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Vitamin D-dependent cathelicidin inhibits Mycobacterium marinum infection in human monocytic cells

      Abstract

      Background

      1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) up-regulates the production of human cathelicidin antimicrobial peptide (CAMP) from monocytes/macrophages infected with Mycobacterium tuberculosis (M. tbc). CAMP facilitates the co-localization of autophagolysosomes with M. tbc, promoting the antimicrobial activity of monocytes. Mycobacterium marinum (M. marinum) is an acid-fast bacillus that causes less severe granulomatous skin lesions compared with M. tbc.

      Objective

      We investigated whether autophagic antimicrobial activity is promoted by 1,25(OH)2D3 or C-terminal of cathelicidin LL-37 in human monocytes upon infection with M. marinum.

      Methods

      Human monocytes (THP-1) were infected with M. marinum. Effects of simultaneous treatments of 1,25(OH)2D3, exogenous LL-37 peptide, autophagolysosome inhibitors, 3-methyladenine or chloroquine, were examined.

      Results

      CAMP was strongly induced by adding 1,25(OH)2D3 to the culture of THP-1 cells. In the absence of 1,25(OH)2D3 M. marinum infection alone did not induce CAMP, however, simultaneous addition of 1,25(OH)2D3 to M. marinum infection accelerated CAMP production more than 1,25(OH)2D3 alone. Proliferation of M. marinum was markedly decreased in the presence of 1,25(OH)2D3 or exogenous LL-37 in THP-1 cells. Co-localization of CAMP with autophagolysosome was evident in 1,25(OH)2D3 and LL-37 treated THP-1 cells after M. marinum infection. Autophagolysosome inhibitors abrogated the antimicrobial effects of 1,25(OH)2D3 and exogenous LL-37 against M. marinum infection in THP-1 cells.

      Conclusions

      Human monocytic cells, whose CAMP production is up-regulated by 1,25(OH)2D3-vitamin D receptor pathway, accelerate antimicrobial function of autophagolysosome in M. marinum infection.

      Abbreviations:

      CAMP (cathelicidin antimicrobial peptide), VDR (vitamin D receptor), 1,25(OH)2D3 (1α,25-dihydroxyvitamin D3), ATG (autophagy-related genes), CQ (chloroquine), 3-MA (3-methyladenine)

