Abstract
BACKGROUND
The objectives of this study were to determine 1) oral hBD2 expression in HIV-infected subjects compared to non-HIV controls, 2) the expression of oral hBD2 in HIV-infected subjects with ART compared with those without ART, and 3) factors associated with the expression of oral hBD2.
METHODS
Oral examination and punched biopsy on buccal mucosa were performed in HIV-infected subjects with and without ART, and non-HIV individuals. The expression of hBD2 mRNA was determined by quantitative real-time PCR. Saliva samples of both un-stimulated and stimulated saliva were collected and analyzed for hBD2 levels using ELISA. Student’s t-test and nonparametric multi-way ANOVA test were used for comparison of measurements between or among groups.
RESULTS
One hundred and fifty-seven HIV-infected subjects were enrolled; 99 on ART (age range 23–57 yr, mean 39 yr), 58 not on ART (age range 20–59 yr, mean 34 yr), and 50 non-HIV controls (age range 19–59 yr, mean 36 yr). The most common ART regimen was 2 NRTIs+1 NNRTI. Salivary levels of hBD2 were significantly increased in HIV infection (p< 0.001). The levels of hBD2 in stimulated saliva were also found to be significantly different between HIV-infected subjects who were and were not on ART (p< 0.001). No significant difference was observed with the expression of hBD2 mRNA.
CONCLUSION
Oral innate immunity is affected by HIV infection and use of ART. Salivary hBD2 levels may be the useful biomarkers to monitor those on long-term ART who are at risk of developing oral infections and malignant transformation.
Keywords: ART, defensins, hBD2, HIV, oral health, oral lesion, risk factor, saliva
Introduction
HIV infection appears to have both direct and indirect effects on systemic and local innate immunity leading to the development of oral opportunistic infections and malignancies (1). Antiretroviral therapy (ART) is the standard treatment of HIV-infection, which consists of a combination of three or four drug groups including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INIs), and fusion inhibitors (FIs) (2). It is well accepted that ART contributed to a global reduction of HIV-associated oral lesions (3–5).
However, it has been reported that prolonged treatment by Azidothymidine (AZT, 3′-azido-3′-deoxythymidine, zidovudine) a mainstay of the NRTI used among HIV-infected subjects, potentially causes malignant transformation of oral epithelia (6). Long-term use of this medication may contribute to an increased risk for non-AIDS related malignancies of different organs including oropharynx that has been observed among HIV-infected subjects even in the ART era (7, 8). Thus, it is important to determine if HIV infection and long-term use of ART adversely affects the local innate immune response that put the subjects at risk of developing oral infections and cancers.
Human β-defensins (hBDs) produced by oral epithelial cells act as part of the innate antimicrobial defense (9). They are expressed in buccal mucosa, gingiva, tongue, salivary glands and other oral regions (10–12). HBDs exhibit broad antimicrobial activities and are believed to be involved in the maintenance of homeostasis in the oral cavity (13, 14). Previous studies reported altered expression of hBDs in oral squamous cell carcinoma (OSCC) suggesting their involvement in carcinogenesis (14, 15) and indicate that hBDs may be useful markers of OSCC.
Thus, the objectives of this study were to determine 1) the expression of hBD2 mRNA and its salivary protein levels in HIV-infected subjects compared with non-HIV controls, 2) the expression of hBD2 mRNA and its salivary protein levels in HIV-infected subjects with ART compared with those without ART, and 3) factors associated with the expression of hBD2 mRNA and its salivary protein levels.
Materials and methods
Subjects
A cross-sectional study was performed in HIV-infected subjects who came to receive ART at the Internal Medicine Clinic at Songklanagarind Hospital and Hat Yai Regional Hospital in southern Thailand. The inclusion criteria of subjects enrolled were i) seropositive for antibody to HIV when tested with a particle agglutination test for antibodies to HIV (SERODIA®-HIV, Fujirebio Inc., Shinjuku-ku, Tokyo, Japan) and enzyme-linked immunosorbent assay (ELISA) (Enzygnost® Anti-HIV ½ Plus, Behring, Behringwerke AG, Marburg, Germany), ii) currently taking ART, and iii) willing to participate in the study. The exclusion criteria were i) severely ill HIV-infected subjects who could not cooperate with the procedures of tissue and saliva samples collection, and 2) HIV-infected subjects who were at risk of prolong bleeding. HIV-infected individuals who came to those hospitals but had not yet started ART, and non-HIV infected volunteer were asked to participate as controls.
