extfieldsofstudy: Chemistry Medicine

text:
MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Abstract Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.

Introduction
Hepatic fibrosis is a result of an abnormal wound healing in response to chronic liver injury from various causes [1,2]. The pathogenesis of hepatic fibrosis is a dynamic and complex process that involves various cell types in the hepatic tissue including the hepatic progenitor cells [3,4]. The chronic hepatocyte injury leads to the activation of resting hepatic stellate cells (HSCs) into myofibroblast-like cells with the characteristic expression of smooth muscle actin filaments [5,6]. The activated hepatic stellate cells initiate a series of signalling and transcriptional events that triggers the pathogenesis of hepatic fibrosis [7,8].
N-Nitrosodimethylamine (NDMA), also known as dimethylnitrosamine (DMN), is a by-product of several industrial processes and a suspected human carcinogen and highly toxic to the liver. It was demonstrated that NDMA-induced model of hepatic fibrosis in rats is an appropriate and suitable animal model for studying biochemical and molecular changes that associated with the pathogenesis of hepatic fibrosis and cirrhosis of human beings [9,10]. This model also depicts several decompensating features of alcoholic fibrosis and cirrhosis such as portal hypertension, ascites, hypoproteinemia and biochemical alterations [11,12]. Recently, NDMA-induced mouse model of liver injury has been used to study the interaction between profibrotic and anti-fibrotic molecules during the development of hepatic fibrosis [13]. The extensive oxidative stress and production of reactive oxygen species (ROS) during detoxification of NDMA triggers acute liver injury and subsequent fibrosis.
Matrix metalloproteinases (MMPs) are a gene family of zinc-and calcium-dependent endopeptidases that are capable of degrading extracellular matrix (ECM) components including all forms of native collagens, and play a key role in remodelling the ECM in both physiological and pathological conditions [14,15]. The major interstitial collagenases that degrade native fibrillar collagens are MMP-1 and MMP-13. However, mice and rats do not possess a homologue to human MMP-1. Thus, MMP-13 is responsible for the metabolic turnover of major fibrillar collagens in mice [16], and transgenic expression of MMP-1 inhibited myocardial fibrosis and prevented heart failure in a mouse model [17].
Connective tissue growth factor (CTGF), a 38-kD cysteine-rich protein, is a multifunctional protein involved cell proliferation and tissue remodelling, and induces by transforming growth factor-b1 (TGF-b1) [18][19][20]. CTGF plays a central role in the pathogenesis of hepatic fibrosis by triggering activation and transformation of quiescent hepatic stellate cells into myofibroblasts, and it also stimulates the production of CTGF itself [21,22]. Activated stellate cells over-produce collagens, fibronectin and laminin under stimulation with CTGF [23]. The two major MMPs, that is MMP-1 and MMP-13, cleave full-length CTGF into the N-terminal and C-terminal fragments of similar molecular weights [19,24]. Due to the absence of MMP-1 in adult mice [25,26], MMP-13 is primarily responsible for the cleavage of full-length CTGF into its fragments that are more potent to induce hepatic fibrogenesis, as it has been reported that the N-terminal fragment of CTGF mediates myofibroblast differentiation and collagen synthesis and the C-terminal fragment stimulates fibroblast proliferation [27]. The N-terminal fragment also acts as downstream mediator of TGF-b1 and increases in fibrotic processes [27,28]. Uchinami et al. [29] reported that MMP-13 contributes to accelerating fibrogenesis by mediating the initial inflammation of the liver. However, the mechanism by which MMP-13 promotes fibrogenesis through mediating the initial inflammation of the liver is not clear. In the current study, we attempted to elucidate some of the mechanisms behind this observation with a specific emphasis to CTGF and its cleaved subunits. NDMA was used to induce the liver injury in wild-type (WT) and MMP-13 knockout (KO) mice for the purpose. Cultured rat hepatic stellate cells were used for in vitro studies.

MP-13 knockout and wild-type mice
The MMP-13 knockout (KO) mice on a 129/Sv genetic background were generated by microinjection of embryonic stem cells into C57BL/6J blastocytes as described before [30,31]. Adult MMP-13 KO mice were back-crossed with C57BL/6J strain for at least five times. MMP-13 KO and wild-type (WT) littermates were generated from the intercross between MMP13 +/À mice, and both male and female mice were used for the experiments. Genotyping was performed by PCR analysis using DNA obtained from tail biopsies of two-week-old pups. The KO mice exhibited a normal lifespan with sufficient fertility and did not exhibit any gross abnormalities after maturation, except growth retardation due to defects in the growth plate during development [30,31]. Histopathological examination of paraffin sections stained with haematoxylin and eosin and Masson's trichrome for collagen from the liver, lung, heart, kidney and uterus showed no pathological alterations or collagen deposition in adult MMP-13 KO mice.
The animal experiments were carried out with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the US National Institutes of Health (NIH Publication No. 86-23, revised 1996) and also in compliance with the Institutional Animal Care and Use Committee (IACUC). Both male and female animals were used for the induction of liver injury. All animals received humane care according to the criteria outlined in the manual.

Induction of liver injury and fibrosis in WT and MMP-13 KO mice
Both WT and MMP-13 KO mice around three months of age, weighing approximately 25 g, were used for the experiment. The animals were divided into four groups of eight animals each. Hepatic fibrosis was induced by serial intraperitoneal (i.p.) injections of N-nitrosodimethylamine (NDMA) in doses of 10 lg/g body weight on three consecutive days of every week over a period of 4 weeks in one group of both WT and MMP-13 KO mice. NDMA was procured from Sigma-Aldrich (St. Louis, MO, USA) and diluted appropriately with 0.15 mol/l sterile NaCl. Control groups received similar injections without NDMA. The injections were given without anaesthesia. All the animals were provided with mice feed and water available ad libitum. A few animals died in both WT and MMP-13 KO NDMA-treated groups due to massive hepatic necrosis. However, the experiment was repeated with additional animals and the total number of animals was eight in each group at the end of the study. The animals were killed on day 28 from the beginning of NDMA administration along with control animals after anesthetization with a mixture of ketamine and xylazine. Blood and liver tissue were collected and processed for biochemical and molecular biological studies.
Evaluation of NDMA-induced liver injury and hepatic fibrosis NDMA-induced hepatic injury and fibrosis were assessed by observing histology of the liver stained with haematoxylin and eosin and Masson's trichrome. The median lobe of the liver tissue was cut into approximately 3-mm slices and fixed in 10% phosphate-buffered formalin. The fixed liver tissues were processed in an automatic tissue processor optimized for liver, embedded in paraffin blocks and cut into sections of 5 lm thickness. haematoxylin and eosin and Masson's trichrome staining were carried out using standard methodology (#K037; Poly Scientific, Bay Shore, NY, USA). The staining intensity was examined using an Olympus microscope (Olympus Corporation, Tokyo, Japan) and photographed. The number of activated stellate cells was evaluated using immunohistochemical staining of a-smooth muscle actin (a-SMA), which is a marker for initiation of hepatic fibrosis. hydrolysed in 6 N HCl in sealed tubes at 110°C for 16 hrs. The hydrolysed samples were evaporated to dryness in a boiling water bath to remove acid, and the residue was dissolved in 5 ml of distilled water. It was then treated with activated charcoal, vortexed well and filtered through Whatman No. 1 filter paper. Hydroxyproline content in the clear filtrate was measured as described before [32]. In brief, 1 ml of sample containing 1-5 lg hydroxyproline was mixed with 1 ml of freshly prepared chloramine-T solution and allowed to stand for 20 min. It was then mixed with 1 ml of 3.15 M perchloric acid and left for 5 min. Next, 1 ml of freshly prepared p-dimethylaminobenzaldehyde was added, vortexed well and incubated in a water bath at 60°C for 20 min. Absorbance of the colour development was measured in a spectrophotometer at 560 nm. The concentration of hydroxyproline in the samples was determined using a known standard of L-hydroxyproline (Sigma-Aldrich) solution. The total collagen content in the liver tissue was calculated by multiplying the hydroxyproline content by the factor 7.46 as described previously [2].

Measurement of ALT and AST
Blood was allowed to clot by standing for 3-5 hrs and serum separated by the conventional method. Serum alanine transaminase (ALT) and aspartate transaminase (AST) were measured using an auto-analyser and presented as International Units per Liter.

Immunohistochemical staining for a-SMA and CTGF
The immunohistochemical staining for a-SMA and CTGF was carried out on paraffin-embedded tissue using a universal staining kit (#85-9943; Invitrogen, Carlsbad, CA, USA). The paraffin liver sections were deparaffinized and hydrated with water. The clear sections were treated with primary antibodies against a-SMA (#18-0106; Invitrogen) or CTGF (#ab6992; Abcam, Cambridge, MA, USA) and non-immune control IgG in a moisturized chamber at 4°C overnight. They were washed three times in cold phosphate-buffered saline (PBS) and incubated with biotinylated anti-rabbit or antimouse secondary antibodies for 1 hr at room temperature. The slides were washed again and treated with horseradish peroxidase-labelled streptavidin and incubated for another 1 hr. Colour was developed using 3% 3-amino-9-ethylcarbazole (AEC). The stained sections were counterstained Mayer's haematoxylin and examined under a Nikon microscope attached with a Spot RT Slider digital camera (Meyer Instruments, Houston, TX, USA) and photographed. The images were quantified using Image-pro discovery software (Media Cybernetics, Silver Spring, MD, USA).