      Keywords

      1. Introduction

      The human cathelicidin antimicrobial peptide (CAMP) has been shown to exhibit broad-spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacterial species. CAMP gene is a direct target of the vitamin D receptor (VDR) and is strongly up-regulated in various cells such as neutrophils, macrophages and epithelial cells by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), an active form of vitamin D [
      • Wang T.T.
      • Nestel F.P.
      • Bourdeau V.
      • Nagai Y.
      • Wang Q.
      • Liao J.
      • et al.
      Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.
      ,
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3.
      ]. CAMP becomes its mature extracellular form of 37 amino acid peptide, LL-37, after truncation by protease, and executes its antimicrobial activity directly against invading pathogens.
      It has been shown that CAMP plays intracellular roles in addition to direct antimicrobial activity against pathogens. Jo et al. have recently shown that 1,25(OH)2D3 can up-regulate the production of CAMP from monocytes/macrophages infected with Mycobacterium tuberculosis (M. tbc), and CAMP induces autophagy and facilitates the co-localization of autolysosomes with M. tbc to promote the antimicrobial activity of monocytes [
      • Yuk J.M.
      • Shin D.M.
      • Lee H.M.
      • Yang C.S.
      • Jin H.S.
      • Kim K.K.
      • et al.
      Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.
      ]. Autophagy is a generic term for all pathways by which cytoplasmic materials are delivered to the lysosome in animal cells or the vacuole in plant and yeast cells [
      • Mizushima N.
      • Komatsu M.
      Autophagy: renovation of cells and tissues.
      ,
      • Levine B.
      • Mizushima N.
      • Virgin H.W.
      Autophagy in immunity and inflammation.
      ]. Autophagy occurs when an autophagosome (a double-membrane vacuole) containing cytoplasmic material, fuses with a lysosome to deliver sequestered material for lysosomal degradation [
      • Virgin H.W.
      • Levine B.
      Autophagy genes in immunity.
      ], and multiple autophagy-related genes (ATG) proteins govern autophagosome formation [
      • Nakatogawa H.
      • Suzuki K.
      • Kamada Y.
      • Ohsumi Y.
      Dynamics and diversity in autophagy mechanisms: lessons from yeast.
      ]. Growing evidence suggests that autophagy contributes to the intracellular killing of M. tbc by facilitating phagolysosome fusion and thereby providing a mechanism to counteract the ability of M. tbc to evade the host response [
      • Fabri M.
      • Modlin R.L.
      A vitamin for autophagy.
      ,
      • Gutierrez M.G.
      • Master S.S.
      • Singh S.B.
      • Taylor G.A.
      • Colombo M.I.
      • Deretic V.
      Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages.
      ,
      • Singh S.B.
      • Davis A.S.
      • Taylor G.A.
      • Deretic V.
      Human IRGM induces autophagy to eliminate intracellular mycobacteria.
      ].
      Mycobacterium marinum (M. marinum) is a nontuberculous photochromogenic mycobacterium species belonging to group I of Runyon's classification [
      • Jarzembowski J.A.
      • Young M.B.
      Nontuberculous mycobacterial infections.
      ], and causes granulomatous skin lesions. The cutaneous infection caused by M. marinum, which mostly affects those who own aquariums or are in contact with fish, is the most common. The lesion, usually at the hand or forearm, is initially nodular but may subsequently ulcerate, whereas the sporotrichoid form is characterized by small nodules along lymphatic ducts [
      • Tortoli E.
      Clinical manifestations of nontuberculous mycobacteria infections.
      ]. M. marinum has been increasingly studied as a model of M. tbc due to its relative safety and its shared mechanisms of pathogenesis [
      • Stamm L.M.
      • Brown E.J.
      Mycobacterium marinum: the generalization and specialization of a pathogenic mycobacterium.
      ,
      • Tobin D.M.
      • Ramakrishnan L.
      Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis.
      ,
      • Collins C.A.
      • De Maziere A.
      • van Dijk S.
      • Carlsson F.
      • Klumperman J.
      • Brown E.J.
      Atg5-independent sequestration of ubiquitinated mycobacteria.
      ]. However it still remains to be determined whether 1,25(OH)2D3 and/or LL-37 can augment antimicrobial activity against M. marinum infection like against M. tbc infection as recently shown. Here we show that antimicrobial activity is promoted by autophagy via 1,25(OH)2D3 or LL-37 in human monocytes infected with M. marinum.

      2. Materials and methods

      2.1 Cells and reagents

      THP-1 cells and U937 cells were purchased (American Type Culture Collection) and maintained in RPMI 1640 (Wako, Osaka, Japan) with 10% FBS (GIBCO, Carlsbad, CA, USA). Autophagy-related gene 5 deficient (ATG5−/−) MEF cells were obtained from Riken BioResource Center, Cell Bank (Ibaraki, Japan) and maintained in DMEM with 10% FBS [
      • Kuma A.
      • Hatano M.
      • Matsui M.
      • Yamamoto A.
      • Nakaya H.
      • Yoshimori T.
      • et al.
      The role of autophagy during the early neonatal starvation period.
      ]. 1,25(OH)2D3 (BIOMOL International) was added to the culture at 10 nM and synthetic LL-37 peptide (Innovagen, Lund, Sweden) was added at 10 μg/ml. Autophagy antagonists 3-methyladenine (3-MA) and chloroquine (CQ) (Sigma–Aldrich, St Louis, MO, USA) were added to the culture at 1 μg/ml and 5 μM, respectively.

