (Fig. 2). Moreover, the uptake perhaps via phagosomes as suggested before (23). However, it of MDP–rhodamine was distinct in BMDMs of wild-type but not more likely that PEPT2 is operating as a transporter of MDP Pept2 knockout mice (Fig. 3B). This latter finding can now ex- because immunoblot analyses failed to detect this protein in plain why, in a previous study (33), we found that PEPT1 was not endosomes (Fig. 5F). required for fluorescent-labeled MDP uptake or MDP-induced PHT1andPHT2havebeendifficulttostudybecauseoftheir signaling in mouse macrophages. intracellular expression, which is localized to endosomal/ There is a paucity of data on how other POTs, such as PEPT2, lysosomal membranes. Although initially cloned from rat brain might transport MDP into the cytosol of cells where, as a NOD (7, 8), a growing body of evidence has shown that PHT1 and ligand, it can activate NF-kB and other immune effector pathways. PHT2 are important modulators of immune responses, primarily Relying upon data from Drosophila phagosomes, an evolutionarily because of their ability to deliver NOD ligands from outside the conserved role was identified for Yin and PEPT2 as respective cell to cytosolic receptors. For example, Lee et al. (22) reported 10 ROLE OF SLC15A IN NOD-MEDIATED IMMUNE RESPONSE IN BMDMs that PHT1 was expressed in the early endosomes of transfected 11. Zhang, Y., J. Sun, Y. Sun, Y. Wang, and Z. He. 2013. Prodrug design targeting HEK293T cells and that NOD-activating ligands most likely en- intestinal PepT1 for improved oral absorption: design and performance. Curr. Drug Metab. 14: 675–687. tered the cells by a clathrin-coated pit pathway. Sasawatari et al. 12. Punitha, A. D., and A. K. Srivastava. 2013. CNS drug targeting: have we trav- (36), in the mouse leukemic monocyte/macrophage RAW264.7 elled in right path? J. Drug Target. 21: 787–800. cell line, found that FLAG-tagged PHT1 was localized to intra- 13. Kufer, T. A., D. J. Banks, and D. J. Philpott. 2006. Innate immune sensing of microbes by Nod proteins. Ann. N. Y. Acad. Sci. 1072: 19–27. cellular vesicular compartments and associated with a lysosomal 14. Chen, G., M. H. Shaw, Y. G. Kim, and G. Nun˜ez. 2009. NOD-like receptors: role membrane marker protein. These authors also observed that cy- in innate immunity and inflammatory disease. Annu. Rev. Pathol. 4: 365–398. 15. Strober, W., P. J. Murray, A. Kitani, and T. Watanabe. 2006. Signalling pathways tokine production was reduced in bone marrow dendritic cells of and molecular interactions of NOD1 and NOD2. Nat. Rev. Immunol. 6: 9–20. Pht1 knockout mice as compared with wild-type animals and that 16. Kim, Y.-G., J. H. Park, M. H. Shaw, L. Franchi, N. Inohara, and G. Nuń˜ez. 2008. these Pht1-deficient mice were more resistant to DSS-induced The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity 28: 246–257. colitis. Finally, Nakamura et al. (5) demonstrated that GFP- 17. Caruso, R., N. Warner, N. Inohara, and G. Nuń˜ez. 2014. NOD1 and NOD2: labeled PHT1 and PHT2 were localized to the endosomes, lyso- signaling, host defense, and inflammatory disease. Immunity 41: 898–908. somes, and endolysosomal tubules of bone marrow dendritic cells 18. Kagan, J. C., T. Su, T. Horng, A. Chow, S. Akira, and R. Medzhitov. 