Measurement of IL-6, hyaluronic acid and TGF-b1 in serum
Interleukin-6 (IL-6) in mouse serum was measured using sandwich ELISA kit (#ab100712; Abcam) following manufacturer's protocol. In brief, 100 ll serum or recombinant IL-6 standard was added to a 96-well plate coated with mouse IL-6 antibody and incubated for 2.5 hrs at room temperature with gentle shaking. After several washing, biotinylated IL-6 second antibody was added followed by HRP-conjugated streptavidin and TMB (tetramethylbenzidine) substrate. The intensity of the coloured product was measured on a microplate plate reader at 450 nm. Hyaluronic acid (HA) levels in mouse serum were determined using an ELISAbased sandwich HA-binding protein assay kit (Chugai Diagnostics Science, Tokyo, Japan) as per the manufacturer's instructions, which follow the method of Chichibu et al. [33]. TGF-b1 levels in mouse serum were determined using a sandwich enzyme assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. In brief, 50 ll of 60-fold diluted serum samples was added to a microplate precoated with TGF-b1 monoclonal antibody followed by 50 ll of assay diluent provided in the kit. The samples were mixed gently and incubated for 2 hrs at room temperature. The wells were washed 3-5 times with wash buffer included in the kit, and 100 ll of TGF-b1 secondary antibody conjugate was added, and incubated for 2 hrs. Finally, 100 ll of TMB substrate solution was added and incubated for 30 min. in the dark for colour development. The reaction was arrested with stop solution, and the intensity of the colour was determined on a microplate reader at 450 nm.
Gelatin zymography for MMP-9 and MMP-2 The activity of MMP-9 and MMP-2 in the liver tissue from WT and MMP-13 KO mice was analysed using gelatin zymography [34]. About 100 mg of fresh liver tissue was homogenized in 1 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and 1% Triton X-100. The homogenates were centrifuged at 5000 9 g for 30 min. at 4°C, and the supernatants were collected. After equalizing the protein concentration, 30 lg protein was loaded to a 10% polyacrylamide gel containing 0.1% bovine skin gelatin (#G8150; Sigma-Aldrich) under non-reducing conditions. The gels were washed twice (30 min./wash) in 2.5% Triton X-100 at room temperature and then incubated overnight at 37°C in 50 mM Tris-HCl buffer (pH 7.6) containing 10 mM CaCl 2 . The gels were stained with Coomassie brilliant blue R-250 for 30 min. and destained until clear transparent bands were developed and photographed. The bands were quantified using Gel-Pro analyzer software (Media Cybernetics).

Western blotting for MMP-9 and MMP-2
Proteins (20 lg/lane) prepared from the liver tissue were resolved on a 7.5% SDS-PAGE under reducing conditions. The proteins were electrotransferred onto an activated polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were incubated with 5% skim milk to block non-specific reactions and reacted overnight on a rocker at 4°C with antibodies against MMP-9 (#19016; Millipore) or MMP-2 (#4022; Cell Signaling Technology, Danvers, MA, USA) that recognize the mouse antigens. After washing in PBS, they were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Biomeda, Foster City, CA, USA) at room temperature for 2 hrs. The membranes were then treated with chemiluminescence reagent (#RPN2232; ECL Prime, GE Healthcare, Piscathaway, NJ, USA) exposed to BioMax XAR autoradiography film (Kodak, New Haven, CT, USA). The membranes were reprobed for b-actin to demonstrate equal loading of protein samples in each lane.

Quantitative real-time PCR for MMP-9
Control and NDMA-treated WT and MMP-13 KO mouse liver tissues were snap-frozen in liquid nitrogen and stored at À80°C. The total RNA was 3823 isolated using RNeasy kit (Qiagen, Valencia, CA, USA). The purity of the isolated RNA was evaluated on a spectrophotometer and with agarose gel electrophoresis. Mouse MMP-9 primer set was designed using Primer 3 software (NHGRI, NIH, USA), and the primer sequences for mouse MMP-9 gene (NM_013599) were as follows: forward 5 0 -CGT CGT GAT CCC CAC TTA CT-3 0 and reverse 5 0 -AAC ACA CAG GGT TTG CCT TC-3 0 . Quantitative PCR was performed using a one-step RT-PCR kit with SYBR green (Bio-Rad, Hercules, CA, USA) on a real-time PCR machine (iCycler iQ5; Bio-Rad). The reaction conditions were set as: cDNA synthesis, 10 min. at 50°C; reverse transcriptase inactivation at 95°C for 5 min.; PCR cycling and detection at 95°C for 10 sec.; and data collection at 56°C for 30 sec. About 100 ng of isolated RNA was used in the experiment.

Real-time PCR and semiquantitative RT-PCR for the major molecules involved in hepatic fibrosis
Total RNA was isolated from the flash-frozen liver tissue employing PureLink RNA Mini Kit (Invitrogen) in combination with Trizol as per manufacturer's instructions. The gene specific primers for CTGF, TGF-b1, a-SMA, type I collagen (a1 chain) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were designed using Beacon Designer software (Premier Biosoft International, Palo Alto, CA, USA). Details of the primer sets used are provided in supplementary Table 1. The same primer sets were used for both real-time PCR and semiquantitative RT-PCR. The cDNA was synthesized with 1-2 lg of isolated total RNA using Sprint RT 8-well strips (Clontech, Mountain View, CA, USA) in a total volume of 20 ll in RNAse-free H 2 O at 42°C for 60 min. Real-time quantitative PCR (qPCR) was carried out with FastStart SYBR Green Master (Roche Applied Science, Indianapolis, IN, USA) on ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The synthesized cDNA was diluted to 20-fold with RNAse-DNAse-free water. The qPCR reaction was carried out with 5 ll of diluted cDNA and 5 ll of SYBR Green master mix containing gene specific primers in a 384well plate (total volume 10 ll). All samples were run in triplicates. The qPCR parameters were set as follows; denature 1 cycle, amplification 45 cycles, melting curve 1 cycle and cooling 1 cycle. All data were normalized to GAPDH gene.
Semiquantitative RT-PCR was performed for all of the above molecules for additional evaluation of their mRNA expression. All of the above primer sets were transcribed with 100 ng of isolated total RNA using SuperScript one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen) on a thermocycler (GeneAMP PCR Systems 9700; Applied Biosystems). The reaction conditions were set as: cDNA synthesis at 50°C for 30 min.; inactivation at 94°C for 2 min.; PCR amplification of 35 cycles; denaturation at 94°C for 20 sec.; annealing at 56°C for 30 sec.; chain extension at 72°C for 45 sec.; and a final chain extension at 72°C for 10 min. The PCR products were resolved on 1.5% agarose gel containing ethidium bromide and examined with a transilluminating UV system (Alpha Innotech Corporation, San Leandro, CA, USA) connected with a computer. The PCR product images were quantified employing Gel-Pro analyzer software (Media Cybernetics).

Western blotting for the major molecules involved in pathogenesis of hepatic fibrosis
To examine the expression of the molecules involved in the pathogenesis of hepatic fibrosis in WT and MMP-13 KO mice after treatment with NDMA, 100 mg of liver tissue was homogenized in 1 ml of cold 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA) and 1% Triton X-100. The protease inhibitors, 0.5 mM phenylmethylsulfonyl fluoride, 5 lg/ml aprotinin and 1 lg/ml pepstatin were added to the buffer just prior to use. The homogenates were centrifuged at 10,000 9 g for 10 min. at 4°C, and the supernatant was collected. Protein concentrations in the supernatants were determined using a protein assay kit (#23227; Pierce Biotechnology, Rockford, IL, USA), and the samples were stored at À20°C until used. The proteins were denatured and resolved on 4-20% SDS-polyacrylamide gradient gel (Bio-Rad) and electrotransferred to an activated PVDF membrane. Non-specific binding was blocked with 5% non-fat dry milk, and the membranes were incubated overnight on a rocker at 4°C with specific antibodies. Antibody to tissue inhibitor of metalloproteinase (TIMP)-1 (#sc-5538) was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies to cleaved TGF-b1 (#ab9758) and a-SMA (#ab5694) were from  Abcam, and the antibody to type I collagen was from Millipore (#AB765P). The polyclonal antibodies that detect the full-length CTGF and its C-terminal fragments were raised in rabbits by injecting bovine serum albumin (BSA)-conjugated oligopeptide corresponding to the amino acid sequence of the CTGF C-terminal domain (I 257 RTPKISKPIK-FELSG 272 C) and previously characterized (24). IgG was isolated from the antisera by DEAE-Sephacel column chromatography and purified. After incubation with the primary antibodies, the membranes were washed thrice and treated with respective HRP-conjugated secondary antibodies (Biomeda) at room temperature for 2 hrs. They were washed again, treated with enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA), exposed to Kodak autoradiography film (BioMax XAR, New Haven, CT, USA), and developed. The membranes were reprobed using Western reprobe buffer (#786-119; Gbiosciences, St. Louis, MO, USA) for GAPDH content with anti-GAPDH monoclonal antibody (#NB300-221; Novus Biologicals, Littleton, CO, USA) to show that equal amounts of protein samples were loaded in all lanes. The Western blotting images were quantified using Gel-Pro analyzer software (Media Cybernetics).

Inhibition of MMP-13 in hepatic stellate cells
To examine whether inhibition of MMP-13 could decrease active CTGF and TGF-b1 protein levels in vitro, liver stellate cell cultures were treated with CL-82198 (N-[4-(4-Morpholinyl) butyl]-2-benzofurancarboxamide hydrochloride) (#ab141576; Abcam), which is a selective inhibitor of MMP-13 [35]. Stellate cells were isolated from about one year old albino rats and cultured as reported before [21]. The cells were cultured in 50:50 mixture of DMEM and Ham's F12 medium (Invitrogen) containing 10% foetal bovine serum (FBS; Invitrogen) and antibiotics (100 units of penicillin G and 100 lg of streptomycin/ml of culture media) in a humidified incubator containing 5% CO 2 on air at 37°C. About 80% confluent cells were harvested using Trypsin-EDTA (Invitrogen) and sub-cultured into 4-well glass microscopic chamber slides (CAT# 154526PK, Lab-Tek, Thermo Fisher Scientific, Waltham, MA, USA) treated with 0.1% collagen solution. Another set was cultured into 100-mm culture dishes for Western blotting analysis. After 48 hrs, the media were replaced with reduced serum medium (2% FBS) containing 10 lM CL-82198 (final concentration) (reported IC 50 = 3.2 lM) [36] and incubated for another 48 hrs. A second set of cultures were prepared in the same way without CL-82198 and served as control. The medium was discarded, washed the slides twice with PBS, and the cells were fixed in 1:1 methanol and ethanol mixture at À20°C for 10 min. The cells were washed with PBS and staining was performed for CTGF. The staining intensity was quantified using Image-Pro Plus software (Media Cybernetics) and represented as square microns.
The treated cells in culture dishes were washed well with cold PBS and harvested with 1 ml of radio-immunoprecipitation assay (RIPA) buffer prepared freshly with protease inhibitors. The cells were centrifuged at 12,000 9 g for 10 min. at 4°C in a micro-centrifuge (Sorvall; Thermo Fisher). The cell pellet was collected and suspended in RIPA buffer containing protease inhibitors, depends on the size of the pellet and sonicated gently in an Omni Ruptor 400 Ultrasonic cell disruptor (Omni International, Kennesaw, GA, USA). The cell suspension was centrifuged at 14,000 9 g for 10 min. at 4°C, and the supernatants were collected. Protein concentration in the supernatant was determined and Western blotting for full-length CTGF and its C-terminal fragments and cleaved TGF-b1 was carried out as described above. Finally, the Western blot membranes were treated with enhanced chemiluminescence reagent (#RPN2232; ECL Prime, GE Healthcare, Piscataway, NJ, USA) and the resultant chemiluminescence was detected using Fuji ImageQuant LAS 4000 (GE Healthcare) and the digitized images were saved on a computer. The membranes were reprobed using Western reprobe buffer (Gbiosciences), and Western blotting was performed for b-actin with antibody (#A5441; Sigma-Aldrich) to demonstrate that equal amount of protein had been loaded in each lane. The Western blotting images were quantified using Gel-Pro analyzer software (Media Cybernetics).

