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Defensins were first identified as antimicrobial peptides and later several lines of evidence suggest that they can interact with host immune cells, thereby playing important roles in both innate and adaptive immune responses against bacterial infection. They are classified into Î±, Î² and Î¸ Defensins (Shi, 2007). This dissertation reports the investigation of the expression of Defensin genes; Î± Defensins 1, 4, 5 and 6 and Î² Defensin 1, 4 and 5 in monocytic cell line, THP-1, using reverse transcriptase polymerase chain reaction (RT-PCR).
First experiments were undertaken to elucidate the effectiveness of RNA isolation from THP-1 cells exposed to LPS for upto 24 hours. Defensins mRNA expression by monocytes is thought to be increased after activation with lipopolysaccharide (LPS) in a dose and time-dependent manner (Duits, et al., 2002). Therefore, the THP-1 cell lines were pre treated with LPS for different times, 6 hours and 24 hours, so as to compare the expressed defensins from these cells with other cells which were not treated with LPS.
4.1 RNA extraction
The RNA was extracted and purified from the THP-1 cell lines using Trizol and a mixture of phenol-chloroform. The RNA yields vary depending on the, the culturing of cells, thoroughness of processing the sample and overall handling of samples as not to degrade the RNA.
4.2 RNA concentration and purity
The concentration and purity of RNA yield was assayed spectrophotometrically using the GeneQuant (Amersham Biosciences) taking the advantage of the ability of nucleic acid to absorb ultraviolet light with absorbance maximum at 260 nm.
Calculation based on the absorbance of 260 nm provides little information about the quality and purity of the sample as the presence of the excess salt, contaminating proteins or the carryover of any solvent may affect the absorbance significantly (Farrell, 2007). Therefore, measurements of A260/A280 ratio and A260/A230 ratio were used as they provide better estimation on the purity of the dilution.
A pure RNA sample has A260/A280 ratio of 2.0 ± 0.1 and A260/A230 ratio should be greater than 1.7 (Farrell, 2007). The A260/A280 ratio for untreated THP-1 cells in this experiment was 1.59, 1.595 for cells exposed to LPS for 6 hours and 1.64, for 24 hours. These results are all below the expected range of A260/A280 ratio (1.9 - 2.1) which indicate that the RNA yield may not be pure. Similarly, the A260/A230 ratio obtained were lower than1.7. This may be due to many reasons. The accuracy of A260 absorbance as an indicator of concentration and A260/A280 as a purity indicator have been found to be interfered by pH of the dilution and is most reliable when measurement are made between 7.5 and 8.5 (Farrell, 2007). In this experiment RNase-free water was used, which pH value was not measured, while it is recommended to use buffered solution as the water often has an acidic pH and can lower the A260/A280 ratio (Ambion, 2009). The TE buffer, Tris and EDTA, could be used as an alternative to the RNase-free water (Ipsogen, 2008).
Lower A260/A280 ratio, less than 1.9, usually indicates protein contamination while isolating the RNA and low A260/A230 ratio is mostly caused by improper drying of sample or carryover of chloroform which may not be removed properly even after washing with 70% ethanol. Therefore, the lower values might be due to the incomplete removal of protein from the cell lysate or carryover of chloroform or phenol used during isolation of RNA. In addition, technical errors while pipetting, preparing and mixing of dilution might also affect the readings. As spectrophotometric method can not discriminate between RNA and DNA, samples have to first be resuspended with RNase-free water (Scheppler, et al., 2000). Therefore, the result might also be affected by improper treatment of sample. Fluorometric assays can be used, as they are more specific and sensitive for quantitation and they can discriminate between RNA and DNA, however, they do not provide information about the extract purity (Kieleczawa, 2006). Droplets of water and bubbles inside cuvettes may interfere with the result and any dust particles cause light scatter at 320 nm and can impact absorbance at 260 nm (Avison, 2006).
Generally when the concentration of RNA in the sample is low then, there are more chances of getting errors in the measurement. Therefore when the reading is significantly low then the dilution should be repeated to obtain a higher value. Usually when A260 value is below 0.1 more sample should be added and when higher than 0.5 then it should be diluted (Avison, 2006). In this experiment A260 obtained was within this range which suggests that the dilution was acceptable. As it is sometimes difficult and impractical to quantitate RNA isolated from small samples that will have lower concentrations after dilution, other methods can be used. For example, "fluorescent dyes such as RiboGreen® exhibit a large fluorescence enhancement when bound to nucleic acids and as little as 1 ng/ml of RNA can be detected and quantitated using RiboGreen with a standard fluorometer, fluorescence microplate reader, or filter fluorometer" (Rapley, 2008). This method is relatively insensitive to non-nucleic acid contaminants commonly found in nucleic acid preparations and so the results are usually not subjected to contaminants. However this method has limitations as the RiboGreen reagent can adsorb to the sides of plastic tubes and has to be stored in the dark (Ambion, 2009).