Ethics
The study protocol was approved by the research committee at the Prince of Songkla University, and at the Ministry of Public Health. All information about the patients and their identity were anonymous. Subjects were given both verbal and written information about the nature of the study and written consent obtained. They were allowed to leave the study at any time during the procedures.
Clinical examination
History taking and oral examination were performed in HIV-subjects with and without ART and non-HIV individuals. Clinical diagnosis of HIV-related oral lesions was made according to the criteria classified by the EC-Clearinghouse (16). The following data were recorded; HIV status, duration of HIV infection, use of ART, duration of ART, CD4+ cell count, HIV viral load, smoking habit and alcohol consumption.
Oral tissue collection
Tissue punch biopsy of 4 mm in diameter was performed under local anesthesia on buccal mucosa of all HIV-infected subjects and non-HIV controls. Immediately after collection, specimen was placed in RNA later (Qiagen Inc., Valencia, CA, USA) and kept at 4 °C until RNA isolation could be performed. RNA samples were used to determine hBD2 mRNA expression.
Saliva collection
Saliva collection was conducted only in the morning between 9:00 a.m.–12:00 a.m. to minimize variation effects. Participants were refrained from eating and tooth-brushing for at least 30 min before saliva collection. All subjects were asked to rinse their mouth with water and spit out, and thereafter swallow before starting the collection procedure comprising of both unstimulated whole saliva using the draining technique and wax-stimulated whole saliva as previously described (17). Saliva samples were kept at −80 °C within 2 h of collection. Samples were later thawed, mixed briefly, and analyzed for hBD2 contents using ELISA (R&D Systems, Minneapolis, MN, USA).
Tissue analysis
Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted from tissues using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s suggestion. cDNA was prepared using 1 μg total RNA with the RETROscript kit (Ambion Inc., Austin, TX, USA). Controls without RT enzyme were included in each experiment. Amplification of the resulting cDNA was carried out with each 50 μl of PCR mixture containing 3 μl cDNA, 1× PCR buffer, 1.5 mM MgCl2, 10 mM dNTP mix, 250 nM each of forward and reverse primers, and 2.5 U of Taq DNA polymerase. Ribosomal phosphoprotein (RPO) was used as a housekeeping control gene to determine the total RNA level. PCR conditions and primer sequences for hBD2 and RPO have been previously described (18).
Quantitative real-time polymerase chain reaction (Q-PCR)
cDNA was analysed using the ABI system (Applied Biosciences, Carlsbad, CA, USA) for quantitative real-time PCR using Brilliant SYBR Green Q-PCR Master Mix (Stratagene, La Jolla, CA, USA). The reaction was set up in a 96-well plate, each well containing 12.5 μl of SYBR Green mix, 2 μl of cDNA, and 2 μM primers. The amplification conditions was initial denaturation at 95 °C for 12 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 57–65 °C for 30 s, and elongation at 72 °C for 60 s. Melt-curve analysis was performed to confirm that the detected signal was that of SYBR Green binding to the expected amplification product and not to the possible primer-dimers. Oligonucleotide primers were designed as previously described (18). In initial experiments, amplification efficiency was determined for all primer pairs. Amplification was performed in duplicate and normalized to the housekeeping gene RPO.
Quantitation of salivary hBD2 level
The levels of hBD2 protein in both unstimulated and stimulated saliva were quantified by ELISA based on matched anti-hBD2 (PeproTech, Rohovot, Israel). Recombinant hBD2 was used as a standard (PeproTech, Rohovot, Israel). Optical density measurements were performed using the Behring ELISA Processor III (Dade Behring Diagnostic Co Ltd., Deerfield, IL, USA). All samples were run in duplicate diluted 1:2.