      2.2 Pathogens and colony-forming unit (CFU) assay

      M. marinum strain NJB0419 was obtained from Japan Anti-Tuberculosis Association (Tokyo, Japan) and cells were infected with M. marinum at multiplicity of infection (MOI) of 1–10. The efficiency of infection was determined by Ziehl-Neelsen staining. Infected cells were treated with 1,25(OH)2D3 or LL37 in triplicate wells, and then collected between 1 and 3 days. Cells were centrifuged at 2000 rpm for 5 min to form a pellet and then the supernatant was aspirated. Intracellular M. marinum were obtained by lysing the cells with 0.5% Triton X-100 in PBS. M. marinum isolates were 10-fold serially diluted and plated on Middlebrook 7H10 (BD Bioscience, San Jose, CA, USA) agar plates supplemented with 10% OADC Enrichment, then incubated at 30 °C for 10 days. Colonies formed were counted as CFU for quantification of the mycobacteria. M. marinum optical density (OD570) is measured by Model 680 Microplate Reader (Bio-Rad, CA, USA).

      2.3 Isolation of total RNA and quantitative RT-PCR

      Total RNA was prepared using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 1 μg of the total RNA using a SuperScript™ III First-Strand Synthesis System (Invitrogen). Quantitative real-time RT-PCR (qRT-PCR) was performed using SYBR Premix DimerEraser™ (TaKaRa, Siga, Japan) in a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The gene-specific primer sequences are shown in Table 1. β-Actin was amplified as an internal control each time qRT-PCR was performed, and the ΔΔCt method was employed to quantify the relative amounts of transcripts. The Student t-test was used for statistical analyses. A p value < 0.05 was considered statistically significant.
      Table 1Primer sequences of assayed genes.
      CAMPForward5′-GGA CCC AGA CAC GCC AAA-3′
      Reverse5′-GCA CAC TGT CTC CTT CAC TGT GA-3′
      IL-12p40Forward5′-CAG CTC GCA GCA AAG CAA-3′
      Reverse5′-GAC GCC ATT CCA CAT GTC ACT-3′
      TNF-αForward5′-TCT CGA ACC CCC GAG TGA CA-3′
      Reverse5′-GGC CCG GCG GTT CA-3′
      IFN-βForward5′-TGC TCT CCT GTT GTG CTT CTC C-3′
      Reverse5′-CAT CTC ATA GAT GGT CAA TGC GG-3′
      β-ActinForward5′-AAG GGA CTT CCT GTA ACA ATG CA-3′
      Reverse5′-CTG GAA CGG TGA AGG TGA CA-3′

      2.4 Enzyme-linked immunosorbent assay (ELISA)

      Interleukin (IL)-12/23 p40, interferon (IFN)-β or tumor necrosis factor (TNF)-α in the supernatants of treated cells was measured using commercially prepared ELISA plates according to the manufacturer's suggestion (IL-12/23 p40 and TNF-α, R&D Systems, Minneapolis, MN, USA; IFN-β, Peprotech, Rocky Hill, NJ, USA).

      2.5 Western blot

      2 × 105 cells were lysed and separated on 15% SDS–PAGE gels and transferred onto a polyvinylidene difluoride membrane as described previously [
      • Sato-Deguchi E.
      • Imafuku S.
      • Chou B.
      • Ishii K.
      • Hiromatsu K.
      • Nakayama J.
      Topical vitamin D3 analogues induce thymic stromal lymphopoietin and cathelicidin in psoriatic skin lesions.
      ]. Membranes were blocked in PBS containing 5% skim milk at room temperature for 1 h. The specific proteins were determined by incubation with specific antibodies against human β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VDR or LL-37 (1:200, Santa Cruz Biotechnology) at 25 °C for 1 h. After washing, the membrane was incubated in a 1:3000 dilution of a secondary antibody (sheep anti-mouse IgG-HRP conjugate; GE Healthcare, Little Chalfont, UK) at room temperature in the washing buffer (PBS containing 0.5% Tween 20) for 30 min. The protein bands were visualized using ECL Western Blotting Detection Reagents (GE Healthcare). Densitometry LAS-3000 software (FUJIFILM, Tokyo, Japan) was used to quantify the LL-37 protein levels, and the levels were normalized to β-actin.