Statistical analysis
Arithmetic mean and standard deviation (S.D.) were calculated for all quantitative data. The data were statistically evaluated using paired Student's t-test, and a value of P < 0.05 was considered as significant. Pearson's correlation coefficient analysis was used to evaluate the correlation between qPCR and semiquantitative PCR for the expression of major molecules involved in the pathogenesis of hepatic fibrosis.

NDMA-induced liver injury and fibrosis
Serum levels of AST and ALT were remarkably increased in WT and MMP-13 KO mice that were administered with NDMA, whereas both control mouse groups untreated with NDMA showed only the baseline levels ( Fig. 1A and B). When the mean serum levels were compared between NDMA-treated WT and MMP-13 KO mice, AST and ALT were significantly higher in WT mouse group than in MMP-13 KO mouse group (P < 0.001) (Fig. 1A and B). Histologically, control WT mice without NDMA treatment showed normal lobular architecture with central veins and radiating hepatic cords (Fig. 1C), and there was no alteration in the liver tissue of control MMP-13 KO mice (Fig. 1D). In contrast, administrations of NDMA to WT mice exhibited extensive hepatic necrosis with haemorrhage and infiltration of mononuclear cells and neutrophils (Fig. 1E). However, NDMA-treated MMP-13 KO mice showed only mild focal haemorrhagic necrosis of hepatocytes (Fig. 1F).

Decrease of collagen content in MMP-13 KO mice liver
Masson's trichrome staining did not show any collagen deposition in the liver parenchyma of either WT or MMP-13 KO control mice ( Fig. 2A and B). However, NDMA-administered WT mice demonstrated deposition of thick collagen fibres (arrows) in the areas of bridging fibrosis (Fig. 2C), whereas such collagen deposition was not present in NDMA-treated MMP-13 KO mice (Fig. 2D).
The total collagen content in the liver tissue determined by estimating hydroxyproline, a characteristic imino acid present in  collagen, was significantly increased (P < 0.001) in WT mice after the administration of NDMA (Fig. 2E). The increase was around 3-fold compared to the total collagen content in WT control mice liver. The total collagen content in NDMA-treated MMP-13 KO mice was also significantly higher (P < 0.01) compared to non-treated MMP-13 KO mice (Fig. 2E). However, compared to the increased collagen level in NDMA-treated WT mice liver, the collagen content in NDMA-treated MMP-13 KO mice was significantly lower (P < 0.001). There was no difference in the collagen content between WT and MMP-KO control mice livers (Fig. 2E).

Activation of hepatic stellate cells is attenuated in MMP-13 KO mice
Immunohistochemical staining was performed for a-SMA to examine the activation of hepatic stellate cells after induction of liver injury by NDMA. As shown in Figure 3A and B, the staining was almost completely absent in control WT and MMP-13 KO mice without the treatment. However, marked staining of a-SMA appeared in stellate cells localized in and around the necrotic and fibrotic areas of the liver from NDMA-treated WT mice (Fig. 3C), although minimal staining was observed in NDMA-treated MMP-13 KO mice (Fig. 3D). Quantitative analysis of the staining intensity of a-SMA demonstrated that the intensity is significantly higher in NDMA-treated WT mice as compared to control non-treated WT mice (P < 0.001) (Fig. 3E). It was also significantly higher in NDMA-treated MMP-13 KO mice than in non-treated MMP-13 KO mice (P < 0.01) (Fig. 3E). However, when the staining intensity was compared between the WT and MMP-13 KO mouse groups, it was significantly lower in the MMP-13 KO mice than in the WT mice (P < 0.001) (Fig. 3E).

Expression of CTGF after NDMA treatment is reduced in MMP-13 KO mice
The expression of CTGF in the livers from control and NDMA-treated WT and MMP-13 KO mice was determined by immunohistochemistry. The staining was absent or negligible in the parenchymal areas of control WT and MMP-13 KO mice, but weak staining was obtained in one row of the peri-central hepatocytes in both WT and MMP-13 KO mice (arrows) (Fig. 4A and B), the finding coinciding with the previous report [22]. On the other hand, marked and intense staining was present by the parenchymal cells in the necrotic and fibrotic areas in NDMA-treated WT mice (Fig. 4C), although only focal minimal staining was observed in the livers from NDMA-treated MMP-13 KO mice (Fig. 4D). The staining intensity of CTGF was significantly higher in NDMA-treated WT mice compared to the untreated WT control mice (P < 0.001) (Fig. 4E). Although the staining intensity was significantly higher in NDMA-treated MMP-13 KO mice compared to the MMP-13 KO control untreated mice (P < 0.01), the intensity in NDMA-treated MMP-13 KO mice was significantly lower than that in the NDMAtreated WT mice (P < 0.001) (Fig. 4E).

Interleukin-6 levels in mouse serum
The acute phase protein, IL-6, was measured in the mouse serum to exclude chronic inflammation during the i.p. procedure. There was no significant difference in IL-6 levels between the groups of animals involved in the study (Fig. 5A). The data ruled out peritonitis or any systemic infection during the course of the study that could affect the outcome of the results.

Serum levels of TGF-b1 and HA decreased in MMP-13 KO mice
TGF-b1 is known to be up-regulated during liver injury and fibrosis [21], and serum HA is a very early indicator of liver damage [6,37]. Thus, we measured serum levels of TGF-b1 and HA in WT and MMP-13 KO mice. As shown in Figure 5B and C, both TGF-b1 and HA levels significantly increased in NDMA-treated WT mice as compared to control untreated WT and MMP-13 KO mice. However, the TGF-b1 and HA levels were decreased 4-fold in NDMA-treated MMP-13 KO mice than in NDMA-treated WT mice (P < 0.001) (Fig. 5B and C). The activity of MMP-9 and MMP-2 were measured by gelatin zymography in the liver samples from WT and MMP-13 KO mice. As shown in Figure 6A and C, MMP-9 activity was significantly increased in NDMA-treated WT mice as compared to control WT mice (P < 0.001) or in NDMA-treated MMP-13 KO mice compared to untreated MMP-KO mice (P < 0.01). However, MMP-9 activity in NDMA-treated MMP-13 KO mice was significantly lower (P < 0.001) compared to the NDMA-treated WT mice (Fig. 6C). Figure 6B depicts the Western blots for MMP-9 and MMP-2 proteins in control WT and MMP-13 KO mice and NDMA-treated mice, showing a similar pattern obtained by the gelatin zymography. MMP-9 protein level appeared to be less in NDMA-treated MMP-13 KO mice as compared to NDMA-treated WT mice (Fig. 6B), and quantitative analysis confirmed the data as shown in Figure 6C. The expression of MMP-9 mRNA in control WT and MMP-13 KO mice and the NDMA-treated mice is shown in Figure 6D.
Expression of MMP-9 mRNA was significantly increased in NDMAtreated WT (P < 0.001) and MMP-13 KO (P < 0.01) mice compared to their respective untreated controls. However, the mean MMP-9 mRNA level in NDMA-treated MMP-13 KO mice was significantly lower (P < 0.001) compared to NDMA-treated WT mice.

Deletion of MMP-13 decreases expression of growth factors and proteins that trigger hepatic fibrosis
To examine the expression of CTGF, TGF-b1, a-SMA and type I collagen, the respective mRNA levels were quantified using real-time PCR in control WT and MMP-13 KO mice and NDMA-treated mice. All of the molecules studied were significantly elevated (P < 0.001) in NDMA-administered WT mice (Fig. 7A). The levels of CTGF and type I collagen were also significantly increased (P < 0.05) in NDMA-treated MMP-13 KO mice as compared to control MMP-13 KO mice, but there was no significant increase in the levels of TGF-b1 or a-SMA (Fig. 7A). The increased mRNA levels of CTGF and type I collagen in NDMA-treated MMP-13 KO mice were significantly (P < 0.001) lower as compared to the NDMA-treated WT mice. Semiquantitative RT-PCR analysis of CTGF, TGF-b1, a-SMA and type I collagen mRNA in the liver specimens from WT and MMP-13 KO confirmed the data of real-time PCR (Fig. 7B).

Deletion of MMP-13 decreases protein levels of major molecules involved in hepatic fibrosis
The protein levels of full-length CTGF and its cleaved products as well as TIMP-1, cleaved TGF-b1, a-SMA and type I collagen were determined by Western blotting. There was an increase in fulllength CTGF in both NDMA-treated WT and MMP-13 KO mice. The increase appeared to be higher in WT mice compared to MMP-13 KO mice (Fig. 8A). The cleaved fractions of CTGF were increased only in NDMA-treated WT mice but not in MMP-13 KO mice, suggesting a role of MMP-13 in the cleavage of CTGF. Figure 8B represents the quantification of Western blot images, which is presented as percentage alteration of the proteins. There was a marked increase (P < 0.001) in protein levels of CTGF, TIMP-1, cleaved TGF-b1, a-SMA and type I collagen in NDMA-treated WT mice as compared to those in control untreated WT mice. Protein levels of TIMP-1 (P < 0.001), full-length CTGF (P < 0.001), TGF-b1 (P < 0.01), a-SMA (P < 0.05) and type I collagen (P < 0.05) were significantly increased in NDMA-treated MMP-13 KO mice as compared to control untreated MMP-13 KO mice. However, the mean protein levels of all these molecules were significantly lower (P < 0.001) (TIMP-1, P < 0.05) in NDMA-treated MMP-13 KO mice compared to NDMA-treated WT mice (Fig. 8B). Among all the five molecules studied, the maximum elevation (3.12-fold) was observed in the case of type I collagen in NDMA-treated WT mice (Fig. 8B). Reprobing the blots for GAPDH demonstrated loading of similar levels of proteins in all lanes.