Statistical analysis
Descriptive statistics were used to analyze breakdown of subjects by status of HIV test and duration of ART received. Chi-square, Student’s t-test, ANOVA, Wilcoxon, Kruskal-Wallis tests were employed to explore possible association between oral hBD2 and HIV/ART status according to the nature of variables. Finally, since the expression of oral hBD2 may be influenced by duration of HIV infection, level of CD4+ cell count, HIV viral load, smoking and drinking behaviors, linear and logistic regression were used to further analyze factors associated with the expression of oral hBD2 mRNA and its salivary protein levels.
Significance level was set at 0.05 for all statistical tests mentioned above. All the analyses were performed using R statistical software (19).
Results
Demographic data and medical status
Tissue and saliva samples were obtained from ninety nine HIV-infected subjects receiving ART (age range 23–57 yr, mean age 39 yr), 58 receiving no ART (age range 20–59 yr, mean age 34 yr), and 50 non-HIV individuals (age range 19–59 yr, mean age 36 yr). All of them were Thai. Most HIV-infected subjects who were on ART received no PI based regimen (n=84, 85%). Different combinations of ART used among HIV-infected subjects were 2NRTIs+1NNRTI (n=82, 83%), 2NRTIs+2PIs (n=7, 7%), 2NRTIs+1PI (n=3, 3%), and others (n=7, 7%). Those who had been taking ART < 3 years were classified as a group with short-term use of ART, and those who had been taking ART for ≥3 years were classified as a group with long-term use of ART, respectively. Various characteristics of the subjects and controls are shown in Table 1.
Table 1.
Demographic data and characteristics of HIV-infected subjects with and without ART and non-HIV individuals
| Variables | HIV-infected subjects
|
non-HIV subjects (n=50) | ||
|---|---|---|---|---|
| No ART (n=58) | with ART (n=99)
|
|||
| Short-term ART (<3 yr) (n=45) |
Long-term ART (≥3 yr) (n=54) |
|||
|
| ||||
| Age | ||||
| Age range (year) | 20–59 | 23–57 | 27–53 | 19–59 |
| Mean age (year) | 34 | 37 | 40 | 36 |
|
| ||||
| Sex | ||||
| Male | 20 (34.5%) | 18 (40%) | 33 (61.1%) | 25 (50.0%) |
| Female | 38 (65.5%) | 27 (60%) | 21 (38.9%) | 25 (50.0%) |
|
| ||||
| Marital status | ||||
| Single | 13 (23.6%) | 5 (15.2%) | 13 (31.7%) | 21 (42.0%) |
| Married | 33 (60.0%) | 17 (51.5%) | 16 (39.0%) | 27 (54.0%) |
| Divorce | 4 (7.3%) | 7 (21.2%) | 7 (17.1%) | 0 (0.0%) |
| Widow | 5 (9.1%) | 4 (12.1%) | 5 (12.2%) | 2 (4.0%) |
|
| ||||
| Highest education | ||||
| Primary school level | 33 (56.9%) | 15 (33.3%) | 12 (22.2%) | 24 (48.0%) |
| Secondary school level | 18 (31.0%) | 16 (35.6%) | 21 (38.9%) | 10 (20.0%) |
| Polytechnic level | 2 (3.4%) | 4 (8.9%) | 10 (18.5%) | 3 (6.0%) |
| University level | 4 (6.9%) | 9 (20.0%) | 9 (16.7%) | 11 (22.0%) |
| Other | 1 (1.7%) | 1 (2.2%) | 2 (3.7%) | 2 (4.0%) |
|
| ||||
| Occupation | ||||
| Employee | 31 (53.4%) | 23 (51.1%) | 21 (38.9%) | 21 (42.0%) |
| Trading | 6 (10.3% | 5 (11.1%) | 2 (3.7%) | 4 (8.0%) |
| Agriculture | 3 (5.2%) | 2 (4.4%) | 1 (1.9%) | 8 (16.0%) |
| Government servant | 0 (0.0%) | 2 (4.4%) | 9 (16.7%) | 2 (4.0%) |
| Student | 1 (1.7%) | 0 (0.0%) | 0 (0.0%) | 8 (16.0%) |