      2.6 Immunofluorescent staining

      Cells were seeded onto glass cover slips in 24-well plates. Cells were fixed in 3.7% paraformaldehyde, incubated in 50 mM glycine for 5 min, permeabilized and blocked with 1.5% BSA for 30 min. Immunostaining was performed using polyclonal anti-LL-37 antibodies (1:1000, Innovagen) or monoclonal anti-LC3 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and LAMP-1 antibodies (1:200, Santa Cruz Biotechnology). Secondary Alexa Fluor 488 and 546-conjugated antibodies were obtained from Invitrogen and used by 1:2000. Cells were washed with PBS, and cover slips were mounted using ProLong® Gold antifade Reagent with DAPI (Invitrogen). Fluorescence images were acquired by BIOREVO BZ-9000 (Keyence, Japan). Presented are representative results observed in the majority of cells from several repeats.

      2.7 Lentiviral shRNA transduction to THP-1 cells

      VDR and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology. RPMI 1640 containing virus (MOI:1) and polybrene (8 μg/ml) was added to THP-1 cells, and seeded onto 24-well plates. The plate was centrifuged at 2000 rpm for 90 min, then RPMI 1640 was added and the cells were incubated at 37 °C overnight. The next day, cells were washed and infected with virus using the same protocol, and incubated for 24 h. Then, medium was changed and the cells were incubated for 72 h. Single colony isolation was performed by growth in 50% methylcellulose mixed-RPMI 1640 containing puromycin (4 μg/ml) for 4–5 weeks. The protocol for transduction into THP-1 cells was a gift from the Department of Virology, Kyushu University School of Medicine, Fukuoka, Japan.

      3. Results

      3.1 LL-37 does not directly inhibit the growth of M. marinum in extracellular culture

      First, we tested the direct antimicrobial activity of the externally added LL-37 (10 μg/ml) in M. marinum in vitro axenic culture in the absence of THP-1 cells. OD570, which reflects the concentration of M. marinum did not show any difference between cultures in the presence or absence of LL-37 and in fact there was no difference in CFU at 72 h, indicating that LL-37 does not directly inhibit the growth of M. marinum in culture medium (Fig. 1A and B ).
      Figure thumbnail gr1
      Fig. 1LL-37 did not directly kill the cultured M. marinum. (A) Growth curves of M. marinum in 7H9 broth medium containing LL-37 (10 μg/ml), or DMSO (0.1%). OD570 was measured at time indicated. Data points represent the mean of five separate experiments. (B) Comparison of CFU at 72 h after the addition of LL-37. M. marinum growth in 7H9 broth was not suppressed by LL-37 directly treatment (n = 3).

      3.2 M. marinum growth in THP-1 cells were suppressed by treatment with 1,25(OH)2D3 or LL-37

      Next, we investigated whether 1,25(OH)2D3 or extracellular LL-37 could suppress M. marinum growth in THP-1 cells. THP-1 cells were infected with M. marinum at MOI of 1 in the presence or absence of 1,25(OH)2D3 (10 nM) or LL-37 (10 μg/ml) and incubated for 24–72 h (Fig. 2A and B ). Intracellular M. marinum growth occurred vigorously in THP-1 cells in the absence of 1,25(OH)2D3 or LL-37. Addition of 1,25(OH)2D3 or LL-37 significantly suppressed M. marinum growth as confirmed by CFU (Fig. 2A). Similar results were obtained using other human monocyte cell line, U937 cells (data not shown). Morphological analysis (Ziehl-Neelsen staining) revealed that numerous live bacilli in the cytoplasm in THP-1 cells at 24 h after infection with M. marinum, while simultaneous treatment of 1,25(OH)2D3 or LL-37 caused marked degradation and decrement of bacilli inside the cells (Fig. 2B).
      Figure thumbnail gr2
      Fig. 2M. marinum growth in THP-1 was suppressed by treatment with 1,25(OH)2D3 or exogenous LL-37. (A) Comparison of CFU 72 h after infection of M. marinum (MOI: 1) in THP-1 cells. M. marinum growth in THP-1 was suppressed by 1,25(OH)2D3 or LL-37 treatment (n = 3, *,**p < 0.01). (B) THP-1 cells did not suppress intracellular M. marinum growth and live bacilli were observed in the cytoplasm. M. marinum bodies (seen in control cells in the leftmost panel) were degraded in the cells cultured with 1,25(OH)2D3 or LL-37 (Ziehl-Neelsen staining).