Inhibition of MMP-13 in hepatic stellate cells decreases CTGF and TGF-b1 protein levels
To obtain more information about the mechanism of decreased liver injury and fibrosis observed in MMP-13 knockout mice, we isolated hepatic stellate cells from rat liver and cultured. The activated stellate cells were treated with CL-82198 to inhibit MMP-13 activity. Immunohistochemical staining for CTGF demonstrated conspicuous staining for CTGF in untreated stellate cells and a marked decrease of staining intensity in the cells treated with CL-82198 (Fig. 9A). Quantitative analysis of CTGF staining intensity depicted around 60% decrease in the treated cells compared to the untreated cells (Fig. 9B). Western blot analysis depicted that blocking of MMP-13 activity resulted in a marked decrease of full-length CTGF and as well as its cleaved fragments and also the active subunit of TGF-b1 indicating MMP-13 is required for the cleavage of both CTGF and TGF-b1 (Fig. 9C).

Discussion
In the present study, we have demonstrated that loss of MMP-13 attenuates NDMA-induced liver injury and fibrosis in a mouse model, and suggested that MMP-13 is involved in the accelerated inflammation by overexpression and activation of CTGF in the impaired liver.
Although the beneficial effects of collagenolytic MMPs such as MMP-1 and MMP-8 on liver fibrosis have been documented [38,39] previous study on a model of the biliary duct ligation-induced cholestasis and fibrosis using MMP-13 KO mice showed a promotional effect of MMP-13 on the initial inflammatory response and subsequent fibrosis [29]. This study provided the data that MMP-13 has a role in the expression of pro-inflammatory mediators such as tumour necrosis factor-a (TNF-a) and Chemokine (C-C motif) ligand 2 (CCL2), which modulate the initial liver damage in their model. The present study not only confirmed the data of promoting effect of MMP-13 on the initial liver damage and fibrosis but also provided the first evidence that CTGF overexpression and activation by MMP-13 are implicated in the process. MMP-13 is a zinc-containing interstitial collagenase capable of degrading native fibrillar collagens and plays a key role in the remodelling of connective tissue matrix under both physiological and pathological conditions [16,40]. In normal and fibrotic livers, expression of collagen types I, III and IV takes place predominantly in non-parenchymal cells [41]. During early fibrogenesis, the hepatic fibrillar collagens are first cleaved by MMP-13 [29] and the partially denatured collagens are subsequently digested by gelatinolytic activities of other MMPs such as MMP-9 [42]. We observed that the mRNA and protein expression of MMP-9 and its activity are markedly increased in NDMA-treated WT mice, and the levels are significantly suppressed in NDMA-treated MMP-13 KO mice. In addition, a-SMA-positive stellate cells were substantially reduced in the liver tissue from NDMA-treated MMP-13 KO mice. Thus, these data strongly suggest that MMP-13 and MMP-9 are involved in the degradation of hepatic ECM in the initial phase of hepatic fibrosis and pave the way for the proliferation of activated hepatic stellate cells that transform into myofibroblasts. The deposition of cross-linked fibrillar collagens, especially collagens types I and III, in the extracellular space of the liver accompanied by defenestration of endothelial cells is the hallmark of hepatic fibrosis [2,8,43]. Previous studies have suggested the implications of CTGF in the pathogenesis of hepatic fibrosis characterized by deposition of fibrillar collagens in the liver [21,[44][45][46]. The full-length CTGF molecule comprises four structural domains, that is modules 1-4, which are susceptible to proteolysis by hepatic stellate cells, yielding C-terminal fragments composed of the modules 3 and 4 or the module 4 alone [46]. Peptide mapping and site-directed mutagenesis study indicated that the sequence I 257 RTPKISKPIKFELSG 272 in the C-terminal region of CTGF is a unique binding domain for integrin avb3 that is sufficient to mediate integrin avb3-and heparan sulphate proteoglycan-dependent (HSPG) stellate cell adhesion [46]. The Nterminal CTGF fragments are reported to be a marker of the fibrotic phenotype in fibrotic diseases such as scleroderma, and both N-terminal and C-terminal fragments of CTGF are suggested to be more potent fibrogenic stimulators compared to the full-length parent molecule [28]. In the present study, we have demonstrated the generation of the C-terminal fragments of CTGF, besides full-length CTGF, in the liver tissues from NDMA-treated WT mice by immunoblotting using the antibody specific to the I 257 RTPKISKPIK-FELSG 272 peptide, and their expression was reduced in NDMA-treated MMP-13 KO mice. Importantly, the inflammation, necrosis, activation of stellate cells, and deposition of collagen fibres were also substantially suppressed in NDMA-treated MMP-13 KO mice. These data suggest that MMP-13 plays a central role in the early inflammatory response through modulation of CTGF expression and processing during the pathogenesis of hepatic fibrosis.
One of the interesting findings in our study is that overproduction of CTGF within the liver tissues of NDMA-treated WT mice was associated with the overexpression of active TGF-b1. TGF-b1 is produced by cells as an inactivated complex form containing latency-associated peptide (LAP) and latent TGF-b-binding protein (LTBP), and activated to the cleaved active TGF-b1 by several pathways, which include the processing by MMP species such as MMP-2 and MMP-9 [47]. In the present study, we could show the activated form of TGF-b1 of 13 kD by immunoblotting of the liver tissues, and a previous study on skin  wound healing in MMP-13 KO mice demonstrated that MMP-13 plays a key role in the activation of latent TGF-b1 [31]. Thus, it may be possible to speculate the implication of MMP-13 in the activation of TGF-b1 within the liver tissues, although the exact activation mechanism of latent TGF-b1 in the present study remains to be clarified. Both CTGF and TGF-b1 exhibit a profound interaction during the pathogenesis of hepatic fibrosis and concurrently elevate in animal models of fibrosis [23,48]. CTGF was originally reported to be transcriptionally induced by TGF-b1 [49]. In human skin fibroblasts, high-level expression of CTGF mRNA and protein is observed only by stimulation with TGF-b1 among several growth factors including TGF-b1, plateletderived growth factor, epidermal growth factor and basic fibroblast growth factor [50]. Nevertheless, the related expression patterns of CTGF and TGF-b1 suggest the possibility that these two molecules act synergistically for the NDMA-induced hepatic injury and fibrosis.
In vitro studies using isolated hepatic stellate cells from rat liver proved that MMP-13 is involved in the cleavage of both CTGF and TGF-b1, thus play a significant role in the pathogenesis of hepatic fibrosis through modulation of the processing of profibrogenic  molecules. CL-82198 is a very specific inhibitor of MMP-13 without affecting the activity of MMP-1 or MMP-9 and blocks 89% of MMP-13 activity at a concentration of 10 lg/ml [35]. As in in vivo studies, inhibition of MMP-13 resulted in a significant decrease of C-terminal fragments of CTGF using the specific antibody that detects C-terminal cleaved subunits. It is important to note that blocking of MMP-13 resulted in a decrease of around 60% of active TGF-b1, which could answer the significant decrease of 38 kD full-length CTGF in the treated cells. It is well established that TGF-b1 regulates the expression of CTGF and TGF-b1 is a requirement for the induction of CTGF [51].
The results of the current in vitro studies further demonstrate that both TGF-b1 and CTGF acts synergistically as in in vivo system and could be the most probable reason for the decrease of 38 kD fulllength CTGF protein in CL-82198 treated hepatic stellate cells.
In summary, we have demonstrated that the NDMA-induced inflammation and injury and subsequent fibrosis of the liver are substantially decreased in MMP-13 KO mice, and these effects of MMP-13 involve the expression and processing of CTGF and TGF-b1. Our study suggests that MMP-13 promotes hepatic damage and fibrosis by modulation of these growth factors within the liver tissues. Thus, blocking of the expression and/or activation of CTGF in early stages of liver injury may pave a way to prevent the progression of hepatic damage and fibrosis.

Author contribution
Joseph George carried out all the major experiments, collected the data, analysed and interpreted the data, and wrote the manuscript. Mikihiro Tsutsumi obtained funding, provided techni-cal and material support, and participated in drafting the manuscript. Mutsumi Tsuchishima involved in research design, analysis and interpretation of the data, and critical review.


extfieldsofstudy: Biology Medicine

text:
Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible.

INTRODUCTION
Finding the primary molecule that was responsible for the initiation of life on Earth is the goal of many studies in the field of evolution. Regarding the "central dogma, " the DNA has been a candidate for the name of the molecule of life. However, fans of the "RNA world theory" explain how life could have been started by the RNAs. The discovery of RNAs with enzymatic activity (Ellington and Szostak, 1990;Robertson and Joyce, 1990;Tuerk and Gold, 1990) and the chemical features of different RNAs-along with the widespread viruses using RNA as their only genetic material-are some clues that help scientists describe the RNA world hypothesis (Pressman et al., 2015). In this theory, it is postulated that RNA and RNA-like molecules, which could fold into a three-dimensional structure with catalytic activities, had played central metabolic roles in the ancient world (Bass and Cech, 1984). Additionally, the double feature of tRNAs to bind with the genetic codes in one loop and their specific binding to amino acids in another stem could further confirm the central role of this molecule in early evolution (Lee et al., 2000;Saito et al., 2001;Murakami et al., 2003;Chumachenko et al., 2009). In this review, we have briefly discussed the importance of the intracellular RNAs in the DNA expansion and its role in shaping the genome to create higher order structures and mechanisms throughout the course of evolution.