| Others | 14 (24.1%) | 12 (26.7%) | 17 (31.5%) | 7 (14.0%) |
| Unemployed | 3 (5.2%) | 1 (2.2%) | 4 (7.4%) | 0 (0.0%) |
|
| ||||
| Income (Baht)/month | ||||
| < 5,000 | 25 (43.1%) | 18 (40.0%) | 13 (24.5%) | 23 (46.0%) |
| 5,000–10,000 | 26 (44.8%) | 17 (37.8%) | 19 (35.8%) | 22 (44.0%) |
| 10,001–20,000 | 7 (12.1%) | 6 (13.3%) | 11 (20.8%) | 5 (10.0%) |
| 20,001–30,000 | 0 (0.0%) | 2 (4.4%) | 6 (11.3%) | 0 (0.0%) |
| > 30,000 | 0 (0.0%) | 2 (4.4%) | 4 (7.5%) | 0 (0.0%) |
|
| ||||
| Risk group | ||||
| Sex with person with HIV | 43 (74.1%) | 30 (66.7%) | 41 (75.9%) | - |
| Commercial sex workers | 4 (6.9%) | 2 (4.4%) | 3 (5.6%) | - |
| MSM | 3 (5.2%) | 2 (4.4%) | 3 (5.6%) | - |
| IVDU | 2 (3.4%) | 7 (15.6%) | 3 (5.6%) | - |
| Blood transfusion | 1 (1.7%) | 0 (0.0%) | 1 (1.9%) | - |
| Other | 5 (8.6%) | 4 (8.9%) | 3 (5.6%) | - |
|
| ||||
| Duration of HIV infection (year) | ||||
| Mean | 3.8 | 4.8 | 8.8 | - |
| Range | 0.1–16 | 0.4–15 | 3–24 | - |
|
| ||||
| Smoking habit | ||||
| Smoker | 39 (67.2%) | 16 (35.6%) | 18 (33.3%) | 34 (68.0%) |
| Non-smoker | 19 (32.8%) | 29 (64.4%) | 36 (66.7%) | 16 (32.0%) |
|
| ||||
| Alcohol consumption | ||||
| Drinker | 37 (63.8%) | 12 (26.7%) | 13 (24.1%) | 34 (68.0%) |
| Non-drinker | 21 (36.2%) | 33 (73.3%) | 41 (75.9%) | 16 (32.0%) |
|
| ||||
| Presence of HIV-related systemic diseases | ||||
| Yes | 15 (25.9%) | 14 (34.1%) | 9 (18.8%) | - |
| No | 43 (74.1%) | 27 (65.9%) | 39 (81.2%) | - |
|
| ||||
| Oral hygiene | ||||
| Good | 1 (1.7%) | 3 (6.7%) | 0 (0.0%) | 1 (2.0%) |
| Fair | 35 (60.3%) | 23 (51.1%) | 31 (57.4%) | 27 (54.0%) |
| Poor | 22 (37.9%) | 19 (42.2%) | 23 (42.6%) | 22 (44.0%) |
|
| ||||
| Total lymphocyte cell counts (cell/mm3) | ||||
| <1000 | 9 (16.7%) | 12 (26.7%) | 6 (11.1%) | - |
| 1000–2000 | 22 (40.7%) | 14 (31.1%) | 14 (25.9%) | - |
| >2000 | 23 (42.6%) | 19 (42.2%) | 34 (63.0%) | - |
|
| ||||
| CD4 cell count (cell/mm3) | ||||
| Mean | 245.5 | 250.1 | 530.7 | - |
| Range | 5–669 | 9–630 | 74–1,600 | - |
|
| ||||
| Viral load (copies) | ||||
| Mean | 782.6 | 21,560 | 5,627 | - |
| Range | 0–30,100 | 50–750,000 | 50–139,000 | - |
Oral health status and salivary flow rates in HIV-infected subjects with and without ART and non-HIV individuals
Prevalence of oral lesions was found to be statistically significant higher in HIV-infected subjects than non-HIV controls (Chi-square test, p<0.001) (Table 2). Significant difference was also observed with respect to ART status of the subjects. Hyperpigmentation was the most common oral lesion seen in all group of subjects. Oral candidiasis and oral hairy leukoplakia were observed in only two and one HIV-infected subjects who received ART, respectively, whereas no oral warts were observed among the subjects. Periodontal pocket depths and bleeding on probing in HIV-infected subjects were found to be statistically significant difference due to ART status. Salivary flow rates of both unstimulated and stimulated saliva subjects were statistically significant lower in HIV-infected than non-HIV individuals (Chi-square test, p<0.001). Unstimulated salivary flow rates were significantly different between those who were and were not on ART.