      3.3 Intracellular CAMP is increased in 1,25(OH)2D3-treated THP-1 cells

      We further investigated whether 1,25(OH)2D3 treatment could induce cathelicidin in THP-1 cells. Analyses by qRT-PCR showed that CAMP mRNA was strongly up regulated in uninfected THP-1 cells, and further up regulated in M. marinum-infected cells following the addition of 1,25(OH)2D3 (10 nM) (Fig. 3A ). In contrast, CAMP was not induced by M. marinum infection alone or M. marinum supplemented with exogenous LL-37. The presence of LL-37 was detected in the culture supernatant of the 1,25(OH)2D3-treated control culture, and was further increased when infected with M. marinum (Fig. 3B). Similar results were observed at 72 h after infection (data not shown). IFN-β, TNF-α and IL-12p40 mRNA expressions were strongly induced by M. marinum infection, and suppressed by simultaneous treatment with 1,25(OH)2D3 or exogenous LL-37 (Fig. 3A). Concentrations of IFN-β, TNF-α, IL-12p40 in the supernatant measured by ELISA showed comparable changes consistent with mRNA data (Fig. 3B). These results suggest an anti-inflammatory function of cathelicidin LL-37 peptide.
      Figure thumbnail gr3
      Fig. 3Intracellular CAMP is increased in 1,25(OH)2D3-treated THP-1 cells 24 h after M. marinum infection. (A) mRNA of cathelicidin is strongly up regulated by 1,25(OH)2D3 in uninfected THP-1 cells. M. marinum infection (MOI:1) in the presence of 1,25(OH)2D3 further promoted the induction of cathelicidin. IFN-β, TNF-α, and IL-12p40 induced by M. marinum infection was suppressed by the addition of 1,25(OH)2D3, or LL-37. (B) Proteins in culture supernatants were measured by Western blotting or ELISA. CAMP expression was measured by Western blotting and M. marinum infected THP-1 cells secreted higher levels of LL-37 in culture supernatant compared to controls. Concentrations of IFN-β, TNF-α, IL-12p40 in the supernatant was measured by ELISA and showed proportional changes consistent with the mRNA data.

      3.4 Autophagy is induced in 1,25(OH)2D3 and LL-37-treated THP-1 cells 24 h after M. marinum infection

      Intracellular CAMP (green) levels increased in 1,25(OH)2D3-treated THP-1 cells, and partly co-localized with LC3 protein (red) located on autophagosome membranes (Fig. 4A ). LC3 (green) and LAMP1 (red), the main glycoprotein in lysosomal membranes, were co-localized in 1,25(OH)2D3 or LL-37-treated THP-1 cells after M. marinum infection (Fig. 4B). These results suggest that autophagolysosome is induced in THP-1 cells by simultaneous treatment with 1,25(OH)2D3 or LL-37 upon infection with M. marinum.
      Figure thumbnail gr4
      Fig. 4Autophagy is induced in 1,25(OH)2D3 and LL-37-treated THP-1 cells 24 h after M. marinum infection. (A) Immunofluorescence staining 24 h after M. marinum infection. Intracellular CAMP (green) was increased in 1,25(OH)2D3-treated THP-1 cells, and partly co-localized with LC3 (red), a protein localized on the autophagosome membrane. (B) Immunofluorescence staining 24 h after M. marinum infection. LC3 (green) and LAMP1 (red), a main glycoprotein in the lysosomal membrane, were co-localized in 1,25(OH)2D3 or LL-37-treated THP-1 cells after M. marinum infection.