4.3 RNA integrity
The purified sample of RNA was checked for integrity and quality using 1% agarose gel electrophoresis. Although the RNA concentration yields were low, they were still sufficient for agarose gel analysis which showed the two ribosomal RNA (rRNA) bands, 28S and 18S, clearly as labeled (Figure 6). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact (Figure 12A) (Rapley, 2008). Partially degraded RNA exhibit a smeared appearance, with a lack of sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA and completely degraded RNA will appear as a very low molecular weight smear. Different degree of smearing can exist (Figure 12B) indicating RNA degradation. The gel electrophoresis result obtained from this experiment (Figure 6) shows no smearing and the 2:1 ratio (28S:18S) was obtained which indicates that the RNA in the samples is intact.
In this same gel, some other bands appeared before the 28S band in the area where usually DNA bands are seen. These may be DNA bands or contamination from other sources. The storage of sample may affect the quality of the sample, however, this can be excluded as in this project the extracted RNA samples were electrophoresed immediately after extraction and quantitation.
Figure 12B: Different degree of RNA degradation. Adapted from Nature, 2009.
Figure 12A: 28S and 18S from intact RNA. Adapted from Ambion, 2009.
Other methods can be used to investigate RNA quality and integrity, for example, Agilent Bioanalyzer and Biorad Experion, can assess the quality and integrates the quantity of RNA in the samples simply and in a short time. Hence, provides detailed information about the condition of RNA samples together with estimation of RNA concentration and purity. Less quantity of sample is required during analysis of RNA and some have the facilities to display the result in a gel-like image and an electropherogram. However due to the expense of the hardware and consumables they are not used frequently (Rapley, 2008).
4.4 Synthesis of complementary DNA (cDNA) and PCR amplification of (cDNA)
The third experiment was used to check for mRNA expression of defensin proteins using RT-PCR through complementary DNA (cDNA) synthesis. The cDNA was used in RT-PCR reaction as templates for subsequent PCR amplification using specific alpha and beta Defensins primers. Controls were also used to validate the test and help to indicate if any incorrect procedures had had taken place (Farrell, 2005). No template controls were included as negative controls which provide a mechanism to control for external contamination or other factors that can result in a non-specific increase of the expression. The most common housekeeping genes used is Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as positive marker in this study, and Î²-actin, has also been used as a housekeeping indicator which are ubiquitously expressed (Nicot et al., 2005). The purpose of analysing expression of the housekeeping gene is because it is thought to remain constant, so normalizing for variations in processing and qualification of the expression. When there is no signal from the positive control, then this indicates that RNA sample is poor and unable to support reverse transcription or amplification of sample (Farrell, 2005). PCR products showing the positions of the different size classes of DNA fragment were clearly visible under ultraviolet irradiation suggesting that RT and RCR had taken place.
4.4.1 Defensins expression in untreated THP-1
As showed in figure 8, the marker worked well and gave a clear band at approximately (~) 250bp. The product length, bands from untreated cells, were compared with the provided product length of each primer. The untreated THP-1 cells showed no Defensins expression except for Î±-Defensin 1 which gave PCR product length of approximately 110bp. The cells also showed faint PCR DNA band with Î±-Defensins 5 and the remaining bands showed PCR product less than 100bp. These bands, less than 100bp, are mostly primer dimers, the product of nonspecific annealing and primer elongation (Abd-Elsalam, 2003). Defensins were expected to be expressed by the THP-1 cells after the treatment with LPS as noted by experiments which reported that Defensins mRNA expression by monocytes is increased after activation with lipopolysaccharide (LPS) (Duits, 2002). These results suggest that Defensins are not or poorly expressed by THP-1 cells which have not been treated by LPS.
4.4.2 Defensins expression in treated THP-1 cells for 6 hours
LPS treated THP-1 cells, for 6 hours shows PCR bands ranging between 100bp and 200bp. By comparing these results with untreated cells results and the expected values (table below) it could be concluded that when THP-1 cells are treated with LPS it is suggestive that they express Defensins mRNA. However, the expression was not to the expected values except for the Î±-Defensin 5 which gave value of ~190bp that is close to the expected value,179bp.