Table 2.
Oral health parameters, salivary flow rates and the expression of hBD2 mRNA and its salivary protein levels in HIV-infected subjects with and without ART and non-HIV individuals
| Variables | HIV status | ART status of HIV-infected subjects | |||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| HIV-infected subjects (n= 157) | Non-HIV individuals (n=50) | P-value | No ART (n=58) | Short-term ART (<3 yr) (n=45) |
Long-term ART (≥3 yr) (n=54) |
P-value | |
|
| |||||||
| Presence of oral lesions | |||||||
| Yes | 110 (70%) | 21 (42%) | < 0.001 | 46 (81%) | 26 (57%) | 38 (70%) | 0.029 |
| No | 47 (30%) | 29 (58%) | 11 (19%) | 20 (43%) | 16 (30%) | ||
|
| |||||||
| Presence of periodontal pockets | |||||||
| Yes | 127 (82%) | 41 (85%) | 0.734 | 51 (89%) | 31 (69%) | 45 (85%) | 0.022 |
| No | 28 (18%) | 7 (15%) | 6 (11%) | 14 (31%) | 8 (15%) | ||
|
| |||||||
| Presence of bleeding on probing | |||||||
| Yes | 146 (94%) | 46 (96%) | 0.736 | 57 (100%) | 38 (83%) | 51 (96%) | < 0.001 |
| No | 10 (6%) | 2 (4%) | 0 (0%) | 8 (17%) | 2 (4%) | ||
|
| |||||||
| Salivary flow rates (ml/min) | |||||||
| - Unstimulated saliva | |||||||
| Range | 0.1–0.4 | 0.2–0.6 | < 0.001 | 0.1–0.4 | 0.1–0.3 | 0.1–0.4 | 0.026 |
| Median | 0.2 | 0.4 | 0.2 | 0.2 | 0.3 | ||
| - Stimulated saliva | |||||||
| Range | 0.9–2.2 | 1.4–2.7 | < 0.001 | 0.9–2.3 | 0.7–2.1 | 1.2–2.4 | 0.089 |
| Median | 1.5 | 1.9 | 1.3 | 1.4 | 1.7 | ||
|
| |||||||
| Expression of hBD2 mRNA | |||||||
| Mean (SD) | 176.55 (503.68) | 101.08 (239.87) | 0.639 | 271.60 (713.91) | 141.34 (371.03) | 109.07 (289.37) | 0.558 |
|
| |||||||
| Salivary hBD2 levels (pg/ml) | |||||||
| - Unstimulated saliva | |||||||
| Mean (SD) | 113.90 (101.26) | 47.79 (21.30) | <0.001* | 107.35 (74.89) | 116.04 (134.49) | 118.03 (93.65) | 0.800** |
| - Stimulated saliva | |||||||
| Mean (SD) | 269.34 (202.28) | 156.32 (92.97) | <0.001* | 157.51 (109.08) | 421.33 (208.96) | 248.51 (187.96) | <0.001** |
Student’s t-test or Wilcoxon rank sum test if non-normally distributed
Kruskal-Wallis test
Expression of hBD2 mRNA and its salivary protein levels in HIV-infected subjects with and without ART and non-HIV individuals
Levels of hBD2 protein in both unstimulated and stimulated saliva were significantly increased in HIV-infected subjects compared to non-HIV individuals (p <0.001) (Table 2). However, no significant difference in the expression of hBD2 mRNA was observed between the two groups. Changes in the expression of hBD2 mRNA and its salivary protein levels were observed among HIV-infected subjects who received ART and those who did not. No significant difference was observed in the expression of hBD2 mRNA with respect to the use of ART. In contrast, levels of hBD2 protein in stimulated saliva were significant higher in HIV-infected subjects who were on ART compared to those who did not take the medication (p < 0.001). The levels were significantly increased in those on short-term ART compared to those who did not take the medication. The levels of hBD2 protein in stimulated saliva seemed to be decreased with long-term use of ART. However, no significant difference was observed with the levels of hBD2 protein in unstimulated saliva.