2008. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon- from mice. However, MDP and Tri-DAP uptake was not directly b. Nat. Immunol. 9: 361–368. studied by these authors (5, 22, 36) but only inferred by examining 19. Zanoni, I., R. Ostuni, L. R. Marek, S. Barresi, R. Barbalat, G. M. Barton, the effect of these bacterial-derived substrates on cytokine pro- F. Granucci, and J. C. Kagan. 2011. CD14 controls the LPS-induced endocytosis of Toll-like receptor 4. Cell 147: 868–880. duction. Moreover, PHT1 and/or PHT2 expression was evaluated 20. Kobayashi, T., T. Tanaka, and N. Toyama-Sorimachi. 2013. How do cells opti- in cell lines (22, 36) or dendritic cells (5) and not in macrophages mize luminal environments of endosomes/lysosomes for efficient inflammatory from native tissue. Our results provide definitive proof that PHT1 responses? J. Biochem. 154: 491–499. Downloaded from 21. Philpott, D. J., M. T. Sorbara, S. J. Robertson, K. Croitoru, and S. E. Girardin. was expressed (Fig. 5C, 5F) and functionally active (Fig. 5D) in 2014. NOD proteins: regulators of inflammation in health and disease. [Pub- mouse endosomes as well as in the endosomes of transfected lished erratum appears in 2014 Nat. Rev. Immunol. 14: 131.] Nat. Rev. Immunol. HEK293 cells (Fig. 5E). The finding that PHT1 is expressed and/ 14: 9–23. 22. Lee, J., I. Tattoli, K. A. Wojtal, S. R. Vavricka, D. J. Philpott, and S. E. Girardin. or functionally active in mouse macrophages and endosomes does 2009. pH-dependent internalization of muramyl peptides from early endosomes not rule out the presence of PHT2 in these cells. However, an enables Nod1 and Nod2 signaling. J. Biol. Chem. 284: 23818–23829.

endosomal presence of PHT2 seems unlikely because one would 23. Charrie`re, G. M., W. E. Ip, S. Dejardin, L. Boyer, A. Sokolovska, http://www.jimmunol.org/ M. P. Cappillino, B. J. Cherayil, D. K. Podolsky, K. S. Kobayashi, N. Silverman, expect the uptake of L-His in Pht1 knockout mice to be increased et al. 2010. Identification of Drosophila Yin and PEPT2 as evolutionarily con- when in the presence of MDP or BAF, which does not occur served phagosome-associated muramyl dipeptide transporters. J. Biol. Chem. (Fig. 5D). 285: 20147–20154. In conclusion, our findings provide a novel paradigm in which 24. Dalmasso, G., H. T. T. Nguyen, L. Charrier-Hisamuddin, Y. Yan, H. Laroui, B. Demoulin, S. V. Sitaraman, and D. Merlin. 2010. PepT1 mediates transport of the cytosolic exposure of MDP, Tri-DAP, and perhaps other pep- the proinflammatory bacterial tripeptide L-Ala-g-D-Glu-meso-DAP in intestinal tidoglycans is optimized by the expression of PEPT2 at plasma epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 299: G687–G696. membranes and PHT1 at endosomal membranes. These findings 25. Song, F., Y. Hu, Y. Wang, D. E. Smith, and H. Jiang. 2018. Functional characterization of human peptide/histidine transporter 1 in stably transfected MDCK offer the potential for new drug development strategies along with cells. Mol. Pharm. 15: 385–393. transporter-targeted therapies for a range of endocrine, inflam- 26. Wang, Y., Y. Hu, P. Li, Y. Weng, N. Kamada, H. Jiang, and D. E. Smith. 