Expected length in base pairs (bp)
Obtained length in base pairs(bp), approximately (~),
for cells treated for 6hrs
Obtained length in base pairs(bp) for cells treated for 24hrs
Table 4: The expected length of Defensins primers (alpha and beta) in base pairs and the approximate obtained length for the cells treated for 6 hrs and 24 hrs.
The lower expression from these cells might be due to insufficient time for the cells to grow or to absorb the LPS although this is highly unlikely. It might also be that the 6 hours is not the ideal time for THP-1 cells to express Defensins in sufficient amount similarly to the finding of un expressed hBD2 by human tracheobronchial epithelial at 6 hrs (Becker, et al., 2000). In addition, other factors including the quality of enzyme and RNA used for reverse transcription and cDNA synthesis are important issues in order to obtain high yields of quality full length cDNA with full gene representation (Abd-Elsalam, 2003). "The optimal primer sequence and appropriate primer concentration are also essential for maximal specificity and efficiency of PCR product" (Abd-Elsalam, 2003). Since that this experiment was conducted under poorly optimized conditions including the primer used, reagent used, Magnesium level and reverse transcriptase, therefore, the result could be possibly affected.
It is also clear that the DNA PCR bands are a bit smeared which might either be due to technical errors or due to the cDNA product that might be degraded or contaminated. Degraded RNA will not generate full-length cDNA and contamination may inhibit the synthesis of cDNA (Farrell, 2005). Generally when performing PCR amplification, extreme care should be exercised to prevent DNA contamination. The GAPDH, positive control, gave a clear band and the negative control gave no band which are the expected results and could conclude that the overall experiment was approached correctly.
4.4.3 Defensins expression in treated THP-1 cells for 24 hours
Cells when treated with LPS for 24 hours show clearer evidence of expression of the Defensins compared with cells treated for 6 hours. However, the expression was still less than the expected values except for the Î±-Defensin 5 which gave a higher than expected value, ~ 220bp, than at 6 hrs, compared to 190bp. Interestingly the Î±-Defensin 5 was expressed more than other Defensin types on the THP-1 cells either when treated for 6 or 24 hours. This suggests that when THP-1 cells are treated with LPS at 100 ng/ml for 6 and up to 24 hours they can express the Î±-Defensin 5. These findings correlate with the study of time course for hBD2 in human tracheobronchial epithelial (hTBE) stimulated with LPS for different hours. The hBD mRNA was highly expressed at 12 and 24 hours (Becker, et al., 2000). It is also clear that the cells expresses Î²-Defensin 5 to a level, 160, closer to the expected value, 172bp which suggests that it might needs more than 24 hours in order to be expressed as expected. Possibly if THP-1 cells were exposed to LPS for longer time it might result in additional Defensins reaching the expected values of mRNA expression.
4.5 The Î±-Defensin 5 expression in THP-1 cells treated with LPS for 24 hours
A further step was done in order to investigate the expression of Î±-Defensin 5 separately. The PCR product was run in triplicate from each type of THP-1 cells on 1% agrose gel. Looking at the results from the third experiment result, it was expected to get bands which indicate the increase in expression as the cells exposed to LPS for longer time. However, the result obtained from the gel showed no significant difference in the expression between untreated cells and those treated for 6 and 24 hours (Figure 10). This could be due to poor quality of PCR templates, primers, reagents or any technical errors.
The densitometry analysis of the PCR bands from the same gel was carried out in order to obtain statistical values which might help in the investigation of the affect of LPS in the expression of Î±-Defensin 5. The result (Table 3 and Figure 11) showed low expression of Defensin at 24 hours compared to the baseline and insignificant increase after 6 hours of LPS which is opposite to the findings in third experiment as well as other studies that shows increase at 24 hours (Becker, et al., 2000). This might be because PCR is inefficient or low amount of cDNA template was used. It might also be due to the effect of ethedium bromide while staining the gel. It has been reported that gel concentration and gel thickness affect the rate of ethidium bromide staining since both retard the rate of diffusion of the stain (Brown, 2002). Moreover, under-staining can greatly affect the densitometry of small DNA bands, below 500bp (Ehrich, et al., 2007). Therefore, carrying out this experiment in optimal conditions including fresh sample and high quality selected primers could provide a better result in showing the effect of LPS in the expression of Î±-Defensin in THP-1 cells in different periods of time.