Logistic regression analysis of hBD2 mRNA expression and its salivary protein levels
On logistic regression, the levels of salivary hBD2 protein seemed to be affected by HIV infection. The levels of hBD2 protein in unstimulated saliva of HIV-infected subjects were significantly different when compared with non-HIV individuals (Table 3). No significant difference in the expression of hBD2 was observed with respect to ART status of the subjects.
Table 3.
Effects of HIV infection and long-term use of ART on the expression of hBD2 mRNA and its salivary protein levels based on logistic regression
| hBD2 | Adjusted odd ratios (95% Confidence Interval)
|
||
|---|---|---|---|
| HIV-positive (HIV-negative as reference) | Short-term ART (No-ART as reference) | Long-term ART (No-ART as reference) | |
|
| |||
| Expression of hBD2 mRNA in buccal mucosa >5 | 0.48 (0.19, 1.20) | 0.77 (0.34, 1.77) | 1.37 (0.61, 3.10) |
|
| |||
| Salivary levels of hBD2 protein | |||
| - Unstimulated saliva >50 | 6.02 (2.69, 13.46) | 0.84 (0.32, 2.19) | 1.19 (0.46, 3.09) |
| - Stimulated saliva >100 | 0.87 (0.39, 1.95) | 0.69 (0.29, 1.67) | 1.44 (0.60, 3.47) |
Factors associated with the expression of hBD2 mRNA and its salivary protein levels
Effects of various variables on the expression of hBD2 mRNA and its salivary protein levels are shown in Table 4. The following factors were found to significantly affect the levels of hBD2 protein in stimulated saliva; duration of ART, duration of HIV infection, HIV viral load, and alcohol consumption. However, those factors were not significantly associated with the expression of hBD2 mRNA.
Table 4.
Effects of long-term use of ART and other variables on the expression of hBD2 mRNA and its salivary protein levels in HIV-infected subjects
| Predictors for the expression | Mean difference between groups
|
|||||
|---|---|---|---|---|---|---|
| hBD2 mRNA | Salivary hBD2 protein | |||||
|
| ||||||
| mRNA | P-value* | Unstimulated saliva | P-value* | Stimulated saliva | P-value* | |
|
| ||||||
| Duration of ART | ||||||
| no vs short-term | 130.26 | 0.228 | 1.36 | 0.763 | −263.82 | 0.059 |
| long vs short-term | −32.27 | 0.635 | 10.52 | 0.733 | −172.82 | <0.001 |
|
| ||||||
| Duration of HIV infection | ||||||
| 5–10 vs < 5 years | −150.19 | 0.134 | 8.47 | 0.684 | −103.09 | 0.006 |
|
| ||||||
| CD4 count | ||||||
| <200 vs >= 200 cell/cm3 | −90.37 | 0.316 | 25.79 | 0.225 | 8.50 | 0.468 |
|
| ||||||
| Viral load | ||||||
| >= 50 vs < 50 | −154.40 | 0.063 | −4.69 | 0.914 | 162.11 | <0.001 |
|
| ||||||
| Smoking | ||||||
| Yes vs No | 64.80 | 0.160 | −6.09 | 0.653 | −22.51 | 0.535 |
|
| ||||||
| Alcohol | ||||||
| Yes vs No | −3.10 | 0.713 | −25.8 | 0.397 | −73.30 | 0.046 |
P-value from ANOVA test, adjusted for other factors
Discussion
This study demonstrated that oral innate immunity was affected by HIV infection and use of ART. The expression of hBD2 mRNA was increased in HIV infected subjects compared to non-HIV individuals. The levels of hBD2 protein in both unstimulated and stimulated saliva were significantly increased with HIV infection. The levels of hBD2 proteins in stimulated saliva were also found to be significantly different between HIV-infected subjects who received ART compared to those who were not on the medication.