      3.5 Blocking VDR by shRNA suppresses intracellular killing of M. marinum but rescued by addition of external LL-37

      VDR-specific shRNA was designed to interfere with the transcription of VDR. Fig. 5A showed a marked reduction of CAMP (18 kDa) levels in VDR-shRNA treated cells in the presence of 10 nM 1,25(OH)2D3 compared to control-shRNA treated cells. A truncated form of CAMP (arrow head, 12 kDa) was abundant in control cells infected with M. marinum, but not in uninfected controls treated with 1,25(OH)2D3. VDR-knock down cells showed increased CFU of M. marinum (Fig. 5B) in the presence of 10 nM 1,25(OH)2D3, suggesting that 1,25(OH)2D3 signaling through VDR is required for the intracellular killing of M. marinum. We further investigated whether reduced antimicrobial activity against M. marinum in shVDR-THP-1 cells can be rescued by adding external LL-37. As expected, addition of LL-37 in shVDR-THP-1 cells recovered the killing activity against M. marinum (Fig. 5B).
      Figure thumbnail gr5
      Fig. 5Blocking VDR by shRNA suppressed intracellular killing of M. marinum. (A) Western blotting of THP-1 cell lysates treated with VDR-shRNA showed marked reduction of CAMP in cells in the presence of 10 nM 1,25(OH)2D3 compared to control-shRNA treated cells. In control cells infected with M. marinum (MOI:1), a truncated form of CAMP (arrow head) was abundantly compared with 1,25(OH)2D3-treated uninfected controls. (B) Cells treated with VDR-shRNA showed increased CFU upon infection with M. marinum (MOI:10) in the presence of 10 nM 1,25(OH)2D3 but rescued by external LL37 (n = 3, *,**p < 0.05).

      3.6 Autophagy antagonists or ATG5 deficiency could not suppress the growth of M. marinum, and addition of external LL-37 did not recover the bactericidal activity

      Next, we tested whether blocking autophagy using autophagy antagonists resulted in the suppression of intracellular killing of M. marinum. Growth of M. marinum in THP-1 cells was facilitated by two different autophagy antagonists, 3-MA or CQ (Fig. 6A ). And this loss of activity cannot be rescued by addition of external LL-37, suggesting that autophagy is required for LL-37 to work. We also assessed the protective ability of autophagy against M. marinum infection using cells from ATG5-deficient mice. Since the ATG5 deficiency in mice is lethal, ATG5−/− mouse embryonic fibroblasts (MEFs) were used. In ATG5−/− MEF, increased M. marinum growth was observed compared to wild type MEF (Fig. 6B).
      Figure thumbnail gr6
      Fig. 6Autophagy antagonists or ATG5 deficiency suppressed intracellular killing of M. marinum. (A) Comparison of CFU 72 h after infection of M. marinum (MOI:10) in THP-1 cells. M. marinum growth in THP-1 was significantly promoted by 3-MA or CQ and cannot be rescued by addition of external LL-37 (n = 3, *, **p < 0.01). (B) Comparison of CFU 72 h after infection of M. marinum (MOI:10) in mouse embryonic fibroblast (MEF) cells. M. marinum growth in ATG5−/− MEFs was significantly increased compared to controls (n = 3, *p < 0.05).