The present study is extremely novel because very little has been investigated into Defensins expression in differentiated THP-1 cells. The notable expression is seen with alpha Defensin -5. The other Defensins species were expressed however, not to the expected value which could be related to many factors including the quality of sample, techniques, reagents non specific amplification or dimers or even the cells themselves might not be ideal for expressing these type of Defensins which could not be compared to other studies as Defensins have yet to be reported for expression in THP-1 cell lines.
The alpha Defensin- 5 which shows an increase in the amount of expression after 6 and 24 hours could be the only type of Defensin to be expressed by THP-1 cell lines. These results suggest that the expression of the Defensins are not the same, even when same dose and cells are used. This was also noted by a study which indicated that hBD-4 expression was up-regulated by infection and in response to phorbol 12-myristate 13-acetate, but not in response to other inflammatory factors that up-regulate the expression of hBD-2 or hBD-3 leading to conclude that the induction and antimicrobial spectrum are not same with different hBDs genes (García, et al., 2001). In another study to investigate the patterns of Defensin mRNA expression in malignant epithelial cell lines, A431 and M-Hela, by RT-PCR, showed that HBD-1 gene is expressed only in A431 cells, the expression of HNP-1, HNP-3, HP-4 and hBD-2mRNA was absent in A431 and M-Hela while HD-6m RNA was expressed in both cell lines. It was also indicated, on the same study, that hBD-2 can be induced by challenge with epidermal growth factor (EGF) (Lisovskiy, et al., 2001). These findings, therefore, enhance the findings of this study which suggest that cells only express one type of Defensin species.
The expression of Defensins may also be induced by other components in addition to LPS. A study reported that viral infection can produce human Î²-defensin-2 (HBD2) and HBD3 from the epithelial cells (Klotman, & Chang, 2006). Interestingly it is recently found that their expression is not only restricted to microorganisms. In a study to determine the acute effects of prolonged exercise on salivary levels of defensin Human Neutrophil Peptide 1-3 (HNP1-3) an increase in the concentrations and secretion rates of HNP1-3 following exercise was reported, however their Salivary antibacterial capacity was not changed (Davison, et al., 2009).
Although the dissertation reported that the alpha Defensin -5 seems to be the Defensin type expressed by THP-1 cells, further confirmation tests have to be done in order to confirm and analyse it including cloning and sequencing techniques or by Southern Blotting method.
The method used for investigating the expression of these genes was RT-PCR which can measure gene expression using as few as one cell compared with Northern Blotting which requires too many cells to be used for cell sorting. It is also provides reliable data due to the efficiency of both RT and PCR (Riedy, et al., 1995). Various laboratory methods have been used to detect defensins expression using method other methods than RT-PCR. For instance, ProteinChip® Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry was used to detect Î²-Defensins in biological fluid. This method offers several technical advantages for detection of Defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size (Diamond, et al., 2001).This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevier. In addition, Western Blot and Enzyme-Linked Immunosorbent Assay (ELISA) can also be used to detect human defensins protein expression (Schneider, et al., 2006). Human beta Defensin 2 can be detected in patient's stool samples using Î²-Defensin 2 ELISA kit and Immunohistochemical tests as it is found that Î² -Defensin-2 deficiency can be observed in the intestinal mucous of individuals with Crohn's disease (Langhorst, et al., 2009). In addition micro arrays, and Serial Analysis of Gene Expression (SAGE) have been also used to investigate gene expression.
As Defensins are known to provide wide range of protection, it is therefore, worth to include studies done to investigate their stimulation in different cells and stimulators and correlate them with diseases. For instance, studies were done to analyze the molecular links between infection and the expression of human Î² -Defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. The study concluded that the three microbe-derived molecules LPS, peptidoglycan (PGN), and streptococcal pyrogenic exotoxin B (SpeB) were all capable of inducing or increasing the expression of hBD-1, 2 and 3 in keratinocytes of whole human skin (Sørensen, 2005).
The experimental approach could be enhanced by considering a variety of other concerns.
4.6 Need for further work
In this study the concentration of the RNA was low, therefore it might have been better to have used other methods of obtaining RNA and to confirm it's purity as the PCR technique is prone to contaminations. Another concern would be to try to optimize the PCR reaction as optimization can minimize use of sample and reagents and maximize reproducibility and reliability. The experiment may also be done in different timing i.e. other than 6 and 24 hours which might not reflect changes of gene expression either between these two time points or before or after the designated time points.