The findings of the present study suggest that HIV infection may impair the function of oral epithelial cells in innate immunity leading to the alterations of the oral hBD2 expression. It is well accepted that HIV infection causes the impairment of both local and systemic immunity (20). Oral epithelial cells, which are part of the local innate immunity (9), may also be affected by the infection. As a consequence, various oral lesions caused by opportunistic infections and malignancies are observed among HIV infected subjects (21–23).
A recent study by Nittayananta et al (17) revealed that ART significantly decreased salivary flow rates of HIV-infected patients. Those who were on long-term use of ART had a greater risk of developing oral lesions than those with short-term ART (17). Thus, both HIV infection and long-term use of ART may have adverse effects on the systemic and local innate immunity resulting in the recurrence of opportunistic infections and increase the risk of developing oral cancers.
Although the incidence of AIDS-defining malignancies (ADMs) including Kaposi’s sarcoma (KS) and non-Hodgkin’s lymphoma (NHL) has declined significantly in the ART era (24), the incidence of other malignancies not known to be associated with immunosuppression (non-ADMs) including OSCC remains significantly higher than in the general population (25, 26). Potential explanations for the higher non-ADM rates include potential oncogenicity of long-term HIV infection or of long-term ART (25). Thus, HIV-infected subjects who are on long-term ART seem to be at risk of developing OSCC (25).
A previous study by Nittayananta et al (17) reported that both unstimulated and stimulated salivary flow rates of the subjects with ART were significantly lower than that of those without ART. Therefore, prevalence of oral Candida carriage is expected to be high among this group of subjects. Oral Candida carriage has been shown to correlate with presence of oral epithelial dysplasia (27). Chronic infection by Candida may cause malignant transformation resulting in the development of OSCC (28). Because HIV-infected subjects receive ART as a life-long therapy, this group of subjects may be susceptible to chronic candidal infection that could potentially lead to malignant transformation. Thus, further studies should be performed to determine the relationship between salivary hBD2 levels and the risk of developing OSCC in HIV-infected subjects on long-term ART.
Besides protective function, hBDs have evolved to be one of the most potent epithelial differentiation and tumor markers (29). Previous studies have reported altered expression of hBDs in cancers suggesting their involvement in carcinogenesis (14, 15). A study by Joly et al (14) reported that hBD1 and hBD2 mRNA expression was significantly lower in OSCC. In contrast, hBD3 was found to be overexpressed in OSCC at both transcriptional and translational levels compared to healthy oral tissue. These results suggest a putative role for hBDs in carcinogenesis and indicate that hBDs may be useful markers of OSCC. However, the specific role of hBDs in carcinogenesis is unknown.
The present study demonstrated that the levels of hBD2 in stimulated saliva were significantly different between HIV-infected subjects and non-HIV individuals. In addition, changes in the levels of hBD2 proteins in stimulated saliva were also observed with the use of ART. Interestingly, the levels of hBD2 protein in stimulated saliva seemed to be increased with short-term use of ART, but decreased with long-term use of the medication. This finding indicates that oral innate immunity may be adversely affected by HIV infection and long-term use of ART. Thus, this antimicrobial peptide may be the useful biomarker to monitor HIV-infected subjects receiving long-term ART who might be susceptible to malignant transformation of the oral mucosa.