TYPES OF RNAs AND INTRACELLULAR INVASIVE RNAs
RNAs had been primarily known as the mediators of the gene expression. However, the different types of RNAs with various roles in the eukaryotic and prokaryotic cells have been discovered. Based on their functions, these molecules can be categorized into four different types: (1) Encoding RNAs that contain the codons for the synthesis of polypeptides.
(2) Structural RNAs [ribonucoeoproteins (RNPs)] that incorporate into the structure of some proteins; thus, they could have played an essential role in maintaining the steady feature and activity of these proteins (Cech and Steitz, 2014). (3) Catalytic RNAs (ribozymes), associated with proteins (RNPs), and mainly involved in the formation of peptide bonds in the peptidyl transferase center of ribosomes, site specific cleavage, ligation of RNAs, and mRNA splicing (Weinger et al., 2004;Keating et al., 2010;Wilson et al., 2016). (4) Regulatory RNAs (riboregulators), which include the non-coding RNAs with various sequences and sizes. These RNAs could regulate the gene expression by targeting mRNAs, leading to the modification of the rRNA, repressions of transposons, and also involved in X-inactivation, chromatin remodeling, and DNA methylation to repress the transcription (Lippman et al., 2004;Esteller, 2011;Cech and Steitz, 2014).
Apart from these functional molecules, the eukaryotic cells also contain RNAs that are exclusively transcribed to be incorporated into the genome by a mechanism called reverse transcription. This process is mainly involved in the construction of telomere (Autexier and Lue, 2006;Lewis and Wuttke, 2012), formation of pseudogenes (Tutar, 2012;Milligan and Lipovich, 2015), and expansion of retrotransposon (Kassiotis and Stoye, 2016). In all these cases, the intracellular RNAs (which we have called "invasive RNAs" in this paper) could be transformed to cDNA in the nucleus and inserted into the genome through the double strand breaks in the DNA. Generally, three types of invasive RNAs can be considered in the eukaryotic cells. Some of these RNAs have been evolved to form specific genomic constructions, such as the telomerase RNA component (TERC), which functions as a template for the extension of telomeres at the end of the eukaryotic chromosomes (Ozturk et al., 2017). Invasive RNAs transcribed from the retrotransposons do not seem to play any pivotal roles in a cells' lifecycle, but have been highly effective during evolution (Cordaux and Batzer, 2009). The DNA might also be attacked by functional RNAs. These RNAs are not naturally invasive, but could be transformed into cDNA by intracellular reverse transcriptase (RTs) and result in the formation of pseudogenes (Tutar, 2012).
The RTs are the key enzymes for RNA invasion. Telomerase and ORF2 (reverse transcriptase produced by retrotransposon) are the two known functional RTs in the eukaryotic cells (Meyer et al., 2017). The role of telomerase is confined to the construction of telomeres by using a specific RNA (TERC) as a template (Lewis and Wuttke, 2012); however, ORF2 uses cytoplasmic RNAs and retroelement transcripts to create pseudogenes and cause retrotransposon expansion respectively (Wei et al., 2001). Interestingly, in some eukaryotes, the retroelement-related RT is responsible for the elongation of the telomere (Biessmann et al., 1992).

INVASIVE RNAs ORIGINATED FROM RETROTRANSPOSONS: STRUCTURAL AND FUNCTIONAL ROLES
Retrotransposons are groups of mobile DNA elements [transposable elements (TEs)] that copy and paste themselves using the RNA molecules (Figure 1). As mentioned in the previous section, these RNA molecules are naturally invasive and are basically transcribed to be randomly inserted into the genome and increase the copy numbers of the retrotransposons (Goodier and Kazazian, 2008). All groups of TEs had been active during the early evolution; however, their selfish and mutagenic movements have resulted in the limitation of their activities to specific types of retrotransposons in the modern human (Marchetto et al., 2013). Long Interspersed Elements (LINE, L1) are the most active retroelement in our cells. It is estimated that the human DNA contains around 500,000 copies of the L1 retrotransposon; however, only 80-100 copies of these elements have maintained their mobility (Goodier and Kazazian, 2008). The structure of a complete L1 element includes a promoter located in the 5 UTR region, an open reading frame (ORF) 1 gene that encodes the RNA binding protein, ORF2 gene that produces a protein with both endonuclease and reverse transcriptase activity in the two different domains, and a 3 UTR providing poly-A-tail for the L1 RNA (Goodier and Kazazian, 2008). The RNA polymerase II apparatus is responsible for the production of the L1 RNA (Burns and Boeke, 2012). The transcribed RNA is then transported to the cytoplasm to produce the ORF1 and ORF2 proteins. This invasive RNA in the complex with ORF1 and ORF2 is transported to the nucleus, where it invades the DNA using endonuclease and reverse transcriptase activity of the ORF2 protein by a mechanism called target prime reverse transcription (TPRT) . The invasive RNAs produced by other retrotransposons (Alu and SVAs) are inserted into the genome through the function of the L1 proteins (Raiz et al., 2012).
It seems that more than the other TEs, the retrotransposons have a greater impact on changing the structure of the DNA and developing specific cellular mechanisms through 100s million years of evolution (Habibi et al., 2015). The retroelement RNA invasions that occurred most often early during evolution have been caused by the genomic expansion and when the DNA is given the space to create structures, such as heterochromatin and centromere (Nigumann et al., 2002) (Figure 2). The human genome project revealed that more than half of our DNA is comprised of non-coding regions. Further evaluation showed FIGURE 1 | Pedigree of Mobile DNA Elements. Transposable elements are categorized in two distinct groups based on their mode of mobilization. DNA transposons move by cut and paste mechanism, however, retrotransposons (retroelements) mobility are mediated by RNAs. Activity of DNA transposons had been fully shut down during evolution, whereas retroelements still show activities in different types of our cells.
FIGURE 2 | Roles of RNA invasion in shaping human genome. Active retroelements in early evolution have been able to actively transpose and increase their copy number by means of their natural invasive RNAs. Retroelements RNAs similar to mRNAs are transcribed and translated by cellular apparatus. The proteins that are encoded by these elements (EN/RT) can bind to cellular RNAs as well as retroelements RNAs, transport them to the nucleus, create DNA breaks, make cDNA, and finally pasting a copy of each RNA in to the genome. Although the movements of these mobile elements are now inhibited remarkably in our cells, 100 million years of their activities have resulted in formation of heterochromatin, centromere, telomere, and pseudogenes. In order to decrease deleterious effects of retrotransposition, inhibitory mechanisms such as DNA methylation, heterochromatinization, and miRNA production have been established by host cell. EN/RT, endonuclease/reverse transcriptase. that these parts of our genome, which mainly construct the heterochromatin, centromeres, telomeres, and gene spacers, include repetitive sequences comprising the remnants of the retrotransposition events (Lander et al., 2001). The importance of the heterochromatin and centromeres in the gene expression, senescence, embryo development, and cell cycle in the eukaryotes has been found in different studies (Eberl et al., 1993;Harmon and Sedat, 2005;Hammoud et al., 2009;Chandra et al., 2015). Therefore, one can conclude that without the actions of these ancient invasive RNAs, our cells would not perform genomic expansion to form the heterochromatin region, centromeres, telomeres, introns, or regulatory elements, and would remain in the prokaryotic phase. On the other hand, the RT enzyme produced by the retroelements could transform the functional cytoplasmic RNAs into invasive molecules to create pseudogenes (Figure 2). This process was essential in the doubling of genes and generation of new genes with different functions throughout the course of evolution (Tutar, 2012). Additionally, it has been shown that the small interfering RNAs transcribed from these pseudogenes might interact with the functional genes in the eukaryotic cells (Ewing, 2017).
Although retrotransposons had been important in the shaping and evolution of the eukaryotic genome, the selfish mobility of these elements would be harmful for DNA integrity and cell viability (Symer et al., 2002). During the retrotransposons' lifecycle, the invasion of the RNAs by means of the endonuclease/RT enzyme could break the genes, disrupt the open-reading frames, and, finally, affect the production of proteins (Dupuy et al., 2001). The excessive activity of endonuclease produced by the retroelements could also induce excessive DNA double strand breaks (Chen et al., 2006). On the other hand, the promoter region of these elements might also be copied and inserted near the genes and thus influence the quantity and quality of the gene expression (Cordaux and Batzer, 2009). Different lines of studies have shown the increased levels of L1 retrotransposition in different types of cancers (Shpyleva et al., 2017), schizophrenia (Bundo et al., 2014;Doyle et al., 2017), autism (Shpyleva et al., 2017), and Rett syndrome (Muotri et al., 2010), emphasizing the pathogenic role of these elements in the human cells. Regarding these potential threats, the eukaryotic cells have developed repressive mechanisms, including epigenetic modifications (DNA methylation, heterochromatinization), miRNAs, and piRNAs expressions, to inhibit and control the activity of the TEs (mainly retrotransposons) (Habibi et al., 2015). All these repressive pathways have other roles at present rather than the retrotransposons repression inside the cells. Therefore, we can emphasize that the embryo development, differential gene expression, cell differentiation, and specifications would not have occurred without the development of repressive mechanisms against intracellular invasive RNAs.

CONCLUSION
Various types of RNAs have been discovered that play a role in the different aspects of the gene expression. Here, we have described another kind of RNAs that are transcribed to invade the DNA and increase their source (retroelement) copy number. These ancient RNAs have a pivotal role in increasing the size of the DNA, establishing heterochromatin, centromeres, telomeres, methylation processes, epigenetic mechanisms, miRNA production, etc. through 100 million years of evolution.
Regardless of the advantageous evolutionary roles; the activities of retrotransposons and their invasive RNAs are highly inhibited in fully differentiated cells (Wissing et al., 2012) since such invasions could be harmful for the genomic integrity of evolved cells. However, different lines of studies showed increased retroelements movements in neural precursor cells (Muotri et al., 2005), embryonic stem cells (Kano et al., 2009) as well as germ cells (Georgiou et al., 2009). One could suggest two ideas for this exceptional high activity of the retrotransposons; (1) in all these cells we are facing to vast changes in epigenetic status of DNA including hypomethylation which could remove the lock of the retroelements and give them chance to increase their movements as side effect of epigenetic changes. These random RNA insertions might result in neurodevelopmental disorders (McConnell et al., 2017) and different kind of cancers (Kano et al., 2009). (2) These increased retrotransposition might do have functional role such as memory storage in neurons (Habibi et al., 2009) and involving in the survival of the organism (Sciamanna et al., 2011). Totally, all these aspects of intracellular invasive RNAs life cycle could show the importance of these elements in creating complex organisms during the evolution.

AUTHOR CONTRIBUTIONS
LH: collected data, wrote the paper, designed and drew figures; HS: collected data.

FUNDING
This article is funded by LH.


s2fieldsofstudy: Biology Medicine

text:
Anti-NKG2D mAb: A New Treatment for Crohn’s Disease?