2018. by guest on September 26, 2021 matory, and autoimmune diseases. Expression and regulation of proton-coupled oligopeptide transporters in colonic tissue and immune cells of mice. Biochem. Pharmacol. 148: 163–173. 27. Wu, S. P., and D. E. Smith. 2013. Impact of intestinal PepT1 on the kinetics and Disclosures dynamics of N-formyl-methionyl-leucyl-phenylalanine, a bacterially-produced chemotactic peptide. Mol. Pharm. 10: 677–684. The authors have no financial conflicts of interest. 28. Merlin, D., M. Si-Tahar, S. V. Sitaraman, K. Eastburn, I. Williams, X. Liu, M. A. Hediger, and J. L. Madara. 2001. Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: transport of bacterial peptides influences References expression of MHC class 1 molecules. Gastroenterology 120: 1666–1679. 1. Smith, D. E., B. Cle´mençon, and M. A. Hediger. 2013. Proton-coupled oligo- 29. Dalmasso, G., H. T. Nguyen, S. A. Ingersoll, S. Ayyadurai, H. Laroui, peptide transporter family SLC15: physiological, pharmacological and patho- M. A. Charania, Y. Yan, S. V. Sitaraman, and D. Merlin. 2011. The PepT1-NOD2 logical implications. Mol. Aspects Med. 34: 323–336. signaling pathway aggravates induced colitis in mice. Gastroenterology 141: 2. Shen, H., D. E. Smith, and F. C. Brosius, III. 2001. Developmental expression of 1334–1345. 30. Wuensch, T., S. Ullrich, S. Schulz, M. Chamaillard, N. Schaltenberg, E. Rath, PEPT1 and PEPT2 in rat small intestine, colon, and kidney. Pediatr. Res. 49: U. Goebel, R. B. Sartor, M. Prager, C. Bu¨ning, et al. 2014. Colonic expression of 789–795. the peptide transporter PEPT1 is downregulated during intestinal inflammation 3. Shen, H., D. E. Smith, T. Yang, Y. G. Huang, J. B. Schnermann, and and is not required for NOD2-dependent immune activation. Inflamm. Bowel F. C. Brosius, III. 1999. Localization of PEPT1 and PEPT2 proton-coupled Dis. 20: 671–684. oligopeptide transporter mRNA and protein in rat kidney. Am. J. Physiol. 276: 31. Charrier, L., A. Driss, Y. Yan, V. Nduati, J. M. Klapproth, S. V. Sitaraman, and F658–F665. D. Merlin. 2006. hPepT1 mediates bacterial tripeptide fMLP uptake in human 4. Teuscher, N. S., A. Novotny, R. F. Keep, and D. E. Smith. 2000. Functional monocytes. Lab. Invest. 86: 490–503. evidence for presence of PEPT2 in rat choroid plexus: studies with gly- 32. Ayyadurai, S., M. A. Charania, B. Xiao, E. Viennois, and D. Merlin. 2013. cylsarcosine. J. Pharmacol. Exp. Ther. 294: 494–499. PepT1 expressed in immune cells has an important role in promoting the im- 5. Nakamura, N., J. R. Lill, Q. Phung, Z. Jiang, C. Bakalarski, A. de Mazie`re, mune response during experimentally induced colitis. Lab. Invest. 93: 888–899. J. Klumperman, M. Schlatter, L. Delamarre, and I. Mellman. 2014. Endosomes 33. Marina-Garcıá, N., L. Franchi, Y. G. Kim, Y. Hu, D. E. Smith, G. J. Boons, and are specialized platforms for bacterial sensing and NOD2 signalling. Nature 509: G. Nuń˜ez. 2009. Clathrin- and dynamin-dependent endocytic pathway regulates 240–244. muramyl dipeptide internalization and NOD2 activation. J. Immunol. 182: 4321– 6. Bissa, B., A. M. Beedle, and R. Govindarajan. 2016. Lysosomal solute carrier 4327. transporters gain momentum in research. Clin. Pharmacol. Ther. 100: 431–436. 34. Sun, D., Y. Wang, F. Tan, D. Fang, Y. Hu, D. E. Smith, and H. Jiang. 2013. 