In this study, no significant association between types of ART and the levels of salivary hBD2 proteins was observed. This may be due to the fact that most patients received the same regimen of 2 NRTIs + 1 NNRTI. AZT, a mainstay of the NRTI prescribed for HIV-infected subjects, is incorporated into DNA causing gene mutations (6). Thus, prolonged treatment by this medication potentially causes malignant transformation of oral epithelia (6). In addition, a previous study demonstrated that AZT has genotoxic effects that may lead to genomic instability in cultured cells (6). These genetic changes have been used to predict the risk of malignant transformation of oral epithelia (30). As HIV-infected subjects switch antiretroviral medications frequently, it would be interesting to determine whether long-term use of any specific medications significantly decrease the levels of salivary hBD2.
Besides HIV infection and ART, the present study demonstrated that various factors known to be associated with the development of oral cancers such as alcohol and HIV viral load, have also been shown to significantly affect the expression of hBD2. It is well accepted that alcohol consumption has strong causative links to OSCC (31). Alcohol serves as a mucosal irritant and may activate members of the cytochrome p450 family enzymes, which is known to activate procarcinogens (32). Of interest, these factors were found to be significantly associated with the expression of hBD2 only at the protein level, but not at the mRNA level. This discrepancy may indicate that the expression of hBD2 is mostly affected by those factors at the posttranscriptional level. In addition, HIV infection and ART may alter message translation or interfere with the protein synthesis.
Infection by human papilloma virus (HPV) is also a risk factor for OSCC (33, 34). Our previous study demonstrated that the prevalence of oral HPV type 16 infection was increased in HIV-infected subjects compared to non-HIV individuals, and long-term use of ART did not seem to decrease the infection (35). Thus, further studies should be performed to determine the association between HPV type 16 infection and the expression of hBD2 that may put the subjects taking ART at risk for developing OSCC.
The strength of this study was that the expression of hBD2 was detected at both transcriptional and translational levels by using quantitative real-time PCR and ELISA, respectively. Although a causal role in tumorigenesis has not been established for hBDs, changes in the salivary levels of hBD2 may have great importance in monitoring HIV-infected subjects who are on long-term ART.
The present study had some limitations. It was conducted as a cross-sectional study. Thus, it lacked the information of changes in the expression of hBD2 overtime in those subjects who were on long-term use of ART. Longitudinal studies should be performed in the future to better demonstrate the effects of long-term use of ART on the alteration of hBD2 expression. In addition, further studies should be conducted to assess the changes in other types of hBDs produced by oral epithelial cells.
In conclusion, the present study demonstrated that oral innate immunity was affected by HIV infection and use of ART. The findings suggest that changes in oral hBD2 expression did occur in HIV-infected subjects who were on long-term ART. These alterations may have a role in carcinogenesis and have the potential to be used as surrogate markers of malignant transformation. Further studies should be performed in order to gain the insights into mechanisms how HIV infection and ART alter the expression of hBD2 in oral tissue and saliva.
Acknowledgments
This study was supported by NIH/NIDCR grant R21-DE-018340. Ms. Korntip Amornthatree was a student in the Biomedical Science Graduate Program at the Faculty of Medicine, Prince of Songkla University. Dr. Sineepat Talungchit was a scholar of the Royal Golden Jubilee PhD Program at the Faculty of Dentistry, Prince of Songkla University, Hat Yai, Thailand. The authors wish to thank Dr. Mostafa Nokta and Dr. Isaac Rodriguez-Chavez for encouragement in initiating this project, Prof. Dr. Sutep Jaruratanasirikul, Prof. Dr. Khachornsakdi Silpapojakul, Assoc. Prof. Dr. Panthip Chayakul, and Dr. Ampaipith Nilmanat for access to HIV-infected patients, and Ms. Welawee Chaiyaphan for technical assistance. The sincere thanks are extended to staffs at Songklanagarind Hospital and Hat Yai Regional Hospital, and subjects in the study for their help and cooperation.
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