Crohn’s disease (CD) and ulcerative colitis (UC) are immunologically-mediated, debilitating conditions resulting from destructive inflammation of the gastrointestinal tract. The pathogenesis of IBD is incompletely understood, but is considered to be the result of an abnormal immune response with a wide range of cell types and proteins involved. Natural Killer Group 2D (NKG2D) is an activating receptor constitutively expressed on human Natural Killer (NK), γδ T, mucosal-associated invariant T (MAIT), CD56+ T, and CD8+ T cells. Activation of NKG2D triggers cellular proliferation, cytokine production, and target cell killing. Research into the NKG2D mechanism of action has primarily been focused on cancer and viral infections where cytotoxicity evasion is a concern. In human inflammatory bowel disease (IBD) this system is less characterized, but the ligands have been shown to be highly expressed during intestinal inflammation and the following receptor activation may contribute to tissue degeneration. A recent phase II clinical trial showed that an antibody against NKG2D induced clinical remission of CD in some patients, suggesting NKG2D and its ligands to be of importance in the pathogenesis of CD. This review will describe the receptor and its ligands in intestinal tissues and the clinical potential of blocking NKG2D in Crohn’s disease.

Introduction
Crohn's disease (CD), an inflammatory bowel disease (IBD), is a complex immunologicallymediated chronic illness that is believed to arise due to a dysregulated immune response to microbiota in the gastrointestinal system. CD is characterized by patchy and transmural inflammation that can occur throughout the gastrointestinal tract, with alternating phases of clinical relapse and remission [1]. CD typically arises between 20 and 30 years of age with symptoms such as abdominal pain, fever, diarrhea with or without bleeding, and weight loss [2]. Despite newer and better therapies, Crohn's disease often presents a heavy everyday burden, sometimes leading to surgery and disability [3,4]. The biologic antibodies in clinical development adopt parallel mechanisms of action with the same or other targets, namely blocking of a variety of cytokines, inhibition of the similar protein-dependent migration mechanism or neutralization of chemokines. However, other mechanisms may be involved in the pathogenesis of IBD [5,6].
The immune activating receptor Natural Killer Group 2D (NKG2D) has been implicated in the pathogenesis of IBD through its presence on intestinal cytotoxic lymphocytes and the increased expression of activating ligands on inflamed tissue [7]. Normally, the NKG2D receptor and its eight different ligands are a part of a system designed to participate in the recognition of "stressed" cells exposed to viruses or tumor growth. NKG2D is then activated on natural killer (NK) or CD8 T cells to eliminate the ligand-bearing cell as an effective defense mechanism as well as producing pro-inflammatory cytokines. For years, this pathway has been a potentially attractive approach to

The Natural Killer Group 2D (NKG2D) Receptor
NKG2D is an activating receptor expressed on human NK, γδ T, mucosal-associated invariant T (MAIT), CD56 + T, and CD8 + T cells [8,15], which can participate in the recognition of inducible "stressed-self" ligands on the surface of target cells. This will include cells acting as intra-epithelial T cells in the intestinal wall [16]. Activation of NKG2D triggers cellular proliferation, cytokine production, and target cell killing [17]. Under normal conditions in the disease-free human system, the receptor is constitutively expressed on all the lymphocytes listed above in both peripheral blood and the intestinal tissues, but also to a low degree on a subset of CD4 + T cells [18].
NKG2D, or CD314, is encoded by the gene KLRK1 (killer cell lectin-like receptor of the subfamily K member 1) and is found on human chromosome 12. The gene sits close to KLRD1 (CD94) and the cluster of KLRC1 (NKG2A), KLRC2 (NKG2C), KLRC3 (NKG2E), and KLRC4 (NKG2F), which are both activating and inhibitory receptors binding to distinct sets of HLA-E residues [19,20]. With just two alleles different by one amino acid, the KLRK1 gene has limited polymorphism and only one isoform encoding a functional protein has been described in humans. All mammals have KLRK1 orthologs, indicating that the gene is highly conserved during evolution and is an important function across species [21]. NKG2D is a type II membrane protein comprising 216 amino acids with a predicted molecular weight of 25,143 kDa. The protein has an N-terminal intracellular region, a transmembrane domain, a membrane-proximal stalk region, and an extracellular region with a single C-type lectin-like domain. KLRK1 is expressed on the cell surface as a disulfide-bonded homodimer with a molecular weight of approximately 42 kDa when analyzed under reducing conditions and approximately 80 kDa under non-reducing conditions. A cysteine residue just outside the transmembrane region forms the disulfide bond joining the two subunits of the homodimer ( Figure 1) [22]. In the adaptive immune system, T cells are mostly dependent on the T cell receptor complex as a result of somatic recombination of genes to recognize and respond to antigens. For NK cells in the innate system, the activation or inhibition of cell signals relies on a wide range of surface receptors where some are shared with subsets of T cells. The NKG2 receptor family are shared between cell types underlining a central role in tumor and pathogen recognition [23]. The frequency of NKG2D + expression is consistently high on NK and CD8 + T cells and consistently low on CD4 + T cells in humans in the steady state, with other subsets showing differentiating expression [7,24]. While the other members of NKG2 family form heterodimers with CD94, the NKG2D receptor forms stable homodimers on the surface when stabilizing non-covalently with its adaptor molecule DAP10 (DNAX-activating protein of 10 kDa) on the inside of the cell membrane. DAP10 is also a disulfide-linked homodimer [18,25] (Figure 1). NKG2D associates with two DAP10 proteins in the transmembrane region via charged residues within the receptor and its adapter subunits and creates a hexameric complex. A charged amino acid residue (aspartic acid) centrally located within the transmembrane region of DAP10, forms a salt bridge with a charged amino acid residue (arginine) in the transmembrane region of NKG2D to stabilize the receptor complex. Upon interaction with its ligands, the complex transmits an activation signal triggering

The NKG2D Ligands
Eight human ligands for NKG2D have been identified: Human Major Histocompatibility Complex (MHC) class I polypeptide-related sequence A (MICA), MICB, and UL16 binding protein (ULBP) 1-6 (also named RAET1E, RAET1G, RAET1H, RAET1I, RAET1L, and RAET1N). All are located on chromosome 6 within the Major Histocompatibility Complex and have various binding affinities for NKG2D ranging from 10 −6 to 10 −9 M, with different homology between ligands down to 25%, and with extensive allelic polymorphisms [26,27]. MICA encodes 79 protein variants and MICB encodes 26 protein variants, while the ULBPs exhibit a lower level of polymorphism [28,29]. MICA, MICB and ULBP4 and 5 share a structure of having a transmembrane anchor in the cell and an intracellular cytoplasmic tail, while ULBP1-3 and 6 have a glycophosphatidylinositol (GPI)-anchored

The NKG2D Ligands
Eight human ligands for NKG2D have been identified: Human Major Histocompatibility Complex (MHC) class I polypeptide-related sequence A (MICA), MICB, and UL16 binding protein (ULBP) 1-6 (also named RAET1E, RAET1G, RAET1H, RAET1I, RAET1L, and RAET1N). All are located on chromosome 6 within the Major Histocompatibility Complex and have various binding affinities for NKG2D ranging from 10 −6 to 10 −9 M, with different homology between ligands down to 25%, and with extensive allelic polymorphisms [26,27]. MICA encodes 79 protein variants and MICB encodes 26 protein variants, while the ULBPs exhibit a lower level of polymorphism [28,29]. MICA, MICB and ULBP4 and 5 share a structure of having a transmembrane anchor in the cell and an intracellular cytoplasmic tail, while ULBP1-3 and 6 have a glycophosphatidylinositol (GPI)-anchored organization. MICA and MICB carry three extracellular immunoglobulin-like domains (α1, α2, α3,) and ULBP1-6 carry two domains (α1, α2). However, unlike class I, the ligands are not covalently bound to a monomorphic β2 microglobulin, and its peptide binding groove is empty and does not present peptides [30] (Figure 1). ULBP2 has the unique feature to be expressed at the cell surface either as a transmembrane or a GPI-anchored protein [31]. Allelic variants of the NKG2D ligands have been reported to result in large differences in the affinity of binding to NKG2D. For instance, MICA alleles with a single amino acid substitution at position 129 in the α2 domain of methionine (M) or valine (V) have been classified as having strong or weak binding affinity for NKG2D, respectively. These variable affinities have been suggested to affect thresholds of NK cell triggering and T cell modulation and consequently influencing clinical phenotypes in autoimmune disorders and malignancies [32][33][34].
NKG2D-ligand complex crystal structures have been described for some of the ligands and the binding of the different proteins seem to be adaptive fit mechanisms in the receptor [35]. Although transcripts are present in some healthy cell types [36], the ligand proteins are rarely present on the cell surface of healthy cells and tissues, but are inducible by virus infection, tumorigenesis, or by stimuli, such as DNA damage, oxidative stress, heat shock, toll-like receptor signaling, or cytokine exposure [16,[37][38][39]. While not present under normal conditions, it seems that every cell type has the ability to express one or several of the ligands under one of the conditions listed above, or by the appropriate stimuli, which can be induced with pro-inflammatory signals. The induction of NKG2D ligand expression is attributed to cellular "stress" and every type of cancer has the same mechanism. Accelerated proliferation other than cancer development can also result in upregulated ligand expression, for instance healthy cells in embryonic tissues and tissues undergoing wound repair [40][41][42]. Increased MICA expression has been reported in tissues with inflammation and autoimmune diseases such as type 1 diabetes, celiac disease, rheumatoid arthritis [43][44][45], in atherosclerosis, where it is found on vascular endothelial cells [45][46][47], and in asthma [11]. MICB has been located on many of the same sites, but the ULBPs are generally underreported in these diseases.
The regulation of NKG2D ligand transcripts is a complex process including epigenetics, RNA degradation, microRNA interference, and regulation of the protein is subject to cleavage from the surface by membrane metalloproteases or intracellular degradation by ubiquitin ligases [38]. This regulation is also different between cell types and maturation statuses.