      4. Discussion

      In this study we demonstrated that 1,25(OH)2D3 treatment reduced M. marinum survival through elevated intracellular and extracellular levels of CAMP protein production in human monocytic cell line THP-1. Strikingly CAMP induction by 1,25(OH)2D3 was almost completely shut down by shRNA VDR knocked down in human monocytic cells line THP-1 and antimicrobial activity of 1,25(OH)2D3 was diminished by shRNA VDR knocked down in THP-1, while antimicrobial activity induced by exogenous LL-37 remains intact with the same shRNA VDR knocked down THP-1 cells. Furthermore addition of LL-37 to shRNA VDR knocked down THP-1 cells recovered antimicrobial activity against M. marinum infection. These results strongly suggest that antimicrobial activity of 1,25(OH)2D3 against M. marinum is mediated by induction of endogenous CAMP, which confirms the previous reports of studies using M. tbc [
      • Yuk J.M.
      • Shin D.M.
      • Lee H.M.
      • Yang C.S.
      • Jin H.S.
      • Kim K.K.
      • et al.
      Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin.
      ].
      In this study we also explored the possible mechanism of antimicrobial activity of CAMP in human monocytic cells against M. marinum infection. First we showed that CAMP induced by 1,25(OH)2D3 co-localizes with autophagolysomes (LL-37/LC3, LC3/LAMP1) in THP-1 cells after infection with M. marinum. In line with this it is of note to mention the appearance of truncated form of CAMP (12 kDa) in 1,25(OH)2D3 treated THP-1 cells after M. marinum infection (Fig. 5A), which is consistent with the findings of CAMP localization in autophagolysosome compartments. Furthermore we found that 3-MA (autophagy inhibitor) and chloroquine (lysosomotropic agent that prevents endosomal acidification and thus inhibits the formation of autophagolysosome) prevent antimicrobial activity induced by CAMP against M. marinum infection in THP-1 cells. In fact autophagy deficient cells (ATG5−/− MEF) failed to show the antimicrobial activity induced by CAMP (endogenous CAMP induced by 1,25(OH)2D3 and exogenously LL-37) against M. marinum infection in THP-1 cells. These results clearly indicate that both endogenous CAMP induced by 1,25(OH)2D3 and exogenously added LL-37 exert antimicrobial activity against M. marinum by induction of autophagolysosome.
      Another finding of particular interest in this study is effect of 1,25(OH)2D3 treatment or exogenous LL-37 on inflammatory cytokine production in THP-1 cells after M. marinum infection. As shown in Fig. 3 we found that treatment with 1,25(OH)2D3 or LL-37 suppress IFNβ, TNFα and IL-12p40, suggesting the anti-inflammatory role of VitD-CAMP in M. marinum infection. The intracellular roles of CAMP have been sought in many aspects, and previous studies using various cell types, including macrophages, dendritic cells [
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      1Alpha,25-dihydroxyvitamin D3 decreases DNA binding of nuclear factor-kappaB in human fibroblasts.
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      ] indicated that vitamin D can inhibit NF-κB signaling, suggesting an immune regulatory function of cathelicidin. Several mechanisms have been proposed, including vitamin D-induced increased levels of IκBα [
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      • Daniel K.C.
      • Adorini L.
      A vitamin D analog down-regulates proinflammatory chemokine production by pancreatic islets inhibiting T cell recruitment and type 1 diabetes development.
      ], which interferes with the binding of NF-κB subunits to promoter regulatory areas [
      • D’Ambrosio D.
      • Cippitelli M.
      • Cocciolo M.G.
      • Mazzeo D.
      • Di Lucia P.
      • Lang R.
      • et al.
      Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.
      ,
      • Harant H.
      • Wolff B.
      • Lindley I.J.
      1Alpha,25-dihydroxyvitamin D3 decreases DNA binding of nuclear factor-kappaB in human fibroblasts.
      ,
      • Deb D.K.
      • Chen Y.
      • Zhang Z.
      • Zhang Y.
      • Szeto F.L.
      • Wong K.E.
      • et al.
      1,25-Dihydroxyvitamin D3 suppresses high glucose-induced angiotensinogen expression in kidney cells by blocking the NF-{kappa}B pathway.
      ]. In addition to this, it has been shown that 1,25(OH)2D3 can reduce the transcription and secretion of protective IFN-γ, IL-12p40 and TNF-α in M. tbc infected peripheral blood mononuclear cells (PBMCs) and macrophages by regulation of RelB [
      • Dong X.
      • Craig T.
      • Xing N.
      • Bachman L.A.
      • Paya C.V.
      • Weih F.
      • et al.
      Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: physiologic and therapeutic implications for dendritic cell function.
      ]. LL-37 suppresses the LPS-induced TNF-α response in PBMCs via NF-κB down regulation and RelB involvement [