7. Yamashita, T., S. Shimada, W. Guo, K. Sato, E. Kohmura, T. Hayakawa, Functional and molecular expression of the proton-coupled oligopeptide trans- T. Takagi, and M. Tohyama. 1997. Cloning and functional expression of a brain porters in spleen and macrophages from mouse and human. Mol. Pharm. 10: peptide/histidine transporter. J. Biol. Chem. 272: 10205–10211. 1409–1416. 8. Sakata, K., T. Yamashita, M. Maeda, Y. Moriyama, S. Shimada, and 35. Shen, H., D. E. Smith, R. F. Keep, J. Xiang, and F. C. Brosius, III. 2003. Targeted M. Tohyama. 2001. Cloning of a lymphatic peptide/histidine transporter. Bio- disruption of the PEPT2 gene markedly reduces dipeptide uptake in choroid chem. J. 356: 53–60. plexus. J. Biol. Chem. 278: 4786–4791. 9. Gong, Y., X. Wu, T. Wang, J. Zhao, X. Liu, Z. Yao, Q. Zhang, and X. Jian. 2017. 36.Sasawatari,S.,T.Okamura,E.Kasumi,K.Tanaka-Furuyama,R.Yanobu- Targeting PEPT1: a novel strategy to improve the antitumor efficacy of doxo- Takanashi, S. Shirasawa, N. Kato, and N. Toyama-Sorimachi. 2011. The rubicin in human hepatocellular carcinoma therapy. Oncotarget 8: 40454–40468. solute carrier family 15A4 regulates TLR9 and NOD1 functions in the in- 10. Tashima, T. 2015. Intriguing possibilities and beneficial aspects of transporter- nate immune system and promotes colitis in mice. Gastroenterology 140: conscious drug design. Bioorg. Med. Chem. 23: 4119–4131. 1513–1525. The Journal of Immunology 11

37. Hu, Y., D. E. Smith, K. Ma, D. Jappar, W. Thomas, and K. M. Hillgren. 2008. 41. Hu, Y., Y. Xie, Y. Wang, X. Chen, and D. E. Smith. 2014. Development and Targeted disruption of peptide transporter Pept1 gene in mice signiﬁcantly re- characterization of a novel mouse line humanized for the intestinal peptide duces dipeptide absorption in intestine. Mol. Pharm. 5: 1122–1130. transporter PEPT1. Mol. Pharm. 11: 3737–3746. 38. Weischenfeldt, J., and B. Porse. 2008. Bone marrow-derived macrophages 42. Takeuchi, F., Y. Ochiai, M. Serizawa, K. Yanai, N. Kuzuya, H. Kajio, S. Honjo, (BMM): isolation and applications. CSH Protoc. 2008: pdb.prot5080. N. Takeda, Y. Kaburagi, K. Yasuda, et al. 2008. Search for type 2 diabetes 39. Ying, W., P. S. Cheruku, F. W. Bazer, S. H. Safe, and B. Zhou. 2013. Investi- susceptibility genes on chromosomes 1q, 3q and 12q. J. Hum. Genet. 53: gation of macrophage polarization using bone marrow derived macrophages. J. 314–324. Vis. Exp. 76: e50323. 43. He, C. F., Y. S. Liu, Y. L. Cheng, J. P. Gao, T. M. Pan, J. W. Han, C. Quan, 40. Hu, Y., Y. Xie, R. F. Keep, and D. E. Smith. 2014. Divergent developmental L. D. Sun, H. F. Zheng, X. B. Zuo, et al. 2010. TNIP1, SLC15A4, ETS1, expression and function of the proton-coupled oligopeptide transporters PepT2 RasGRP3 and IKZF1 are associated with clinical features of systemic lupus and PhT1 in regional brain slices of mouse and rat. J. Neurochem. 129: 955–965. erythematosus in a Chinese Han population. Lupus 19: 1181–1186. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

Fusion Protein Vector-GFP A B mPHT1-GFP

FIGURE S1. Fluorescence microscopy of mPht1-transfected HEK293 cells. (A) mock cells were transfected by the vector pcDNA3.1-CT-GFP and (B) mPht1-containing cells were transfected by the vector pcDNA3.1-mPht1/CT-GFP (40x magnification).