Immunological Pathway of NKG2D
NKG2D serves as a sensor for recognition of "induced self" for the detection and removal of hyper-proliferative cells, transformed cells, or cells infected by pathogens. As with the ligands, signaling and regulation by the NKG2D receptor in NK cells and T cells is complex and incompletely understood. While NKG2D is expressed constitutively on essentially all resting human NK cells and CD8 + T cells [48], engagement of NKG2D alone is not sufficient to trigger cell-mediated cytotoxicity or cytokine production [49,50]. The simultaneous engagement of NKG2D and other costimulatory receptors, such as CD335 (NKp46) or CD244 can, under the right circumstances, result in cytolytic activity in resting NK cells [49]. NK cells can also be primed by culture in IL-2 or IL-15-resembling some inflammatory sites-and engagement of NKG2D alone will then be sufficient to initiate degranulation and cytokine production. The result can be increased killing of the ligand-expressing cells and/or production of cytokines, such as IFN-γ, TNF-α, and GM-CSF enhancing the inflammatory environment [51][52][53].
As described, each NKG2D homodimer associates with two DAP10 homodimers to form a hexameric receptor complex ( Figure 1) [25]. The related DAP12 adaptor protein was previously described as another transducer in this mechanism, but the protein, which signals through an ITAM region, does not form stable complexes with human NKG2D, unlike in mouse, thus identifying a species difference, and was reviewed in [54]. The adaptor molecule of NKG2D, DAP10 has a YINM motif, which recruits a p85 PI3 kinase and Vav-1 signaling complex [22,55]. Once DAP10 is activated, and when the complex is binding to a ligand, the stimulation leads to a PI3K-dependent Akt phosphorylation (alternatively through Vav1), which presumably activates cell survival pathways, NKG2D-mediated calcium release, cytokine production, and cytotoxicity [17]. IL-15 seems to be an essential cytokine for the phosphorylation of the YINM motif on DAP10 leading to the activation of PI3K or Vav-1 [23]. The mechanisms can be distinct, with both PI3K and Vav-1 recruiting Grb2 for downstream signaling, but Vav-1 uses the PLCγ2→Ca 2+ or SLP-76 pathway for granule release and cytotoxicity, while PI3K signals through MEK→ERK or directly via Ca 2+ increases. DAP10 phosphorylation can also result in cytotoxicity through JNK→cJunN. If PI3K signals Akt instead, the result seems to be increased survival of the lymphocyte. The release of pro-inflammatory cytokines is, instead, believed to happen in the DAP10→JAK2→STAT5 pathway [17] (Figure 2). 18,1997 5 of 17 NKG2D-mediated calcium release, cytokine production, and cytotoxicity [17]. IL-15 seems to be an essential cytokine for the phosphorylation of the YINM motif on DAP10 leading to the activation of PI3K or Vav-1 [23]. The mechanisms can be distinct, with both PI3K and Vav-1 recruiting Grb2 for downstream signaling, but Vav-1 uses the PLCγ2→Ca 2+ or SLP-76 pathway for granule release and cytotoxicity, while PI3K signals through MEK→ERK or directly via Ca 2+ increases. DAP10 phosphorylation can also result in cytotoxicity through JNK→cJunN. If PI3K signals Akt instead, the result seems to be increased survival of the lymphocyte. The release of pro-inflammatory cytokines is, instead, believed to happen in the DAP10→JAK2→STAT5 pathway [17] (Figure 2). In addition to the function of being a potent activating receptor, NKG2D is reportedly involved in the normal development of NK and T cell function, and even in B cells maturation from hemapoietic precursors. These potential regulatory roles are reviewed elsewhere, but should be observed through the development of the blocking antibody against NKG2D [56]. In addition to the function of being a potent activating receptor, NKG2D is reportedly involved in the normal development of NK and T cell function, and even in B cells maturation from hemapoietic precursors. These potential regulatory roles are reviewed elsewhere, but should be observed through the development of the blocking antibody against NKG2D [56].

Cancer Evasion and Infection Control
Most knowledge about NKG2D and its ligands comes from cancer and infectious disease research. NKG2D ligands are induced in cells infected with intracellular bacteria and viruses and can, for instance, be induced on dendritic cells responding to microbial pathogens [38]. Pathogens can seemingly induce expression of any of the ligands in a range of different cell types without general rules and it might be dependent on other external factors as well. However, viruses have evolved complicated mechanisms to prevent a given cell from expressing ligands or to truncate the protein to make it soluble or captured intracellularly, underlining the importance of the NKG2D receptor activation. Soluble ligand protein can then antagonize NKG2D to avoid detection by T or NK cells [57,58]. The diversity of NKG2D ligand genes and their polymorphism is likely driven by the pathogens' evolving mechanisms to escape detection by immune cells through NKG2D pathway.
NKG2D ligands are expressed on all cell types by cancers [59]. In some cases, ligands may be induced due to the genomic instability of the transformed cells, resulting in activation of DNA repair pathways [60], but the factors causing NKG2D ligand expression depend on the transformed cells, as well as exposure to cytokines and other factors in the microenvironment [38]. Hyper-proliferation of transformed cells might be another reason for induction caused by activation of transcription factors [42]. Like viruses, primary tumors frequently develop mechanisms for avoiding detection and elimination by lymphocytes. These include systemic release of soluble NKG2D ligands [61,62]. Additionally, when NK or T cells encounter cells bearing NKG2D ligands, a mechanism down-modulates the receptor [63,64]. In addition, tumor-derived factors, such as anti-inflammatory TGF-β, can cause down-regulation of the NKG2D receptor on NK cells and T cells [65][66][67]. These pathways are further reviewed in [59].

NKG2D in Crohn's Disease
NKG2D on lymphocytes and its ligands MICA, MICB, and ULBP1-6 modulate T and NK cell activity and may contribute to IBD pathogenesis. However, only a few studies have addressed the relation of NKG2D to IBD and especially the ligands are poorly characterized [7,12,[68][69][70][71][72].
NKG2D is expressed to a high degree on NK and CD8 + T cells in CD also, but can also be detected on CD4 + T cells in inflamed CD intestine [7,73]. The expression level on CD4 + T cells have been shown to increase with Crohn's disease in the lamina propria compared with controls and patients with ulcerative colitis. CD4 + NKG2D + T cells with a Th1 cytokine profile and expressing perforin were increased in the periphery and in the mucosa in CD. CD4 + NKG2D + T cell clones were functionally active through MICA-NKG2D interactions, producing interferon-γ and killing targets expressing MICA [7]. This defines a subset of CD4 + T cells shown to have cytotoxic and inflammatory properties. Other T cell types can show heterogeneous results between patients. The differences in the NKG2D expression with disease stage might be caused by ligand-induced internalization or due to differences in cytokines in the mucosal gut tissue, as several cytokines are known to either increase [74][75][76][77] or decrease [65][66][67]78,79] NKG2D on NK and T cell subsets. CD56 + αβ T and γδ T cells have previously been found to express NKG2D differently [80] and the balance between these two cell types might be a modulating factor between self-tolerance and autoimmunity [81]. Our group has found that NKG2D expression on γδ T cells (CD45 + CD3 + γδTCR + ) correlate negatively with C-reactive Protein (CRP), while CD56 + T cells (CD45 + CD3 + γδTCR − CD56 + ) correlate positively. This might be connected to the finding that NKG2D expression on γδ T cells also correlates negatively to the cells production of GM-CSF, but positively to the release of IL-10, both measured on the protein level. The expression level of NKG2D on CD56 + T cells correlate to the two cytokines in the complete opposite way, which implies that pro-and anti-inflammatory cytokines influence the NKG2D molecular pathway [73]. Notably, γδ T cells constitute~40% of the intraepithelial lymphocytes (IEL) [82,83], and might have a protective role in IBD [84]. CD56 + αβ T cells are activated T cells and may participate in the inflammatory response. Unlike conventional naïve T cells, IELs do not need priming and they will immediately release cytokines or mediate killing of infected target cells. CD8 + T and γδ T cells are most common IELs [85]. These activated T cell subsets or all available activated NK cells can release interferon-gamma (IFN-γ) and use cytotoxic killing without prior cell activation, but just by stimulation of FcRs or NK receptors such as CD94 or NKG2D by stressed or infected cells. Inhibitory lymphocyte receptors, like the heavily-studied PD-1, will ensure that self will be preserved under immunosurveillance by these innate subsets and non-self without ligands for inhibitory receptors will be killed if activated through NKG2D [86].
There could be a tendency toward the down-regulation of the NKG2D receptor on lymphocytes in inflamed CD tissue compared to non-inflamed CD tissue and normal controls. Down-regulation of NKG2D protein is not due to a decrease in NKG2D mRNA levels in CD patients versus normal controls. This lower NKG2D expression in inflamed tissue likely results from either the cytokine balance as mentioned or by increased ligand expression in the inflamed mucosal gut tissue, causing ligand-induced down-regulation of NKG2D [87][88][89].

Upregulated Ligands in Crohn's Disease
Weak MICA expression has been reported on the cell surface of some healthy cell types including epithelial cells in the gut [90,91]. Increased MICA expression has also been reported in autoimmune diseases, such as type 1 diabetes, celiac disease, rheumatoid arthritis, and atherosclerosis, where it is found on vascular endothelial cells [43][44][45][46][47]. Additionally, associations between MICA alleles and thyroid disease and Addison's disease have been observed, pointing to these factors of innate immunity contributing to the pathogenesis of autoimmune disorders [92,93]. Increased levels of MICA and/or MICB have also been observed on epithelial cells and monocytes in CD patients, where they may trigger cytokine release and cytolytic activity [7,12,68,94]. Allez et al. described how increased MICA expression could prime NKG2D + CD4 + T cells to kill the ligand bearing cells while also producing high levels of IFN-γ and IL-15 [7]. Analyses of ULBP1-6 expression in human CD are incomplete, but ULBP1 and ULBP2 expression have been reported on intestinal monocytes from pediatric IBD patients. This study identified an increase of MICA/B + , ULBP1 + , and ULBP2 + cells from mucosal infiltrates in tissue sections from active disease only, not when disease was in remission or in normal controls [12]. The fact that MICA is typically more expressed than MICB could be due to gene promotor polymorphisms [95], and whether this is also the case for the ULBPs needs to be addressed. mRNA differences for MICA in CD patients compared to normal controls have previously been reported [96]. Additionally, several reports have linked polymorphisms in MICA and MICB to increased prevalence of IBD [70,97,98], and other polymorphisms in MICA that, on the contrary, protects from ulcerative colitis, have been found [69].
We have recently shown the presence of all eight ligands on intestinal monocytes and B cells in CD patients by flow cytometry, as well as on endothelial and epithelial cells [73]. To be able to show this, we had to produce antibodies for ULBP3-6, which could explain the few studies on these ligands. The level of expression was highly heterogeneous and differed widely between cell types. No difference in the average expression levels was detected between MICA and MICB, and the ULBPs, but at the mRNA level, the ULBPs were generally more produced. The presence on epithelial cells could be part of the pathogenesis of CD, if lymphocytes eliminate the gut mucosal wall because of ligand presence in the inflammatory microenvironment. The expression of some of the ligands was tended to increase with higher concentrations of pro-inflammatory cytokines like IL-1β and TNF-α.
A better characterization of the expression pattern and functional role of NKG2D ligands in CD is warranted to improve the understanding of the mechanism-of-action by NKG2D blockade therapy and the pathway itself.