      • Dong X.
      • Lutz W.
      • Schroeder T.M.
      • Bachman L.A.
      • Westendorf J.J.
      • Kumar R.
      • et al.
      Regulation of relB in dendritic cells by means of modulated association of vitamin D receptor and histone deacetylase 3 with the promoter.
      ,
      • D’Ambrosio D.
      • Cippitelli M.
      • Cocciolo M.G.
      • Mazzeo D.
      • Di Lucia P.
      • Lang R.
      • et al.
      Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.
      ]. Activation of autophagy by 1,25(OH)2D3 or LL-37 may also limit proinflammatory cytokine production by targeting inflammasome or inflammasome-independent modulation of cytokine response [
      • Crisan T.O.
      • Plantinga T.S.
      • van de Veerdonk F.L.
      • Farcas M.F.
      • Stoffels M.
      • Kullberg B.J.
      • et al.
      Inflammasome-independent modulation of cytokine response by autophagy in human cells.
      ,
      • Nakahira K.
      • Haspel J.A.
      • Rathinam V.A.
      • Lee S.J.
      • Dolinay T.
      • Lam H.C.
      • et al.
      Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.
      ,
      • Shi C.S.
      • Shenderov K.
      • Huang N.N.
      • Kabat J.
      • Abu-Asab M.
      • Fitzgerald K.A.
      • et al.
      Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction.
      ]. Exogenous LL-37 suppressed the LPS-induced TNF-α and chemokine MCP-1 responses in peripheral blood mononuclear cells (PBMCs) [
      • Dong X.
      • Lutz W.
      • Schroeder T.M.
      • Bachman L.A.
      • Westendorf J.J.
      • Kumar R.
      • et al.
      Regulation of relB in dendritic cells by means of modulated association of vitamin D receptor and histone deacetylase 3 with the promoter.
      ,
      • D’Ambrosio D.
      • Cippitelli M.
      • Cocciolo M.G.
      • Mazzeo D.
      • Di Lucia P.
      • Lang R.
      • et al.
      Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.
      ,
      • Wang G.
      • Elliott M.
      • Cogen A.L.
      • Ezell E.L.
      • Gallo R.L.
      • Hancock R.E.
      Structure, dynamics, and antimicrobial and immune modulatory activities of human LL-23 and its single-residue variants mutated on the basis of homologous primate cathelicidins.
      ]. LL-37 also functions as an immune regulator to control inappropriate immune responses to pathogen infection such as septic shock. Furthermore, LL-37 inhibits the binding of LPS and lipopolysaccharide-binding protein (LBP) thereby suppressing the production of proinflammatory cytokines such as TNF-α [
      • Giarratana N.
      • Penna G.
      • Amuchastegui S.
      • Mariani R.
      • Daniel K.C.
      • Adorini L.
      A vitamin D analog down-regulates proinflammatory chemokine production by pancreatic islets inhibiting T cell recruitment and type 1 diabetes development.
      ,
      • Deb D.K.
      • Chen Y.
      • Zhang Z.
      • Zhang Y.
      • Szeto F.L.
      • Wong K.E.
      • et al.
      1,25-Dihydroxyvitamin D3 suppresses high glucose-induced angiotensinogen expression in kidney cells by blocking the NF-{kappa}B pathway.
      ]. In line with these studies, our current results of CAMP in M. marinum infected THP-1 cells also suggest that cathelicidin may have unique suppressive role on innate immune mediator such as IFNα, TNFα and IL-12, although further studies will be needed to explore the precise mechanism of this anti-inflammatory role of CAMP in human monocytic cells.
      In summary we showed that both intracellular/extracellular CAMP/LL-37 suppress M. marinum infection via autophagolysosome in human monocytes and 1,25(OH)2D3 plays an important role in M. marinum infection. 1,25(OH)2D3 insufficiency is known to cause infection and relapse of tuberculosis [
      • Luong K.
      • Nguyen L.T.
      Impact of vitamin D in the treatment of tuberculosis.
      ]. Current study demonstrates that 1,25(OH)2D3 has an important protective role against the infection with another mycobacterium species, M. marinum, in addition to M. tbc. Analogs of 1,25(OH)2D3, such as calcipotriol, is used for the treatment of dermatologic diseases such as psoriasis. Also, ultraviolet-light (UV) therapy is a common practice for various dermatologic diseases. Our results might suggest that topical 1,25(OH)2D3 analogs as well as UV therapy could be useful supplemental therapies for superficial infection of M. marinum in combination with regular antibiotic therapy. Considering that activated Vit D3 induced CAMP increases antimicrobial activity via autophagolysosome, while down regulating pro-inflammatory cytokine production, topical therapy with active Vit D3 may benefit to the prevention of immunohistopathological tissue damages.

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