NKG2D Pathway Contribution to CD Pathology
Loss of tolerance towards microbiota crossing the epithelial barrier is likely the initiation of CD, and the pathology accelerates and persists due to the increased amount of immune cells and the mix of cytokines and chemokines creating inflammation. The pathologic response could start with the innate immune system and would then be mediated by antigen presenting cells (APCs) to activate adaptive immunity. Increased signaling or ineffective processing and clearance by epithelial cells, monocytes, neutrophils or dendritic cells (DCs) could lead to an exaggerated cytokine release resulting in an overly aggressive acquired T cell environment [99]. The inflammatory environment in turn activates other cells like cytotoxic Innate like lymphocytes (ILCs), NK cells, CD8 + T and CD4 + T cells capable of killing epithelial cells and other APCs and thus increasing inflammation. The cytotoxic cells can recognize the stress-induced ligands for activating receptors. This pathway could lead to degradation of the ligand-bearing epithelium and result in more microbial influx generating even more inflammation [100,101]. Translational research linked the properties of NKG2D-activated lymphocytes to the potential contribution to pathogenesis in Crohn's disease. CD4 + T cells expressing NKG2D has been observed to be increased in CD. Like innate lymphocytes, these cells are able to kill NKG2D ligand-bearing cells and produce pro-inflammatory cytokines like IL-17, TNF-α and IFN-γ [7]. It has been shown that most of the oligoclonal expansion of mucosal T cells in CD consists of NKG2D-expressing CD4 + T cells [102]. This expansion will increase concentration of pathogenic TNF-α and IL-17 [103], which has been proven to contribute to disease by the registration of a biologic working through the IL-23/Th17 pathway [104,105]. The implication of CD4 + NKG2D + T cells in gut inflammation has been further shown in a murine model of transfer-induced colitis. In these studies, administration of a specific NKG2D blocking antibody decreased NKG2D expression on CD4 + T cells, blocked the receptor on the other lymphocyte subsets and attenuated the development of colitis, highlighting NKG2D as a possible therapeutic target in IBD [9,10]. Additionally, from coeliac disease it was shown how NKG2D modulates the disease through the cytotoxicity of NK and CD8 + T cells, indicating potential for a blockage in autoimmune diseases [47,106].
By blocking the NKG2D receptor with an antibody in human patients, the ligands will be physically unable to bind resulting in a reduced cytotoxic microenvironment and reduced killing of target cells in CD [107]. Our group also suggests an additional effect, namely a reduced ability of NKG2D-expressing lymphocytes to cross a ligand-expressing endothelial barrier if blocked by anti-NKG2D antibody [73]. Microvascular intestinal endothelial cells expressing MICA covered a transwell with activated human CD8 + T cells above. When attracted through the monolayer by a chemoattractant, blocking of NKG2D resulted in significant inhibition of T cells migrating through. In that way, fewer activated lymphocytes bearing NKG2D could enter the lamina propria of affected intestinal sites.
Besides an increased cytotoxic microenvironment and granular release by lymphocytes activated by a NKG2D ligand interaction, the receptor can also be shown to contribute to the inflammatory balance. Using the previously described explant assay method [108], we added anti-NKG2D mAb to CD intestinal biopsies and observed a tendency towards a decreased cytokine production by the mucosal cells when the NKG2D pathway was blocked (Figure 3). bearing NKG2D could enter the lamina propria of affected intestinal sites.
Besides an increased cytotoxic microenvironment and granular release by lymphocytes activated by a NKG2D ligand interaction, the receptor can also be shown to contribute to the inflammatory balance. Using the previously described explant assay method [108], we added anti-NKG2D mAb to CD intestinal biopsies and observed a tendency towards a decreased cytokine production by the mucosal cells when the NKG2D pathway was blocked (Figure 3).

Phase IIa Results
Animal experiments suggest a link between NKG2D and IBD, as NKG2D blockade can attenuate disease progression in certain mouse models of colitis [9,10]. In CD patients, a recent phase IIa study using a human blocking antibody against NKG2D showed significantly increased clinical remission after 12 weeks, suggesting that NKG2D-ligand interactions are viable therapeutic targets [13]. In the randomized, double-blind, parallel-group, placebo-controlled, single-dose, phase 2a trial, 78 CD patients were given either placebo or 2 mg/kg anti-NKG2D mAb and followed for 24 weeks. The primary endpoint was clinical response at four weeks. The trial was stopped prematurely primarily due to a slow recruitment rate and a negative futility analysis of the primary endpoint. However, a significant effect was achieved on the secondary endpoints at week 12 for both the Crohn's disease activity index (CDAI) score and the Harvey Bradshaw (HBI) score (∆CDAI = −55; p < 0.1 and ∆HBI = −2.7; p < 0.1). In patients naïve to biologic treatment, the CDAI difference from placebo was significant from week 1 through week 12 (p < 0.1). An exposure-response analysis for patients with CDAI > 330 was published suggesting that higher doses and repeated dosing may optimize therapeutic benefit. The failure to reach the primary end point could be due to the dose applied was too low in combination with the rather low number of patients included, but occupancy of the receptor did show to be over 80% for the first eight weeks, falling to~20% by week 12. Higher doses have been applied in rheumatoid arthritis and a dose response study had not been performed in Crohn's patients before initiation of the study. Furthermore, multiple dosing in the first 4-8 weeks should have been considered similar to dosage regimens for anti-TNF treatment in Crohn's disease. The drug was well-tolerated with no evidence of immunogenicity. The findings of this small sized randomized study encourage to further randomized studies in Crohn's disease with larger sample size, optimizing dose regimens and maybe prolongation of primary endpoint evaluation to 6-8 weeks.

Phase IIb Initiation
Two new clinical studies with the anti-NKG2D biologic have been initiated; a phase IIb investigating the safety and efficacy of the drug in participants with moderately to severely active Crohn's disease including 450 patients for 22 weeks with subcutaneous doses up to 400 mg induction and 200 mg every four weeks [109], and a safety study in healthy Japanese and Caucasian male participants also testing tolerability, pharmacokinetics, and pharmacodynamics following subcutaneous drug injections [110].

Future Perspectives
The introduction of anti-TNF-α monoclonal antibodies (mAb) (e.g., Infliximab) was a great advance in CD, leading to improved remission and maintenance hereof in patients with insufficient, or lack of, effect of immunomodulators [111]. The drug has low adverse effects, but only 60-70% of patients will respond and, of responders,~40% will lose effect within the first year [112].
Targeting and blocking NKG2D would be another new mechanism of action for moderate to severe Crohn's disease patients. Administration of a blocking antibody against NKG2D has been shown to significantly increase clinical remission in CD patients [107], most likely a result of the NKG2D blockade leading to abrogation of lymphocyte cytotoxicity and cytokine production, but it might also influence migration, recruitment, and retention of inflammatory cells into affected tissue [113]. Tissue-specific homing involves tethering, activation, and firm adhesion steps [114], and the activation might be targeted here. Activation of intestine-derived T cells has been shown to increase their migration [115]. In multiple sclerosis, a CD4 + T cell NKG2D-dependent migration mechanism has been observed [116]. It is therefore possible that the NKG2D and NKG2D-ligand interaction may provide an activating signal to the NK and T cells promoting successful migration, as we have proposed as a supporting mechanism [73]. In this way, anti-NKG2D antibody may uniquely interfere with both intestinal inflammation and lymphocyte homing, the two main processes targeted by current biological therapies for CD and ulcerative colitis [117,118]. Both the mechanism of cytotoxicity and the possible effect on migration should be investigated further. It would be of special interest to know which cells in the CD intestine are targeted for elimination through this pathway and which ligands play integrant roles.
More research is needed into the mechanism of action of anti-NKG2D and its therapeutic effect, especially from a genetic perspective. Single nucleotide polymorphism (SNP) is implicated in the extensive polymorphism of the ligands and relates to the binding strength of the receptor and handling of pathogens [119,120]. The single SNP at the protein level that exists for KLRK1 can also produce differences in NKG2D function. NKG2D can vary in the intensity of cell surface expression due to genetic polymorphisms. The functional consequences of polymorphisms in NKG2D and NKG2D ligands may be cooperative or counteracting. The interaction of the variants could be highly important for the outcome of NKG2D signaling and disease associations of the NKG2D signaling pathway [119,121]. This could result in diverse efficacy between patient populations exposed to the antibody.
It could be of great interest to further investigate each NKG2D ligand to explore the exact differences between them and how they are regulated. Evolutionarily, eight ligands to one receptor indicate the importance of the interaction or differences in mechanism. The blocking of a specific ligand instead of the receptor could induce different results in CD patients.
Patients with moderate to severe Crohn's disease, who have failed conventional treatment, and maybe some biologics, could be candidates for treatment with a drug with a new mode of action. Given the natural mechanism of NKG2D-ligand interactions in the human system, special attention should be paid to any adverse effects concerning malignancies and infections in the development and use of a blocking antibody for the use in CD.

Concluding Remarks
The NKG2D pathway poses an attractive new treatment option for Crohn's disease. The receptor prevalence is high, its biology well-described, and it is straightforward to block the activity through a monoclonal antibody-presumably with limited adverse effects. This could potentially abrogate lymphocyte destruction of the gut tissue and the system's contribution to the inflammatory pathogenesis. However, surprisingly little is known about the ligand prevalence and biology, especially in autoimmune diseases. Excellent commercial antibodies for all eight ligands are now available and further research should be directed towards the characterization of ligand-expressing cell types in IBD and the mechanism of action through this potent pathway.

Conflicts of Interest:
The authors declare no conflict of interest.